Lecture-1

Introduction, Carbohydrates – importance &classification

Biochemistry, as the name implies, is the chemistry of living organisms.
Living organisms, whether they are microorganisms, plants or animals are basically
made up of the same chemical components. Biochemistry is the study of the way in
which these components are synthesized and utilized by the organisms in their life
processes. It bridges the gap between the conventional chemistry and biology.
In other words, life is nothing but thousands of ordered chemical reactions or
chemistry is the logic of all biological phenomena.
History of biochemistry
During 17th and 18th centuries, important foundations were laid in many fields of
biology.
 The 19th century observed the development of concepts - the cell theory by
Schleiden and Schwann, Mendel’s study of inheritance and Darwin’s
theory of evolution.
 The real push to biochemistry was given in 1828 when total synthesis of urea
from lead cyanate and ammonia was achieved by Wohler who thus initiated
the synthesis of organic compound from inorganic compound.
 Louis Pasteur, during 1857, did a great deal of work on fermentations and
pointed out the central importance of enzymes in this process.
 The breakthrough in enzyme research and hence, biochemistry was made in
1897 by Edward Buchner when he extracted enzyme from yeast cells in
crude form which could ferment a sugar molecule into alcohol.
 Neuberg introduced the term biochemistry in 1903.
The early part of 20th century witnessed a sudden outburst of knowledge in chemical
analysis, separation methods, electronic instrumentation for biological studies (X-
ray diffraction, electron microscope, etc) which ultimately resulted in understanding
the structure and function of several key molecules involved in life processes such as
proteins, enzymes, DNA and RNA.
 In 1926, James Sumner established the protein nature of enzyme. He was
responsible for the isolation and crystallization of urease, which provided a
breakthrough in studying of the properties of specific enzymes.
 The first metabolic pathway elucidated was the glycolytic pathway during the
first half of the 20th century by Embden and Meyerhof. Otto Warburg, Cori
 and Parnas also made very important contributions relating to glycolytic
pathway.
 Krebs established the citric acid and urea cycles during 1930-40.
 In 1940, Lipmann described the central role of ATP in biological systems.
 The biochemistry of nucleic acids entered into a phase of exponential growth
after the establishment of the structure of DNA in 1953 by Watson and Crick
followed by the discovery of DNA polymerase by Kornberg in 1956.
From 1960 onwards, biochemistry plunged into an interdisciplinary phase sharing much
in common with biology and molecular genetics.
 Frederick Sanger’s contributions in the sequencing of protein in 1953
and nucleic acid in 1977 were responsible for further developments in the
field of protein and nucleic acid research.
The growth of biochemistry and molecular biology was phenomenal during the past two
decades.
 The development of recombinant DNA research by Snell and coworkers
during 1980 allowed for further growth and emergence of a new field, the
genetic engineering.
Thus there was progressive evolution of biology to biochemistry and then to molecular
biology, genetic engineering and biotechnology.

CARBOHYDRATES

Compounds with empirical formula, (CH2O)n, were called as carbohydrates

(hydrates of carbons). With the discoveries of many diverse carbohydrates it was noticed
that many, but not all, carbohydrates have the above empirical formula; some also
contain nitrogen, phosphorus or sulfur. There are some carbohydrates (derivatives) that
do not possess (CH2O)n. On the other hand, there are a few non-carbohydrate
compounds like lactic acid with empirical formula (CH2O)n. Hence, carbohydrates are
chemically defined as polyhydroxy aldehydes or ketones, their derivatives and
their polymers.
Occurrence and importance
 The carbohydrates comprise one of the major groups of naturally occurring
biomolecules. This is mainly because; the light energy from the sun is converted
into chemical energy by plants through primary production and is transferred to
sugars and carbohydrate derivatives.
 The dry substance of plants is composed of 50-80% of carbohydrates. The
structural material in plants is mainly cellulose and related hemicelluloses.
 Starch is the important form of storage polysaccharide in plants.
 Pectins and sugars such as sucrose and glucose are also plant constituents.
 Many non-carbohydrate organic molecules are found conjugated with sugars in
the form of glycosides.
 The carbohydrates in animals are mostly found in combination with proteins as
glycoproteins, as well as other compounds.
 The storage form of carbohydrates, glycogen, found in liver and muscles, the
blood group substances, mucins, ground substance between cells in the
form of mucopolysaccharides are few examples of carbohydrates playing
important roles in animals.
 Chitin found in the exo-skeleton of lower animals, is a polymer of N-acetyl
glucosamine.
Carbohydrates are also universally found in other polymeric substances. For
example,
o Fats are fatty acid esters of a sugar alcohol, glycerol.
o Ribose and deoxyribose are constituent of nucleic acids.
Moreover, in all living forms, the energy needed for mechanical work and chemical
reactions are derived from carbohydrates.
 Adenosine triphosphate and related substances that contain ribose as a
constituent are key substances in energy storage and transfer.
 The carbon skeletons of almost all organic molecules are derived from
carbohydrates.
 Besides, the carbohydrates are the basic raw material of many important
industries including sugar and sugar products, starch products, paper and
wood pulp, textiles, plastics, food processing and fermentation.
CLASSIFICATION
Carbohydrates are classified into three major groups:
 Monosaccharides
 Oligosaccharides
 Polysaccharides
Classification of carbohydrates

Monosaccharides (Simple Oligosaccharides Polysaccharides

sugars) (Glycans)
Low molecular weight Contain 2-10 Contain many
carbohydrates and monosaccharides joined by monosaccharides joined by
cannot be hydrolysed glycosidic bonds. Low glycosidic bonds. They can
further molecular weight be hydrolysed by enzymes
carbohydrates which can be or acids.
hydrolysed by enzymes or
acids to yield
monosaccharides
Crystalline, soluble in water, Powdery or crystalline, Insoluble in water,
and sweet in taste. soluble in water tasteless, linear or
and sweet in taste branched

Classified into triose, Classified into disaccharide, Classified into

tetrose, pentose, hexose trisaccharide, homoglycans and
and heptose depending tetrasaccharide and heteroglycans depending
upon the number of carbon pentasaccharide depending upon the kind of
atoms. They may be either upon the number of monosaccharides present.
aldoses or ketoses monosaccharides they Depending upon the
depending upon whether contain. function, they are classified
they contain a free aldehyde as storage and structural
or ketone group, polysaccharides.
respectively
All monosaccharides are Some of them are reducing Non reducing in nature and
reducing in nature and some of them are non give deep blue (amylose)
reducing in nature. or red colour (amylopectin)
with iodine.

Monosaccharides:
Monosaccharides are the simplest form that cannot be hydrolyzed further into
smaller units. They are classified into a) simple monosaccharides b) derived
monosaccharides
Simple monosaccharides are further classified
 based on the type of functional group and
 the number of carbon atoms they possess.
Derived monosaccharides include the derivatives of simple monosaccharides such as
oxidation products, reduction products, substitution products and esters
Classification of monosaccharides
Monosacchar No. of Aldose Ketose Occurrence
ides carbon
atoms
Simple
Triose 3 D-Glycerose Dihydroxy Intermediary meta-
acetone bolites in glucose
metabolism
Tetrose 4 D-Erythrose D-Erythrulose
Pentose 5 D-Ribose D-Ribulose Ribose is a constituent
of nucleic acid
L-Arabinose - Occurs in oligosac-
charides
D-Xylose D-Xylulose Gum arabic, cherry
gums, wood gums,
proteoglycans
Hexose 6 D-Glucose D-Fructose Fruit juices and cane
sugar
 D-Galactose - Lactose, constituent
of lipids
D-Mannose - Plant mannosans
and glycoproteins
Heptose 7 - D- Intermediate in
Sedoheptulose carbohydrate
metabolism
Derived
Deoxysugar 5 2-Deoxyribose - DNA
6 L-Rhamnose - Component of cell wall
Aminosugar 6 D-Glucosamine - A major component of
polysaccharide found in
insects and
crustaceans (chitin)
Polyol 6 Sorbitol - Berries
6 Mannitol - Commercially prepared
from mannose and
fructose
Aldonic acid 6 Gluconic acid - -
Uronic acid 6 Glucuronic acid - Constituent of
chondroitin sulfate
6 Galacturonic - Constituent of pectin
acid

Aldaric acid 6 Glucaric acid - Oxidation product of

(Saccharic glucose
acid)
6 Mucic acid - Oxidation product of
galactose

Oligosaccharides:
They contain two to ten monosaccharide units joined by glycosidic linkages that
can be easily hydrolyzed.
Polysaccharides:
 They are high molecular weight polymers containing more than ten
monosaccharides. They are either linear or branched in structure.
Polysaccharides are further classified based on
a) the kind of monosaccharides present as:
 Homopolysaccharides when made from a single kind of monosaccharide.
Eg starch, cellulose, inulin, glycogen, chitin
 Heteropolysaccharides are made up of more than one type of
monosaccharides. Eg. Hemicellulose, Mucopolysaccharides – Chondroitin
sulphate, Hyaluronic acid Heparin and Keratan sulphate
b) functional aspect as:
 Storage Polysaccharide eg. Starch, glycogen, inulin, Galactomannan
 Structural Polysaccharide eg.Cellulose, Chitin, Hemicellulose
 Lecture: 2
OCCURRENCE AND STRUCTURE OF MONOSACCHARIDES
The simplest monosaccharide that possesses a hydroxyl group and a carbonyl
group with an asymmetric carbon atom is the aldotriose -glyceraldehyde. (A carbon is
said to be asymmetric if four different groups or atoms are attached to it. The carbon is
also called as a chiral center).
 Glyceraldehyde is considered as a reference compound and it exists in two optically
active forms, D and L
The two families of monosaccharides, D-and L occur based on the configuration of D
and L glyceraldehydes. In general, the D-family of sugars occur in nature.
 For monosaccharides with two or more asymmetric carbons, the prefixes D or L refer
to the configuration of the penultimate carbon (i.e, the asymmetric carbon farthest
from the carbonyl carbon).
 If the hydroxyl group on the penultimate carbon is on the right-hand side of the
carbon chain when the aldehyde or ketone group is written at the top of the formula
it belongs to the D family and if on the left hand side it belongs to L family. The D
or L has nothing to do with optical activity. D sugars may be dextro- or levorotatory.
 The important monosaccharides containing aldehyde group belonging to the D family
are
 the aldotetrose - D-erythrose
 the aldopentoses - D-ribose, D-arabinose and D-xylose
 the aldohexoses - D-glucose, D-mannose and D-galactose
 The important monosaccharide belonging to the L-family is L-arabinose.
 The important ketoses are
 Ketotriose - dihydroxy acetone (It is optically inactive since there is no asymmetric
carbon);
 the ketotetrose - D-erythrulose;
 the ketopentoses - D-ribulose and D-xylulose
 the ketohexose - D-fructose

Cyclic structure of Monosaccharides:

The monosaccharides exist either in cyclic or acyclic form. There are many
evidences to show that the pentose and hexose monosaccharides are present in cyclic
form. The evidences are 1. Glucose and other aldoses fail to give the Schiff 's test for
aldehydes. 2. Solid glucose is quite inert to oxygen whereas aldehydes are easily auto-
oxidizable. 3. Glucose and other aldoses do not form bisulfite or aldehyde ammonia
compound. 4.Glucose pentaacetate does not react with hydroxylamine. 5. Presence of
two forms of glucose with different physical and chemical properties. 6. X-ray analysis
definitely proves the existence of the ring structure and also the size of the ring. 7.
Mutarotation.

 When an aldehyde or a ketone group is present in a molecule that also

possesses hydroxyl groups, an intramolecular arrangement may occur to form a
hemiacetal or a hemiketal, respectively. This intramolecular hemiacetal or hemiketal is
the basis for the cyclic structure of the sugars. Hence, Haworth (an English chemist)
proposed a cyclic hemiacetal structure that accounts completely for its chemical
properties

Cyclic Fischer Projection of -D- Haworth Projection of -D-

Glucose Glucose

 Two types of ring structures are possible, the five-membered furanose and the six-
membered pyranose ring if the carbonyl group interact with hydroxyl group. These
names are derived from the parent compounds 'furan' and 'pyran'.
 The most common ring structure for aldohexoses is the pyranose ring structure
that involves the first carbonyl carbon and the hydroxyl group attached to the fifth
carbon.
 The furanose ring structure is formed by interaction of carbonyl carbon with the
hydroxyl group attached to the fourth carbon. This furanose form is less stable than
the pyranose strucure and is not very common among aldohexoses.
 Very seldom is a seven-membered ring formed.
 Fructose exists in solution and in compounds as a furanose; however, in the
crystalline state only the pyranose ring is believed to exist.
 Ribose occurs as the furanose structure in many important biological compounds.
 A new asymmetric carbon is introduced in the molecule due to this rearrangement.
As a result of this new asymmetric centre, two isomers are formed.
 Isomeric forms of monosaccharides that differ only in their configuration about the
hemiacetal or hemiketal carbon atom are called anomers and the carbon is referred
as anomeric carbon.
 When the newly formed hydroxyl group in C1and the ring are on the same
orientation, it is  - anomer.
 When the newly formed hydroxyl group in C1 and the ring are on opposite
orientation, it is  - anomer.
While writing the cyclic form (Haworth) of monosaccharides it is sometimes
difficult to judge whether an OH group should be above or below the plane of the ring.
A few rules can be followed for writing Haworth's structure for carbohydrates.
 Write the oxygen at the upper right hand corner of the ring structure (pyranose) and
the carbons clockwise around the ring. At the fifth carbon it is necessary to rotate the
bond to 90o to make the ring closure. For the D-family of sugars, it is customary to
write the terminal CH2OH above the plane of the ring.
 If the hydroxyl group or hydrogen atom occurs on the right-hand side of the
carbon chain in the linear structure it is placed below the plane of the ring in the
cyclic structure.
 Conversely, if the hydroxyl group or hydrogen atom is on the left-hand side of
the carbon chain, it is placed above the plane of the ring in the structure formula

Conformational structure:
The six-membered pyranose ring is not actually planar, as suggested by Haworth, but
assume usually the stable chair conformation.
 Chair form of -D-Glucose

 The substituents are represented either axially or equatorially.

 The axial substituents project almost parallel with the vertical axis through the
ring
 The equatorial substituents project roughly perpendicular to this axis.
 Substituents in the equatorial positions are less sterically hindered by
neighbouring substituents. Conformations with their bulky substituents in
equatorial positions are favoured.
Derived monosaccharides
The important functional groups present in monosaccharides are hydroxyl and
carbonyl groups. The hydroxyl group forms esters, usually with phosphoric acid or is
replaced by a hydrogen or amino group. The carbonyl group undergoes reduction or
oxidation to produce number of derived monosaccharides.
a) Deoxysugars
 In sugars, the hydroxyl group is replaced by a hydrogen to produce deoxy sugars
(devoid of oxygen).
 The important deoxy sugar is 2-deoxy ribose that occurs in deoxy ribonucleic
acid.
 Other important deoxy sugars are L-fucose and L. rhamnose. The substitution of
the hydroxyl group at C-6 of L. galactose or L.mannose with hydrogen produces
fucose or rhamnose respectively.
 L-fucose occurs in the cell wall polysaccharides namely hemicelluloses and L-
rhamnose occurs in pectic polysaccharides namely rhamnogalacturonan. These
deoxy sugars are also found in the complex oligosaccharide components of
glycoproteins and glycolipids.

b) Amino sugars
 The hydroxyl group, usually at C-2, is replaced by an amino group to produce
aminosugars such as glucosamine, galactosamine and mannosamine.
 The amino group may be condensed with acetic acid to produce N-acetyl amino
sugars, for example, N-acetyl glucosamine.
  This glucosamine derivative is important constituent of many structural polymers
(chitin, bacterial cell wall polysaccharides etc.)

c) Polyols (alditols)
 Both aldoses and ketoses are reduced to polyhydric alcohols (polyols) when
treated with enzymes, sodium amalgam, and hydrogen under high pressure with
catalyst or sodium borohydride.
 Each aldose yields the corresponding alcohol upon reduction
 A ketose forms two alcohols because of the appearance of a new asymmetric
carbon atom in the process.
By this reduction process, the following sugars give rise to their respective alcohols
under specified conditions.
Glucose Sorbitol
Fructose Sorbitol and mannitol
Mannose Mannitol
Glyceraldehyde Glycerol
Erythrose Erythritol
Ribose Ribitol
Galactose Dulcitol

 Polyols occur in many plant products.

 Sorbitol was first isolated from the berries of mountain ash (Sorbus
aucuparia).
 Commercially sorbitol is manufactured by the hydrogenation of glucose.
 Mannitol occurs in many terrestrial and marine plants.
 Potential food applications of polyols include confectionery products,
bakery products, deserts, jams and marmalade.
 Sorbitol is an excellent moisture conditioner and is used in pharmaceutical
preparations such as elixirs and syrups.
 Sorbitol, as a humectant in creams and lotions helps to stabilize the water
content, providing better moisture control.
 The use of sorbitol or xylitol in toothpaste and mouthwashes is highly
desirable.
d) Oxidation products
When aldoses are oxidized under proper conditions with different types of
oxidizing agents, three types of acids are produced, namely aldonic acids, uronic
acids and aldaric acids or saccharic acids.
Aldonic acid
 Oxidation of an aldose with bromine water at neutral pH converts the
aldehyde group (C1) to a carboxyl group yields Aldonic acid.
 Hydrobromous acid formed by the reaction of water with bromine acts as an
oxidizing agent.
 Ketoses are not readily oxidized by bromine water.
 Aldoses are not only oxidized by bromine water but also by the alkaline iodine
solution.
Uronic acid
 When aldoses are oxidised with hydrogen peroxide (H2O2) uronic acids are
formed.
 In this reaction only primary alcohol group(C6) is oxidized to carboxyl group,
whereas the aldehyde group remains unchanged.
 Uronic acids are constituents of pectic polysaccharides.
Aldaric or saccharic acid
 When aldoses are oxidised with nitric acid, saccharic acids are formed.
 Both aldehyde (C1)and primary alcohol groups (C6) are oxidised to carboxyl
groups.
 Glucose on oxidation with nitric acid produces glucaric or glucosaccharic
acid.
 The aldaric acid produced from galactose is called as mucic acid.
 Lecture 3
STRUCTURE OF DISACCHARIDES & POLYSACCHARIDES
Composition, sources and properties of common disccharides
Disaccharides Constituent Linkage Source Properties
monosaccharides
Reducing disaccharides
Maltose -D-glucose+ (14) Germinating Forms osazone with
-D-glucose cereal and phenylhydrazine.
malt Fermentable by enzyme
maltase present in yeast.
Hydrolysed to two
molecules of D-glucose.
Undergoes mutarotation.
Lactose -D-glucose+ (14) Milk. In trace It shows reactions of
-D-glucose amounts it reducing sugars including
can be seen mutarotation.
in urine Decomposed by alkali.
during Not fermentable by yeast.
pregnancy Hydrolysed to one
molecule of galactose and
one molecule of glucose
by acids and the enzyme
lactase.
Non-reducing disaccharides
Sucrose -D-glucose+ ,(12) Sugar beet, Fermentable. Hydrolysed
-D-fructose sugarcane, by dilute acids or enzyme
sorghum and invertase (sucrase) to one
carrot roots molecule of glucose and
one molecule of fructose.
Relatively stable to
reaction with dilute alkali.
Trehalose -D-glucose+ ,(11) Fungi and It is hydrolysable by acids
-D-glucose yeast. It is to glucose with difficulty.
 stored as a Not hydrolysed by
reserve food enzymes.
supply in
insect's
hemolymph

The oligosaccharides commonly encountered in nature belong to disaccharides.

 The physiologically important disaccharides are maltose, lactose, trehalose and
sucrose.
 Disaccharides consist of two monosaccharides joined covalently by an O-
glycosidic bond.
 The hydroxyl group formed as a result of hemiacetal formation is highly reactive
when compared to other hydroxyl groups.
 This hydroxyl group present in one monosaccharide reacts with any one of the
hydroxyl groups attached to C-1, C-2, C-3, C-4, or C-6 of another
monosaccharide to produce 1→1, 1→2, 1→3, 1→4, and 1→6 linked
disaccharides.
 When only one anomeric carbon is involved in glycosidic bond formation,
reducing disaccharides are formed.
 If both anomeric carbon atoms of monosaccharides are involved in glycosidic
bond formation that results in the formation of a non-reducing disaccharides such
as trehalose (aldosyl-aldosyl disaccharide) or sucrose (aldosyl-ketosyl
disaccharide)'.
 In the case of reducing disaccharides, one end of the molecule having free
anomeric carbon is called reducing end and the other end, where the anomeric
carbon is involved in glycosidic bond, is called as non-reducing end
Reducing disaccharides
Maltose
 Maltose is a disaccharide made up of two glucose residue joined by a
glycosidic linkage between C-1 of one glucose residue and C-4 of the other.
 The configuration of the anomeric carbon of glucose involved in the linkage is
and hence the glycosidic linkage is (1→ 4).
 The anomeric carbon atom of the second glucose is free and therefore maltose is
a reducing sugar.
  The second glucose residue is capable of existing in or configuration
 Maltose has been recorded occasionally in plants. It is usually obtained as a
product of the enzyme hydrolysis of starch during germination or malting
process.

Maltose

Lactose
 Lactose is a reducing disaccharide found only in milk.
 It is made up of galactose at the non-reducing end and glucose at the reducing
end.
 They are connected by a (1→ 4) linkage

Lactose

Non-reducing disaccharides
Trehalose
  Trehalose, a non-reducing disaccharide., occurs as a major constituent of the
circulating fluid (hemolymph) of insects and serves as an energy storage
compound.
 It is also present to a limited extent in the fat body of a variety of insects.
 It gives twice the amount of energy as that of glucose and at the same time
maintains the osmotic balance.
 It has been described as an important adaptation of insects engaged in flight.
 The anomeric carbons of both glucose moieties are involved in the formation of
glycosidic bond.
Sucrose
 Sucrose, a sugar of commercial importance, is widely distributed in higher plants.
 Sugarcane and sugar beet are the sole commercial sources.
 It is made up of glucose and fructose.
 The anomeric carbon atom of glucose (C-1) and fructose (C-2) are involved in
linkage and is therefore a non-reducing disaccharide
 Sucrose is a major intermediate product of photosynthesis and it is the
principal form in which sugar is transported from the leaves to other portions of
plants via their vascular systems.

Sucrose

Invert sugar

 The hydrolysis of sucrose when followed polarimetrically the optical rotation

changes from positive (dextro-) to negative (levo-).
 The dextrorotatory sucrose on hydrolysis yield levorotatory mixture of glucose
and fructose.
  The levorotaion is due to the presence of fructose which is by itself more
levorotatory (-92 ) than dextrorototary glucose (+52.2).
 This phenomenon is called inversion and the mixture of glucose and fructose is
called invert sugar.
 This reaction is catalysed by the enzyme invertase.
 Invert sugar is more sweeter than sucrose.
 Honey contains plenty of invert sugar and therefore is very sweet.

Sucrosyl oligosaccharides

 The degree of polymerization (DP) of sucrosyl oligosaccharides normally

ranges from 3 to 9.
 Though sucrose is found at higher concentration in all plants, members of the
sucrosyl oligosaccharides occur at least in traces in each plant family.
 The main accumulation of surosyl oligosaccharides is found in storage organs
such as roots, rhizomes and seeds.
 The important members of sucrosyl oligosaccharides are raffinose (DP-3),
stachyose (DP-4), verbascose (DP-5) and ajugose (DP-6).
 All sucrosyl oligosaccharides are non-reducing in nature .

Raffinose

 It occupies the second position next to sucrose in abundance in the plant

kingdom.
 Raffinose occurs only at low concentration in the leaves of leguminous
plants, but accumulates in the storage organs such as seeds and roots.
 Most of the leguminous seeds contain these oligosaccharides in large
amounts.
 Bengal gram has higher amounts of raffinose.
 Red gam and green gram have significantly high amounts of verbascose
and stachyose than Bengal gram and black gram.
 These sucrosyl oligosaccharides are responsible for flatulence following
the consumption of these legumes.
 It serve as reserve material.
  It also contributes to frost resistance

Polysaccharides
The polysaccharides found in nature either serve a structural function (structural
polysaccharides) or play a role as a stored form of energy (storage polysaccharides).

Storage polysaccharides

 Starch, galactomanans and inulin are important storage polysaccharides in

plants.

Starch
 The principal food-reserve polysaccharide in the plant kingdom is starch.
 It forms the major source of carbohydrate in the human diet.
 Starch has been found in some protozoa, bacteria and algae. But the major
source is plants where it occurs in the seeds, fruits, leaves, tubers and bulbs in
varying amount from a few percent to over 74%.
 Starch is an alpha-glucan that has structurally distinct components called
amylose and amylopectin.
 A third component referred as the intermediate fraction has also been identified
in some starches.
 Starch molecules are organized into quasicrystalline macromolecular aggregates
called granules.
 The shape of the granules are characteristics of the source of the starch.
 The two components, amylose and amylopectin, vary in amount among the
different sources from less than 2% of amylose in waxy rice or waxy maize to
about 80% amylose in amylomaize.
 The majority of starches contain 15 to 35% of amylose.
 The ratio of amylose and amylopectin is a function of the enzymes, granulosis
bound starch synthase (GBSS) and soluble starch synthase (SSS).
 GBSS is able to synthesise amylose in a form that is not a substrate for
branching enzyme to form amylopectin.
  Waxy mutants containing only amylopectin lack the GBSS but still contain
soluble starch synthase.

 Amylose is made up of - D-glucose units linked mostly in a linear way by

4 linkages
 It has a molecular weight of 150,000 to 1,000,000 depending on its
biological origin.
 It has been shown that amylose has some elements of nonlinearity.
 It consists of a mixture of linear molecules with limited, long-chain
branching involving 1 -6 linkages.
 The branches contain several hundred glucose residues.
 Amylose gives a characteristic blue color with iodine due to the ability of
the iodine to occupy a position in the interior of a helical coil of glucose
units.
 Pure amylose binds 20% of iodine at 20 C

Amylopectin
 Amylopectin is a branched, water-insoluble polymer comprised of thousands
of D-glucose residues.
 The main chain of amylopectin consists of D-glucose residues joined by
(1  4) glycosidic bonds.
 Side chains of glucose residues are attached to the main chain by (1 6)
glycosidic bonds.
 Each chain contains 15-25 glucose residues joined by (1->4) bonds.
 It contains 94-96% 1 4 and 4-6% - 1 6 linkages.
 The molecular weight of amylopectin is in the order of 107 - 108.
 Robin and co-workers have proposed a model for amylopectin
 In this model, A and B chains are linear and have degree of polymerization as 15
and 45 respectively.
 The B chain form the backbone of the amylopectin molecule and extend over two
or more clusters.
 Each cluster of A chain are primarily responsible for the crystalline regions within
the granule.
 The intercrystalline regions occur at regular intervals (60 - 70 A) containing the
majority of - 1 - 6 linkages.
 The amylopectin molecule is 100 - 150 A in diameter and 1200-4000 A long.
 Within the granule, amylose may be located between amylopectin molecules
and associated with the linear regions of the amylopectin molecule.
 Amylopectin produces a purple to red color with iodine.
Inulin
 Inulin is a non-digestible fructosyl oligosaccharide found naturally in more than
36000 types of plants.
 It is a storage polysaccharide found in onion, garlic, chicory, artichoke,
asparagus, banana, wheat and rye.
 It consists of mainly,if not exclusively, of - 2->1 fructosyl-fructose links
 A starting glucose moiety can be present,but is not necessary.
 Inulin is asoluble fibre that helps maintain normal bowel function,decreases
constipation,lowers cholesrerol and triglycerides.
 It is used for fat replacement and fibre enrichment in processed foods.

Structural polysaccharides
Cellulose

 Cellulose is the most abundant organic substance found in nature.

 It is the principal constituent of cell walls in higher plants.
 It occurs in almost pure form (98%) in cotton fibres and to a lessor extent in flax
(80%), jute (60-70%), wood (40-50%) and cereal straws (30-43%).
 It is linear, unbranched homoglycan of 10,000 to 15,000 D-glucose units joined
by -1 4 linkages
 The structure of cellulose can be represented as a series of glucopyranose rings
in the chair conformation.
  The most stable conformation for the polymer is the chair turned 180 relative to
the adjacent glucose residues yielding a straight extended chain.
 Celluose molecules within the plant cell walls are organized into biological units
of structure known as microfibrils.
 A microfibril consists of a bundle of cellulose molecules arranged with its long
axis parallel to that of the others.
 This arrangement permits the formation of intramolecular hydrogen bonding
between the hydroxyl group of C-3 of one glucose residue and the pyranose ring
oxygen atom of the next glucose residue.
 This hydrogen bond impart a double bond character to the glycosidic bond and
impedes the rotation of adjacent glucose residues around the glycosidic bond.
 Within the microfibril, the adjacent cellulose molecules are linked by
intermolecular hydrogen bond between C-6 hydroxyl group of one molecule and
the glycosidic bond oxygen atom of adjacent cellulose molecule
 The cross section of the microfibril consists of a central crystalline core of about
5–30 nm short diameters.
 The central crystalline core contains around 50-100 cellulose molecules which
are arranged in perfect three dimensional array and exhibits a crystalline
structure.
 Surrounding this crystalline core is a region of paracrystalline matrix which
contains about 100 polysaccharide molecules of cellulose and hemicellulose
 This region does not have perfect three-dimensional order and water molecules
are able to penetrate the paracrystalline region but not the crystalline core.

Chemical properties of carbohydrates

Monosaccharides
Reactions of monosaccharides are due to the presence of hydroxyl (-OH) and
the potentially free aldehyde (-CHO) or keto ( >C=O) groups.
Reaction with alkali
Dilute alkali
 Sugars in weak alkaline solutions undergo isomerization to form 1,2-enediol
followed by the formation of a mixture of sugars.
Strong alkali
Under strong alkaline conditions sugar undergo caramelization reactions.
Reducing property of sugars

 Sugars are classified as either reducing or non-reducing depending upon the

presence of potentially free aldehyde or keto groups.
 The reducing property is mainly due to the ability of these sugars to reduce metal
ions such as copper or silver to form insoluble cuprous oxide, under alkaline
condition.
 The aldehyde group of aldoses is oxidized to carboxylic acid. This
reducingproperty is the basis for qualitative (Fehling's, Benedict's, Barfoed's and
Nylander's tests) and quantitative reactions.
 All monosaccharides are reducing. In the case of oligosaccharides, if the
molecule possesses a free aldehyde or ketone group it belongs to reducing
sugar (maltose and lactose).
 If the reducing groups are involved in the formation of glycosodic linkage., the
sugar belongs to the non- reducing group (trehalose, sucrose, raffinose and
stachyose).

Reaction with phenylhydrazine

 When reducing sugars are heated with phenylhydrazine at pH 4.7 a yellow

precipitate is obtained.
 The precipitated compound is called as osazone. One molecule of reducing
sugar reacts with three molecules of phenylhydrazine.
 D-mannose and D-fructose form same type of osazone as that of D-glucose
since the configuration of C-3, C-4, C-5 and C-6 is same for all the three sugars.
 The osazone of D-galactose is different.
 Different sugars form osazone at different rates. For example, D-fructose forms
osazone more readily than D-glucose.
 The osazones are crystalline solids with characteristic shapes, decomposition
points and specific optical rotations.
 The time of formation and crystalline shape of osazone is utilized for identification
of sugars.
  If methyl phenylhydrazine is used instead of phenylhydrazine in the preparation
of osazone, only ketoses react.
 This reaction serves to distinguish between aldose and ketose sugars.

Reaction with acids

 Monosaccharides are generally stable to hot dilute mineral acids though ketoses
are appreciably decomposed by prolonged action.
 Heating a solution of hexoses in a strong non-oxidising acidic conditions,
hydroxyl methyl furfural is formed.
 The hydroxymethyl furfural from hexose is usually oxidized further to other
products When phenolic compounds such as resorcinol, -naphthol or anthrone
are added, mixture of coloured compounds are formed
 The molisch test used for detecting carbohydrate in solution is based on this
principle.
 When conc. H2SO4 is added slowly to a carbohydrate solution containing -
naphthol, a pink color is produced at the juncture.
 The heat generated during the reaction hydrolyse and dehydrate it to produce
furfural or hydroxymethyl furfural which then react with -naphthol to produce
the pink color.
 Lecture 4
Muta rotation, optical activity and physical properties of sugars
A. Isomerism
In organic chemistry, isomerism is defined as the existence of more than one
compound with the same molecular formula. A close observation of the structure of
monosaccharides (hexoses) indicate that they possess same molecular formula
(C 6 H 12 O 6 ) but with different physical and chemical properties. There are different types
of isomerism
• D-glucose and D-fructose differ in the position of carbonyl group (aldehyde and
ketone group). These two compounds are functional isomers.
• Another type of isomerism exhibited by compounds possessing asymmetric
carbon atom like monosaccharides, is stereoisomerism. These stereoisomers
differ in the spatial arrangement of atoms or groups.There are two types of
stereoisomerisms - geometrical and optical isomerism.
 Geometrical isomers (cis-trans) differ in the spatial arrangement of atoms
across a double bond. Geometrical isomerism is not noticed among
carbohydrates.
 Optical isomers differ in the arrangement of atoms around an asymmetric
carbon atom. The number of possible optical isomers can be calculated using the
formula 2n where n=number of asymmetric carbon atoms. For example, glucose
contains four asymmetric carbon atoms and the possible optical isomers of
glucose are 24 = 16.
Epimers, enantiomers and diastereomers:
 Epimers are monosaccharides differing in configuration around a single carbon
atom other than the carbonyl carbon. e.g. Mannose and glucose are epimers
with respect to carbon 2. Galactose and glucose are epimers with respect to
carbon 4.
 Enantiomers are non- superimposable mirror images of each other. They differ
in the ability to rotate the plane polarized light. A solution of one enantiomer
rotates the plane of such light to the right, and a solution of the other to the left.
D-glucose and L-glucose are examples of enantiomers.
 Diastereomers are stereoisomers that are not mirror images of each other. D-
glucose, Dmannose, D-galactose and other members of aldohexose are
diastereoisomers.
B. Optical activity
A ray of ordinary light vibrates in all directions at right angles to the direction in
which the ray is travelling. When this light is passed through a Nicol prism, the emerged
light vibrates in only one direction and such light is called as a 'plane polarized light '
When a beam of plane polarized light is passed through a sugar solution, that is
optically active, the plane-polarized light will be rotated either to the right (clockwise) or
to the left (anticlockwise).
• When the plane polarized light is rotated to the right, the compound is
dextrorotatory and is written as (+).
• If the plane polarized light is rotated to the left, the compound is levorotatory (-)

Optical activity is measured using polarimeter. Optical activity varies with the
concentration of the sugar solution and length of the polarimeter tube where sugar
solution is placed.
Specific rotation (α) of a sugar molecule is calculated by the formula :

Observed rotation
( α ) = -----------------------------------------------------------------
Length of tube (dm) x concentration
where T= temperature and D = D line of spectrum.

The specific rotation of some important sugars are given below:

D - glucose (dextrose) + 52.2 D - fructose (levulose) -92.0 D - galactose + 80.5
D – mannose + 14.6 L - arabinose + 104.5 Sucrose + 66.5

C. Mutarotation

 Mutarotation refers to the change in optical rotation when an aqueous sugar

solution is allowed to stand.
 Sugars having potential free aldehyde or keto group exhibit mutarotation.
 Many sugars exist in two crystalline forms. For example, when D-glucose is
dissolved in water and allowed to crystallize out by evaporation of water, one form
of D-glucose is obtained. If D-glucose is crystallized from acetic acid or pyridine,
another form of D-glucose is obtained. These two forms exhibit different physical
and chemical properties.
 A freshly prepared aqueous solution of α-D glucose has a specific rotation of
+113o. If the solution of α- D-glucose is allowed to stand, the specific rotation
changes to +52.2o.
 Similarly, a fresh solution of β- D-glucose has a specific rotation of +19o which
changes to +52.2o on standing.
 This change in optical rotation is called mutarotation. On standing in solution, the
hemiacetal ring opens and reformed to give a mixture of - and 
-D-glucose
having a specific rotation of +52.2o.
 Lecture 5
Chemical properties of carbohydrates
Monosaccharides
Reactions of monosaccharides are due to the presence of hydroxyl (-OH) and
the potentially free aldehyde (-CHO) or keto ( >C=O) groups.

Reaction with alkali

Dilute alkali
• Sugars in weak alkaline solutions undergo isomerization to form 1,2-enediol
followed by the formation of a mixture of sugars.

Strong alkali
Under strong alkaline conditions sugar undergo caramelization reactions.

Reducing property of sugars

• Sugars are classified as either reducing or non-reducing depending upon the
presence of potentially free aldehyde or keto groups.
• The reducing property is mainly due to the ability of these sugars to reduce
metal ions such as copper or silver to form insoluble cuprous oxide, under
alkaline condition.
• The aldehyde group of aldoses is oxidized to carboxylic acid.
• This reducing property is the basis for qualitative (Fehling's, Benedict's, Barfoed's
and Nylander's tests) and quantitative reactions.
• All monosaccharides are reducing. In the case of oligosaccharides, if the
molecule possesses a free aldehyde or ketone group it belongs to reducing
sugar (maltose and lactose).
• If the reducing groups are involved in the formation of glycosodic linkage.,
the sugar belongs to the non- reducing group (trehalose, sucrose, raffinose and
stachyose).

Reaction with phenylhydrazine

• When reducing sugars are heated with phenylhydrazine at pH 4.7 a yellow

precipitate is obtained.
 • The precipitated compound is called as osazone.
• One molecule of reducing sugar reacts with three molecules of phenylhydrazine.
• D-mannose and D-fructose form same type of osazone as that of D-glucose
since the configuration of C-3, C-4, C-5 and C-6 is same for all the three sugars.
• This reaction serves to distinguish between aldose and ketose sugars.

Reaction with acids

• Heating a solution of hexoses in a strong non-oxidising acidic conditions,
hydroxyl methyl furfural is formed.
• The hydroxymethyl furfural from hexose is usually oxidized further to other
products When phenolic compounds such as resorcinol, -naphthol or
anthrone are added, mixture of coloured compounds are formed
• The molisch test used for detecting carbohydrate in solution is based on this
principle.
• When conc. H 2 SO 4 is added slowly to a carbohydrate solution containing -
naphthol, a pink color is produced at the juncture.
• The heat generated during the reaction hydrolyse and dehydrate it to produce
furfural or hydroxymethyl furfural which then react with 
-naphthol to produce
the pink color.
Lecture 6
Lipids - introduction, importance and classification
Occurrence and importance
 The word lipids is derived from the Greek word 'lipos' meaning fat.
 Lipids are chemically heterogenous group of compounds that are insoluble in
water but soluble in non-polar solvents such as chloroform.
 Lipids occur in plants and animals as storage and structural components
 Structural lipids present in animals and plants are in the form of meat and
vegetables respectively.
 Storage fats occur in milk and adipose tissue of farm animals and in seed oils
 Fats supply over twice as much energy per unit weight as proteins or
carbohydrates.
 Lipids are anhydrous due to non-polar nature and represent more energy than
carbohydrates which are heavily hydrated due to polar nature.
 The presence of lipids in diet contributes considerably to palatability.
 Lipids contribute palatability in two ways. They induce olfactory responses,
namely, taste in the mouth and aroma through nose.
 Secondly, they contribute to the texture of food and is responsible for the mouth-
feel.
 Lipids also supply the essential fatty acids which are not synthesised in human
beings but are essential for growth.
 Lipids are essential for the effective absorption of fatsoluble vitamins A, D,
E and K from intestine.
 Many enzymes require lipid molecules for maximal activity. Examples are
microsomal enzyme, glucose 6-phosphatase and mitochondrial enzyme, -
hydroxybutyrate dehydrogenase.
 Adrenal corticosteroids, sex hormones and vitamin D3 (Cholecalciferol) are
synthesized from lipid derivative- cholesterol.
 Much of the lipid of mammals is located subcutaneously and acts as insulation
against excessive heat loss to the environment.
 The subcutaneous lipid deposits also insulate the important organs against
mechanical trauma.
 Classification
Lipids are broadly classified into simple, compound and derived lipids

Classification of Lipids
Lipids
Simple Lipids Compound Lipids Derived Lipids
Esters of fatty acids with glycerol Esters Containing chemical Substances derived from
and monohydric alcohols. groups in addition to alcohol simple and compound lipids
and fatty acids. by hydrolysis. Alcohols, fatty
acids, aldehydes, ketones,
sterols and hydrocarbons.
Depending upon the constituent Depending upon the chemical
alcohols they are further subdivided groups they are further
into fats or oils and waxes. subdivided into phospholipids,
glycolipids, sulpholipids and
lipoproteins.
Fats, also termed as Phospholipids contain
triacylglycerols are esters of fatty phosphate group. Phopholipids
acids with glycerol e.g. Plants- are further grouped as
vegetable oils; Animals-ghee and glycerophospholopids e.g.,
butter Lecithin if the constituting
alcohol is glycerol or as
sphingophospholipids if the
alcohol is sphingosine e.g.,
sphingomyelin.
Waxes are esters of fatty acids and Glycolipids contain hexose
alcohols other than glycerol e.g., units preferably galactose
Plant wax-carnauba wax; alongwith fatty acids and
alocohol e.g. Cerebrosides.
Insect wax-beeswax; Plant sulpholipids contain
sulfated hexose with fatty acids
and alcohol
Animal wax – lanolin Lipoproteins contain protein
subunits along with lipids.
Depending upon density and
lipid compound they are further
classified as VLDL, LDL and
HDL.
 SEED PRODUCTION IN MAIZE

Maize is common millet of India with wider industrial and household utility. It
is used a feed, food and raw material in soft drink industry. Botanically it is known
as Zea mays and belongs to the family poaceae.

Floral biology

Botanical name : Zea mays

Chromosome number : 2n=20

Botanical Family : Poaceae

Inflorescence : Panicle cob, as the crop is monoceious in nature

Type of flowers : Female : Cob (axillary inflorescence in the middle

portion of plants)
Male : Tassel (terminal inflorescence)

Husk : Enlarged leaf sheaths from each node, forming a

protective covering around the inflorescence.

Pollination : Cross pollination

Special character : Protandry

Flowering pattern : Top to bottom (Tassel) Bottom to top (Cob)

Anthesis : Pollen shedding begins 1 to 3 days before the silk

emerge from the cob.

Fertilization : Within 12 to 18 hrs after silk emergence

The entire silk is receptive. Silk will be pinkish
and sticky at the beginning (receptive) after
fertilization it will be chocolate / brown colour.
No. of pollen in tassel : 2,50,00,000

Pollen viability : 12-18h

Silk receptive : 8-10 days

Male flower anthesis : 6.00 am to 8.00 a.m

Duration of flowering : 2-14 days

Tassel Cob

Husk Silk

Seed
Types and Methods of seed production in maize
In maize, open pollinated varieties, synthetics, composites and hybrids are
available.
a. Open pollinated varieties
Raise the varieties under isolation of 400 m in foundation seed stage and 200 m
in certified seed stage and allow the plants to openly pollinate among
themselves and set seed.
b. Synthetics
In cross pollinated species, a variety obtained by in mating in all possible
combinations, a number of lines (>5) that combine well with each other. COBC 1
(Baby corn).
c. Composite varieties
These are produced by open pollination among a number of outstanding strains
usually not selected for combining ability with each other e.g. K1, Jawahar,
Vikram, Sona, Amber, CO 1 and Kisan.
d. Inbreds
It is relatively true breeding strain resulting from repeated selfing (5 times.)

Varietal seed production technique

Open pollination under isolation is the common method of varietal seed

production.

Stages of seed multiplication

In maize seed (varieties composites and synthetics) is multiplied adopting

three generation system, as breeder seed, foundation seed and certified seed as
the crop is highly cross pollinated crop , where the chances for genetic
contamination is high.

Popular varieties

In Tamil Nadu, CO1, K1, COH3, COH4, are the popular varieties for grain
purpose, while African tall is a fodder maize.COBC1 is a variety identified for salad
purpose.

Season

The best season for production is June - July, November- December and
January – February and the flowering should not coincide either with rain or high
RH and the maturation should coincide with dry weather. The temperature of 37°C
is favourable for better seed setting.

Land requirement

The land required for open pollinated variety, composites and synthetics
should be fertile and problem soils will lead to low pollen fertility and will adversely
affect the quality and the seed set will be poor. The previous crop should not be the
same crop to avoid the occurrence of volunteer plants and if to be the same crop it
has to be the same variety and should be certified and has to be accepted for
certification. The field should not have any volunteer plants.
Isolation distance and Modification of isolation distance
Composite, Synthetics and OPV = (FS:CS 400 : 200 m)
Differential blooming dates are permitted for modifying isolation distance provided
5.0% or more of the plants in the seed parent do not have receptive silks when
more than 0.50% of plants in the adjacent field (s) within the isolation distance are
shedding pollen.

Distances less than 200 meters may be modified by planting border rows of
male parent, if the kernel colour and the texture of the contaminant are the same
as that of seed parent. The number of border rows shall be determined by the size
of the field and isolation distance from the contaminant.

Selection of Seed

For production of foundation seed, breeder seed is used as the base

material, while for certified seed, foundation seed should be used as the base
material. The seed used should be from authenticated source with tag and bill. The
required seed rate will be 20kg /ha or 8kg/ acre.

Pre sowing seed treatment

The seeds are given with any one of the seed treatment or in combination.
Seeds are soaked in 2% KH 2 PO 4 for 16h with a seed to solution ratio of 1:0.06
and are dried back to their original moisture content of 8-9% .This management
could be used both for dryland agriculture as well as gardenland.

Seeds are also treated with 5% carbofuran 3G to protect the seed from
shoofly infection. Seed treatment with chlorpyriphos @4 ml /kg is also
recommended against the attack by shootfly.

Seeds are dry dressed with bavistin @2g/kg of seed to protect against seed
borne pathogens and soil borne pathogen.

Seeds are also treated with azospirillum @50g/kg of seed to fix atmospheric
N. Any one of these treatment or combination of treatment is adopted for better
productivity.

Seeds are also treated with polycoat @ 3g/kg of seed diluted in 5ml of water
to invigourate the seed towards better marketability and production. Pink coloured
polycoat performed better than other colour polymers. On adoption of sequence of
treatment physiological should be followed with physical seed treatment.

Sowing

The seed are sown at a spacing of 45 x 10 cm or 60 x 20 cm at a depth of 2-

4 cm based on the specific features of the variety. Nursery production will not be
suited to this crop. In the main field seeds are sown either in ridges and furrows or
under beds and channels. The seedlings are thinned and gap filled should be done
7-8 days after sowing.

Plant spacing Row spacing

Seed rate
Varieties : 20 kg /ha

Nutrient application

At last ploughing apply 12.5 tonnes of compost per hectare

Fertilizers(varieties) 150:75:75
 Basal 40:75:40 NPK kg/ha
 1st top 20 DAS 50:0 :0 kg/ha
 2nd top 40 DAS 60:0:35 kg/ha.

Micronutrients
2% DAP is sprayed at 50% flowering stage to enhance uniform flowering and
increased seed set
If Zn deficiency is found apply 20 kg of zinc sulphate / ha.
If Fe deficiency is found apply 12.5 kg /ha micronutrient mixture

 The crop is mostly affected by micronutrient deficiencies by N,P,Mg,Mn,Zn,Fe

and K. Apply 12.5kg of micro nutrients in furrows and the mixture in the soil.

Weeding

Application of atrazine @ 500g per ha as pre-emergence herbicide control

the growth of weeds upto 20-25 days.(If pulses is used as intercrop do not use
atrazine) One hand weeding at 17-18 days after sowing keep the field free of
weeds.Weeding after boot leaf stage is not economical and shade will also
minimize the weed flora . On organic production, 2 hand weeding at seedling stage
and other at boot leaf formation will keep the field weed free.

Irrigation

The crop should be irrigated once in 10-15days for enhanced seed set and
formation of bolder grains. The critical stages of irrigation are primordial initiation
stage, vegetative stage , flowering, milky and maturation stage. If the irrigation is
withheld in these stages seed set will be poor and seed size will be reduced.
Pest and disease management

Shoot fly Monocrotophos 0.03%

Stem borer Rogar 0.3% / Carbaryl 50 WP 1kg.per heactre on 20th day
Lesion nematodes Carbofuran 3 G@30kg./ha.in seed holes at the time
of sowing.
Downy mildew Mancozeb @ 1kg/ha.
Leaf spot Mancozeb or captan @ 1kg/ha

Cob borer Apply carbaryl 10% dust @ 25kg/ha. At milky stage repeat it
15 days thereafter.(50 lts. Spray fluid per ha)

Roguing

It is specific to seed crop and is done from seedling stage to harvesting stage
based on the phenotypic characters. Off types can be identified through stem
colour,plant structure, number of leaves ,auricles, nodal colour, tassel colour,sheath
colour ,grain colour etc. The field standard for seed crop is as follows

Seed Certification

Number of Inspections

A minimum of two inspections shall be made at flowering and another during

flowering.

Field Standards

General: Maize field should be isolated from contaminants as follows

Contaminants Minimum distance(meters)

Foundation Certified
stage stage
Fields of other varieties 400 200
Fields of same variety not confirming to varietal 400 200
purity requirements for certification and teosinte
 In maize hybrid alone increasing the border row and minimising the isolation
is permitted

Specific standard: These are verified at the final inspection

Factor Maximum
permitted (%)

FS CS

Off types plants that have shed are or shedding pollen at anyone 1.0 1.0
of the inspections during flowering when 5%or more of the plants
in the seed field have receptive silks .

Preharvest sanitation spray

Spraying of endosulphan @ 0.07% and bavistin@10g /lit 10 days prior to

harvest prevent the seed weevil ( Sitophilus oryzae) infestation at storage.

Seed maturation

•14-20 DAA milky stages (starch in fluid stage)

• 35 DAA : Soft dough stage

• 45 DAA : Glazad dough stage

• 55 DAA : Ripe dough stage

Symptom of Physiological maturation

• Cob sheath turn straw yellow colour

• The funicular degeneration

• Formation of dunken layer

• Moisture content of seed 35%

 Matured cob Dunken layer

Harvesting

The crop attains physiological maturity 30-35 days after 50% flowering and
the seed moisture at this stage will be around 25-30%. The crop is harvested as
cob harvesting when the sheath of cob dries and attains straw yellow color. The
crop is harvested as once over harvest for seed purpose.

Dehusking

After harvest manually the sheath are removed, which is known as

dehusking.
Cob sorting

Based on the kernel arrangements on the shank as irregular discoloured,

diseased and ill filling the Cobs are sorted out and cobs with characteristic kernel
colour and shank colour and regular row arrangements are selected for seed
purpose. The kernel discolouration should not 10% for certification.

Zenia and metazenia

The discolouration in cobs may be due to disease infection or genetic

contamination. The effect of foreign pollen on kernel colour is known as Zenia,
metazenia effect which causes genetic contamination in the seed lot. Zenia is the
effect of foreign pollen of same generation and metazenia is the effect of foreign
pollen in next generation.
Shelling

The cobs are dried under sun and threshed with fliable stick for extraction of
seeds the moisture content of seed at the time of threshing will be 15-18%.On
large scale production cob shellers are used, but care should be given to avoid
mechanical damage, which in turn will reduce the seed quality and storability.

Drying

The seeds are dried to 8 to10 % moisture content either under sun or
adopting mechanical driers for long term storage as the seeds is orthodox in
nature.

Processing

Mechanical grading can be done with cleaner cum grader, which will remove
the undersized immature and chaffy seeds .The middle screen size should be
18/64” round perforated sieves. The size can vary depending on the variety from
14/64 to 20/64 inch round perforated sieves.

Seed treatment

The seeds are infested with several storage pests, to protect against these
pests the seeds are given protective treatment with bavistin @2g/kg of seed with
carbaryl @200mg/kg of seed as slurry treatment. Bifenthrin @5mg /kg of seed or
diflubenzuran @ 200 ppm per kg of seed or imidachlopride @ 3 ml per kg of seed is
also recommended for better seeds storage .

Seed packing

Seeds are packed in gunny bag for short term storage while in HDPE and
polylined gunny bag for long term storage.

Storage

The treated seed can be stored up to 12 months provided the seeds are not
infected with storage pests. Seed can be stored up to 3 years if the seeds are
packed in moisture containers and are stored at low temperature .The godown
should be kept clean as the possibility of secondary infestation with Trifolium (red
flour weevil ) is much in these crop. The major problem in storage is incidence of
grain weevil which will powder the seed material in a short period.

Seed yield: 3 to 4.0 tones

Seed standard

The processed seed should have the following seed quality characters both
for certification and labeling.

A. Seed ears inspected after harvest shall not contains in excess of 1.0% of offtype
ears including the ears with off-coloured kernels.

B. Shelling

Shelling of the seed ears is to be done after obtaining approval from the
Certification Agency

Factor Standards for each class

FOUNDATION CERTIFIED
Pure seed ( maximum) 98.0% 98.0%
Inertmatter(maximum) 2.0% 2.0%
Other crop seed (maximum) 5/kg 10/kg
Weed seed None None
Other distinguishable varieties based on 10/kg (by 20/kg (by
kernel colour and texture (max) number) number)
Germination ( Minimum) 90% 90%
Moisture (maximum) 12.0% 12.0%
For vapour proof container (maximum) 8.0% 8.0%

Mid storage correction

The seeds loose their quality during storage due to deterioration and pest
infestation, when the germination falls below 5-10 % of the required standard the
seeds are imposed with midstorage correction, where the seeds are soaked in
double the volume of 10-4 M solution of potassium dihydrogen phosphate
(3.6mg/lit of water) for 6 hours and the seeds are dried back to original moisture
content (8-9%).
 Lecture - 7 -9
Plant fatty acids
 Fatty acids are carboxylic acids with hydrocarbon chains of 2 to 36 carbons.
 More than 200 fatty acids have been isolated from higher and lower plants.
 Of these, only a few are present in large quantities in most plant lipids. These are
referred as major fatty acids.
 Fatty acids present in smaller proportions are called as minor fatty acids.
 Major and minor fatty acids are usually biosynthesised by analogous pathways.
 Fatty acids that occur only in a few plant species are called as unusual fatty
acids.

Major fatty acids

 The major fatty acids are saturated or unsaturated with an unbranched carbon
chain.
 The saturated fatty acids are lauric (dodecanoic), myristic (tetradecanoic),
palmitic (hexadecanoic) and stearic (octadecanoic) acid
 The unsaturated fatty acids are oleic (9-octadecenoic), linoleic (9,12-
octadecadienoic) and -linolenic (9,12,15- octadecatrienoic) acid.
 They are usually found in the lipids from all parts of plants
 The structure of fatty acids are written as a symbol of two numbers separated by
a colon: the first number denotes the carbon atoms in the chain and the second
number denotes the number of unsaturation centres.
 The positions of double bonds are specified by superscript numbers following
(delta).
9, 12
 Thus 18:2 ( ) indicates an eighteen carbon fatty acid with two double bonds
between C-9 and C-10, and between C-12 and C-13.
 The double bonds of all naturally occurring unsaturated fatty acids are in the cis
configuration.
 The non-polar hydrocarbon chain accounts for the poor solubility of fatty acids in
water.

Minor fatty acids

The fatty acid composition of cow's and goat's milk are characterised by a high
content of short and medium chain saturated fatty acids.
 Some minor fatty acids
Common name Carbon skeleton Systematic name
Butyric 4:0 Butanoic
Caproic 6:0 Hexanoic
Caprylic 8:0 Octanoic
Capric 10:0 Decanoic

Unusual fatty acids

 The unusual fatty acids are found only in few individual species or genus or a
whole family.
 Castor bean (Ricinus communis) seed oil is rich in ricinoleic acid (90%) which
is 12-hydroxy oleic acid CH3(CH2)5-CH(OH)-CH2-CH=CH-(CH2)7-COOH.
 Rape seed (Brassica napus) is rich in erucic acid (cis-13-docosenoic acid
CH3(CH2)7-CH=CH-(CH2)11-COOH).
 Hydnocarpic and chaulmoogric acids are found in chaulmoogra oil which is
used in the treatment of leprosy.
Essential fatty acids
 Human body is unable to synthesise all fatty acids found in the body.
 Those fatty acids that are not synthesised in the body but required for normal
body growth and maintenance are called as essential fatty acids.
 These fatty acids are to be supplied through diet.
 Linoleic and linolenic acids are essential fatty acids
 The longer chain fatty acids can be synthesised by the body from dietary linoleic
and -linolenic acids.
 Arachidonic acid is essential but it can be synthesised by the body from
linolenic acid. It is also present in the meat
 Linoleic acid is grouped under n-6 family because the 6th carbon from methyl
end possesses the double bond.
 Other fatty acids that are synthesised in the body from linoleic acid such as
linolenic and arachidonic acids also belong to n-6 family.
 -Linolenic acid belongs to n-3 family and is an essential fatty acid.
 The third carbon from the methyl end possess the double bond
  The organs and tissues that perform the more routine and generalized functions
such as adipose tissue, liver, muscle, kidney and the reproductive organs tend to
have membranes in which n-6 family of polyunsaturated fatty acids predominate.
 Nervous tissue and retina of the eye have a larger proportion of the longer chain
acids with 5 or 6 double bonds predominantly of the n-3 family.
 Fish oils and spirulina are rich in fatty acids of n-3 family.
 Arachidonic acid serves as precursor for the synthesis of
prostaglandins,thrombaxanes and prostacyclins.
 These fatty acid derivatives are called as 'eicosanoid' meaning 20 C compounds.
 The main source of these eicosanoids are the membrane phospholipids from
which they are released by the action of phospholipase-A.
 Phosphatidyl inositol which contains a high concentration of arachidonic acid in
carbon-2 of glycerol provides a major store of eicosanoid precursors.
 Phosphatidyl inositol is an important constituent of cell membrane phospholipids;
upon stimulation by a suitable animal hormone it is cleaved into diacylglycerol
and inositol phosphate, both of which act as internal signals or second
messengers

Simple lipids
 Lipids containing only fatty acids and glycerol or long chain alcohols
(monohydric) are called as simple lipids which include fats, oils and waxes.
Fats and oils
 Triacylglycerols are the simplest lipids constructed from fatty acids and glycerol.
 They are also referred as triglycerides, fats or neutral fats.
 Triacylglycerols are composed of three fatty acids esterified to the three
hydroxyl groups of glycerol

 When all the 3 fatty acid molecules are of the same kind the triacylglycerol is said
to be simple triacylglycerol.
 Mixed triacylglycerol possesses two or more different fatty acids.
 Triacylglycerol that are solid at room temperature are called as fats
 Liquid triacylglycerols are called as oils.
 Neutral fats or oils are mostly composed of mixed triacyl glycerol.
  Fats are usually rich in saturated fatty acids and the unsaturated fatty acids
predominate in oils.
 Most oil-producing plants store their lipids in the form of triacylglycerols.

Storage fats or oils

 Triacylglycerols are widely distributed in the plant kingdom. They are found both
in vegetative as well as reproductive tissues.
 Triacylglycerols are normally stored in the endosperm of the seed although
some plants store appreciable quantities of fat in the fleshy fruit mesocarp, for
example, avocado.
 Some plants like the oil palm, store oils in both the mesocarp (Palm oil) and the
endosperm (Palm kernel oil).
 The oil present as droplets in the cytoplasm of the seed cells.
 These droplets are called as oil bodies and are surrounded by a membrane
composed of phospholipids and protein.
 Most of the common edible oils (groundnut, sunflower, gingelly, soybean,
safflower, rice bran) contain limited number of the common fatty acids such as
palmitic, stearic, oleic, linoleic and linolenic acids.
 Palm kernel and coconut oils contain higher amount of medium chain saturated
fatty acids.
 Seed oils contain small amount of phospholipids, carotenoids, tocopherols,
tocotrienols and plant sterols depending on the species of plant and degree of
processing.
Structural or hidden fats in plants
 The leaves of higher plants contain upto 7% of their dry weight as fats;
 Some of them are present as surface lipids, the others as components of leaf
cells, especially in the chloroplast membrane.
 The fatty acid composition of plant membrane lipids is very simple.
 Six fatty acids- palmitic, palmitoleic, stearic, oleic,linoleic and -linolenic
generally account for over 90% of the total fatty acids.
Waxes
 Waxes are esters of long-chain saturated and unsaturated fatty acids with
long chain alcohol.
  The carbon number of fatty acids vary from 14 to 34 and that alcohol from 16 to
30.
For example, beeswax is an ester of palmitic acid with a 30 carbon alcohol, triacontanol
 Waxes are the chief storage form of metabolic fuel in marine phytoplanktons.
 Biological waxes find a variety of applications in the pharmaceutical, cosmetic
and other industries.
 Lanolin from lamb's wool, beeswax, carnauba wax, spermaceti oil from whales
are widely used in the manufacture of lotions, ointments and polishes.
 Waxes are not easily hydrolysed like fats or digested by lipases.

Liquid wax - Jojoba oil

 About 50% of the seed dry weight of jojoba consists of a liquid wax which is
unique in the plant kingdom and is similar to sperm whale oil.
 The wax is made up of straight chain esters with an average total chain length of
42 carbons
 Jojoba wax has a wide range of industrial uses including cosmetics,
pharmaceuticals, extenders for plastics, printers ink, gear oil additives and
various lubricants.
 The oil is highly stable and can be stored for years without becoming rancid.
Cuticular wax
 The outermost surface of the cell walls of epidermal cells are covered with a
hydrophobic cuticle which contains wax called cuticular wax.
 The main components of cuticular waxes are hydrocarbon (odd chain alkanes)
and its derivatives, wax esters, free aldehydes, free acids, free alcohols and
other components like mono esters of phenolic acids and aliphatic alcohols.
 The main function of the cuticular wax is to reduce the excessive losses and
gains of water by the underlying tissue.
 It also helps in protecting the tissues from chemical, physical and biological
attack.

Compound lipids
Compound lipids contain certain chemical groups in addition to alcohol and fatty
acids.
 These group of lipids include glycerophospholipids, sphingo phospholipids,
glycolipids, sulpholipids and lipoproteins.
Glycerophospholipids
 The important structural lipid in biological membrane is glycero phospholipid
which contains glycerol, fatty acids phosphoric acid and a nitrogenous base.
 The general structure of a glycerophospholipid is given below

 Without alcoholic residue (X), it is called as phosphatidic acid

 Depending on the alcoholic residue attached to phosphatidic acid, they are
named as
i. Phosphatidyl choline (lecithin)
ii. Phosphatidyl ethanolamine (cephalin)
iii. Phosphatidyl serine
iv. Phosphatidyl inositol
v. Phosphatidyl glycerol (which include monophosphatidyl glycerol
and diphosphatidyl glycerol or cardiolipin).
Phosphatidyl choline (lecithin)
 Lecithin contains glycerol, fatty acids, phosphoric acid and a nitrogenous
base, choline
 Lecithins are widely distributed in the membranes of cells having both metabolic
and structural functions.
 Dipalmityl lecithin is a very effective surface active agent preventing
adherence due to surface tension of the inner surfaces of the lungs.
 Most phospholipids have a saturated fatty acid in the C1 position but an
unsaturated fatty acid in the C2 position.
Phosphatidyl ethanolamine (cephalin)
 The cephalin differs from lecithin only in the nitrogenous group where
ethanolamine is present instead of choline
Phosphatidyl serine
 The hydroxyl group of the amino acid L-serine is esterified to the phosphatidic
acid
Phosphatidyl inositol
 Phosphatidyl inositol is an important constituent of cell membrane
phospholipids;
  upon stimulation by a suitable animal hormone it is cleaved into diacylglycerol
and inositol phosphate, both of which act as internal signals or second
messengers.
Phosphatidyl glycerol and diphosphatidyl glycerol (Cardiolipin)
 Cardiolipin is a phospholipid that is found in membranes of mitochondria.
 It is formed from phosphatidylglycerol
Sphingophospholipids
 The phosphate and fatty acids are attached to the alcohol sphingosine instead
of glycerol in sphingophospholipids.
 The fatty acids are attached through an amide linkage rather than the ester
linkage.
 The base present is normally choline.
 The structure of the parent compound sphingosine and phytosphingosine are
shown below
 C-1, C-2 and C-3 of the sphingosine or phytosphingosine bear functional
groups,-OH, -NH2 and -OH respectively, which are structurally homologous with
the three hydroxyl groups of glycerol.
 Carbon 4 to 18 in sphingosine and C-5 to 18 in phytosphinogsine resembles that
of a fatty acid.
 When a fatty acid is attached by an amide linkage to the -NH2, group the
resulting compound is a ceramide which is similar to diacyl glycerol
 Ceramide is the fundamental structural unit common to all sphingophospholipids
 Sphingophospholipids are found in the seeds of several plant species.
 There is a range of molecular species among the phospholipid sub groups which
differ from one another in the fatty acid composition
 All the sub groups of phospholipids are found in plant photosynthetic tissue
 Animal phospholipids contain mostly fatty acids with chain length between 16
and 20. The predominant fatty acids are palmitic, stearic, oleic, linoleic and
arachidonic.
 Plant leaf phospholipids have a more limited range with very few fatty acids
greater than C-18.
 The approximate proportion of each phospholipid expressed as a percentage of
the total phospholipid present is phosphatidyl choline, 45%; phosphatidyl
ethanolamine, 10%;
  Trace amounts of phosphatidyl serine, phosphatidyl inositol, 8%;
monophosphatidyl glycerol, 35%, diphosphatidylglycerol, 2%.
 The diphosphatidyl glycerol is present in the inner mitochondrial membrane.
 The phospholipids are only minor components of seed lipids in which
triacylglycerol predominate.
 The most abundant mammalian phospholipid is phosphatidyl choline.
 The phospholipids carry an electrical charge and interact with water. They are
called as polar or hydrophilic molecules and also as amphiphilic molecules.
 The sphingomyelins, the main sphingophospholipids of animals, are not present
in plants.
Glycolipids and sulpholipids
 Glycolipids are structurally characterised by the presence of one or more
monosaccharide residues and the absence of a phosphate.
 They are O-glycoside of either sphingosine or glycerol derivative. The
monosaccharides commonly attached are D-glucose, D-galactose or N-acetyl
D-galactosamine.
 Monogalactosyl diglycerides and digalactosyl diglycerides have been shown to
be present in a wide variety of higher plant tissues
 The 3 position of 1, 2-diacylglycerol is linked to 6- sulpho-6-deoxy D-glucose by
an -glycosidic bond in plant sulpholipid
 The predominant fatty acid present in sulpholipid is linolenic acid.
 The sulpholipid is mostly present in chloroplasts, predominantly in the
membranes of thylakoid.
 Cerebrosides are composed of a monosaccharide residue glycosidically linked
to C-1 of an N-acylated sphingosine derivative.
 The monosaccharide is D-glucose in plants and D-galactose in animals.
Lipoprotein
 Protein molecules associated with triacylglycerol, cholesterol or phospholipids
are called lipoproteins.
 Triacylglycerols derived from intestinal absorption or from the liver are not
transported in the free form in circulating blood plasma, but move as
chylomicrons, as very low density lipoproteins (VLDL) or as free fatty acids
(FFA) - albumin complexes.
  Besides, two more physiologically important groups of lipoproteins are low
density lipoprotein (LDL) and high density lipoprotein (HDL).
 The major lipid components of chylomicrons and VLDL are triacylglycerol,
whereas the predominant lipids in LDL and HDL are cholesterol and
phospholipid respectively.
 The protein part of lipoprotein is known as apoprotein.
 Lipoproteins occur in milk, egg-yolk and also as components of cell
membranes

Sterols
 The characteristic structure of sterol is their steroid nucleus consisting of four
fused rings, three with six carbons (Phenanthrene) and one with five carbons
(cyclopentane).
 This parent structure is known as perhydro cyclopentano phenanthrene.
 The steroid nucleus is almost planar and relatively rigid.
 Steroids with methyl groups attached to carbons 10 and 13 and 8-10 carbon
atoms in the side chain at position 17, an alcoholic group at position 3 and a
double bond between carbons 5 and 6 are classified as sterols.
 Cholesterol is the most abundant sterol in animals.
 Cholesterol is a major component of animal plasma membranes and occurs
in lesser amounts in the membranes of their subcellular organelles.
 Its polar OH group gives it a weak amphiphilic character, whereas its fused
ring system provides it with greater stability than other membrane lipids.
 Cholesterol is therefore an important determinant of membrane properties.
 It is also abundant in blood plasma lipoproteins where 70% of it is esterified to
long chain fatty acids to form cholesteryl esters.
 Plants contain little cholesterol. Rather, the most common sterol components of
their membranes are stigmasterol and -sitosterol which differ from
cholesterol only in their aliphatic side chains.
 Yeast and fungi have another sterol named ergosterol which has a double bond
between C7 and C8.
 In animal system, cholesterol functions as a precursor of various
physiologically important compounds such as vitamin D, bile acids, female
sex hormones and corticosteroids.
  In plants, cholesterol functions as an intermediate compound in the synthesis of
various phytosteroids such as saponins, cardiac glycosides,
phytoecdysteroids and brassinosteroids.

Brassinosteroids
 In 1979, a novel plant growth regulating steroidal substance called
brassinolide was isolated from rape (Brassica napus) pollen
 More than 24 compounds are known (designated as BR1, BR2).
 Pollen is the richest source.
 Brassinosterols are active at concentration much lower (nM to pM range) than
those of other types of hormones.
 Brassinosterols elicit a pronounced stem elongation response in dwarf pea
epicotyls, mung bean epicotyls that are sensitive also to gibberellic acids but not
auxins.
 Brassinosteroids are thought by some to be a new class of plant hormones.
 The evidences are
i. They are widely distributed in the plant kingdom.
ii. They have an effect at extremely low concentration.
iii. They have a range of effects which are different from the other classes of
plant hormones.
iv. They can be applied to one part of the plant and transported to another
where in very low amounts elicit a biological response.
 They are widely distributed including dicots, monocots, gymnosperms and algae,
and in various plant parts such as pollen, leaves, flowers, seeds, shoots and
stems.
 Among the naturally occurring brassinosteroids, brassinolide and
castasterone are considered to be the most important because of their wide
distribution as well as their potent physiological activity.
Physiological effects of brassinosteroids
i. Promotion of ethylene biosynthesis by stimulating ACC synthase activity.
ii. Promote elongation of vegetative tissue in a wide variety of plants at very
low concentration.
iii. They are powerful inhibitors of root growth and development (via ethylene).
 iv. They have been shown to interfere with ecdysteroids at their site of action,
and are thus the first true antiecdysteroids.
v. They enhance resistance to chilling, disease, herbicides and salt stress,
promote germination and decrease fruit abortion and drop.
Practical application of BR
 Large scale field trials in China and Japan over a six year period have shown that
24-epibrassinolide, an alternative to brassinolide, increased the production of
agronomic and horticultural crops (wheat, corn, tobacco, watermelon and
cucumber).
 Environmental stresses were also seem to be allievated by treatment with
brassinolide.
Properties of fat
Physical
 Fats are greasy to touch and leave an oily impression on paper.
 They are insoluble in water and soluble in organic solvents.
 Pure triacylglycerols are tasteless, odourless, colourless and neutral in reaction.
 They have lesser specific gravity (density) than water and therefore float in
water.
 Though fats are insoluble in water, they can be broken down into minute
dropletsand dispersed in water. This is called emulsification.
 A satisfactory emulsion is one highly stable and contains very minute droplets
with diameter less than 0.5 m.
 Examples of naturally occurring emulsions are milk and yolk of egg. But they
are not mere fat droplets in water.
 They contain hydrophilic colloidal particles such as proteins, carbohydrates and
phospholipids which act as stabilizing agents.
 Emulsification greatly increases the surface area of the fat and this is an
essential requisite for digestion of fat in the intestine.
Chemical
 The most important chemical reaction of neutral fat is their hydrolysis to yield
three molecules Alkali hydrolysis (saponification)The process of alkali hydrolysis is
called 'saponification'
  The alkali salt of fatty acid resulting from saponification is soap.
 The soaps we use for washing consists of Na or K salts of fatty acids like
palmitic, stearic and oleic acid.
 The potassium soaps are soft and soluble whereas the sodium soaps are hard
and less soluble in water.
Enzyme hydrolysis
 Hydrolysis of triacylglycerol may be accomplished enzymatically through the
action of lipases.
 Lipases are widespread in both plants and animals.
Rancidity
 Development of disagreeable odour and taste in fat or oil upon storage is called
rancidity.
 Rancidity reactions may be due to hydrolysis of ester bonds (hydrolytic
rancidity) or due to oxidation of unsaturated fatty acids (oxidative rancidity).
Hydrolytic rancidity
 This involves partial hydrolysis of the triacylglycerol to mono and
diacylglycerol.
 The hydrolysis is hastened by the presence of moisture, warmth and lipases
present in fats or air.
 In fats like butter which contains a high percentage of volatile fatty
acids,hydrolytic rancidity produces disagreeable odour and taste due to the
liberation of the volatile butyric acid.
 Butter becomes rancid more easily in summer.
Oxidative rancidity
 The unsaturated fatty acids are oxidised at the double bonds to form peroxides,
which then decompose to form aldehydes and acids of objectionable odour and
taste.
Hydrogenation
 The degree of unsaturation of the fatty acids present in triacylglycerol
determines whether a fat is liquid or solid at room temperature.
 The presence of more unsaturated fatty acids lower the melting point.
 The presence of highly unsaturated fatty acids makes the oil more susceptible to
oxidative deterioration.
 The objective of hydrogenation is to reduce the degree of unsaturation and to
increase the melting point of the oil.
 The oil can be selectively hydrogenated by careful choice of catalyst and
temperature.
 Hydrogenation of unsaturated fats in the presence of a catalyst is known as
hardening.
 Normally the process of hydrogenation is partial so as to get desired
characteristics and to avoid products with high melting points.
 Hydrogenation is carried out in a closed container in the presence of finely
powdered catalyst (0.05 - 0.2% of nickel) at temperature as high as 180oC.
 The catalyst is usually removed by filtration.
 During hydrogenation process a proportion of the cis double bonds are
isomerized to trans double bonds and there is also migration of double
bonds.
 The hydrogenation process has made it possible to extend the food uses of a
number of vegetable oils and marine oils whose melting points are too low.
Constants of fats and oils
 Since fats and oils form essential nutrient of human diet, it is necessary to
identify a pure fat or to determine the proportion of different types of fat or
oil mixed as adulterant in edible oils and fats like butter and ghee.
 With an adequate knowledge of the characteristic composition of fats or oils, it is
possible to identify the fat or oil under investigation.
 The chemical constants also give an idea about the nature of fatty acids
present in fats or oils.
 Even though gas chromatographic method is available to identify and quantify
the fatty acids present in fat or oil, the physical and chemical constants are still
used in routine public health laboratories where such sophisticated facilities are
lacking.
 HYBRID SEED PRODUCTION IN MAIZE

Crossing technique : Manual emasculation by detasseling

Detasseling : Removal of male inflorescence from the

monoecious crop

Time for detasseling : The time taken for shedding of pollen from the
tassel in 1-2 days after emergence. Hence the
tassel should be removed before the shedding of
pollen.

Detasseling
Detasseling is the removal of tassel from female parent. Detasseling is done
when the tassel emerged out of the boot leaf, but before anthesis have shed pollen.
Anthers take 2-4 days to dehisce after complete emergence. Only in few cases, the
anthers start dehisce before its complete emergence. In such case detasseling
should be done earlier. Detasseling is done every day from the emergence of tassel
upto 14 days.

Method
 Hold the stem below the boot leaf in left hand and
the base of the basal in right hand and pull it out
in a single pull.

 Grasp entire tassel so that all the pollen parts are

fully removed.

 Do not break or remove leaves as removal will

reduce yields and will result in lower quality of
seed.

Precautions to be adopted during detasseling

 No part should be left on the plant as it causes contamination.

 It should be uniform process done daily in the morning in a particular direction.

 Donot break the top leaves as the field may be reduced due to the earning of

source material to accumulate in sink [seed ] as removal of 1 leaf course 1.5%

loss 2 leaves 5.9% loss and 3 leaves 14% loss in yield.

 Detassel only after the entire tassel has come out and immature detasseling

may lead to reduced yield and contamination.

 Mark the male rows with marker to avoid mistake in detasseling

 Look out for shedders [shedding tassel] in female rows as the may cause

contamination.

 After pulling out the tassel drop it there itself and bury in soil. Otherwise late

emerging pollen from detasseled tassel may cause contamination.

 Do not carry the tassel through the field as any fall of pollen may lead to

contamination.

 Donot practice, improper, immature and incomplete detasseling.

 Improper detasseling: A portion of the tassel is remaining in the plant while

detasseling.

 Immature detasseling: Carrying out detasseling work when the tassel is

within the leaves.

 Incomplete detasseling: The tassel is remaining in lower or unseen or

unaccounted in within the whole of leaves.

 There should not be any shedding tassel.

 Shedding tassel: Either full or part of tassel remain in female line after

detasseling and shedding pollen which may contaminate the genetic purity of

the crop.
System of Hybrid seed production

 Detasseling ( Manual creation of male sterility )

Types of hybrids
Single cross hybrid
It is a cross between 2 inbreds. A x B. A genotype will be detasseled and
crossed with B genotypes.

 COH 1- UMI 29 x UMI 51

 COH 2- UMI 810 x UMI 90
 CoH(M) 5-UMI 285 X UMI 61

Double cross

 It is a cross between two single crosses.

 It is a cross between 2 hybrids (A x B) x (C x D) (A x B) single cross

hybrid will be produced by detasseling A and by crossing with B (C x D)

hybrid will be produced by detasseling C and crossing with D.

 Then (A x B) will be detasseled and crossed with (C x D) hybrid.

 Example

Ganga 2 : (CM 109 x CM 110) x (CM 202 x CM 111)

Ganga 101 : (CM 103 x CM 104) x (CM 201 x CM 206)

COH3 : ( UMI 101 x UMI 130 ) x (UMI 90 x UMI 285 )

Three way cross

 It is a cross between a single cross and an inbred.

 It is first generation resulting from the crossing of on approved inbred line

and a certified open pollinated variety A x variety)

 A will be detasseled and allowed for crossing in the variety.
Example Ganga -5 (CM 202 x CM 111) x CM 500.
COH (M) 4 : (UMI 90 x UMI 285) x UMI 112

Double top crosses : The first generation resulting from the controlled
crossing of a certified single cross and a certified
open pollinated variety.
: (A x B) x variety
: (Ax B) will be detasseled and crossed with a variety

Seed production technology

Season - November- December, Mid July, Jan. Feb and Sep. Oct

Isolation distance
Foundation seed (m) Certified seed (m)
1. Inbreds 400 -
2. Single cross hybrid 400 -
Field standards for isolation (modification based on situation)
For (foundation single crosses and hybrid of certified class)

Foundation Certified stage

stage

• Same kernal color 400 200

• Different kernal colour 600 300

• Field of single cross / inbreds not 400 200

confirming to varietal purity

• Single cross with same male parent 5 5

confirming to varietal purity

• Single cross with other male parent 400 200

not confirming to varietal purity

 Differential blooming dates are permitted for modifying isolation distance

provided 5.0% or more of the plants in the seed parent do not have receptive
silk when more than 0.20% of the plants in the adjacent field within the
prescribed isolation distance are having shedding pollen.
 In hybrid seed production (certified seed stage) alone the isolation distance (less
than 200 meter) can be modified by increasing the border rows of male parent,
if the kernal colour and texture of the contaminant are the same as that of the
seed parent.

The number of border rows to be planted all around the seed field to modify
isolation distance less than 200 m shell also be determined by the size of the field
and its distance from the contaminant as shown below.
 Area in ha. Isolation distance Border rows
(m)

< 4 ha 200 1

< 4 ha 150 5

< 4 ha 100 9

< 4 ha 50 13

10-12 ha 180 1

10-12 ha 130 5

10-12 ha 80 9

10-12 ha 30 13

> 16 ha 165 1

> 16 ha 115 5

> 16 ha 65 9

> 16 ha 15 13

Seed production stages and production of parental lines / hybrids

Stage of seed Single Double Three way Double top Top

cross cross cross cross cross

Breeder seed A, B A, B, C, D A, B, C A, B, A, variety

variety

Foundation A, B (AxB) (AxB), C (AxB) A, variety

seed (CxD) variety

Certified seed AXB (AxB) x (AxB) x (AxB) x Ax variety

(CxD) variety variety
Spacing
Seeds are sown in ridges and furrows
Hybrids : 60x 25 cm

Seed rate : Female : 7 -10 kg ha-1

: Male : 3 -4 kg ha-1

Spacing : Female : 60 x 20 to 75 x 30 depending on the

area.

Male :45 x 30 cm

Planting ratio
Single cross 4:2
Double cross 6:2
3 way cross 6:2
Border rows a. Inbreds & single cross - 4 rows
b. Others - 3 rows

Fertilizer

NPK kg / ha : 200 : 100 : 100

Basal : 100 : 100 : 50

1st Top : 50 : 0 : 0 (20th days -vegetative phase)

2nd Top : 50 : 0 : 50 (Boot leaf stage at 45 days)

Foliar : DAP 2% at 50% flowering

In Zn deficient soil : ZnSO 4 @ 25 kg ha-1

Roguing
Should be done periodically based on position of cob, colour of silk,
arrangements of seeds in cob, leaves etc. Shedding tassels are to be removed in
roguing . It refers to the tassels in female parents rows, shedding pollen or that has
shed pollen in hybrid maize plots. During field inspection a tassel whose main spike
or any side branch or both have shed pollen or shedding pollen in more than 5 cm
of branch length is counted as a shedding tassel during inspection the shedding
tassels are taken into count for acceptance or rejection of production plot.

Field standard (%)

FS CS
Off types 0.2 0.5
Shedding tassel 0.5 1.0 (when receptive silk
is 5% or more)

Inseparable other crop : Nil (both stage)

Objectionable weed : Nil (both stage)

Designated diseases : Nil (both stage)

Field standards –specific

Specific factors Certified stage

Off types shedding pollen when 5 % or more of seed 0 .50 %
parent in receptive silk
Seed parent shedding pollen when 5 % of the seed 1.0 %
parent is having receptive silk
Total of pollen shedding tassel including tassel that 2 .0 %
had shed pollen for all 3 inspections conducted during
flowering on different dates
Off types in seed parent at final inspection 0 .5 %

Number of inspection : Four

(Seed certification officers) : One : Before flowering

: Three : During flowering

Harvest

 Harvest when the moisture content falls to 20-25%

 Harvest male first and remove from the field and then harvest female
Threshing
a. Dehusking - The husks are removed manually.
b. Cob sorting - Remove ill filled, diseased cobs and cobs having
kernel colour variation.
Zenia
The direct/visible effects of pollen on endosperm and related tissues in the
formation of a seed colour. e.g. seed colour. In maize, the gene present in sperm
cell contributes in the expression of colour of hybrid seeds.
Matazenia
Is the effect of pollen on the maternal tissues of fruit.
Shelling
Cob sorting should be the first operation it is a post harvest, evaluation for genetic
purity. The sheath is removed and check for kernel colour, shank colour, diseased
cobs, kernel arrangement. The cobs are shelled either mechanically or manually at
15-18% moisture content. Improper shelling leads to48% damage to kenel Growth
of storage fungal Pericarp damage. Crack on pericarp can be identified by FeCl 3 or
Tz test. Shelling is done mechanically using cob sheller and manually by rubbing
with stones.
Drying
Seeds are dried to 12% moisture content.
Grading
Grade the seeds using 18/64" (7.28 mm) sieve.
Seed treatment
Slurry treat the seeds with 8% moisture content either with captan or thiram
75% W.P. @ 70 g/100 kg with 0.5 litre of water. Treated seeds can be stored for 1
year in cloth bag.
Others: As in varietal seed production

Seed yield : 2.5 - 3.6 t/ha

Seed standard inbred, varieties and hybrids

Hybrids

Parameters Inbreds FS CS

1. Physical purity (%) (min) 98 98 98

2. Inert matter (%) (max) 2 2 2
3. Other crop seed (max) 5 /kg 5 kg-1 10 kg-1
4. ODV seeds (max) 5/kg 5 kg-1 10 kg-1
5 Germination % (min) 80 80 90
6. Moisture content (%) (max)
a. Moisture pervious 12 12 12
b. Moisture vapour proof 8 8 8

Production of Synthetic cultivars

Breeding of cereal and other agronomic crops has contributed significantly to
the growth of agribusiness worldwide. In normally self fertilized crops, new
variability may be created by hybridisation, followed by the selection of desired
cultivars in which desirable characteristics from two or more parents are combined.
The type of hybrid cultivar obtained will depend upon the genetic background of the
chosen parents as well on the method of selection used. A similar situation arises
when new variability is artificially induced through mutations.
In pure-line theory of classic plant breeding, a pure line is defined as all the
descendants of single homozygous individual by continued self-fertilization,
resulting in a homogeneous cultivar. Hybridization, however, results in significant
heterogenity. The multiplication of such heterogenous progeny in bulk to select
homozygous individuals would be gigantic task. Most modern hybrid cultivars are,
therefore, selected at an early stage (F 2 ) as subsequent lines and probably
released at the F 8 and F 12 generations. These are obviously not as homogeneous
as a pure line.
 Cultivars can also be selected by producing multilines. Whereas normal line
selection seeks to produce a new cultivar on the basis of one line or a few lines
that are very similar, multiline cultivars are essentially different from each other in
their characteristics, such as resistance to pests and diseases or environmental
stresses. Thus, by incorporating different sources of resistance, the newly
synthesized cultivar is buffered against changes brought about by virulent
pathognes. These cultivars are however, not very stable compared to those
produced by the conventional methods of selection. A change in the prevalence of
a virulent pathogen may eliminate certain lines from the cultivar. It is, therefore,
necessary to return the cultivar to the plant breeder for its reconstitution. This may
be advantageous, because it enables plant breeders to substitute new sources of
resistance in the material.
Alternatively, the plant breeder can create a composite cross by bulking the
F 2 generations of several crosses. The composite is allowed to develop for several
generations during which natural selection may occur. If the composite is grown at
more than one location, a locally adapted cultivar may be developed in time. The
composite constitutes a gene pool from which the plant breeder can select a
cultivar with desirable characteristics for further multiplication.
An alternative to the composite is the synthetic or artificial method of plant
breeding in which a number of lines are put together by the plant breeder in
predetermined proportions. A synthetic line generally has a limited life, because the
proportions of the constituent lines are likely to change over number of
generations. The plant breeder must plan for seed production of limited generation
basis. This system can be extended by using mixtures of cultivars claimed to be
advantageous in some species over a single cultivar, especially if different resistant
genes are present in each cultivar. This method adds to the cost of mixing, which
can be reduced by growing a seed crop for one or two generations after mixing
before using it for crop production.
A hybrid cultivar results from a controlled cross between a male and female
parent, the seed being harvested from female parent only and used for crop
production.
 In self fertilized crop species, it is easy to produce hybrid cultivars if male
sterile lines are available that can be used as female parents. There are certain
substsnces that act as a gametocides, destroying the pollen of desired female
parent, or as inhibitors that prevent pollen produced by the female parent from
effecting fertilization. The advantage of the synthetic hybrid cultivar lies in
heterosis. Special expensive measures are required to produce seed that is
harvested from the female parent only. The resultant heterosis therefore must have
a profitable effect to compensate for the cost of production of synthetic hybrid
cultivars in the self pollinating crop species.
In the cross pollinated crop species, plant breeders look for parent plants
that have good combining ability. These plants, when allowed to multiply together,
produce a desirable combination of characteristics. Cross fertilization results in
greater heterozygosity in these plants than in the self fertilized plants and therefore
less homogeneity. Each generation of an open pollinated cultivar is thus a mixture
of hybrids. The open pollinated cultivars are generally grown for a limited number
of generations and returned to the plant breeder’s maintenance material after each
cycle of seed production to produce commercial quantities of seeds.
Putting together a large number of parent plants and allowing random
pollination to occur can create composites. A composite in a cross fertilized species
is generally the product of the first generation of such random pollination.
Production of synthetic cultivars begins with a limited number of specific
parents, which are permitted to interpollinate. The number of generations of
multiplication is strictly limited so as to recreate the synthetic/artificial cultivar at
the end of each multiplication cycle. As with the self fertilized species, synthetic
hybrid cultivars of cross fertilized species are created by controlling pollination to
ensure that seed is produced from a desired crossing. This can be achieved by the
following methods.
1) By emasculating the female parent, as is done in monoecious plants like maize,
by removing the male flowers before the release of pollens.
2) By using male sterility in the female line, so as to avoid the physical removal of
male flowers.
3) By using self incompatibility. In this system, the seed crop is harvested as a
whole, since all plants are contributing and receiving pollen. The self
incompatibility, however, is not always complete, and there may be production of
some inbred plants. With the excessive production of such plants, the advantage of
heterosis in the subsequent crop is diminished.
The advantage of the synthetic hybrid cultivar in cross pollinated species is
not restricted only to heterosis. Most hybrids are based upon inbred lines. Normally,
cross fertilized plants require inbreeding for several generations to reduce
heterozygosity and to include desirable genes in synthetic cultivars. A controlled
cross between two such inbreds produces heterosis and desirable combination of
genes in the form of a synthetic cultivar.
The major disadvantage of the production of synthetic cultivars is the higher
cost of plant breeding and seed production, requiring considerable time consuming
work to produce desirable inbreds, which alone can be used to synthesize new
artificial hybrids. The final seed crop is not fully productive when male sterility or
emasculation is used, because only the female parent is harvested for seed.
Therefore various other hybrids have been produced. The hybrid resulting
from the cross of two inbred lines is a single cross, whereas the F1 resulting from
the cross of two single cross hybrids as parents is known as a double cross. In a
three way cross, an inbred is mated with an f1 hybrid. A top cross is the F1
resulting from a cross between an inbred or a single cross and an open pollinated
cultivar. All of the forms of hybrid cultivars require a particular cycle of seed
production to produce the seed used in crop production.
 SEED PRODUCTION TECHNIQUES IN PADDY VARIETIES

Phenology
Botanical Name : Oryza sativa
Chromosome number [2n] : 24
Family : Poaceae
Inflorescence : Panicle
Pollination : Self-Pollination
Panicle Emergence : 4 –5 days after boot leaf
emergence
Flower Opening Pattern : Tip of primary & secondary
branches and proceeds
downward
Duration of Flowering : 6-8 days
Time of Anthesis : 7.00 –10.00 A.M
Speciality with flowering : Flower remain open for 10
minutes and afterwards it
closes.
Anther dehiscence : Either before or after flower
opening [independent of
spikelet opening]
Temperature favorable for flowering : 24 -280C
Favourable RH for flowering : 70-80%
Difference between day and
Night temperature : 8-100c
Stigma receptivity : 3 days
Pollen viability : 10 minutes

Varietal seed production

Stages of seed production
In paddy depending on the demand 3 or 4 or 5 stages of seed
multiplications are permitted under seed certification programme as follows.
 Breeder seed - foundation seed - certified seed
 Breeder seed - foundation seed stage 1- foundation seed stage 2 –
certified seed
 Breeder seed - foundation seed stage 1- foundation seed stage 2 -
certified seed 1- certified seed 2

Land requirement
The land should be free of volunteer plants (crop of previous season occur
in this season) and the same crop or the other varieties of the same crop should
not have been grown for the previous season, if it is the same crop it (previous)
should be the same variety that has been certified. This selection is highly
important for maintenance of genetic purity. They should have adequate
irrigation and drainage facilities and the problem soils are not suitable for seed
production.

Isolation
The crop should have 3meters of isolation at all sides of the seed
production plot for maintenance of genetic purity.

Selection of seed
Seed should be from an authenticated source (SAU, NSC, State
Department).For production of certified seed, foundation seed (FS) should be
used as source seed which should be purchased with bill and tag (white for FS
seed)

Seasons practiced at Tamil Nadu

In Tamil Nadu the availability of water in cannals, depends on the
monsoon. Based on this in different districts, different sowing seasons are
adapted as follows:
Month of sowing Seasons Duration of varieties
December - January Navarai Below 120 days
April – May Sornavari Below 120 days
April – May Early kar Below 120 days
May – June Kar Below 120 days

June – July Kuruvai Below 120 days

July - August Early samba 130 -135 days

August Samba 130-135 & above 150 days
Late samba / thaladi /
September – October 130 - 135 days
pishanam
November – October Late thaladi 115 -120 days
November - October Late pishanam 130 -135 days

Selection of season
Season should be selected based on duration of the variety and the water
availability.

VARIETIES SEASON DURATION POPULAR

VARIETIES
Shout duration November- December Below 120 days TKM9 ,CO 36,
varieties (Karthikai –Margazhi) ADT 36
Medium duration November 130-135 days Bhavani ,CO43,
varieties (Iyyppasi- Karthikai)
Long duration August More than 135 White Ponni,
varieties (Adi-Avani) days
Upland rice July –August ---on All durations but MDU1,PKM1
receipt variety specific Co 43,IR 20
of showers .TKM9 and
IR 50 should be sown
Before 15th of July (dire
seeding)
Rainfed rice June-July and Septemb Specific to ADT 38 ADT39
– location (Medium Duration
October Varieties)

Seed Rate
It varies with varieties and type of cultivation.

Variety / type of cultivation Seed rate

LOW LAND CULTIVATION (transplanting)

Short duration varieties 60 kg /ha
Medium duration varieties 40kg /ha
Long duration varieties 30kg/ha

For low land cultivation by broadcasting 80-100 kg/ha

For rainfed rice 75-100 kg/ha

Seed Management Technique

Dormancy
Paddy exhibits dormancy which varies for duration of 0-30/45days
depending on the variety. This could be broken by either soaking in KNO 3 0.5 %
for 16 hr or soaking in 0.1N HNO 3 for 16 hrs. However the duration and
concentration vary with varieties (e.g.) ADT36 exhibit 20-30 days of dormancy
period from days to physiological maturity period which could be broken by
soaking the seeds in 0.5%KNO3 for 16 hrs. Practically the intervening duration
between the harvesting, and threshing, and further drying will remove the
dormancy.

Seed Upgradation Technique (Egg Floatation Technique)

Either before processing or after storage or due to improper processing
Paddy seed may have less vigorous seed such as immature, ill filled and
insect damaged seed which may adversely affect the planting value of the seed.
Removal of this seed will favour better establishment and higher production
potential. These seed may be removed by adaptation of a simple water
floatation technique based on specific gravity using salt water as a dissecting
solution for separation of good quality seed from low quality seed, and egg is
used as an indicator for specification of specific gravity measurement of 1.03
(120g of salt in 1000ml of water)

Methodology
A bucket of potable water has to be taken and in that water o fresh egg
which sinks to the bottom has to be taken. To the potable water with egg
outside slowly the common salt was added to a level at which the egg floats at
top exposing 2.5 cm of its shell outside (check the egg floatation now and then
on addition of salt to the solution). The egg is removed and the paddy seed are
dropped into the solution which separates as sinker and floater .the sinkers are
good seeds while the floaters are less vigorous and dead seeds. The floaters are
removed and used as feed and sinkers are used for further multiplication.

Caution
 Egg is only for measurement of specific gravity and has no work to do
with separation.
 If the density of water is more, more portion of egg will float if less egg
will be inside the solution.
 If the density of water is more loss of quality seed may occur ,lesser
density the separation will not be perfect

Sprouting of seeds (pre germination)

Paddy seeds are sown at nursery in pre germinated condition for better
establishment for supply of oxygen at waterlogged condition. Seeds are soaked
in big tough for 24 h in gunny bags tied loosely for easy transmission of water
and for ensuring soaking of each and every seed. Seeds are then tied tightly and
incubated in dark for 12h (overnight). White protrusion of radices by the seed
exposed to outside expresses the pre germination of seeds and these seeds are
sown in nursery by broadcasting.
Hardening and other seed management techniques
 In case of implementation of fortification treatment, seed could be soaked
in equal volume of water to ensure that none of the solution is left
unimbibied by the seed
 For dry land and upland paddy, seed hardening with KCl (1%) and
pelleting with Azospirillum (600g /ha) could be adopted (e.g.) MDU 1,
Paramagudi1.
 Seed colour variation occurs due to bacterial infection at later stages of
maturation. Seed coloring with polycoat @3g kg-1of seed could improve
the initial quality and marketability of such discolored seed.
 Polymer coating of Seed also will help to identify the brand name of seed
and to identify the varietal variation among the cultivars by even the
illiterate labours.

Nursery Management
For raising one hectare of paddy, 20 cent (800m2) nursery is needed. The
area should be prepared by floating the area one or two days before ploughing
and allowed the water to soak in. The soil should be kept at shallow sub
emergence. Before ploughing the water should be allowed to a depth of 2.5cm
.Then the land is ploughed and brought to a puddling condition. The optimum
size of the nursery bed will be 2.5 meters broad and with channels of 30cm
width in between. In paddy, on raising more varieties in a same place separate
irrigation channels are to be prepared for each variety to avoid the admixture
of seeds and to maintain the genetic purity.

Nutrient Management
Before the last puddling apply 40kg of DAP and if not readily available
apply straight fertilizers@16 kg of urea and 120kg of super phosphate.
Basal application is required (DAP) if the seedlings are to be pulled out at
20 to 25 days after sowing. If the seedling are to be pulled out after 25 days
application of DAP is done 10 days prior to pulling out of the seedling.
 Basal application of phosphorus to the nursery enables the seedling to
store phosphorus and utilize it even in later stages of growth and application of
DAP to the nursery is highly economical.

Sowing
A thin film of water should be maintained in the nursery, and the sprouted
seeds of paddy should be sown uniformly on the seed bed.

Water Management
 Drain the water 18 to 24 hours after sowing and if there are pockets
where water is stagnating, drain it into the channel as germination will be
affected in the places where the water is being stagnated
 Allow the water to saturate the soil from the third to fifth day
 From the fifth day onwards increase the quantity of water to a depth of
1.5 cm depending on the height of the seedling
 Afterwards, maintain the water level to a depth of 2.5 cm

Weed Management
Apply any one of the pre emergence herbicides viz. butachlor 2l per
ha,thiobencarb@2l/ ha, pendimithalin @ 2.5l/ha on 8th day after sowing to
control weeds in the low land nursery. Keep a thin film of water and allow it to
disappear. Avoid drainage of water. This will control germinating weeds.

Pest Management (NURSERY)

INSECTS /DISEASE CONTROL MEASURES

Army worm Spray Cholophyriphos 20EC 80ml or endosulphan

35 EC80ml during the evening
Thrips Phosphamidon85 WSC 25 ml(or)Monocrotophos 36
WSC 40ml (or) Endopsulfan 35 EC 80 Ml

Green leaf hopper As above or maintain 2.5 cm of water in the nursery and
broadcastanyone of the following
Carbofuran3g3.5kg or Phorate 10G1.0kg or Quinalphos 5g
2.0kg
Case worm Mix kerosene in standing water and remove the cases and
destroy and spray Monocrotophos 36 WSC 40ml (or)
Quinalphos 25 EC 80 ml

White tip nematode Sun drying of seeds for two days at 6h interval
Rice root nematode Carbofuran3g at 3.5kg / 20cents
Diseases
Blast Spray with insecticide Copper oxy chloride100g or
Mancozeb 80 g
Brown spot Carbendazim 40 g
Tungro disease Aplly carbofuran 3g at the rate of 3.5 kg ten days after
sowing or spray two rounds of Monocrotophos 36 WSC
40ml or Phosphamidon 85 WSC 25 ml

Age of transplanting
The age of transplanting vary with varieties as follows
DURATION OF VARIETIES AGE OF TRANSPLANTING
Short duration varieties 18-22days
Medium duration varieties 25-30days
Long duration varieties 35-40days

Pulling out of seedling

 Pull out the seedling at appropriate time
 Do not remove the adhering soil with a hard surface
 Tie the seedling in convenient size for easy handling
 Do not allow the seedling to dry

Main field preparation

 Puddle the soil well
 Apply 12.5tonnes of FYM or compost per ha
 Incorporate green manure into the field by in situ ploughing
 Dig the corners and prepare the bunds well with plastering for effective
stagnation of water
 Apply the phosphorus and potasic fertilizers at last ploughing for effective
availability of nutrients to plants
 Keep a thin film of water at the time of transplanting and raise the water
level to 2.5 cm on the next day

Fertilizer Requirement

CROP DURATION FERTILIZER REQUIRMENT ( Kg / ha )

 Nitrogen ( N ) Phosphorus (P ) Potash ( K)
Short duration 120 38 38
Long and medium duration 150 50 50
Bio-fertilizer Azolla @ 1t/ha 3-5 days after weeding

Transplanting
 Dip the root in phosphamidon 0.02 % against rice root nematode 20
minutes prior to planting
 Plant the seedling at optimum spacing and optimum depth
 Transplant the seedling at 4-5 leaf stage

Details on transplanting

Specifications Duration of cultivars

Short Medium Long
No. of seedling per hill 2-3 2 2
Depth of planting (cm) 3 3 3
Spacing ( cm) 20 x10 20 x15 20 x20
No. of hills/m2 50 33 25
Breeder Adopt double row planting with a spacing of 15 x 10 cm
seed multiplication for easy roughing

- Adjust the sowing in such a way that harvesting does not coincide with rain

Weed Management
Pre emergence herbicide

Use butachlor 2.5l/ha or thiobencarb 2.5l/ha fluchloralin2l/ha or

pendimethalin3l/ha as pre emergence on third day and is to be followed by hand
weeding on 30-35days. On the failure of pre emergence application, hand weed
at 15 days and spray 24Dsodium salt with a high volume sprayer 3 weeks after
transplanting when the weds are in3-4 leaf stage

Gap Filling
It is to be taken up between 7-10days after transplanting

Pest and disease management

Insects Control measures

Stem borer Fenthion100EC @ 500ml

Thrips Phosphamidon85 WSC @ 300ml

Brown plant hopper MonocrotophosWSC @ 500ml
Leaf folder Endosulfan 35EC @ 60ml
Stemborer (white ear 2 %) Quinalphos 25EC @ 1000ml

Mealy bug Phosphamidon85 WSC @ 300ml

Earhead bug Quinalphos 25EC @1000ml

Rice root nematode Carbofuran3g 16.25kgin standing water

Diseases

Blast Carbendazim @ 250g/ha

Brown plant hopper Mancozeb @ 1000g/ha

Sheath rot Carbendazim @ 250g/ha

 Sheath blight Difolatan @ 200
Bacterial leaf blight Streptomycine
Sulphate+Tetracycline@300g+Copper
Oxychloride @ 1250g/Ha

Grain discolouration Mancozeb@1000g/ha

Water Maintenance of Paddy

 5cm of water should be stand in the field. Normally once ion 2 days for
loamy soils and once in 3 days for clay soils.
 Excess water leads to yellowing of plant. So drain the water
 The critical stages of irrigation are primordial initiation, booting,
heading and flowering

Top Dressing
Apply 25% of N and k as basal and remaining 75 % in 3 split doses at
active tillering, panicle initiation, and at heading stage in equal proportion of
1:1.

Foliar Spray
 Spray FeSO 4 0.5% to prevent yellowing of plants in calcarious soils.
 Spray DAP 2% to enhance seed set in paddy cultivars (BEST).
 Spray GA 3 three times at panicle initiation stage for complete exertion
of panicle (hybrids).
 Spray panchakavya 1% for organic seed production to enhance seed
set.
 Spray 0.5 % zinc sulphate thrice during crop growth on 20th 30th and
40th day of planting for short duration varieties or 30th 40th and 50th day
for medium and long duration varieties in case of zinc deficient soils.

Rouging
 Is important to maintain for maintenance of genetic purity.
  Remove all off types (deviant of the variety) and rouges (variant of the
variety).
 Remove when suspected is the thumb rule of roughing.
 Rouging should be done from the sowing up to harvest and remove the
as and when it come across.

Physiological maturity
Seeds attain maturity with the visual symptom of turning of ear heads
to golden yellow color and when the ear heads exhibit drooping
symptomsi.e 28 days after 50% flowering in short and 31 days in
medium and 35 in long duration.
 When 80% of the plants are exhibiting the symptom the crop is ready
for harvest
The moisture content of the seed will be 18-20-%.

Pre-harvest Sanitation Spray

Ten days prior to harvesting spray endosulphan 30EC 70ml / ha against
storage pests. Spraying of 10 % prosopis leaf extract is recommended against
grain discolouration.

Harvesting
 Lodged plants should not be selected for seed purpose.
 Withhold irrigation one week before harvest.
 Delayed harvest may lead to heavy shattering
 Bundled plants should be stacked as ear heads facing outside to avoid
heat damage.
 Threshed produce should be clean and free of admixture in cracks and
crevices.
 Birds scaring are also practiced in places of requirement.
Threshing
 Thresh the seed by beating the plants on a hard surface ,but take care
that the seeds are not mechanically damaged.
  In tractor and machine threshing avoid mechanical damage by proper
adjustment of speed/machine setting.
 Thresh at proper moisture content to avoid crushing / cracking (16-17 per
cent).
 Clean the floor, equipment, containers to avoid genetic and physical
mixture.

Winnowing and Drying

Threshed produce are cleaned and winnowed to remove the dirt and other
unwanted physical material. Winnowing should be done in a cleaned surface.
The seeds are dried in a threshing floor with adequate stirring which is known as
tempering. The seeds are dried to 13 % moisture for better storage .On drying
in a threshing avoid drying between 12 noon to 2pm to avoid the ill effects of
ultra violet rays of noon sun. Through not for bulk for prolonged storage this
practice should be adopted. Seeds are also can be dried in mechanical driers in
places of high humidity like areas of sea shore.

Grading
The bulk seeds are normally processed through seed cleaner cum
grader and the seeds of middle sieve are selected for seed purpose.

Size of seed Sieve size

Long slender (Ponni, whitePonni) = 1/16 x 3/4 " (1.3mm x 19 mm)
Slender - IR 50 = 1/15 x 3/4" "
Medium slender (IR 20, CO 43) = 1/14 x 3/4" (1.5 mm x 19 mm)
Short bold (ADT 36, 37,38,39,
TKM 9,Ponmani) = 1/13 x 3/4" (1.8 mm x 19 mm)

Seed Treatment
Normally paddy seeds are not treated with chemicals owing to their
economic utility. But for long term storage, treat it with captan or thiram or
bavistin @ 2-4g / kg of seed, Halogen mixture treatment (Chlorine based
halogen mixture @3 g /kg of seed) is a eco-friendly treatment. As a prophylactic
measure seed can be fumigated with celphos @ 3-6g/m3. But the moisture
content of the seed should not be above 10-12% which may interfere with the
seed quality in terms of germination.

Seed Yield
The yield of crop varies from 3000 to 7000 kg /ha depending on
genotypes, location, season management practices and pest infestation.

Storage
Paddy is a good storer. Generally paddy seeds store well up to 12-36
months depending on the genotypes but heavy infestation of storage pests
reduce the storability of seed even to a month or two. For prolonged storage
HDPE and polylined gunny bags are used, while for normal storage jute canvas
bags are used. However the bags should not be stirred for more than 8 bags
height to avoid pressure on seeds of lost bag which may cause damage to the
seed. Polythene bags of 700 gauge is not highly preferable for paddy as the
sharp edge may pierce the bag and convert moisture vapor proof container as
moisture pervious container.

Mid storage Correction

Seeds from storage are given with mid storage correction when the seed
standard reduce to 5-10% lesser than recommended. The seeds are soaked in
double the volume of disodium phosphate solution (3.60g dissolved in 100l of
water) for 16h and the seeds are dried back to original moisture content (12-13
percent).

Seed Certification
Land Requirement
The previous crop should not be the same crop and if to be the same crop
it has to be the same variety and should be certified and has to be accepted for
certification. The field should not have any volunteer plants.
Number of Inspections
A minimum of two inspections is needed, one at the time of flowering
and another at the time of or before harvest.

Field Standards
General: Paddy field should be isolated from contaminants as follows
Contaminants Minimum distance(meters)
Foundation stage Certified stage
Fields of other varieties 3 3
Fields of same variety not 3 3
confirming to varietal purity
requirements for certification

Specific standard: These are verified at the final inspection

Factor Maximum permitted (%)
Off types 0.050 0.20
Objectionable weed plants* 0.010 0.020
*Objectionable weeds are Wild rice (Oryza sativa L.var.fatua Prain
(Syn.O.sativa L.f. spontanea Rosch.)

Seed Standard
Factor Standards for each class
FOUNDATIO CERTIFIED
Pure seed ( maximum) 98.0% 98.0%
Inert matter (maximum) 2.0% 2.0%
Huskless seed (maximum) 2.0% 2.0%
Other crop seed (maximum) 10/kg 10/kg
Other distinguishable varieties (maximum) 10/kg 10/kg
Total weed seed (maximum) 10/kg 10/kg
Objectionable weed seed (maximum ) 2/kg 2/kg
Seeds infected with paddy bunt 0.10% (By 0.50% (By number)
(Neovossia horrida (Tak.) ( maximum) number)
Germination ( Minimum) 80% 80%
Moisture (maximum) 13.0% 13.0%
For vapour proof containers (maximum) 8.0% 8.05%

Paddy Bunt
 Lecture 10
Physical constants
i. Specific gravity
 Since different oils have different specific gravity, any variation from normal value
shows mixture of oils.
ii. Refractive index
 Fats have definite angles of refraction.
 Variation from the normal value indicates adulteration of fats or oils.

iii. Solidification point or setting point

 Solidification point is the temperature at which the fat after being melted, sets
back to solid or just solidifies.
 Each fat has a specific solidification point.

Chemical constants
i. Saponification number
 It is defined as milligrams of KOH required to saponify 1 gm of fat or oil.
 Saponification number is high for fat or oil containing low molecular weight
or short chain fatty acids and vice versa.
 It gives a clue about the molecular weight and size of the fatty acid in the fat or
oil.
ii. Iodine Number
 It is defined as the number of grams of iodine taken up by 100 grams of fat
or oil.
 Iodine number is a measure of the degree of unsaturation of the fatty acid.
 Since the quantity of the iodine absorbed by the fat or oil can be measured
accurately, it is possible to calculate the relative unsaturation of fats or oil.
iii. Reichert-Meisel number (R.M.number)
 This is a measure of the volatile soluble fatty acids.
 It is confined to butter and coconut oil.
 It is defined as the number of millilitres of 0.1 N alkali required to neutralise
the soluble volatile fatty aicds contained in 5 gm of fat.
 The determination of Reichert-Meisel number is important to the food chemist
because it helps to detect the adulteration in butter and ghee.
  Reichert-Meisel value is reduced when animal fat is used as adulterant in butter
or ghee.
iv. Polanski number
 Ghee may be adulterated by the addition of insoluble, non-volatile fatty acids
(by addition of animal fat).
 This can be tested by finding out the Polanski number.
 It is defined as the number of millilitres of 0.1 N potassium hydroxide
solution required to neutralise the insoluble fatty acids (not volatile with
steam distillation) obtained from 5 gm of fat.
v. Acetyl number
 It is defined as the amount in millilitres of potassium hydroxide solution
required to neutralise the acetic acid obtained by saponification of 1 gm of
fat or oil after acetylation.
 Some fatty acids contain hydroxyl groups. In order to determine the
proportion of these, they are acetylated by means of acetic anhydride.
 This results in the introduction of acetyl groups in the place of free hydroxyl
groups.
 The acetic acid in combination with fat can be determined by titration of the
liberated acetic acid from acetylated fat or oil with standard alkali.
 Acetyl number is thus a measure of the number of hydroxyl groups present
in fat or oil.
vi. Acid number
 It is defined as the milligram of potassium hydroxide required to neutralise
the free fatty acids present in one gram of fat or oil.
 Acid number indicates the amount of free fatty acids present in fat or oil.
 The free fatty acid content increases with age of the fat or oil.
Molecular aggregation of phospholipids
 Glycerophospholipids are virtually insoluble in water.
 Depending on the precise conditions and the nature of lipids used, three types
of lipid aggregates can form when amphipathic lipids are mixed with water.
Micelles
 Free fatty acids, lysophospholipids and sodium dodecyl sulphate (SDS) form
micelle.
  Micelles are relatively small spherical structures involving a few dozen to few
thousand molecules arranged so that their hydrophobic regions aggregate in
the interior excluding water and their hydrophilic head groups are at the
surface in contact with water.
 This molecular arrangement eliminates unfavourable contacts between water
and the hydrophobic tails
Bilayer
 A second type of lipid aggregate in water is the bilayer in which two lipid
monolayers combine to form a two dimensional sheet.
 The hydrophobic portions in each monolayer interact excluding water.
 The hydrophilic head groups interct with water at the two surfaces of the bilayer
lipid bilayers form the structural basis of biological membranes

Liposomes
 The third type of lipid aggregate is formed when a lipid bilayer folds back on
itself to form a hollow sphere called a liposome or vesicle.
 These bilayer vesicles enclose water creating a separate aqueous compartment
Biological membranes
 Proteins and polar lipids account for mass of biological membranes.
 The relative proportions of protein and lipid differ in different membranes,
reflecting the diversity of biological roles.
 Amphipathic molecules form a lipid bilayer with the non polar region of lipids
facing outward.
 In this lipid bilayer, globular proteins are embedded at regular intervals held by
hydrophobic interactions.
 Some proteins protrude from one or other face of the membrane (peripheral
proteins); some span its entire width (integral proteins).
 The individual lipid and protein subunits in a membrane form a fluid mosaic
 The membrane is fluid because the interactions among lipids, between lipids and
proteins are non covalent, leaving individual lipid and protein molecules free to
move laterally.
 One of the key functions of a membrane is to control the passage of
substances across it.
 They are said to be selectively permeable. The different membranes of the cell
have different selective permeabilities.
 Lecture 11
AMINO ACIDS AND PROTEINS

The word "Protein" was coined by J.J. Berzelius in 1838 and was derived from the
Greek word "Proteios" meaning the ‘first rank’.
 Proteins are macromolecular polymers composed of amino acids as the basic
unit linked by peptide bonds.
 Amino acids are the fundamental structural units of all proteins.
 These biopolymers contain carbon, hydrogen, oxygen, nitrogen and sulphur.
 The elementary composition of most proteins is very similar; approximate
percentages are C=50-55, H=6-8, O=20-23, N=15-18 and S=Traces
Occurrence
 Proteins are found in all living cells.
 They form essential constituent of protoplasm, cell membrane and nuclear
material.
 They may be present as simple proteins or complexes with lipids or nucleic
acids.
 Proteins from different tissues such as muscle, bone, brain, blood and other
biological fluids differ in composition and properties.
 In cereal and leguminous plants, seeds contain comparatively higher amounts of
protein than stem, leaves and flowers.
 Tuber crops usually contain less amounts of protein in all parts.
 Enzymes are specialized proteins with catalytic activities and are present in all
living organisms.
 Proteins serve as regulators of metabolic reactions, directly as components of
enzymes and indirectly in the form of chemical messengers known as
hormones as well as receptors for hormones.
 They regulate and integrate the numerous physiological and metabolic
processes in the body.
 Proteins are the center of action in many biological processes.

Amino acids
 All proteins are formed from 20 different amino acids. All the amino acids have
trivial or common names based on the source from which they were first isolated or
based on their properties. For eg.
Asparagine was named so, as it was isolated from asparagus and glycine was
so named because of its sweet taste (Greek:'glykos' meaning sweet).
All the 20 amino acids, except proline, found in proteins have an amino group
and a carboxyl group attached to the same carbon atom, namely the -carbon. They
differ only in the side chains (R groups). The 20 amino acids found in proteins are
referred as the standard or normal or protein amino acids.
There are many other amino acids found in nature but do not occur in proteins.
They are referred as non-protein amino acids.

Classification of protein amino acids

The protein amino acids are classified according to the chemical nature of their
R groups as aliphatic, aromatic, heterocyclic and sulphur containing amino acids.
More meaningful classification of amino acids is based on the polarity of the R groups.
The polarity of the R groups varies widely from totally non-polar to highly polar. The 20
amino acids are classified into four main classes whose structures, three-letter and one-
letter symbols are given below
a) Amino acids with non-polar or hydrophobic, aliphatic R groups
 This group of amino acids includes glycine, alanine, valine, leucine,
isoleucine and proline. The hydrocarbon R groups are non-polar and
hydrophobic.
 The side chains of alanine, valine, leucine and isoleucine are important in
promoting hydrophobic interactions within protein structures.
 The minimal steric hindrance of the glycine side chain (hydrogen) allows
more flexibility than other amino acids.
 On the other hand, the imino group of proline is held in a rigid conformation
and reduces the structural flexibility of the protein.
b) Amino acids with non-polar aromatic R groups
 This group includes phenylalanine, tyrosine and tryptophan .
 All these amino acids participate in hydrophobic interactions, which is
stronger than aliphatic R groups because of stacking one another.
 Tyrosine and tryptophan are more polar than phenylalanine due to the
presence of hydroxyl group in tyrosine and nitrogen in the indole ring of
tryptophan.
 The absorption of ultraviolet (UV) light at 280 nm by tyrosine, tryptophan and
to a lesser extent by phenylalanine is responsible for the characteristic strong
absorbance of light by proteins. This property is exploited in the
characterization and quantification of proteins.
c) Amino acids with polar, uncharged R groups
 This group of amino acids includes serine, threonine, cysteine, methionine,
asparagine and glutamine .
 The hydroxyl group of serine and threonine, the sulphur atom of cysteine and
methionine and the amide group of asparagine and glutamine, contribute to
the polarity.
 The R groups of these amino acids are more hydrophilic than the non-polar
amino acids.
d) Amino acids with charged R groups
 Acidic: The two amino acids with acidic R groups are aspartic and glutamic
acids. These amino acids have a net negative charge at pH 7.0.
 Basic: This group includes lysine, arginine and histidine . The R groups have
a net positive charge at pH 7.0. The lysine has a second -amino group;
arginine has a positively charged guanidino group; and histidine has an
imidazole group.
Properties of amino acids
Physical
 Amino acids are white crystalline substances.
 Most of them are soluble in water and insoluble in non-polar organic solvents
(e.g., chloroform and ether).
 Aliphatic and aromatic amino acids particularly those having several carbon
atoms have limited solubility in water but readily soluble in polar organic solvents.
  They have high melting points varying from 200-300oC or even more.
 They are tasteless, sweet or bitter.
 Some are having good flavour. Sodium glutamate is a valuable flavouring agent
and is used in the preparation of certain dishes and sauces.

Amphoteric nature of amino acids

o Amino acids are amphoteric compounds, as they contain both acidic (COOH)
and basic (NH2) groups.
o They can react with both alkalies and acids to form salts.
o In acid solution amino acids carry positive charges and hence they move
towards cathode in an electric field.
o In alkaline solution, the amino acids carry negative charges and therefore move
towards anode.
o When an amino acid is dissolved in water, it exists as inner salt carrying both
positive and negative charges.This occurs as a result of dissociation of carboxyl
group to release the H+ ion, which passes from the carboxyl to the amino group.
The amino acids possessing both positive and negative charges are called
zwitterions.
o The zwitterion reacts as an acid with a base by liberating a proton (H+) from
the NH3+ group and as a result possesses a net negative charge.
o On the other hand, zwitterions reacts with an acid as base, combining with the
proton (H+) of the acid resulting in the formation of a compound having a net
positive charge. These reactions are reversible.
o The pH at which the amino acid has no tendency to move either towards positive
or negative electrode is called isoelectric pH or isoelectric point.
o At isoelectric pH, the amino acid molecule bears a net charge of zero.
Isomerism
 All amino acids except proline, found in protein are -amino acids because NH2
group is attached to the -carbon atom, which is next to the COOH group.
 Examination of the structure of amino acids reveals that except glycine, all other
amino acids possess asymmetric carbon atom at the alpha position.
 Because of the presence of asymmetric carbon atom, amino acids exist in
optically active forms.
  For example, in the steric configuration for serine, the carboxyl group is written
on the top, while the amino group is written to the left in the case of L-serine and
to the right in the case of D-serine This distinction will hold good for all the amino
acids having asymmetric carbon atoms.

 'D' and 'L' do not refer to the optical rotation, but to the steric configuration of
amino group to the right and left side of the carboxyl group.
 The direction of optical rotation of amino acid is indicated by the symbol + or -,
which follows the designation 'D' or 'L'.
 The steric configuration and optical rotation of an amino acid may be
simultaneously expressed as D (+) or D (-) and L (+) or L (-).
 L-forms are more common than D-forms and most of the naturally occurring
amino acids are L-amino acids.

Chemical properties
a) Reactions due to amino group
Reaction with formaldehyde (Formal titration)
 Amino acid exists as zwitterion in aqueous medium. If an amino acid solution is
treated with excess of neutralized formaldehyde solution, the amino group
combines with formaldehyde forming dimethylol amino acid which is an amino
acid formaldehyde complex.
 Hence the amino group is protected and the proton released is titrated against
alkali.
 This method is used to find out the amount of total free amino acids in plant
samples.
Reaction with nitrous acid
Nitrous acid reacts with the amino group of amino acids to form the corresponding
hydroxyacids and liberate nitrogen gas.
Reaction with ninhydrin
 Ninhydrin is a strong oxidizing agent.
 When a solution of amino acid is boiled with ninhydrin, the amino acid is
oxidatively deaminated to produce ammonia and a ketoacid.
 The keto acid is decarboxylated to produce an aldehyde with one carbon atom
less than the parent amino acid.
 The net reaction is that ninhydrin oxidatively deaminates and decarboxylates -
amino acids to CO2, NH3 and an aldehyde.
 The reduced ninhydrin then reacts with the liberated ammonia and another
molecule of intact ninhydrin to produce a purple coloured compound known as
Ruhemann's purple.
 This ninhydrin reaction is employed in the quantitative determination of amino
acids.
 Proteins and peptides that have free amino group(s) (in the side chain) will also
react and give colour with ninhydrin.

b) Reactions due to carboxyl group

Decarboxylation
 The carboxyl group of amino acids is decarboxylated to yield the corresponding
amines. Thus, the vasoconstrictor agent, histamine is produced from histidine.
 Histamine stimulates the flow of gastric juice into the stomach and the
dilation and constriction of specific blood vessels.
 Excess reaction to histamine causes the symptoms of asthma and various
allergic reactions.
Essential amino acids
 Most of the prokaryotic and many eukaryotic organisms (plants) are capable of
synthesizing all the amino acids present in the protein. But higher animals
including man possess this ability only for certain amino acids.
 The amino acids, which are needed for normal functioning of the body but
cannot be synthesized from metabolic intermediates, are called essential
amino acids.
 These must be obtained from the diet and a deficiency in any one of the amino
acids prevents growth and may even cause death.
  Methionine, Arginine, Threonine, Tryptophan, Valine, Isoleucine, Leucine,
Phenylalanine, Histidine, and Lysine are the essential amino acids
(Remember MATTVILPHLy).
Peptide
 Amino acids are linked together by formation of covalent bonds.
 The covalent bond is formed between the -carboxyl group of one amino acid
and the -amino group of the next amino acid.
 The bond so formed between the carboxyl and the amino groups, after
elimination of a water molecule is called as a peptide bond and the compound
formed is a peptide.
 The peptide formed between two amino acids is a dipeptide; three amino acids
is a tripeptide; few amino acids are an oligopeptide and many amino acids is a
polypeptide.
 In writing the peptide structure, the amino terminal (N-terminal) amino acid is
written first and carboxyl terminal (C-terminal) amino acid written last.

Peptides of physiological interest

 Glutathione is a commonly occurring tripeptide ( -glutamyl cysteinyl glycine)

in
many living organisms.

 It has a role in detoxification of toxic compounds in physiological system.

 The nanapeptides (nine amino acids), oxytocin and vasopressin are important
animal peptide hormones.
 Oxytocin induces labor in pregnant women and controls contraction of uterine
muscle.
 Vasopressin plays a role in control of blood pressure by regulating the
contraction of smooth muscles.
 A dipeptide L-aspartyl-L-phenylalanine, is of commercial importance. This
dipeptide is about 200 times sweeter than cane sugar. The methyl ester of this
dipeptide is called as aspartame and marketed as an artificial sweetener for
diabetics.
 Lecture 12
Proteins
Classification of protein
Proteins are classified based on their
 Solubility and composition
 Function
 Shape & size
A. Classification based on solubility and composition
According to this classification, proteins are divided into three main groups as
simple, conjugated and derived proteins.
(i) Simple proteins
 Simple proteins yield on hydrolysis, only amino acids.
 These proteins are further classified based on their solubility in different solvents
as well as their heat coagulability.

Albumins
 Albumins are readily soluble in water, dilute acids and alkalies
 coagulated by heat.
 Seed proteins contain albumin in lesser quantities.
 Albumins may be precipitated out from solution using high salt concentration, a
process called 'salting out'.
 They are deficient in glycine.
 Serum albumin and ovalbumin (egg white) are examples.
Globulins
 Globulins are insoluble or sparingly soluble in water, but their solubility is
greatly increased by the addition of neutral salts such as sodium chloride.
These proteins are coagulated by heat.
 They are deficient in methionine.
 Serum globulin, fibrinogen, myosin of muscle and globulins of pulses are
examples.
Prolamins
 Prolamins are insoluble in water but soluble in 70-80% aqueous alcohol.
 Upon hydrolysis they yield much proline and amide nitrogen, hence the name
prolamin.
  They are deficient in lysine.
 Gliadin of wheat and zein of corn are examples of prolamins.
Glutelins
 Glutelins are insoluble in water and absolute alcohol but soluble in dilute
alkalies and acids.
 They are plant proteins e.g., glutenin of wheat.
Histones
 Histones are small and stable basic proteins
 They contain fairly large amounts of basic amino acid, histidine.
 They are soluble in water, but insoluble in ammonium hydroxide.
 They are not readily coagulated by heat.
 They occur in globin of hemoglobin and nucleoproteins.
Protamines
 Protamines are the simplest of the proteins.
 They are soluble in water and are not coagulated by heat.
 They are basic in nature due to the presence of large quantities of arginine.
 Protamines are found in association with nucleic acid in the sperm cells of
certain fish.
 Tyrosine and tryptophan are usually absent in protamines.
Albuminoids
 These are characterized by great stability and insolubility in water and salt
solutions.
 These are called albuminoids because they are essentially similar to albumin and
globulins.
 They are highly resistant to proteolytic enzymes.
 They are fibrous in nature and form most of the supporting structures of animals.
 They occur as chief constituent of exoskeleton structure such as hair, horn and
nails.

ii. Conjugated or compound proteins

 These are simple proteins combined with some non-protein substances
known as prosthetic groups.
 The nature of the non-protein or prosthetic groups is the basis for the sub
classification of conjugated proteins.
Nucleoproteins
 Nucleoproteins are simple basic proteins (protamines or histones) in salt
combination with nucleic acids as the prosthetic group.
 They are the important constituents of nuclei and chromatin.
Mucoproteins
 These proteins are composed of simple proteins in combination with
carbohydrates like mucopolysaccharides, which include hyaluronic acid and
chondroitin sulphates.
 On hydrolysis, mucopolysaccharides yield more than 4% of amino-sugars,
hexosamine and uronic acid e.g., ovomucoid from egg white.
 Soluble mucoproteins are neither readily denatured by heat nor easily
precipitated by common protein precipitants like trichloroacetic acid or picric acid.
 The term glycoproteins is restricted to those proteins that contain small
amounts of carbohydrate usually less than 4% hexosamine.
Chromoproteins
 These are proteins containing coloured prosthetic groups e.g., haemoglobin,
flavoprotein and cytochrome.
Lipoproteins
 These are proteins conjugated with lipids such as neutral fat, phospholipids
and cholesterol
Metalloproteins
 These are metal-binding proteins.
 A -globulin, termed transferrin is capable of combining with iron, copper and
zinc.
 This protein constitutes 3% of the total plasma protein.
 Another example is ceruloplasmin, which contains copper.
Phosphoproteins
 These are proteins containing phosphoric acid.
 Phosphoric acid is linked to the hydroxyl group of certain amino acids like serine
in the protein e.g., casein of milk.
iii. Derived proteins
 These are proteins derived by partial to complete hydrolysis from the simple or
conjugated proteins by the action of acids, alkalies or enzymes.
  They include two types of derivatives, primary-derived proteins and
secondary-derived proteins.
Primary-derived proteins
 These protein derivatives are formed by processes causing only slight changes
in the protein molecule and its properties.
 There is little or no hydrolytic cleavage of peptide bonds.
Proteans
 Proteans are insoluble products formed by the action of water, dilute acids and
enzymes.
 These are particularly formed from globulins but are insoluble in dilute salt
solutions
 e.g., myosan from myosin, fibrin from fibrinogen.
Metaproteins
 These are formed by the action of acids and alkalies upon protein.
 They are insoluble in neutral solvents.
Coagulated proteins
 Coagulated proteins are insoluble products formed by the action of heat or
alcohol on natural proteins
 e.g., cooked meat and cooked albumin.
Secondary-derived proteins
 These proteins are formed in the progressive hydrolytic cleavage of the peptide
bonds of protein molecule.
 They are roughly grouped into proteoses, peptones and peptides according
to average molecular weight.
 Proteoses are hydrolytic products of proteins, which are soluble in water and are
not coagulated by heat.
 Peptones are hydrolytic products, which have simpler structure than proteoses.
 They are soluble in water and are not coagulated by heat.
 Peptides are composed of relatively few amino acids.
 They are water-soluble and not coagulated by heat.
 The complete hydrolytic decomposition of the natural protein molecule into amino
acids generally progresses through successive stages as follows:
Protein -----> Protean ------- Metaprotein
Proteoses ------>Peptones ------->Peptides ------ amino acids
b. Classification of proteins based on function
Proteins are classified based on their functions as:
Catalytic proteins – Enzymes
 The most striking characteristic feature of these proteins is their ability to
function within the living cells as biocatalysts.
 These biocatalysts are called as enzymes.
 Enzymes represent the largest class.
 Nearly 2000 different kinds of enzymes are known, each catalyzing a different
kind of reaction.
 They enhance the reaction rates a million fold.
Regulatory proteins - Hormones
 These are polypeptides and small proteins found in relatively lower
concentrations in animal kingdom but play highly important regulatory role in
maintaining order in complex metabolic reactions
 e.g., growth hormone, insulin etc.
Protective proteins - Antibodies
 These proteins have protective defense function.
 These proteins combine with foreign protein and other substances and fight
against certain diseases.
 e.g., immunoglobulin.
 These proteins are produced in the spleen and lymphatic cells in response to
foreign substances called antigen.
 The newly formed protein is called antibody which specifically combines with the
antigen which triggered its synthesis thereby prevents the development of
diseases.
 Fibrin present in the blood is also a protective protein.
Storage proteins
 It is a major class of proteins which has the function of storing amino acids as
nutrients and as building blocks for the growing embryo.
 Storage proteins are source of essential amino acids, which cannot be
synthesized by human beings.
 The major storage protein in pulses is globulins and prolamins in cereals.
 In rice the major storage protein is glutelins.
 Albumin of egg and casein of milk are also storage proteins.
Transport proteins
 Some proteins are capable of binding and transporting specific types of
molecules through blood.
 Haemoglobin is a conjugated protein composed of colourless basic protein, the
globin and ferroprotoporphyrin or haem.
 It has the capacity to bind with oxygen and transport through blood to various
tissues.
 Myoglobin, a related protein, transports oxygen in muscle.
 Lipids bind to serum proteins like albumin and transported as lipoproteins in the
blood.
Toxic proteins
 Some of the proteins are toxic in nature.
 Ricin present in castor bean is extremely toxic to higher animals in very small
amounts.
 Enzyme inhibitors such as trypsin inhibitor bind to digestive enzyme and
prevent the availability of the protein.
 Lectin, a toxic protein present in legumes, agglutinates red blood cells.
 A bacterial toxin causes cholera, which is a protein.
 Snake venom is protein in nature.
Structural proteins
 These proteins serve as structural materials or as important components of
extra cellular fluid.
 Examples of structural proteins are myosin of muscles, keratin of skin and
hair and collagen of connective tissue.
 Carbohydrates, fats, minerals and other cellular components are organized
around such structural proteins that form the molecular framework of living
material.
Contractile proteins
 Proteins like actin and myosin function as essential elements in contractile
system of skeletal muscle.
Secretary proteins
 Fibroin is a protein secreted by spiders and silkworms to form webs and
cocoons.
Exotic proteins
  Antarctic fishes live in -1.9oC waters, well below the temperature at which their
blood is expected to freeze.
 These fishes are prevented from freezing by antifreeze glycoproteins present
in their body.
C. Classification based on size and shape
Based on size and shape, the proteins are also subdivided into globular and
fibrous proteins.
 Globular proteins are mostly water-soluble and fragile in nature e.g., enzymes,
hormones and antibodies.
 Fibrous proteins are tough and water-insoluble.
 They are used to build a variety of materials that support and protect specific
tissues, e.g., skin, hair, fingernails and keratin
 Lecture 13 - 14
Conformation of proteins
Conformation of a protein refers to the three-dimensional structure in its
native state. There are many different possible conformations for a molecule as large as
a protein. A protein can perform its function only when it is in its native condition. Due
to the complexity of three-dimensional structures, the structure of protein is discussed at
different levels of its organization.
Four levels of structural organization can be distinguished in proteins:
1. Primary
2. Secondary
3. Tertiary
4. Quaternary
Primary structure
 Primary structure of protein refers to the number of amino acids and the order
in which they are covalently linked together.
 It also refers to the location of disulfide bridges, if there are any, in a polypeptide
chain.
 The peptide bond is covalent in nature, quiet stable and referred as backbone
of the protein.
 They can be disrupted by chemical or enzymatic hydrolysis but are not directly
influenced by salt concentration, change in pH or solvent.
 Frederick Sanger in 1953 determined the complete amino acid sequence of
insulin for the first time.

The important steps involved in determining the primary structure of protein are
 Determination of number of (chemically different) polypeptide chains or subunits
in the protein.
 Separation of polypeptide chains if more than one are present in a protein.
 Determination of the amino acid sequence of the subunits.
 Elucidation of the position of the disulfide bonds, if any, between and within the
subunits.
1. Determination of number of polypeptides or subunits
Determination of the number of C-terminal or N-terminal amino acids will indicate
the number of polypeptides in a protein.
 H2N -------------------> COOH
N-terminal C-terminal

N-terminal identification
 Fluoro dinitro benzene (FDNB), known as Sanger's reagent, was used to
identify the N-terminal amino acid.
 This reagent was replaced by dansyl chloride and Edman's reagent (phenyl
isothiocyanate, PITC).
 Edman's reagent is also used to determine the amino acid sequence of a
polypeptide chain from the N-terminal by subjecting the polypeptide to repeated
cycles of Edman degradation.
 After every cycle, the newly liberated phenylthiohydantoin (PTH) amino acid
was identified
 The sequence of peptides containing 30-40 amino acids can be determined
using a sequencer by adopting the Edman's degradation method.
C-terminal identification
C-terminal amino acid can be determined by methods similar to those used for
the N-terminal acid.
 Hydrazine is used to find out the C-terminal amino acid.
 It reacts with the carbonyl group of each peptide bond except C-terminal
amino acid.
 The bond is cleaved and each amino acid derivative is released as the hydrazide
derivative (hydrazinolysis).
 Since the carboxyl group of C-terminal amino acid is not involved in a peptide
bond, it remains in the mixture as the only unmodified amino acid
 After chromatographic separation and comparison with the standards, the C-
terminal amino acid can be identified.
 Carboxypeptidases are used for enzymic determination of the C-terminal amio
acid.
Separation and purification of polypeptide chains
 Determination of C-terminal and/or N-terminal amino acids reveals the presence
of one or more polypeptide chains in a protein.
 If the protein contains more than one polypeptide chain, separation of
polypeptide chain is essential.
  If the polypeptide chains are connected by disulfide bond, they are cleaved to
separate the individual peptide chains.
 The polypeptide is treated with 2-mercaptoethanol (HS-CH2-CH2OH) so that
reductive cleavage occurs and the polypeptide chains are separated.
 The resulting free-SH groups are usually alkylated by treatment with iodoacetic
acid
 After cleaving the disulfide links using mercaptoethanol, subunits are dissociated
using denaturing agents such as urea or guinidinum ion or detergents such
as sodium dodecyl sulphate (SDS).
 The dissociated subunits are then separated using ion exchange or gel filtration
chromatographic method.
Amino acid sequencing of polypeptides
 The amino acid sequence in polypeptides with 30-40 amino acids can be
determined by Edman reaction.
 For polypeptides containing more than 40 amino acids, both enzymatic and
chemical methods are employed to break polypeptide chains into smaller
peptides.
 The enzyme, trypsin hydrolyses the peptide bond on the carboxyl side of the
basic amino acid residues of lysine or arginine.
 The chemical reagent, cyanogens bromide cleaves peptide bond on the
carboxyl side of methionine residues.
 The hydrolyzed peptides are separated and the amino acid sequence is
determined by Edman reaction.
 The hydrolysis of the original polypeptide by two different methods separately
gives overlapping regions, from which the sequence is derived
Secondary structure
 Secondary structure refers to the steric relationship of amino acids that are
close to one another in the linear sequence.
 The folding of a linear polypeptide chain occurs to form a specific coiled
structure.
 Such coiling or folding is maintained by hydrogen bonds and hydrogen bond is
the only bond responsible for secondary structure.
 X-ray studies of several polypeptides by Linus Pauling and Robert Corey
revealed that the peptide group has a rigid, planar structure which is a
 consequence of resonance interactions that give the peptide bond a 40%
double bond character.
 Peptide groups mostly assume the transconformation in which successive C2
atoms are on opposite sides of peptide bond joining them.
 The cis configuration creates steric interference.
 If a polypeptide chain is twisted by the same amount each of its C atoms, it
assumes a helical conformation
Helix structure
 The -helix is the most stable arrangement of polypeptides
 The helix structure of proteins is stabilized by intramolecular hydrogen
bonding.
 In this structure, hydrogen bonds are formed between the C=O group of one
peptide bond and the N-H group of another after 3 amino acid units.
 The polypeptide chain constituted by L-amino acids form a right-handed helix,
whereas the polypeptide chains made up of D-amino acids form a left-handed
helix.
 In the -helical conformation, all the side chains lie outside the helix whereas
C, N, O and H of the peptide bond lie in the same plane.
 Certain amino acids tend to disrupt the -helix. Among these are proline (the
N atoms is part of the rigid ring and no rotation of the N-C bond can occur) and
amino acid with charged or bulk R groups that either electrostatically or
physically interferes with helix formation.
The -pleated sheet structure
 Pauling and Corey also proposed a second ordered structure, the -pleated
sheet for polypeptide.
 This structure is a result of intermolecular hydrogen bonding between the
polypeptide chains to form a sheet like arrangement.
 There are two ways in which proteins chains can form the pleated sheet
structure.
 One is with the chains running in the same direction i.e. the -COOH or NH2
ends of the polypeptide chains lying all at the top or all at the bottom of the sheet.
This is called parallel pleated-sheet structure.
 In another type, known as antiparallel -pleated sheet structure, the
polypeptide chains alternate in such a way that the -COOH end of the one
 polypeptide is next to the -NH2 end of the other i.e. polypeptide chains run in
opposite directions.
The random coil
 Regions of proteins that are not identifiably organized as helices or pleated
sheets are said to be present in random coil conformation.
 Considerable portion of the protein may be present in this conformation.
 The term 'random' is unfortunate which imply less biological significance than
more highly repeating regions.
 But in terms of biological function, the regions of random coil are of equal
importance to those of helix and pleated sheet.
Tertiary structure
 Tertiary structure refers to the steric relationship of amino acid residues that
are far apart in the linear sequence.
 This leads to the twisting of polypeptide chains into specific loops and bends
which are maintained chiefly by five kinds of bonds.
Hydrogen bonds
 Hydrogen bonds are formed between the side chain (R group) of amino acids
having a hydrogen donor group and an acceptor group

Salt-linkages (electrostatic forces; ionic bonds)

 Salt linkages are due to the interaction between amino groups of basic amino
acids and the carboxyl group of acidic amino acids present in the R group
Disulfide bonds (S-S linkages)
 The S-S linkages are formed by the oxidation of sulfhydryl (-SH) group of two
cysteine side chains

Hydrophobic bonds
 Hydrophobic bonds are formed as a result of interaction between non-polar side
chains

Dipole-dipole interaction
 This interaction occurs between polar unionized side chains
 The folding of a polypeptide chain due to different covalent and non-covalent
interactions is shown below.
 Out of the above bonds, the disulfide bond (covalent bond) is the strongest
and cannot be affected by solvent, pH, temperature and salts whereas the
above conditions.
 The disulfide bond can be split and reformed by oxidation/reduction respectively
 The tertiary structure gains special importance in the case of enzymes.
Domain
 Domains are structurally independent units that have the characteristics of a
small globular protein.
 Domains often have a specific function such as the binding of a small
molecule.
 A long peptide strand of a protein will often fold into multiple, compact
semiindependent folded regions or domains.
 Each domain having a characteristic spherical geometry with a hydrophobic
core and polar surface very much like the tertiary structure of a whole globular
protein
 The domains of a multidomain protein are often interconnected by a segment of
polypeptide chain lacking regular secondary structure.
 In enzymes with more than one substrate or allosteric effector sites the
different binding sites are often located in different domains.
 In multifunctional proteins, the different domains perform different tasks.

Quaternary structure
 Proteins that have more than one subunit or polypeptide chains will exhibit
quaternary structure.
 Quaternary structure refers to a functional protein aggregate (organization)
formed by interpolypeptide linkage of subunits or polypeptide chains.
 These subunits are held together by noncovalent surface interaction between
the polar side chains.
 Proteins formed like above are termed oligomers and the individual polypeptide
chains are variously termed protomers, monomers or subunits.
 The most common oligomeric proteins contain two or four protomers and are
termed dimers or tetramers, respectively.
 Myoglobin has no quaternary structure since, it is composed of a single
polypeptide chain.
  Hemoglobin molecule, which consists of four separate polypeptide chains,
exhibits quaternary structure.

A schematic of hemoglobin.

The ribbon parts represent the protein globin; the four green parts are the heme groups.

 Quaternary structure may influence the activity of enzymes.

 Some enzymes are active only in their quaternary state and become inactive
when split into smaller units.
 Other enzymes are inactive in the quaternary state and are activated only when
they are dissociated to form monomeric state.
Physical and chemical properties of proteins
Physical
 Pure proteins are generally tasteless, though the predominant taste of protein
hydrolysates is bitter.
 Pure proteins are odourless.
 Because of the large size of the molecules, proteins exhibit many properties that
are colloidal in nature.
 Proteins, like amino acids, are amphoteric and contain both acidic and basic
groups.
 They possess electrically charged groups and hence migrate in an electric
field.
 Many proteins are labile and readily modified by alterations in pH, UV radiation,
heat and by many organic solvents.
 The absorption spectrum of protein is maximum at 280 nm due to the
presence of tyrosine and tryptophan, which are the strongest chromophores
in that region.
 Hence the absorbance of the protein at this wavelength is adapted for its
determination.
Denaturation of protein
 The comparatively weak forces responsible for maintaining secondary, tertiary
and quaternary structure of proteins are readily disrupted with resulting loss of
biological activity.
 This disruption of native structure is termed denaturation.
 Physically, denaturation is viewed as randomizing the conformation of a
polypeptide chain without affecting its primary structure
 Physical and chemical factors are involved in the denaturation of protein
a) Heat and UV radiation supply kinetic energy to protein molecules causing th
atoms to vibrate rapidly, thus disrupting the relatively weak hydrogen bonds
and salt linkages. This results in denaturation of protein leading to coagulation.
Enzymes easily digest denatured or coagulated proteins.
b) Organic solvents such as ethyl alcohol and acetone are capable of forming
intermolecular hydrogen bonds with protein disrupting the intramolecular
hydrogen bonding. This causes precipitation of protein.
b) Acidic and basic reagents cause changes in pH, which alter the charges
present on the side chain of protein disrupting the salt linkages.
c) Salts of heavy metal ions (Hg2+, Pb2+) form very strong bonds with
carboxylate anions of aspartate and glutamate thus disturbing the salt
linkages. This property makes some of the heavy metal salts suitable for use
as antiseptics.
Renaturation
 Renaturation refers to the attainment of an original, regular three-
dimensional functional protein after its denaturation.
 When active pancreatic ribonuclease A is treated with 8M urea or
mercaptoethanol, it is converted to an inactive, denatured molecule.
 When urea or mercaptoethanol is removed, it attains its native (active)
conformation.
Chemical
Colour reactions of proteins
 The colour reactions of proteins are of importance in the qualitative detection and
quantitative estimation of proteins and their constituent amino acids.
 Biuret test is extensively used as a test to detect proteins in biological materials.
Biuret reaction
  A compound, which is having more than one peptide bond when treated with
Biuret reagent, produces a violet colour. This is due to the formation of
coordination complex between four nitrogen atoms of two polypeptide
chains and one copper atom

Xanthoproteic reaction
 Addition of concentrated nitric acid to protein produces yellow colour on
heating, the colour changes to orange when the solution is made alkaline. The
yellow stains upon the skin caused by nitric acid are the result of this
xanthoproteic reaction. This is due to the nitration of the phenyl rings of
aromatic amino acids.
Hopkins-Cole reaction
 Indole ring of tryptophan reacts with glacial acetic acid in the presence of
concentrated sulphuric acid and forms a purple coloured product. Glacial
acetic acid reacts with concentrated sulphuric acid and forms glyoxalic acid,
which in turn reacts with indole ring of tryptophan in the presence of sulphuric
acid forming a purple coloured product.
 SEED PRODUCTION IN PEARL MILLET

Bajra is common minor millet of India with wider industrial and household
utility. It is used a feed, food and raw material in soft drink industry. Botanically it
is known as Pennisetum typhoides L. and belongs to the family poaceae.

Floral biology

It is a highly cross-pollinated crop. The pollinating agent is wind. The flowers

are protogynous. The spike emerges about 10 weeks after sowing, The styles begin
to protrude 2-3 days later first at the top of the inflorescence and proceeds. They
take two days to complete the entire spike. Exerted stigma remains receptive for
12-24 hours. Anthers usually emerge after the styles are dry. The anther
emergence starts from middle of the spike and proceeds upwards and downwards.
Anthesis occurs throughout the day and night with the peak between 8.00 p.m. to
2.00 a.m.

Protogynus

Stigma Anther

Popular variety : co7, co 8

Synthetics : If more than 5 parental lines are combined ,which are having general

combining ability e.g. CO 7, ICMS 7703

Composite: 3-5 inbreds with no general combining ability are mixed and

multiplied. WCC 75.(ICRISAT).

Land requirement

Seed field offered for certification should not have been grown with bajra in
the previous season. However if it was grown, the field should be irrigated 3 weeks
before sowing to destroy the germinating seeds.

Field Standards for isolation

Bajra field should be isolated from contaminants as follows

Contaminants Minimum distance(m)

Foundation Certified
stage stage

Fields of other varieties 400 200

Fields of same variety not confirming to 200 100

varietal purity requirements for certification

In bajra differential blooming dates for modifying the isolation distance is not
permitted

Selection of Seed

 For production of foundation seed, breeder seed is used as the base material
while for certified seed, foundation seed should be used as the base
material .

 The seed used should be from authenticated source with tag and bill.

 The required seed rate will be 18kg /ha or 3-4kg/ acre.

Presowing seed treatment

 The seeds are given with any one of the seed treatment or in combination.

 Seeds are soaked in 2% KH 2 PO 4 or 0.5% brassinolide for 16h with a seed

to solution ratio of 1:0.06 and are dried back to their original moisture
 content of 8-9% .This management could be used both for dryland
agriculture as well as garden land.

 As an ecofriendly treatment seeds are also fortified or hardened with 1%

prosopis and pungam leaf extract for 16h with a seed to solution ratio of
1:0.06 and are dried back to their original moisture content of 8-9%

 Seeds are treated with metalaxyl @6g/kg of seed to prevent the infestation
by downy mildew.

 Seeds are also treated with 5% carbofuran 3G to protect the seed from
shoofly infection. Seed treatment with chlorpyriphos @4 ml /kg is also
recommended against the attack by shoofly.

 Seeds are dry dressed with bavistin @2g/kg of seed to protect against seed
borne pathogens and soil borne pathogen.

 Seeds are also treated with azospirillum @50g/kg of seed to fix atmospheric
N. Any one of these treatment or combination of treatment is adopted for
better productivity.

 On adoption of sequence of treatment physiological should be followed with

physical seed treatment.

Sowing

 The seed are sown at a spacing of 45 x 20 cm at a depth of 2-4cm as the

plant has adventitious root system.

 In some places seeds are also raised in nursery and transplanted to the main
field at an age of 20 -25 days.

 In the main field seeds are sown either in ridges and furrows or under beds
and channels.

 The seedlings are thinned or transplanted at 20-25 days after sowing and
gapfilling should be done 10-15 days after sowing.
Nutrient application

 At last ploughing apply 12.5 tonnes of compost per hectare. The fertilizer
requirement of seed crop is 100:50:50 kg of NPK, in which 50:50:50 kg /ha
of NPK is applied as basal, while 50kg of N is applied after 30-35 days after
sowing at tillering phase .

 The seed crop is also sprayed with 2% DAP at primordial initiation stage and
twice thereafter at 10 days interval to enhance uniform flowering and
increased seed set.

Weeding

Application of atrazine @ 10ml per litre as pre-emergence herbicide controls

the growth of weeds upto 20-25 days. One hand weeding at the time of primordial
initiation keep the field free of weeds. Weeding after boot leaf stage is not
economical and shade will also minimize the weed flora. On organic production, 2
hand weeding at seedling stage and other at boot leaf formation will keep the field
weed free.

Irrigation

 The crop should be irrigated once in a week for enhanced seed set and
formation of bolder grains .

 The critical stages of irrigation are primordial initiation stage, vegetative

stage ,milky and maturation stage. If the irrigation is withheld in these
stages seed set will be poor and seed size will be reduced.

Pest and disease management

Common pests Management techniques

Shootfly Monocrotophos 0.03%

Stemborer Rogar 0.3%

Downy mildew Metalaxil @ 500gor ridonil MZ WP 2@2kg/ha

Mancozeb@ 1kg/ha.
 Earhead bugs Endosulphan 0.07%

Black mould Endosulphan 0.07% + Bavistin @10g /lit.

Green ear /Smut/Ergot Spray carbendazim @500g/ac in 2stages 10 and 50

flowering

Rust Spray with wettable sulphur @2.5g/ha on initiation

symptom and 10 days thereafter..

Green ear Smut Ergot

Roguing

It is specific to seed crop and is done from seedling stage to harvesting stage
based on the phenotypic characters. Off types can be identified through stem
colour, plant structure, number of leaves, auricles, nodal colour, grain colour etc.
The field standard for seed crop is as follows

Specific standard: These are verified at the final inspection

Factor Maximum permitted

(%)

FS CS
Off types at any one inspection and after flowering 0.050 0.10

Plants infected by downy mildew/ green ear disease 0.050 0.10

any one inspection

Ergot earheads at final inspection ** 0.020 0.040

Earheads infected with grain smut at final inspection 0.050 0.100

** Even if the infection is within the limit seeds are graded with brine
solution to become eligible for certification.

Seed Certification

Number of Inspections

A minimum of three inspections shall be made as follows:

1. The first inspection shall be made before flowering preferably within 30 days
after planting in order to verify isolation, volunteer plants, off types, downy mildew
incidence and other relevant factors.

2. The second inspection shall be made during 50% flowering to check isolation, off
types, downy mildew incidence /green ear and other relevant factors

3. The third inspection shall be made at maturity and prior to harvesting and in
order to determine the incidence of downy mildew /green ear disease, ergot, grain
smut and to verify true nature of plant and other relevant factors

Pre harvest sanitation spray

Spraying of endosulphan @ 0.07% and bavistin@10g /lit 10 days prior to

harvest prevent the seed weevil (Sitophilus oryzae) infestation at storage.

Harvesting

The crop attains physiological maturity 30-35 days after 50% flowering and
the seed moisture at this stage will be around 25-30%. This stage can be easily be
identified by the formation of dunken layer at the place of attachment to the ear
head. The ear heads are harvested when 80 % of the ear heads are physiologically
matured, where the moisture content will be around 20 %.The crop is commercially
harvested as once over harvest but harvesting of ear heads as 2or 3 picking will
preserve the seed quality as matured seeds are not over exposed to the changes in
environmental conditions.

Special techniques

Selection of first formed 5-6 tillers for seed purpose ensures seeds quality.
Ear heads also exhibit positional polymorphism where seeds of middle are better in
seed quality. This type of selection will be useful in long term storage of seeds

Threshing

The ear heads are dried under sun and threshed with fliable stick for
extraction of seeds. The moisture content of seed at the time of threshing will be
15-18%.On large scale production LCT threshers are used, but care should be given
to avoid mechanical damage, which in turn will reduce the seed quality and
storability.

Drying

The seeds are dried to 8 to10 % moisture content either under sun or
adopting mechanical driers for long term storage as the seeds is orthodox in
nature.

Processing

Mechanical grading can be done with cleaner cum grader, which will remove
the undersized immature and chaffy seeds .The middle screen size should be 4/64”
round perforated sieves. The size can vary depending on the variety. (For WCC 75
5/64”sieve is used).
Seed yield: 3500- 4000 kg/ha

Seed treatment

The seeds are infested with several storage pests, to protect against these
pests the seeds are given protective treatment with bavistin @2g/kg of seed with
carbaryl @200mg/kg of seed as slurry treatment. Bifenthrin @5mg /kg of seed is
also recommended for better seeds storage

Seed packing

Seeds are packed in gunny bag for short term storage while in HDPE and
polylined gunny bag for long term storage.

Storage

The treated seed can be stored up to 12 months provided the seeds are not
infected with storage pests. Seed can be stored up to 3 years if the seeds are
packed in moisture containers and are stored at low temperature .The godown
should be kept clean as the possibility of secondary infestation with Trifolium (red
flour weevil ) is much in these crop. The major problem in storage is incidence of
grain weevil which will powder the seed material in a short period.

Seed standard

The processed seed should have the following seed quality characters both
for certification and labeling.

Seed Standard

Factor Standards for each class

FOUNDATION CERTIFIED

Pure seed ( maximum) 98.0% 98.0%

Inert matter(maximum) 2.0% 2.0%

Other crop seed (maximum) 10/kg 20/kg

Weed seed 10/kg 20/kg

Ergot, sclerotia, seed entirely or partially 0.020% 0.040%

modified as sclerotia, broken or ergotted (by number) (by number)
seed (maximum)

Germination ( Minimum) 75% 75%

Moisture (maximum) 12.0% 12.0%

For vapour proof container (maximum) 8.0% 8.0%

Mid storage correction

The seeds loose their quality during storage due to deterioration and pest
infestation, when the germination falls below 5-10 % of the required standard the
seeds are imposed with midstorage correction, where the seeds are soaked in
double the volume of 10-4 M solution of potassium dihydrogen phosphate
(3.6mg/lit of water) for 6 hours and the seeds are dried back to original moisture
content (8-9%).

HYBRID SEED PRODUCTION

Breeding Technique for hybrid

seed production : Cytoplasmic genetic male sterility
system (CGMS)

History of bajra hybrid

Seed production : The first report on CGMS line was

made by Burton and his co workers at
Tifton Georgia USA. The line is
Tift 23A.
Popular hybrid

Hybrid Female Male

KM 1 MS 5141 A J 104
KM 2 MS 5141 A K 560 -D-230
X4 MS 5141 A PT 1921
X5 PB 111A PT 1921
X6 732 A PT 3095
X7 111A PT 1890
H B1 Tift 23A(USA) BIL -3B
HB 3 Tift 23A(USA) J 104
HB 5 Tift 23A(USA) K 559
UCH 11 732 A PT 3075 (TNAU)
COH(cu) 8 732 A PT 4450

Commercial Hybrid Seed Production

Isolation : Foundation seed : 1000 m

Certified seed : 200 m

Season : Irrigated : March – April, June - July

January – February
Rainfed : October – November

Seed rate : A line : 6 kg ha-1

B line : 2 kg ha-1

Main field preparation : Ridges and furrows

Planting ratio : Foundation Seed : 4:2

Certified Seed : 6:2
Pusa 23 - 8 : 2

Border rows : Foundation Seed : 8 (B line)

Certified Seed : 4 (R line)
Spacing : A line : 45 x 20 cm
B line : 45 x solid row.
Nursery : Seedling can also be raised in raised bed
nursery and can transplanted
to the main field at 20-25 days of aging.
Manures & Fertilizers

Nursery : 750 kg / 7.5 cents for transplanting in one ha.

Mainfield : Compost : 12.t ton/ha NPK 100:50:50 kg ha-1

Basal : 50:50:50 kg ha-1
Top : 50:0:0 kg ha-1 (At tillering phase

Foliar spray : DAP 1% at peak flowering to enhance flowering

and seed set.

Steps for synchronization of flowering

 Withholding irrigation
 Application DAP 1%
 Staggered sowing
 Jerking
Jerking

It is done 20-25 days after transplanting or 30-40 days after direct sowing.
The early formed earheads of the first tillers are pulled out or removed which will
result in uniform flowering of all the tillers.

Specialty with bajra in synchronization

The synchronization problem is less in bajra due to

 Tillering habit
 Supply of continuous pollen
 Lesser pollen weight
 Flight capacity of pollen
 Pollen viability & stigma receptivity are longer.
Roguing : Done in both lines

• A line : seek for offtypes pollen shedder and

partials
• R line : Seek for early flowering plants,
rouges and diseased plants.

Character of offtypes : Variation in leaf colour, leaf waviness,

grain colour earhead, shape, size, etc.

No. of field inspection : Three

• Seedling stage
• Tillering stage
• Grain formation stage.

Field standards

Standards Maximum permitted (%)

FS CS
Offtypes 0.05 0.10
Pollen shedders 0.05 0.10
Downy mildew diseased plants 0.05 0.10
Earheads affected by ergot 0.02 0.04

Harvesting Technique : • Due to tillering habit, harvest the

panicle / earhead in 2 picking (to
avoid delayed harvest)
• Select 5-7 tillers for seed purpose.

Processing : • Grade with 4/64” round perforated

metal sieve as middle screen
• Use OSAW cleaner cum grader
Seed Treatment : Thiram / Bavistin @3g kg-1 seed
Seed storage : • Cloth bag for short term storage
(12 months)
• 700 gauge polyethylene bag – long
term storage (> 24 months)

Mid storage correction : HDH with Na 2 PO 4 10-4m for 4h.

Seed standards

Standards Permitted (%)

FS CS
Physical purity (Maximum) 98 98
Inert matter (Maximum) 2 2
Other crop seed (Maximum) 10 / kg 10 / kg
Weed seed (Maximum) 10 / kg 10 / kg
Ergot effected seeds (Maximum) by 0.020 % 0.040%
number
Germination 75 75
Moisture content - Moisture pervious 12 12
Moisture impervious 5 5

Seed yield : 3200 - 3250 kg / ha

 SEED PRODUCTION IN COTTON VARIETIES AND HYBRIDS

Cotton botanically as Gossypium sp. is a fibre yielding crop. It is known as

the queen of fiber crops. It serves as a cash crop to the farmer as the lint serves as
the raw material for the textile industry .The seed is used both for multiplication
and as animal feed.The success of commercial crop depends on the quality of the
basic seed.

Floral biology

Simple, solitary, terminal, extra axillary, petals yellow to cream in colour,

hermaphrodite, bracteoles called as epicalyx, three in number, free and deeply
serrated and persistent at the base of the flower. Nectary gland is present on each
bracteole. Calyx five, united, cup shaped corolla five, polypetalous, a purple spot is
present on the inner side of the claw of the petal (petal spot) in some species.
Androecium forming a staminal column (monadelphous) bearing numerous anthers.
Ovary superior penta carpellary, style slender, passes through staminal column with
three to five lobed stigma, ovules many in axile placentation.
 There is much variation in case of flower opening. Asiatic cotton opens
between 8 and 10. a.m. American cotton opens much earlier. Temperature affects
the flower opening. After flower opening the cream yellow colour of corolla turns
pink within a day and later changes to red. The receptivity of the stigma is 8 to 10
a.m.

Cotton is an often cross-pollinated crop where the extend of cross-pollination

is > 60%. In cotton 4 different species are in popular usage, viz. G. arboreum (eg.
K 10) G. herbaceum (e.g. Uppam) G. hirsutum (e.g. MCU varieties) and G.
barbadense (e.g. Suvin and Suguna).

Method of Seed Production

Varieties: Under isolation, by open pollination, the varieties are multiplied. For
nucleus seed production, selfing of flowers is done with cotton (lint) dipped in clay
or red earth.

Hybrids: In cotton both inter and intraspecific hybrids are available.

Interspecific Hybrid :

Varalakshmi : Lakshmi x SB298 E (G. hirsutum x G. barbadense)

DCH 32 / Jayalakshmi : DS 28 x SB 425 (G. hirsutum x G. barbadense)

TCHB213 : TCH 1218 x TCB 209

Intraspecific hybrid : Suguna, Savitha (T7 x M12)

Tool employed for hybrid

 The hybrid seed production in cotton is achieved through emasculation and
dusting technique, which is the physical removal of male organ (staminal column)
from the female parent.

1. Emasculation and dusting

At the time of flower initiation in female line, the flowers that are going to open
next day are selected and the petals are removed between 3-6 pm. With the help of
nail or needle, the total staminal (pollen + anther + anther tube) column are
removed. Then the flowers are covered with a definite colour cover for easy
identification of the emasculated flowers. In the morning between 9 am -12 noon,
which is the anthesis time, the flowers of selected male parent are plugged and
dusted on the stigma of the emasculated flower on opening the cover. It is again
covered with different coloured cover to avoid pollination with other pollen and to
identify the emasculated and dusted flower from the rest. The pollen from a single
flower is enough to dust 4-5 female flowers. The pollen receptivity of the stigma is
for 46 hours.

For easy identification of selfed boll from emasculated and dusted boll the bract
can be removed while emasculating owing to the little contribution of bract to seed
set and seed yield.
Particulars of varieties/hybrids

Varieties Parentage Season Irrigated / Seed yield

Rainfed (kg/ha)
Varieties
MCU 5 Multiple cross Aug- January Irrigated 1850
MCU7 X ray irradiation of x Jan- Feb. to Irrigated (Rice 1330
L 1143 EE May - fallows)
June(summer)
MCU 11 MCU 5 x Egyptian Aug - Irrigated 2200
hirsutum hybrid September
derivative
LRA 5166 Laxmi x Reba B.50 x Sep-October to Rainfed 725
AC 122 Jan - February
K10 K9 x 11876 hybrid Sep-October to Rainfed 726
derivative Jan - February
K11 (0794-1-DX 11876) x Oct- March Rainfed 1100
(0794-D x 11450)
Multiple Hybrid
derivative
SVPR 1 MCU 7 x AC 129/2 February - July Summer - 15-16 Qtl. Of
Irrigated kapas /ha
Hybrids
Suvin Hyrbid derivative Aug - February Irrigated 1020
from the cross
Sujatha x St.Vincent
Jalyalaxmi Interspecific hybird of Aug-February Irrigated 2880
DS 28 G. hirsutum x
SB 425 (VF) G.
barbadense
TCHB 213 Interspecific hybird of Aug-February Irrigated 2215
TCH 1218 G. hirsutum
x TCB 209 G.
barbadense
Savitha T7 x M 12 (Intra Aug-February Irrigated 1800
hirsutum hybrid)
HB 224 It is an interspecific Aug-February Irrigated 2000
hybrid involving
G. hirsutum x
G. barbadense
Steps in hybridizing technique

 Emasculate and dust as far as possible buds appearing during the first six weeks
of reproduction phase to ensure good setting and development of bolls.

 Restrict your emasculation each day evening from 3 pm to 6 pm and pollination

in morning between 9-12 noon to ensure highest purity of hybrid seeds.
Emasculation should be complete and perfect.

 Choose optimum size of bud and avoid young or too old buds for emasculation.

 Cover the male buds with paper bags, previous evening for their use next day.

 Emasculated buds may be covered preferably with butter papers.

 Do not forget to tie a thread to the pedicel of the bud immediately after
pollination.

 Close your crossing programme after 9th week (from commencement of

crossing) and remove all buds and flowers appearing subsequently to facilitate
the development of crossed bolls.

 Nip the top and side shoots to stop further vertical and horizontal growth.

 Light irritations should be given as and when required. Excessive or scanty or

inadequate irrigations should be avoided especially during crossing and boll
development period.

 Continue irrigation till last picking of the crossed bolls. Frequency of irrigation
depends on weather factors like rainfall, temperature and wind velocity.

 Pick up the ripe and completely opened bolls along with threads and collect in
baskets for second sorting. Bolls without threads may be bulk harvested as
female seed cotton.

 Crossed bolls collected in baskets may be sorted out for second time to verify
that they are crossed bolls. Then collect the crossed seed cotton and store in
gunny bags carefully marked as crossed bolls.

 Rain touch cotton or hard locks be picked and kept separately to avoid poor
germination of hybrid seeds.
 Store the crossed seed cotton in a cool dry place till it is handed over to processing
unit.

Use of Genetic male sterility

Hybrids are also produced by employing genetic male sterility system in

cotton, where the female parent will segregate into 50:50 ratio of male sterile and
male fertile plants. The male fertile plants are removed and the male sterile plants
are crossed with concerned male line.

E.g. Suguna: Gregg x K 3400

Land requirement

The field should be fertile and formed into ridges and furrows. Black cotton
soils are highly preferable than other soils. Land should be free from volunteer
plants and designated diseases especially the wilt disease.

Season

Winter crop : Aug - Sep

Summer crop : Feb - March

Seeds and Sowing

Seeds should be obtained from an authenticated source with tag and bill.

Pre-sowing management

The seeds can be hardened with 1% prosopis and pungam leaf extract for
rainfed/summer sowing to resist water stress problem.Use of delinted seed is better
than fuzzy seed to avoid diseased and injured seed.

Seed rate

Varieties : 15 kg/ha (fuzzy seed) 7.5 kg/ha (delinted seed)

Hybrids : 3.75 kg/ha (Jayalakshmi), 1 kg (TCHB 213)

Male : 2 kg /ha and Female 4 kg /ha.

Seed treatment

Treat the seeds with azospirillum at 3 packets (600 g/ha) and 2 kg of

azospirillum / ha mixed with 25 kg of FYM and 25 kg of soil and applied on the seed
line. This saves 25 % nitrogen besides increasing yield.

Spacing – Varieties Hybrids

1. Long duration :90 x 30 cm ♀ : 120 x 60 cm

2. Short duration :60 x 30 cm ♂ : 90 x 60 cm

Hybrids - Planting ratio

8:2 but here it is block system where flowers of 2 parts of male is sufficient
to dust 8 parts of female parent.

Isolation (m)

Foundation seed Certified seed

Varieties 50 30

Hybrids 50 30

Manures and fertilizers

Compost : 12.5 tons/ha

Total : 100:50:25 NPK kg/ha

Basal : 50:50:25 NPK kg/ha

Top dressing : 25:0:0 NPK kg/ha

(40-45 days after sowing)

25:0:0 NPK kg/ha (70-75 days after sowing)

Foliar spray

Spray DAP 2% (for female parents, spray on 60,70,80 and 90th days after
sowing. (Soak 5 kg of DAP in 25 liters of water over night and supernatant liquid
should be taken and mixed with 475 liters of water for spraying 1 hectare).

Micronutrient application
 Mix 12.5 kg of micronutrient mixture formulated by the Department of
Agriculture Tamil Nadu with enough sand to make a total quantity of 50 kg for one
hectare.

NAA application

Spray 40 ppm of NAA (40 mg of NAA dissolved in 1 liter of water) at 40 /

45th day using high volume spray liquid in 1125 liter /ha. Repeat the same dose
after 15 days of first spray.

Topping

Topping arrests terminal growth by nipping the terminal 10-12th node for
controlling excessive vegetative growth.

Rouging

The crop should be rouged for off types, selfed plants, from vegetative phase
to harvest phase depending on plant stature, leaf size, leaf colour, hairiness, stem
colour, flower colour, petal spot, pollen colour, number of sympodia, boll size, boll
shape, pittedness etc. to maintain genetic purity.

Field standards

Maximum permitted (%)

Foundation seed Certified seed

Varieties Hybrids Varieties Hybrids

Off types 0.1 0.1 0.2 0.5

Irrigation management

Once in 10 days. Critical periods are boll formation to boll maturation stages.

Specific problems

Boll shedding will occur either due to extreme dry climate or lesser
frequency of irrigation or physiological disorder.

By spraying 40 ppm of NAA and cycocel at 20ppm, this can be minimized.

Harvesting
 The seed attains physiological maturation 45 days after anthesis.

 The initiations of hair line cracks on the dried bolls are the physical
symptoms of physiological maturation.

 At that time, the moisture content will be 30-35%.

 The bolls are harvested as pickings in cotton.

 Due to continuous flowering habit once over harvest is not practiced.

 As and when the bolls burst with hairline cracks the bolls are collected and
dried.

 Normally five to seven pickings can be practiced in a crop.

 But early 4-5 pickings are recommended for seed purpose.

 Harvest in the morning hours upto 10 to 11 a.m. only when there is moisture
so that dry leaves and bracts do not stick to the kapas and lower the market
value.

 Pick kapas from well burst bolls only.

 Remove only the kapas from the bolls and leave the bracts on the plants.
  As kapas is picked, sort out good puffy ones and keep separately.

 Keep stained, discoloured and insect attacked kapas separately.

Kapas sorting

Kapas is sorted manually to pick good quality seeds. Hard locks are to be
removed (Kapas without proper bursting and lint is light yellow in colour), since
these kapas mostly result in poor quality seeds, due to boll worm or other insect
attack.

Skewed bolls or ill filled or nonviable seeds are formed if stigmatic lobes are
not pollinated.

Ginning and certification

 Gin the crossed kapas in separate gins erected in authorized seed processing
units or farm gins under the close supervision of the authorities concerned to
ensure purity and avoid seed damage.

 Sieve the seed in two types of mesh to remove small, shrivelled seeds,
broken seeds and clean perfectly from any dirt or dust.

 After ginning, the seeds should be dried well and cleaned by hand picking.
After cleaning, certification agency will take sample for testing germination
and genetic purity test. Minimum germination 65% and genetic purity 90%
should be maintained.

 Certified seeds would be bagged in one kg bag, sealed and details regarding
its origin, germination, physical purity per cent and genetical purity percent,
besides season of production are passed on to sale agencies or respective
producers for commercial sale.

 Uncertified seeds would be procured by the concerned Department or Agency

at the market rate for the ordinary cotton seeds for further multiplication.
This step is essential to avoid unauthorised sale of substandard uncertified
seed.

Processing
 The ginned seeds (or) the fuzzy seeds are graded by hand picking and by
pressing on wire-mesh sieves to remove the under sized seeds and dust.

Acid delinting

 Fuzzy seeds will clog with one another. So for easy handling the seeds are
delinted using H 2 SO 4 @ 100 ml/kg of seed for 2-3 minutes.

 After acid treatment, the seed should be washed thoroughly for 3 to 4 times
with fresh water.

 From the floaters, mature seeds without any visible damage can be picked
and added to the sinkers.

Acid delinting machine

Procedure
 Weighed quantity of fuzzy seeds is taken in a plastic container and required
quantity of the acid is added. Stir well with wooden rod till a shiny black colour
appears (Tar like) wash with more of water (5-6 times) and shade dry the seed to
reduce the moisture content to 12% before further handling.

Processing of delinted seed

The free flowing delinted seeds can be graded using 10/64" round perforated
metal sieve, which is recommended as standard sieve in OSAW cleaner cum grader
for cotton.

The seed can also be graded by specific gravity method by using floatation
technique using water. The seeds will separate into floaters and sinkers. The
sinkers are good seeds. From floaters, reddish (immature) and damaged (seed with
insect hole) are removed. The brownish seeds which are good seeds are hand
picked and used for sowing.

Seed standards

Characters Foundation Certified seed

seed

Physical purity % (min) 98 98

Inert Matter % (max) 2.0 2.0

Other crop seeds (max) 5 kg-1 10 kg-1

Weed seeds (max) 5 kg-1 10 kg-1

Genetic purity (%) 100 100

Germination (min) % ( variety) 65 65

Germination (min) % ( hybrid) 75 75

Moisture content (max) %

a. Moisture pervious 10 10

b. Moisture vapour proof 6 6

Seed storage
 The seeds can be stored upto 8-9 months in moisture pervious container and
upto 12-15 months in moisture vapour proof containers.

The seed treatment with thiram @ 2.5 kg-1 or chlorine based halogen mixture
@ 3g kg-1 will protect the seed from storage fungi Aspergillus spp and preserve the
storability.

Mid storage correction

 The fuzzy and delinted seeds can be soaked in double the volume of 10-4
molar solution of Na 2 HPO 4 for 2 and 1 hr respectively ( 3.59 g / 100 l of
water.)

 Then the seeds are shade and sun dried to bring back to the moisture
content of 10-12%. The mid storage correction improves the planting value
of old seeds.

 Dead seeds may be removed by soaking acid delinted cotton seeds in

monolayer for 3 h and drying back to original moisture content.

 The seeds when put into potable water will separate into sinkers and floaters.
Dead seeds become buoyant and float.
 Lecture 15
ENZYMES
One of the unique characteristics of a living cell is its ability to permit complex
reactions to proceed rapidly at the temperature of the surrounding environment.
 The principal agents which participate in the remarkable transformations in the
cell belong to a group of proteins named enzymes. In the absence of enzymes
in the cell, these reactions would proceed too slowly.
 Enzymes are proteins specialised to catalyse biological reactions with the
following characteristics.
Characteristics of enzymes
 Enzymes being proteins exhibit all properties of proteins.
 They have their specific isoelectric points at which they are least soluble.
 Like proteins, they can be denatured by changes in pH and temperature.
 The enzyme-catalysed reactions occur below 100oC, at atmospheric pressure
and nearby neutral pH.
 Enzymes undergo physical changes during the reaction but revert to their
original form at the end of the reaction.
 Enzymes exhibit enormous catalytic power.The rates of enzymatically
catalysed reactions are 106 - 1012 times greater than those of the corresponding
uncatalysed reactions and several times greater than those of the corresponding
chemically catalysed reactions.
 For example the carbonic anhydrase enzyme catalyses the conversion of
carbondioxide to carbonic acid.
CO2 + H2O . H2CO3
 In this reaction, each enzyme molecule can hydrate 105 molecules of CO2 per
second.
 Enzyme activity is regulated in a variety of ways, ranging from controls over
the amount of enzyme protein synthesised by the cell or modulation of
activity through reversible interaction with metabolic inhibitors and activators
or through isoenzymes.
Specificity of the enzymes
 One of the characteristic feature which distinguishes enzymes from catalysts is
their specificity.
  Enzymes are specific in the reaction catalysed and in their choice of
substrates.
 It usually catalyses a single chemical reaction or a set of closely related reactions
Three kinds of specificities are observed.
i. Absolute specificity
 When enzymes catalyse only one particular reaction they are said to exhibit
absolute specificity.
 e.g. Urease acts only on urea.
ii. Group specificity
 Enzymes acting on a group of substances that possess a particular type of
linkage common to that group of substances are said to exhibit group specificity.
 Amylase hydrolyses the group of substances like starch, dextrin and glycogen,
which have the same type of glycosidic linkages (α1,4).
iii. Optical specificity
 Almost all enzymes show a high degree of optical specificity.
 There are certain enzymes which catalyse the hydrolysis of same group of
substances possessing same optical activity
 Eg. D-amino acid oxidase acts on D-amino acid and L-amino acid oxidase
acts on L-amino acid.
 Maltase catalyses the hydrolysis of α-but not β- glycosides.
Classification of enzymes
 In olden days enzymes have been named by adding the suffix -ase to the
name of
the substrate (the molecule on which the enzyme acts).
 Ex. Urease (Substrate urea) Arginase (Substrate arginine)
 Recent studies on the mechanism of enzyme catalysed reactions have led to
a more rational classification of enzymes.
 The International Union of Biochemistry (IUB) established a commission on
enzyme nomenclature to adopt a systematic classification and nomenclature of
all the existing and yet to be discovered enzymes.
 This system is based on the substrate and reaction specificity.
 Although, this International Union of Biochemistry system is complex, it is
precise, descriptive and informative.
  IUB system classifies enzymes into six major classes (should be written in
specific order only)
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
 Again each class is divided into subclasses according to the type of reaction
catalysed.
 Each enzyme is assigned a recommended name usually a short for everyday
use, a systematic name which identify the reaction it catalyses and a
classification number which is used where accurate and unambiguous
identification of an enzyme is required.
I. Oxidoreductases
 Enzymes catalysing oxido-reductions between two substrates, S and S'.

S reduced + S' oxidised → S oxidised + S' reduced

Example:
CH3-CH2-OH + NAD+ -------- →
CH3-CHO + NADH + H+
(reduced) (oxidised) (oxidised) (reduced)
Enzyme: Recommended name Alcohol dehydrogenase
Systematic name Alcohol:NAD+ oxido-reductase
Enzyme Commission number E.C.1.1.1.1
First digit 1 indicates oxido-reductase (Major class)
Second digit 1 indicates enzymes acting on CH-OH group of donors (Sub-
class)
Third digit 1 indicates NAD+ as the electron acceptor (Sub-sub class)
Fourth digit 1 indicates the specific enzyme
II Transferases
 Enzymes catalysing the transfer of a functional group (G) other than
hydrogen between substrates.
S - G + S' ---------> S' - G + S
Example: Phosphorylation of glucose by hexokinase
 Glucose + ATP -------> Glucose – 6- Phosphate + ADP
Enzyme : Recommended name: Hexokinase
Systematic name: ATP:D-hexose, 6- phosphotransferase
Enzyme commission No: 2.7.1.1
2→ Transferase group (major class)
7→ Transfer of phosphate group (sub-class)
1→ Alcohol group as acceptor of phosphate group (Sub-sub-class)
1→ Hexokinase
III Hydrolases
 Enzymes catalysing hydrolysis of ester, peptide or glycosidic bonds.
 Example
Acetyl choline + H2O -----------> Acetic acid + Choline

Enzyme: Acetyl choline esterase

Systematic name : Choline:acetyl hydrolase
E.C : 3.1.1.8
IV Lyases
 Enzymes catalysing the removal of groups from substrates by mechanism
other than hydrolysis leaving a double bond in one of the products.
 Example: convertion of malic acid to fumaric acid by fumarase
COOH – CH(OH) – CH2-COOH --------> COOH – CH = CH –COOH + H2O
Malic acid Fumaric acid
Enzyme : Fumarase (Fumarate hydratase)
Systematic name: L. Malate hydrolyase
E.C.No.4.2.1.2
V. Isomerases
 Enzymes catalysing interconversion of optical, geometrical or positional
isomers
Example
All-trans retinal -----------> 11 cis-retinal

Enzyme Retinene isomerase

Systematic name : All-trans retinene:11-cis isomerase
 E.C.No. 5.2.1.3
VI. Ligases
 Enzymes catalysing the joining together of two compounds with the
hydrolysis of a high energy compound.
 Example

ATP ADP + Pi
Glutamic acid + NH3 ----------> Glutamine

Enzyme: Glutamine synthetase

L.Glutamate: Ammonia ligase
E.C.6.3.1.2
 Lecture 16
MECHANISM OF ENZYME ACTION
 A chemical reaction such as A ----> P takes place because a certain fraction of
the substrate possesses enough energy to attain an activated condition
called the transition state.
 This transition state is at the top of the energy barrier separating the reactants
and products.
 The rate of a given chemical reaction is proportional to the concentration of
this transition state species.
 The energy of activation is the amount of energy required to bring all the
molecules in 1 mole of a substance at a given temperature to the transition state.
 Enzymes combine transiently with the substrate to produce a transition state
intermediate having a lower energy of activation than the uncatalysed reaction.
Thus, they accelerate chemical reactions by lowering the energy of activation
Example
H2O2 ---------> H2O + (O)
Catalase
Reaction condition Activation energy (KCal mol-1)
Uncatalysed 18
Catalysed by colloidal Pt 13
Catalysed by catalase 7

It is generally believed that the catalytic reactions occur in at least two steps.

Step 1: A molecule of enzyme(E) and a molecule of substrate(S) collide and react

to form an intermediate called the enzyme-substrate complex (ES).
Step 2: The decomposition of ES complex to give product(s) and the active enzyme
[S] + [E] ----------> [ES] ---------> P+[E]

The formation of an ES complex affords a lower activation energy.

Active site
 The substrate binding site in the enzyme is referred as active site.
 The functional groups that are essential for the formation of ES complex occur at
a specific location on the surface of the enzyme molecule.
  This section of enzyme where substrate binding and transformation of
substrate to product occurs is called as active site.
 Many attempts have been made to implicate specific amino acid residues
(side chain or R groups) as being part of the active site of various enzymes.
 Some of the amino acids occurring at the active site of enzymes are hydroxyl
group of serine, sulfhydryl group of cysteine, imidazole group of histidine and
carboxyl groupof aspartic acid.
Two theories were proposed to explain the mechanism of enzyme action.
1. Fischer’s lock and key theory (Rigid template model)
 During 1890, Emil Fischer proposed this theory
 According to this, the active site possesses a unique conformation which is
complementary to the structure of the substrate thus enabling the two
molecules to fit together in much the same way as a key fits into a lock

 An unfortunate feature of this model is the implied rigidity of the catalytic site.
2. Koshland’s induced-fit theory
 Koshland had advocated a theory to account for the specificity of enzymes.
 He postulated that the essential functional groups on the active site of the free
enzyme are not in their optimal positions for promoting catalysis.
 When the substrate molecule is bound by the enzyme, the catalytic groups
assume favourable geometrical position to form the transition state.
 The enzyme molecule is unstable in this active conformation and tends to revert
to its free form in the absence of substrate.
 In the induced fit model, the substrate induces a conformational change in the
enzyme which aligns the amino acid residues or other groups for substrate
binding, catalysis or both.

Factors affecting enzymatic reaction

The factors that mainly influence any enzyme-catalysed reaction are:
1. Substrate concentration
2. Enzyme concentration
3. Temperature
4. pH
5. Inhibitors
Other factors such as state of enzyme (oxidation), time and activators also affect
enzyme-catalysed reaction to certain extent.
Substrate concentration
 Keeping the factors such as pH, temperature and enzyme concentration at
optimum levels, if the substrate concentration is increased, the velocity of
the reaction recorded a rectangular hyperbola.
 At very low substrate concentration the initial reaction velocity (v) is nearly
proportional to the substrate concentration (first order kinetics).
 However, if the substrate concentration is increased the rate of increase slows
down (mixed order kinetics).
 With a further increase in the substrate concentration the reaction rate
approaches a constant (zero order-reaction where velocity is independent of
substrate concentration).
 At initial point, eventhough the substrate molecules are present in excess than
enzyme on molar basis, not all the enzyme molecules present combine with the
substrate.
 Hence, increasing the substrate concentration will increase the amount of
enzyme associated with substrate as ES and thus v will depend on [S].
 At Vmax, all the enzyme molecules are saturated with substrate molecules so
that further increase in [S] cannot result in increased reaction rate.
 Michaelis-Menten derived an equation to explain this type of behaviour.
 Vmax [S]
v = -------------
Km + [S]

[S] = Substrate concentration Vmax = Maximum velocity

v = Velocity of the reaction

At half maximal velocity [S] = Km

i.e Vmax Vmax [S]

-------- = -------------
2 Km+[S]

Km + [S] Vmax [S]

----------- = ----------
2 Vmax

Km + [S] = 2 [S]

Km = 2 [S] – [S] = [S]

Hence, Michaelis - Menten constant, Km, is defined as the substrate concentration

at half maximal velocity and is expressed as mole per litre.
 The Michaelis-Menten equation can be algebraically transformed into more
useful way to plot the experimental data.
 Lineweaver and Burk have taken the reciprocal of both [S] and v of the Michaelis-
Menten equation to give
1 Km 1 1
--- = ------ ------ + -------
v Vmax [S] Vmax

 A plot of 1/v versus 1/ [S] (the double reciprocal) yields a straight line.
 This line intercept X-axis at -1/Km and Y-axis at 1/Vmax.
 The slope of the line is Km/Vmax.
 The Lineweaver-Burk plot has the great advantage of allowing more accurate
determination of Vmax and Km

Significance of Km
i. Km value may vary with substrate.
 ii. An enzyme whose Km is very low will have a high degree of affinity for its
substrate

Enzyme concentration
 When compared to substrate concentration, the concentration of enzyme is
always very very low on molar basis.
 Hence, increasing the enzyme concentration will always increase the
reaction rate

Temperature
 The velocity of enzyme-catalysed reactions roughly doubles with a 10oC rise
in temperature over a limited range of temperature
 Enzymes, being proteins, are denatured by heat and become inactive as the
temperature increases beyond a certain point.
 Most of the enzymes are inactivated at temperatures above 60oC.
 The temperature at which the reaction rate is maximum is known as optimum
temperature
pH
 Most enzymes have a characteristic pH at which their activity is maximum;
above or below this pH, the activity declines
 The pH affects the ionic state of the enzyme and frequently that of the substrate
also.
 If a negatively charged enzyme (E-) reacts with a positively charged substrate
(SH+), ESH is formed.
 At low pH values, E- will be protonated and ESH is not formed.
 Similarly, at very high pH values SH+ will ionize and lose its positive charge.

E- + SH+ ------> ESH

acidic pH

E- + SH+ ------> EH + SH+ --------> No ESH formation

alkaline pH

SH+ ----------> S + H+ + E- --------> No ESH formation

  Another important factor is the change in conformation (denaturation) of
enzyme at extreme pH values.

Inhibitors
 Compounds that have the ability to combine with certain enzymes but do not
serve as substrates and therefore block catalysis are called inhibitors.
 The important type of inhibitors are competitive and noncompetitive
inhibitors.

Competitive inhibitor
 Any compound which possessess a close structural resemblance to a
particular substrate and which competes with that of substrate for the
same active site on the enzyme is called as competitive inhibitor.
 The inhibitor is not acted upon by the enzyme and so remains bound to the
enzyme preventing the substrate to bind.
 This is a reversible process.
 It depends upon the relative concentration of substrate and inhibitor.
 Competitive inhibition can be completely reversed by addition of large excess
of substrate

high inhibitor concn.

E+I ----------------------> EI
<----------------------
high substrate concn.

Eg. the enzyme, succinate dehydrogenase converts succinate to fumarate.

For this reaction, malonic acid is a competitive inhibitor as it structurally resembles
that of succinate
 In case of competitive inhibition, Km is increased but Vmax is not altered.
Non-competitive inhibitor
 Non-competitive inhibitors bind to a site other than the active site on the
enzyme often to deform the enzyme, so that, it does not form the ES complex
at its normal rate.
 Once formed, the ES complex does not decompose at the normal rate to yield
products.
 These effects are not reversed by increasing the substrate concentration.
E + I -------> EI
ES + I ------> ESI
 Some enzymes possessing an essential -SH group are non-competitively
inhibited by heavy metal ions (Hg2+, Pb2+).
 Some metalloenzymes are inhibited non competitively by metal chelating
agents like ethylene diamine tetraacetic acid(EDTA).
 Inhibitors are used as tools to probe the mechanism of enzyme - catalysed
reactions and as therapeutic agents.
 In case of noncompetitive inhibition, Vmax is lowered but Km is not altered

Uncompetitive inhibitor
 In case of uncompetitive inhibition, the inhibitor binds only to free enzyme and
not to the enzyme substrate [ES] complex
 Lecture 18
APOENZYMES, COENZYMES AND COFACTORS, ISOZYMES

A complete, catalytically active enzyme together with its coenzyme and/or

metal ions is called holoenzyme.
 The protein part of an enzyme is called apoenzyme or apoprotein.
 Enzymes require an additional non-protein component to carry out its catalytic
functions.
 Generally these non-protein components are called as cofactors.
 The cofactors may be either one or more inorganic ions such as Fe2+, Mg2+,
Mn2+ and Zn2+ or a complex organic molecules called coenzymes.
 A coenzyme or metal ion that is covalently bound to the enzyme protein is called
prosthetic group.
 Some enzymes require both coenzyme and one or more metal ions for their
activity
 Coenzymes function as transient carriers of specific functional groups

Cofactors
 Metals are required as cofactors in approximately two thirds of all enzymes.
 Metalloenzymes contain a definite quantity of functional metal ion that is
retained
throughout whereas metal-activated enzymes bind metals less tightly but require
added metals.
 The distinction between metalloenzymes and metal activated enzymes thus rests
on the affinity of a particular enzyme for its metal ion.
 The mechanisms whereby metal ions perform their function appear to be similar
both in metalloenzymes and metal activated enzymes.
 Metals participate through their ability to act as Lewis acids and through
chelate formation. Eg. For metal functioning as a Lewis acid is the zinc in
carbonic anhydrase.
 The metal can also promote catalysis by binding substrate at the site of
bond cleavage. In carboxypeptidase, the carbonyl oxygen is chelated to the
zinc.
The iron-sulfur enzymes are unique class of metalloenzymes in which the active centre
consists of one or more clusters of sulfur-bridged iron chelates. These are of greater
importance in plant systems
Isoenzymes

 Enzymes which exist in multiple forms within a single species of organism

or even in a single cell are called isoenzymes or isozymes.

 Such multiple forms can be detected and separated by gel electrophoresis of cell
extracts.

 Since they are coded by different genes, they differ in amino acid
composition and thus in their isoelectric pH values.

 Lactate dehydrogenase is an example for the isoenzymes which occur as five

different forms in the tissues of the human and other vertebrates.

 All the five isozymes catalyze the same reaction.

Lactate + NAD+ ----------. Pyruvate + NADH + H+

 They have the molecular weight of about 134,000 and contain four polypeptides.

 The five isozymes consist of five different combinations of two different kinds of
polypeptides M and H.

 Kinetic study of lactate dehydrogenase isozymes has revealed that although they
catalyze the same reaction, they differ significantly in their Km values for
their substrates as well as Vmax values.

 The two polypeptide chains in LDH are coded by two different genes.

 Skeletal muscle contains four identical M chains and designated as M4; whereas
heart muscle contains four identical H chains and designated as H4.
 LDH of other tissues are a mixture of the five possible forms H4, H3M, H2M2,
HM3 and M4.

 A determination of the relative amounts of the five LDH isozymes and the
total concentration of LDH in a serum sample can provide valuable diagnostic
information about which tissues have been damaged and the extent of the
damage.
 CHILLI (Capsicum frutescense)

Chillies widely used as vegetable and spice is an often cross pollinated crop,
where the extend of cross pollination is upto 7 to 36 per cent. It belongs to the
family solanaceae. It is also known as hot pepper and botanically it is known as
capsicum annuum. The quality seed production techniques of chillies comprises of
the following steps.

Botany: Often Cross pollinated vegetable. The flower is protogynous. Anther

dehisces only half to 5 ½ hr after stigma becomes receptive. Anthesis in chilli
occurs between 6.00 and 9.00 hr. Flower remains open for 2 to 3 days, receptivity
of stigma was the highest at the day of flower anthesis.

Method of seed production : Seed to seed

Stages of seed production : Breeder seedFoundation seedCertified seed.

Varieties: K.1, K.2, K.3, Co.1, Co.2, PKM.1, MDU.1, Bagyalakshmi.

Hybrids: KT.1, (Pusa Deepti), Solar Hybrid 1, Solar Hybrid 2. Early Bounty, Indira,
Lario, Hira, Bharat.

Season : June-July, February-March, September- October.

Land requirement : There are no land requirement as of previous crops, but the
land should be free from volunteer plants. Generally areas affected by wilt or root
rot may be avoided. Crop rotation must be followed to avoid endemic Solanaceous
pests.

Isolation requirement : Minimum isolation distance of 400 M for foundation and

hybrid seed and 200 M for certified seed production are necessary.

Seed rate : Seed required for one hectare is 500 g to 1 kg for variety; for hybrids
- Female = 200 g and male = 50 g

Nursery : Sow the seeds in raised nursery bed of 20 cm height, in rows of 5 cm

gap and covered with sand. Eight and ten nursery beds will be sufficient to
transplant one acre. Apply 2 kg of DAP 10 days before pulling out of seedling.

Transplanting: The seedlings of 30-35 days old are ready for transplanting.
Transplanting may be done on the ridges in the evening.

Foliar spray: To arrest the flower drop, NAA (Planofix) can be sprayed @ 4ml/L.
Very light irrigation is also done arrest the flower drop.

Manuring : Apply 50 tones of FYM/ha for irrigated crop. Basal 0:70:70 kg of NPK
and 50 kg of N at 15 days after transplanting and 50 kg N at 45th days after
transplanting.

Roguing: Field inspection and roughing should be done both for varieties and
hybrid at different stages based on the plant height and its stature, flower colour
and pod characters. The plants affected with leaf blight, anthracnose and viral
diseases should be removed from the seed field.
Pest and disease management: The important pest attacking chilli and capsicum
are thrips, aphids, pod or fruit borer and mites. The thrips and aphids can be
controlled by spraying Dimecron (systemic pesticide), pod borer can be controlled
by spraying Nuvacron and the mites can be controlled by spraying Kelthane. The
major diseases affecting the plants are die back or fruit rot, powdery mildew and
bacterial leaf spot. Spray Dithane M-45 for control of die back, Karathane for
powdery mildew and Agromycin for leaf spot disease control.

Hybrid seed production: The crossing operation can be performed as per the
methods outlined for tomato and brinjal hybrid seed production. However, hand
emasculation and pollination is somewhat difficult since the flowers are minute.
Hence use of male sterile lines can also be employed for hybrid seed production.

Harvesting and processing: Harvesting should

be done in different pickings. First and last one or
two pickings can be harvested for vegetable
purpose. The well ripened fruits with deep, red
colour alone should be collected in each picking.
After harvest, fruit rot infected fruits are to be
discarded. The harvested pods are to be dried
under shade for one (or) two days and then under sun for another 2 or 3 days.
Before drying pods are to be selected for true to type and graded for seed
extraction. The seed are extracted from graded dried pods. The pods are taken in
gunny bag and beaten with pliable bamboo sticks. The seeds are cleaned by
winnowing and dried to 10% moisture content over tarpaulin. Then seeds are
processed with BSS 8 wiremesh screens. For large scale seed extraction, the TNAU
model chilli seed extractor may be used.

Seed Yield : 50-80 Kg/ha

Seed Certification

Number of Inspections

A minimum of three inspections shall be made as follows:

1. The first inspection shall be made before flowering on order to verify isolation,
volunteer plants, and other relevant factors,

2. The second inspection shall be made during flowering to check isolation, offtypes
and other relevant factors

3. The third inspection shall be made at maturity and prior to harvesting to verify true
nature of plant and other relevant factors

Specific standards:

Factors Foundation Certified

Off types 0.1% 0.2%

Designated diseased 0.1% 0.5%

plant

The designated diseases are caused by Collertotictum capsici and leaf blight
caused by Alternaria solari.

Seed standards (Variety & Hybrid)

Factors Foundation Certified

Pure seed 98% 98%
Inert matter 2% 2%
Other crop seeds 5/kg 10/kg
Weed seeds 5/kg 10/kg
Germination 60% 60%
Moisture content 8% 8%
For VP Container 6% 6%
 BHENDI (Abelmoschus esculentus)

Botany: Anthesis is between 9 and 10 hr and is preceded by maximum anther

dehiscence between 8 and 9 hr. The stigma remains receptive on the day of
anthesis. Bhendi is an often cross pollinated crop. Cross pollination to an extent of
12 per cent is due to protogynous.

Method of seed production : Seed to seed

Stages of seed production : Breeder seed  Foundation seed  Certified

seed.

Varieties : Co.1, MDU.1, Parbhani Kranti, Arka Anamika, Pusa A-4, Pusa Sawani

Hybrids:CO2,CO 3, Mahyco hybrid, Shoba

Season : June-July, September- October and February- March

Land requirement : Select field on which bhendi crop was not grown in the
previous season, unless the crop was of the same variety and certified. Field should
be free from wild bhendi (Abelmoschus sp.)
Isolation requirement: Seed field must be isolated from other varieties at least
by 400 M for foundation and hybrid seed production and 200 M for certified seed
production.

Seed rate : Varieties : 8-10 kg/ha

Hybrids : 4 kg/ha (Female); 1 kg/ha (Male)

Manuring: Apply 12.5 tons of FYM/ha before ploughing. Apply 150:75:75 kg

NPK/ha, of which 50% of the N should be applied as top dressing in two split doses
at flowering and 10 days later.

Planting ratio: For hybrid seed production, female and male parents are normally
planted in the ratio of 4:1.

Roguing: Minimum of three inspections for varieties and 4 inspections for hybrids,
one at vegetative, two at flowering and one at fruit maturity stages. The rouging
should be based on the plant characters, hairiness, fruit character like fruit colour,
number of ridges, fruit length etc., and the off type and mosaic attacked plants
should be removed from the seed field. Wild bhendi if present should be removed
before flowering.

Pest and disease management: The major pest attacking bhendi are jassids,
aphids and white fly, which can be controlled by spraying Rogar or Dimecron or
Endosulphon. The pod borer and red spider mites can be controlled by spraying
Endosulphon and Kelthane, respectively. The diseases such as yellow vein mosaic
and powdery mildew can be controlled by spraying systemic insecticides and
Karathane, respectively.

Hybrid seed production: In bhendi, since the flowers are large in size, hand
emasculation and pollination is the best suitable method for seed production. The
emasculation and dusting can be done as per the methods outlined in tomato. The
male and female parents are raised in blocks at the ratio of 9:1 (Female: Male).

Harvesting: Fruits should be harvested when they have dried (30-35 days after
crossing). The pods which expose hairline crack and turn to brown colour on drying
alone are cut using sickle manually.
Threshing:

The pods are dried and threshed using pliable sticks. Separated seeds are
winnowed to remove plant debris and dried over a tarpaulin to 10% moisture
content. Dried seeds are subject to water floatation in which, good seeds sink while
poor seeds float. The floaters are removed, while sinkers are dried under shade
followed by sun drying. Then the seed are cleaned, dried and treated with Captan/
Thiram.

Processing: Seeds are to be processed with BSS 7 wiremesh sieve.

Seed Yield: 1000-1200 Kg/ha

Specific standards:

Factors Foundation Certified

Off types 0.1% 0.2%

Objectionable weed None None

Disease affected plants 0.1% 0.5%

Objectionable weed: wild Abelmoschus sp.

A.moschatus A. manihot

Designated diseases: Yellow Vein Clearing Mosaic (Hybiscus virus-1)

Seed standards

Factors Foundation Certified

Pure seed 99% 99%

Inert matter 1% 1%

Other crop seed None 5/kg

Total weed seed None None

Objectionable weed None None

Other Distinguishable 10/kg 20/kg

Varieties (ODV)

Germination 65% 65%

Moisture 10% 10%

For VP Container 8% 8%

Questions
1.Chilli is an often Cross pollinated vegetable. (True/False)
2. The flower of chilli is
a. Protandry b. Terminal
c. Axillary d. Protogyny
3. _______ arrests flower drop in chilli.
a. NAA b. GA3
c. MH d. Ethrel
4. Off type allowed during certified seed production in chilli is _______
a. 0.5% b. 0.02%
c. 0.2% d. 2%
5. Isolation requirement for foundation and certified seed production
in chilli is_________
a. 400 & 200 M b. 100 & 200 M
c. 600 & 200 M d. 600 & 400 M
6. Bhendi is an often cross pollinated crop. (True/False)
7. Cross pollination in bhendi is due to protogynous.

8. Isolation requirement for foundation and certified seed production

in Abelmoschus esculentus is_________
a. 600 & 100 M b. 200 & 100 M
c. 300 & 200 M d. 400 & 200 M
9. Planting ratio for hybrid seed production in Abelmoschus esculentus is
a. 8:1 b. 4:1
c. 12:1 d. 10:1
10. Seeds of bhendi is processed with ___________ wiremess sieve.
a. BSS 10 b. BSS 3
c. BSS 7 d. BSS 15
11. Objectionable weed in bhendi is _____________
a. Manihot esculentus b. A.moschatus
c. Coleus forskohlli d. None
12. Designated diseases of bhendi is yellow vein mosaic.
13. Other Distinguishable Varieties (ODV) allowed in bhendi is _____
a. 5/kg b. 20/kg
c. 10/kg d. 40 %
 Lecture 19 -24

METABOLISM OF CARBOHYDRATE

Introduction
 Carbohydrates are major sources of energy for living organisms.
 The chief source of carbohydrate in human food is starch, which is the storage form
of glucose in plants.
 Plants may store relatively large amounts of starch within their own cells in time of
abundant supply, to be used later by the plant itself when there is a demand for energy
production.
 Glycogen is the glucose storage polysaccharide of animals.
 It accounts for upto 10% of the mass of the liver and one percent of the mass of the
muscle.
 Glycogen is larger and highly branched than amylopectin.
 By the action of several enzymes, such as -amylase, -amylase, amylo (16)
glucosidase and (14) glucosidase, starch and glycogen from dietary intake are
degraded finally to glucose.
 Carbohydrate is utilized by cells mainly in the form of glucose.
 The three principal monosaccharides resulting from the digestive processes are
glucose, fructose and galactose.
 Both fructose and galactose are readily converted to glucose by the liver.
 Pentose sugars such as xylose, arabinose and ribose may be present in the diet, but
their fate after absorption is obscure.
 Since glucose is the compound formed from starch and glycogen, the carbohydrate
metabolism commences with this monosaccharide.
The major metabolic processes in carbohydrates are:
i. Glycolysis:
Glycolysis is the sequence of reactions that convert glucose into pyruvate with
the concomitant trapping of the energy as ATP.
ii. The citric acid cycle:
It is the final common oxidative pathway for carbohydrates, fats and
proteins. It is also a source of precursors for biosynthesis of various biomolecules. The
acetyl CoA that enters in this pathway is completely oxidised to carbon dioxide and
water with concomitant production of reducing equivalents, namely NADH and FADH2.
 iii. The hexose monophosphate shunt:
It is an alternative pathway to the glycolytic pathway and the citric acid cycle for
the oxidation of glucose to carbon dioxide and water with the generation of reduced
nicotinamide adenine dinucleotide phosphate (NADPH) molecules and ribose 5-
phosphate.
iv. Gluconeogenesis:
It is a biosynthetic pathway that generates glucose from non-carbohydrate
precursors.
v. Glycogenesis:
It is a pathway by which glycogen is synthesised from glucose.
vi. Glycogenolysis:
Glycolysis
 Glycolysis, also called as Embden-Meyerhof-Parnas pathway (EMP pathway),
consists of a series of reactions through which glucose is converted to pyruvate with the
concomitant production of relatively small amounts of adenosine triphosphate (ATP).
 It is derived from the Greek stem 'glykys' meaning sweet and 'lysis' meaning
splitting.
 It is the primary pathway occurring in the cytoplasm of all the tissues of biological
systems.
 All the enzymes responsible for the catalysis are found in the extra-mitochondrial
soluble fraction of the cells (cytoplasm).
In plants, glucose and fructose are the main monosaccharides catabolised by
glycolysis although others are also converted into these sugars.
 Glucose entering the glycolysis is derived from starch or sucrose, and fructose is
derived from sucrose.
 The starch is either from seeds or chloroplasts of matured plants.
 Glycolysis normally takes place in the presence of O2 in higher plant cells.
The enzymes in the cytoplasm catalyse the reactions involved in the conversion of
glucose to pyruvate .
The series of reactions indicated take place in 3 stages.
Stage 1: Conversion of glucose to fructose 1,6-bisphosphate
 The formation of fructose 1,6-bisphosphate takes place in three steps catalysed by
enzymes.
 The purpose of these reactions is to form a compound that can be readily cleaved into
phosphorylated three carbon units from which, through a series of reactions, ATP is
formed.
 After the first phosphorylation reaction to form glucose 6-phosphate, isomerisation of
glucose 6-phosphate to fructose-6-phosphate occurs which is conversion of an aldose
into a ketose.
 A second phosphorylation reaction follows the isomerization, catalysed by
phosphofructokinase resulting in the formation of fructose 1,6-bisphosphate.
 Phosphofructokinase is the key enzyme in the control of glycolysis.
Stage 2: Conversion of fructose 1,6-bisphosphate to 3-phosphoglycerate.
 The splitting of fructose 1,6-bisphosphate occurs in the second stage of glycolysis
resulting in the formation of a molecule of glyceraldehyde 3-phosphate and a molecule
of dihydroxyacetone phosphate catalysed by aldolase.
 The dihydroxyacetone phosphate is isomerised to glyceraldehyde 3-phosphate by
phosphotriose isomerase. The isomerisation reaction is rapid and reversible.
 In the next step, glyceraldehyde 3- phosphate is oxidised to 1,3-bisphosphoglycerate
catalyzed by glyceraldehyde 3-phosphate dehydrogenase.
 The product is further converted into 3-phosphoglycerate and a molecule of ATP is
formed. The phosphorylation of ADP to ATP is called substrate level phosphorylation
since the phosphate group from a substrate molecule is transferred to ADP.
Stage 3: Formation of pyruvate
 An intramolecular rearrangement of the phosphoryl group occurs resulting in the
formation of 2-phosphoglycerate from 3-phosphoglycerate catalyzed by
phosphoglycerate mutase.
 The 2-phosphoglycerate formed undergoes dehydration forming
phosphoenolpyruvate which gives rise to pyruvate and a molecule of ATP (substrate level
phosphorylation).
 The reaction is irreversible and catalyzed by pyruvate kinase.
The net reaction in the transformation of glucose to pyruvate is

Glucose + 2 Pi + 2ADP + 2 NAD+ --- 2 pyruvate + 2 ATP + 2 NADH + 2 H+ +

H 2O
Once pyruvate is formed, further degradation is determined by the presence or
absence of oxygen.
Under anaerobic conditions, in one of the pathways, pyruvate undergoes reduction
yielding lactic acid.
The formation of lactic acid is very rare in plants with exception of potato tubers
maintained under anaerobic condition and some green algae.
In the second pathway, pyruvate is converted to ethyl alcohol and carbon dioxide.
The alcoholic fermentation is the basis of the beer and wine-making industries.
Under aerobic conditions, pyruvate is oxidatively decarboxylated to acetyl
CoA which is then completely oxidised to CO2 and water through the citric acid cycle

Energetics of glycolysis
From glucose, two molecules of glyceraldehyde 3-phosphate are formed in the
second stage of glycolysis from which two molecules of pyruvate are obtained as end
products of glycolysis. Hence energetic of glycolysis is calculated by taking into account
two molecules of glyceraldehyde 3-phosphate.
Energetics of glycolysis
Stages/steps Enzyme Method of high energy No. of
bond formation ATP
formed
Stage 1
Formation of1,3- Glyceraldehyde3- Respiratory chain oxidation of 5
bisphospho glycerate phosphate 2 NADH
from glyceraldehydes dehydrogenase
3-phosphate
Stage 2
Formation of 3 Phosphoglycerate Phosphorylation at subtrate 2
phosphoglycerate from kinase level
1,3 bisphospho
glycerate
Stage 3
Formation of pyruvate Pyruvate kinase Phosphorylation at subrate 2
from phosphoenol level
pyruvate
Allowance for consumption of ATP by reactions catalysed by hexokinase 2
and phosphofructokinase.
Number of ATP molecules generated by the catabolism of one molecule of 7
glucose under aerobic conditions.
Number of ATP molecules generated by the catabolism of one molecule 2
of glucose under anaerobic conditions.

Significance of glycolysis
 Glycolysis is an almost universal central pathway of glucose catabolism occurring
in the cytoplasm of all the tissues of biological systems leading to generation of energy
in the form of ATP for vital activities.
 It is the pathway through which the largest flux of carbon occurs in most cells.
 Some plant tissues which are modified for the storage of starch such as potato
tubers and some plants adapted to growth in inundated water such as water cress derive
most of their energy from glycolysis.
 In plants, glycolysis is the key metabolic component of the respiratory process,
which generates energy in the form of ATP in cells where photosynthesis is not taking
place.
 Many types of anaerobic microorganisms are entirely dependent on glycolysis.
 Mammalian tissues such as renal medulla and brain solely dependent on glycolysis
for major sources of metabolic energy.
The tricarboxylic acid cycle
 In 1937, Sir Hans Krebs, an English biochemist proposed a pathway consisting of a
cycle of reactions through which acetyl CoA is converted to carbon dioxide and water
and hence the cycle was named as Kreb's cycle.
 All the enzymes catalyzing the reactions of this cycle occur inside mitochondria
(mitochondrial matrix) in contrast with those of glycolysis, which occur in the cytosol.
Before pyruvate can enter the citric acid cycle, it must be oxidatively
decarboxylated to acetyl CoA (active acetate).
Three different enzymes working sequentially in a multienzyme complex catalyse this
reaction.
This formation of acetyl CoA from pyruvate by alpha-oxidative decarboxylation occurs
in the mitochondrion following the formation of pyruvate in the cytosol during glycolysis.

The reaction involves six cofactors: coenzyme A, NAD+, lipoic acid, FAD, thiamine

pyrophosphate (TPP) and Mg2+.

TPP, FAD
CH3-CO-COOH+CoASH+NAD+ CH3-CO-S-

CoA+NADH+H++CO2

Lipoate, Mg2

Reactions of the TCA cycle

Acetyl CoA, derived mainly from the oxidation of carbohydrates, lipids and
proteins, combines with oxaloacetate to form citrate which is the first reaction of the
citric acid cycle. Subsequently, citrate is oxidised in a series of reactions liberating
carbon dioxide and reducing equivalents (NADH, FADH2).

The oxaloacetate is regenerated and functions therefore in a catalytic manner in the

oxidation of acetyl CoA to two molecules of carbon dioxide.
The citric acid cycle has eight steps as described below:
i. Formation of citrate
The first step is the reaction between the four-carbon unit, oxaloacetate and the
two-carbon unit, acetyl CoA resulting in the formation of citrate and coenzyme A
catalysed by citrate synthase. The coenzyme A formed in this reaction is recycled.
ii. Formation of isocitrate via cis-aconitate
The isomerization of citrate to isocitrate catalysed by aconitase occurs in two
steps with the formation of cis-aconitate as an intermediate. This formation of isocitrate
involves both dehydration and hydration. The result is an interchange of hydrogen and a
hydroxyl group. In this reaction, fluoroacetate acts as an inhibitor to the enzyme,
aconitase.
iii. Oxidation of isocitrate to -ketoglutarate
The enzyme, isocitrate dehydrogenase oxidatively decarboxylates isocitrate to
-ketoglutarate with simultaneous liberation of carbon dioxide. The intermediate in this
reaction is oxalosuccinate, an unstable -ketoacid. While bound to the enzyme, it loses
carbon dioxide to form -ketoglutarate. There are two different forms of isocitrate

dehydrogenase (isozymes), one requiring NAD+ and other requiring NADP+.

iv. Oxidation of -ketoglutarate to succinyl CoA
-Ketoglutarate, undergoes oxidative decarboxylation forming succinyl-CoA and
carbon dioxide in the presence of -ketoglutarate dehydrogenase complex, an
assembly consisting of three kinds of enzymes. The mechanism of this reaction is very
similar to the reaction catalyzed by pyruvate dehydrogenase complex. This reaction
is irreversible. Arsenite acts as an inhibitor of TCA cycle by inhibiting the action of -
ketoglutarate dehydrogenase complex.
v. Conversion of succinyl CoA to succinate
Succinate is formed in a reversible reaction from succinyl CoA catalysed by the
enzyme, succinyl CoA synthetase or succinate thiokinase with the simultaneous
formation of GTP and coenzyme A. Succinate thiokinase utilises GDP in animal tissues
whereas it uses ADP predominantly in plants and bacteria. The formation of GTP in
this reaction is a substrate level phosphorylation reaction.
vi. Formation of fumarate by oxidation of succinate
The succinate formed from succinyl CoA is oxidised to fumarate by succinate
dehydrogenase with the participation of FAD. Malonate, an analogue of succinate
being a strong competitive inhibitor of succinate dehydrogenase, blocks the citric acid
cycle.
vii. Formation of malate by hydration of fumarate
The reversible hydration of fumarate to L-malate is catalysed by fumarase.
viii. Oxidation of malate to oxaloacetate
This reaction forms the last reaction of the citric acid cycle. NAD-linked malate
dehydrogenase catalyses the oxidation of L-malate to oxaloacetate.
Energetics of tricarboxylic acid cycle
From one molecule of glucose, two molecules of pyruvate are formed which in
turn give rise to two molecules of acetyl CoA. When two molecules of acetyl-CoA
undergo oxidation through TCA cycle, the following number of high-energy bonds
(ATPs) are produced.
Significance of the TCA cycle
i) The major significance of the citric acid cycle is to act as the final common pathway
for the oxidation of carbohydrates, lipids and proteins, since glucose, fatty acids
and many amino acids are all metabolised to acetyl CoA.
ii) This cycle serves as the mechanism by which much of the free energy liberated
during the oxidation of carbohydrate, lipids and amino acids is made available.
iii) TCA cycle is of further significance since it has dual or amphibolic role thus
providing precursor compounds for biosynthesis of other biomolecules (amino
acids, fatty acids, and glucose.
Glyoxylate cycle
 Plants, especially seedlings, can use acetate as the only source of carbon for all
carbon compounds they produce.
 Acetyl CoA, which enters the TCA cycle, is completely oxidised to two molecules of
CO2. Thus it would not be possible for the cycle to produce the massive amounts

biosynthetic precursors needed for acetate based growth unless alternative reactions
were possible.
 Plants and bacteria employ a modification of the TCA cycle called the glyoxylate
cycle to produce four carbon dicarboxylic acids from acetyl CoA. The glyoxylate
cycle bypasses the decarboxylations of the TCA cycle.
 The enzymes of the glyoxylate cycle in plants are present in glyoxysomes.
Isocitrate lyase and malate synthase are the additional enzymes required for this
cycle in addition to TCA cycle enzymes.
 Glyoxysomes do not contain all the enzymes needed for the glyoxylate cycle. The
enzymes succinate dehydrogenase, fumarase and malate dehydrogenase are absent.
 Hence glyoxysomes, with the help of mitochondria run their cycle Succinate
molecules formed in glyoxysomes are transported to mitochondria where it is converted
to oxaloacetate with the help of TCA cycle enzymes. The oxaloacetate is then
converted to asparate and transported to glyoxysomes where it is transaminated to
oxaloacetate.
 The oxaloacetate is converted to malate through glyoxylate cycle. The malate then
enters the cytosol and converted into glucose via gluconeogenesis pathway.

The existence of glyoxylate cycle is important for the germinating seeds where
photosynthesis is not possible. Triacylglycerols rich in oilseeds are degraded to acetyl
CoA. Glyoxysomes formed during germination convert the acetyl CoA to oxaloacetate,
which is then utilised for the conversion to glucose through gluconeogenesis. Once the
growing seedling begins their photosynthesis to produce carbohydrates, the
glyoxysomes disappear.
Electron transport chain and oxidative phosphorylation
 The mitochondrion is the aerobic organelle in which the final stage of the oxidation of
food occurs.
 It is the site of the citric acid cycle, fatty acid oxidation and oxidative
phosphorylation, processes that are responsible for the formation of ATP under aerobic
condition.
 The two most important energy transductions in the biological systems are the
oxidative phosphorylation (ATP synthesis driven by electron transfer to oxygen)
and photophosphorylation (ATP synthesis driven by light).

 Oxidative phosphorylation is the process in which ATP molecules are formed as a

result of the transfer of electrons from the reducing equivalents, NADH or FADH2

(produced by glycolysis, the citric acid cycle and fatty acid oxidation) to oxygen by a
series of electron carriers in the form of a chain located in the inner membrane of
mitochondria. This is the final reaction sequence of respiration.
 Since the electrons are transferred by a series of electron carriers in the form of a
chain, it is known as electron transport chain (ETC).

 In plants, ATP is mainly derived through photosynthesis utilizing the energy derived
from the sun. In non-photosynthetic tissues, ATPs are derived through respiration.

The electrons are transferred along a set of cytochromes in the form of a chain
in steps from the more electronegative components (NADH/FADH2) to the more

electropositive oxygen.
The respiratory chain consists of a number of protein complexes that are
remarkably complicated in nature. They are known as NADH- ubiquinone reductase,
succinate-ubiquinone reductase, ubiquinone-cytochrome c reductase and cytochrome
c oxidase These complexes are also called as NADH dehydrogenase, succinate
dehydrogenase, cytochrome b-c complex and cytochrome c oxidase
respectively or as complexes I - IV.
All the three reductases are also known as iron-sulphur proteins since they
contain Fe-S centres as their critical components. Iron in these enzyme complexes can

exist in two forms as Fe2+ and Fe3+. Each cytochrome in its oxidised form (Fe3+)

accepts one electron and becomes reduced to Fe2+ form. Fe2+ donates electron to the
next carrier.

Oxidation of one molecule of NADH results in generation of 2.5 molecules of ATP

whereas oxidation of one molecule of FADH2 generates 1.5 molecules of ATP.

Sites of ATP formation

When electrons are transported along the respiratory chain, due to high amount of
energy released, ATP molecules are synthesised at the following three sites.
i) transfer of electrons from NADH to ubiquinone via flavoprotein
(FMN).
ii) transfer of electrons from cyt b to cyt c.
iii) transfer of electrons from cyt a to cyt a3.

Mechanism of ATP formation

 Two principal hypotheses have been proposed for the mechanism of oxidative
phosphorylation.
i. Chemical hypothesis
ii. Chemiosmotic theory
Chemical hypothesis
Many attempts have been made since 1920 to identify an energy-rich metabolite
linking oxidation and phosphorylation. No such intermediates was isolated and in 1960,
Peter Mitchell suggested that no possibility of existence of such an intermediate
compound. So, the chemical hypothesis has become discredited.
Chemiosmotic theory

The chemiosmotic theory states that the coupling of oxidation to phosphorylation

is indirect. According to this, the hydrogen ions (protons) generated by the oxidation of
components in the respiratory chain are ejected to the outside (matrix) of the inner
membrane. The electrochemical potential difference resulting from the asymmetric

distribution of the hydrogen ions (protons or H+) is used to drive a membrane-located

ATP synthase which in the presence of Pi + ADP forms ATP.
Inhibitors of respiratory chain
Inhibitors, which inhibit respiratory chain, may be grouped as follows:
i. Inhibitors of electron transfer
ii. Inhibitors of ATP synthase
iii. Uncouplers of oxidative phosphorylation
Inhibitors that arrest respiration by blocking the respiratory chain act at three sites.
Compounds such as barbiturates, amytal, rotenone prevent the transfer of
electron from FeS centre to ubiquinone. Carboxin specifically inhibits transfer of
reducing equivalents from succinate dehydrogenase to ubiquinone.
Antimycin A blocks electron transfer from cytochrome b to cytochrome c1.

Substances such as cyanide (CN-), azide (N3-) and carbon monoxide inhibit

cytochrome c oxidase by binding to heme group and are extremely poisonous.

Oligomycin inhibits ATP synthase.
In the presence of the uncouplers such as dicoumarol and 2,4-dinitrophenol,
oxidation proceeds without phosphorylation (dissociation of oxidation in the respiratory
chain from phosphorylation) releasing energy in the form of heat rather than in the form
of ATP.
The hexose monophosphate shunt
The hexose monophosphate shunt (HMP shunt), also called as pentose
phosphate pathway (PPP) and phosphogluconate pathway is an alternate pathway for
the oxidation of glucose. In 1930, Otto Warburg discovered the first evidence for the
existence of this pathway, which was later, elucidated in 1950 by Frank Dickens group.
The pathway is important during the hours of darkness and in non-photosynthetic
tissues such as differentiating tissues and germinating seeds. In animal system, it
occurs in certain tissues, notably liver, lactating mammary gland and adipose tissue in
addition to the Embden - Meyerhof pathway. The enzymes of the shunt pathway are
found in the extra mitochondrial soluble portion of the cell. It is in effect, a multicyclic
process whereby three molecules of glucose 6-phosphate give rise to three molecules of
CO2 and three 5-carbon residues. The latter are rearranged to regenerate two

molecules of glucose 6-phosphate and one molecule of glyceraldehyde-3-phosphate.

Since two molecules of glyceraldehyde 3-phosphate can regenerate a molecule of
glucose 6-phosphate by reactions, which are essentially a reversal of glycolysis, the
pathway can account for the complete oxidation of glucose. Here oxidation is achieved
by dehydrogenation using NADP and not NAD as in Embden-Meyerhof's glycolytic
pathway. This pathway consists of a series of reactions taking place in three stages
Stage I. Formation of NADPH and ribulose 5-phosphate
The first three reactions of the pathway, catalysed by glucose-6-phosphate
dehydrogenase, phosphogluconolactonase and phosphogluconate dehydrogenase
ultimately result in the formation of ribulose 5-phosphate and NADPH.
Stage II.
In this stage, the ribulose 5-phosphate is converted to ribose 5-phosphate by
ribulose 5-phosphate isomerase and then to xylulose-5 phosphate by ribulose 5-
phosphate epimerase. The ribose 5-phosphate is essential precursor in the biosynthesis
of nucleotides.
Stage III.
In the third stage, three molecules of the 5-carbon sugars are converted to two
molecules of 6-carbon sugars and one molecule of 3-carbon sugar, glyceraldehyde 3-
phosphate catalysed by two enzymes, transaldolase and transketolase.
 Transketolase catalyses the transfer of a C2 unit from xylulose 5-phosphate to

ribose 5-phosphate yielding glyceraldehyde 3-phosphate and sedoheptulose 7-

phosphate.
Transaldolase catalyses the transfer of a three carbon unit from sedoheptulose
7-phosphate to glyceraldehyde 3-phosphate yielding erythrose 4-phosphate and fructose
6-phosphate.
Control of the HMP shunt
Ribose 5-phosphate and NADPH are the principal products of the HMP shunt. In
this pathway, excess amount of ribose 5-phosphate is converted into glycolytic
intermediates when the need for NADPH exceeds that of ribose 5-phosphate in
nucleotide biosynthesis.
If ribose 5-phosphate is needed more than NADPH, fructose 6-phosphate and
glyceraldehyde 3-phosphate are used for the synthesis of ribose 5-phosphate by
reversal of the transaldolase and transketolase reactions.
The rate of NADPH formation in the pathway is controlled by the rate of the
glucose 6-phosphate dehydrogenase reaction.
Metabolic significance of the HMP Shunt
i.Major function of HMP shunt appears to be the production of reduced NADP (NADPH)
required by anabolic (synthetic) processes such as fatty acid synthesis outside the
mitochondria .
ii.The pathway provides ribose for nucleotide and nucleic acid synthesis.
iii.It also provides erythrose required for the synthesis of phenolics and other aromatic
compounds through shikimate pathway.
Glucose 6-phosphate can be used as a substrate either for glycolysis or for the
pentose phosphate pathway. On the basis of the cell's needs, it makes this choice for
biosynthesis and for energy from catabolism. If glucose 6-phosphate is channeled into
glycolysis, ATP is produced in abundance; but if it is channeled into pentose phosphate
pathway. NADPH and ribose 5-phosphate are produced. The fate of glucose 6-
phosphate is determined to a large extent of phosphofructokinase and glucose-6 P.
There are four principal possibilities in which, depending upon the cell's need, HMP
shunt operates.
i. More ribose 5-phosphate than NADPH is required
Most of the glucose 6-phosphate is converted into fructose 6-phosphate and
glyceraldehyde 3-phosphate by the glycolytic pathway. Two molecules of fructose 6-
phosphate and one molecule of glyceraldehyde 3-phosphate are converted into three
molecules of ribose 5-phosphate by a reversal of reactions catalysed by transaldolase
and transketolase reactions.
ii.. Both ribose 5-phosphate and NADPH are needed by the cell
In this, the first four reactions of the pentose phosphate pathway predominate.
Ribose 5-phosphate is the principal product of the metabolism and NADPH is also
produced. The net reaction for these processes is

Glucose 6 P + 2 NADP+ + H2O -------- Ribose 5-Phosphate + CO2 + 2 NADPH + H+

3. More NADPH than ribose 5-phosphate is needed by the cell

Under this situation, glucose 6-phosphate is completely oxidized to carbon
dioxide. Three reactions are active. First, two NADPH and one ribose 5-phosphate are
formed by the oxidative branch of the pentose phosphate pathway. Then, ribose 5-
phosphate is converted into fructose 6-phosphate and glyceraldehyde 3-phosphate by
transketolase and transaldolase. In the final reaction, glucose 6-phosphate is
resynthesised from fructose 6-phosphate and glyceraldehyde 3-phosphate by the
gluconeogenic pathway. The sum of these reactions is

Glucose 6-phosphate + 12 NADPH+ + 7H2O ------- 6 CO2 + 12 NADPH + 12H+ + Pi

iv. Both NADPH and ATP are needed by the cell.

In this, fructose 6-phosphate and glyceraldehyde 3-phosphate derived from
ribose 5-phosphate enter the glycolytic pathway and form pyruvate. ATP and NADPH
are concomitantly generated and five of the six carbons of glucose 6-phosphate emerge
in pyruvate.

3 Glucose 6-phosphate + 6 NADP+ + 5NAD+ + 5 Pi + 10ADP ------ 5 pyruvate + 3

CO2 + 6NADPH + 5NADH + 10ATP + 2H2O + 10H+

Comparative account of glycolysis and HMP shunt

These two major pathways are meant for the catabolism of glucose. They have
little in common, e.g. the presence of metabolites like glucose 6-phosphate. The major
differences are
i. ATP is not generated in the HMP pathway, whereas in glycolysis, ATP molecules
are generated.
ii. Pentose phosphates are generated in the HMP pathway but not in glycolysis.
iii. NADH is produced in glycolytic pathway whereas NADPH is produced in HMP shunt.
 BHENDI (Abelmoschus esculentus)

Botany: Anthesis is between 9 and 10 hr and is preceded by maximum anther

dehiscence between 8 and 9 hr. The stigma remains receptive on the day of
anthesis. Bhendi is an often cross pollinated crop. Cross pollination to an extent of
12 per cent is due to protogynous.

Method of seed production : Seed to seed

Stages of seed production : Breeder seed  Foundation seed  Certified

seed.

Varieties : Co.1, MDU.1, Parbhani Kranti, Arka Anamika, Pusa A-4, Pusa Sawani

Hybrids:CO2,CO 3, Mahyco hybrid, Shoba

Season : June-July, September- October and February- March

Land requirement : Select field on which bhendi crop was not grown in the
previous season, unless the crop was of the same variety and certified. Field should
be free from wild bhendi (Abelmoschus sp.)
Isolation requirement: Seed field must be isolated from other varieties at least
by 400 M for foundation and hybrid seed production and 200 M for certified seed
production.

Seed rate : Varieties : 8-10 kg/ha

Hybrids : 4 kg/ha (Female); 1 kg/ha (Male)

Manuring: Apply 12.5 tons of FYM/ha before ploughing. Apply 150:75:75 kg

NPK/ha, of which 50% of the N should be applied as top dressing in two split doses
at flowering and 10 days later.

Planting ratio: For hybrid seed production, female and male parents are normally
planted in the ratio of 4:1.

Roguing: Minimum of three inspections for varieties and 4 inspections for hybrids,
one at vegetative, two at flowering and one at fruit maturity stages. The rouging
should be based on the plant characters, hairiness, fruit character like fruit colour,
number of ridges, fruit length etc., and the off type and mosaic attacked plants
should be removed from the seed field. Wild bhendi if present should be removed
before flowering.

Pest and disease management: The major pest attacking bhendi are jassids,
aphids and white fly, which can be controlled by spraying Rogar or Dimecron or
Endosulphon. The pod borer and red spider mites can be controlled by spraying
Endosulphon and Kelthane, respectively. The diseases such as yellow vein mosaic
and powdery mildew can be controlled by spraying systemic insecticides and
Karathane, respectively.

Hybrid seed production: In bhendi, since the flowers are large in size, hand
emasculation and pollination is the best suitable method for seed production. The
emasculation and dusting can be done as per the methods outlined in tomato. The
male and female parents are raised in blocks at the ratio of 9:1 (Female: Male).

Harvesting: Fruits should be harvested when they have dried (30-35 days after
crossing). The pods which expose hairline crack and turn to brown colour on drying
alone are cut using sickle manually.
Threshing:

The pods are dried and threshed using pliable sticks. Separated seeds are
winnowed to remove plant debris and dried over a tarpaulin to 10% moisture
content. Dried seeds are subject to water floatation in which, good seeds sink while
poor seeds float. The floaters are removed, while sinkers are dried under shade
followed by sun drying. Then the seed are cleaned, dried and treated with Captan/
Thiram.

Processing: Seeds are to be processed with BSS 7 wiremesh sieve.

Seed Yield: 1000-1200 Kg/ha

Specific standards:

Factors Foundation Certified

Off types 0.1% 0.2%

Objectionable weed None None

Disease affected plants 0.1% 0.5%

Objectionable weed: wild Abelmoschus sp.

A.moschatus A. manihot

Designated diseases: Yellow Vein Clearing Mosaic (Hybiscus virus-1)

Seed standards

Factors Foundation Certified

Pure seed 99% 99%

Inert matter 1% 1%

Other crop seed None 5/kg

Total weed seed None None

Objectionable weed None None

Other Distinguishable 10/kg 20/kg

Varieties (ODV)

Germination 65% 65%

Moisture 10% 10%

For VP Container 8% 8%

Questions
1.Chilli is an often Cross pollinated vegetable. (True/False)
2. The flower of chilli is
a. Protandry b. Terminal
c. Axillary d. Protogyny
3. _______ arrests flower drop in chilli.
a. NAA b. GA3
c. MH d. Ethrel
4. Off type allowed during certified seed production in chilli is _______
a. 0.5% b. 0.02%
c. 0.2% d. 2%
5. Isolation requirement for foundation and certified seed production
in chilli is_________
a. 400 & 200 M b. 100 & 200 M
c. 600 & 200 M d. 600 & 400 M
6. Bhendi is an often cross pollinated crop. (True/False)
7. Cross pollination in bhendi is due to protogynous.

8. Isolation requirement for foundation and certified seed production

in Abelmoschus esculentus is_________
a. 600 & 100 M b. 200 & 100 M
c. 300 & 200 M d. 400 & 200 M
9. Planting ratio for hybrid seed production in Abelmoschus esculentus is
a. 8:1 b. 4:1
c. 12:1 d. 10:1
10. Seeds of bhendi is processed with ___________ wiremess sieve.
a. BSS 10 b. BSS 3
c. BSS 7 d. BSS 15
11. Objectionable weed in bhendi is _____________
a. Manihot esculentus b. A.moschatus
c. Coleus forskohlli d. None
12. Designated diseases of bhendi is yellow vein mosaic.
13. Other Distinguishable Varieties (ODV) allowed in bhendi is _____
a. 5/kg b. 20/kg
c. 10/kg d. 40 %
 ONION (Allium cepa)

Onion is one of the most important

commercial vegetable crops in India.
Maharastra, Gujarat, Uttra Pradesh, Orissa
and Andhra Pradesh are the major onion
growing states. The total annual area is
estimated to be about 3 lakhs hectare and
production is about 35.37 lakh tonnes. It is
grown mainly in rabi season. Three crops
viz., Kharif, late Kharif and rabi are taken in
Nasik division of Maharashtra whereas
Gujarat, Andhra Pradesh, Rajasthan, Punjab, Harayana, Madhya Pradesh,
Karnataka and Tamil Nadu take up two crops that is Kharif and rabi. Kharif onion is
a recent introduction in Northern, Eastern and Central India.

Botany

Onion is the biennial crop and takes two full seasons to produce seeds. In the
first year bulbs are formed and in the second year stalks develop and seed are
produced. It is a long-day plant. The day length influences bulb onion, but has
little effect on induction of seeding. It appears to be day-neutral for seed
production. It requires cool conditions during early development of the bulb crop
and again prior to and during early growth of seed stalk. Varieties bolt readily at 10
to 15 degree C. In the early stages of growth, a good supply of moisture is required
and temperatures should be fairly cool. During bulbing, harvesting and curing of
seed, fairly high temperatures and low humidity is desirable. Seed production is
widely adapted to temperate and sub-tropical regions.
Stages of seed production : BS – FS - CS

Varieties

A. RED
1. Punjab Selection PAU, Ludhiana
2. Pusa Ratna NBPGR, New Delhi
3. Pusa Red IARI, New Delhi
4. Pusa Madhavi IARI, New Delhi
5. N-2-4-1 MPAU, Rahuri
6. Arka Niketan IIHR, Bangalore
7. Arka Kalyan IIHR, Bangalore
8. Agrifound Dark Red NHRDF, Nasik
9. Agrifound Light Red NHRDF, Nasik
B. WHITE
1. N-257-9-1 MPAU, Rahuri
2. Pusa White Round IARI, New Delhi
3. Pusa White Flat IARI, New Delhi
4. Punjab-48 PAU, Ludhiana
C. Aggregatum Onion
1. CO 5 TNAU, CBE

• Bellary Red, Rampur local, and Kalyanpur,

Season

The optimum sowing season is middle of June to Middle of July in the plains.

Isolation Requirements

Onion is largely cross-pollinated crop with up to 93 per cent natural crossing

but some self-pollination does occur. It is chiefly pollinated by honey-bees. For pure
seed production, the seed fields must be isolated from fields of other varieties of
onion and fields of the same variety not conforming to varietal purity requirements
for certification atleast by 1000 metres for foundation seed production and 500
metres for certified seed production.

Method of Seed Production

There are two methods of seed production

1. Seed to seed method: In this method, the first season bulb crop is left to over-
winter in the field so as to produce seed in the following season.
2. Bulb to seed method: The bulbs produced in the previous season are lifted,
selected, stored and replanted to produce seed in the second year.
Mostly the bulb to seed method is used for seed production because of the following
advantages over the seed to seed method.
a) It permits selections of "true-to-type" and healthy bulbs for seed production.
b) Seed yields are comparatively very high. The seed to seed method, however,
can be practiced for varieties having a poor keeping quality.
Bulb to seed method
A. Bulb Production stage

a) Climatic requirement
Though it is possible to produce bulbs in different climatic conditions, mild
climate is reported to be very good. For better bulb production a temperature of
15.5 to 210C and about 70% relative humidity required.

b) Land requirement
Fields in which onion was grown should be selected unless it was of the same
variety and was certified. The onion can be grown on various types but it grows
best in soils which are able to retain moisture for longer time. Heavy soils do not
permit proper bulb development and many times bulbs are misshapen. 6-8 pH
range are considered better for onion.

c) Isolation requirement
Onion is highly cross pollinated crop with upto 93% natural crossing. It is mostly
pollinated by honeybees. For pure seed production the seed fields must be
isolated from field of other varieties of onion of the dame colour at least by 1000
 meters for foundation seed and 500 m for certified seed. The isolation distance
between colour particularly white and red colour must be much more which
needs to be decided.

d) Seed rate
8-10 Kg per hectare

e) Sowing and transplanting time

Season Sowing Transplanting

Kharif June-July July-August
Rabi Oct-Nov Dec-Jan

In kharif 6-7 weeks old seedling and in rabi 8-9 weeks seedlings should be
transplanted. Over aged nursery should not be planted otherwise premature
bolting may be there.

f) Manures and fertilizers

FYM 50 tonnes
CAN 400Kg
Or
Urea 200kg
Super phosphate 300 Kg
Muriate of Potash 100 Kg
Nitrogen should be applied as basal and top dressing in two splits. Top
dressing may not be delayed otherwise thick necks may be a problem.

g) Spacing
15 x 10 cm. More spacing between plants results in thick necked plants.

h) Irrigation
Irrigation should be given at fortnightly interval or weekly interval as the case
may be. Field should not be left dry for long otherwise splitting problem is more.
i) Weeding
2-3 weedings and hoeings are done. Stomp @ 3.51 / ha may be applied 3
days after transplanting to manage the weeds economically. One weeding by hand
is, however, necessary.

j) Plant protection
Malathion @ 0.1% along with tritone against thrips. 4-5 spraying may be
necessary. Indofil M45 @ 0.25% along with tritone against purple blotch and
stemphylium blight, 5-6 sprayings may be done.

k) Roguing
Remove off type plants on difference in colour of leaves or plant type.
Remove resprouted plants or premature bolters.

l) Harvesting
Harvesting the crops one week after 50% of tops falling and keep in windrow
upto 3-5 days for field curing. After that bulbs are cured in shade to remove fields
heat before keeping in store. In kharif bulbs are ready for harvesting within 90-100
days after transplanting while tops are still erect. Bulbs are allowed for field curing
upto 3-5 days then again cured were in shade or in field depending upon the
temperature for 12-15 days. Tops are cut leaving 2.5 cm neck.
B. Seed Production Stage

a) Selection of bulb
True to type bulbs are selected based on colour, size and shape kept in
ventilated storage in rabi crop and in kharif crop bulbs are planted after curing
for 15 days. 4-6 cm size bulbs are selected for getting good crop.

b) Climate
Conditioning of plants / bulbs is necessary for seed stalks formation.
Temperature of 4.50c to 140C are favourable for this conditioning. Longer this
prevails, more stalks each plant will produce and more flowers will be in each
umbel. Low humidity gives good seed development. While plants are in
flowering clear bright sunny days are necessary for good insect activity.

c) Bulb rate
25 quintal / ha

d) Spacing
45 x 30 cm

e) Fertilizer and manures

200 kg urea / ha.50% as basal and rest as top dressing
300 Kg super phosphate (single) / ha
100 Kg muriate of potash / ha
f) Irrigation
Irrigation at an interval of 15 days in winter and 7-10 days in summer is
necessary for proper seed development. Fields should not be kept saturated for
long as this facilities development of diseases.

g) Rouging
Remove plants based on foliage, colour inflorescence and flower characters.

h) Plant protection
1) Spray Indofil M45 @ 0.25% against purple blotch and stemphylium blight.
2) Endosulfan @ 0.20% against thrips and head borer.

i) Harvesting and curing

When capsules become brown and seeds inside become black the umbels are
then cured and dried.
j) Threshing and cleaning
Threshing is done manually. Pre-cleaning is done by brushing machine and
scalper. Cleaning and grading are done by Air screen cleaner by using 1/14x1/2
as grading screen and then upgrading is done by gravity separators.

k) Drying and Packing

 Seeds are dried upto 6-8% moisture depending upon packaging requirements. If
seeds are required to be packed in Aluminium foil and other moisture proof
containers, seed are dried upto 6% otherwise upto 8%.

l) Seed yield
5-7 q / ha

Certification Standards
I. Field Standards
A. General requirements
1. Isolation
Onion seed fields shall be isolated from the contaminants shown in column 1
of the Table below by the distance specified in columns 2,3,4 and 5 of the
said Table:
Contaminants Minimum distance (meters)
Mother bulb Seed Production stage
production stage
Foundation Certified Foundation Certified
2 3 4 5
Fields of other varieties 5 5 1000 500
Fields of the same 5 5 1000 500
variety not conforming
to varietal purity
requirement for
certification

B. Specific requirements
Factors Maximum permitted
Foundation Certified
* Bulbs not conforming to the 0.10% (by 0.20% (by
varietal characteristics number) number)
* Off types 0.10% 0.20%
 *

* Maximum permitted at second inspection at mother bulb production stage.

** Maximum permitted at and after flowering at seed production stage.

II. Seed Standards

Factors Standards for each class

Foundation Certified
Pure seed (minimum) 98.0% 98.0%
Inert Matter (maximum) 2.0% 2.0%
Other crop seed (maximum) 5 / Kg 10 / Kg
Weed seeds (maximum) 5 / Kg 10 / Kg
Germination (minimum) 70% 70%
Moisture (maximum) 8.0% 8.0%
For vapour-proof containers 6.0% 6.0%
(maximum)

Problems and Prospects of certification in onion seed production

Following are the problems and remedial measures in certification of onion
seed:
a. Unawareness about the notified varieties by the farmers
Many improved and notified varieties have not been demonstrated fully with
the farmers as such farmers still prefer old varieties. The extension agencies in the
state may therefore take up demonstration so as to allow farmers to know about
the new improved varieties.

b. Unawareness about the advantage of certified seed over truthful seed

In cereals and some other seeds the seed production and distribution
programme are properly organized. Farmers have been demonstrated with the
advantage of using certified seed. In vegetables particularly in small seed such
demonstrations or extension education programmes have not been carried out.
Farmers thus are not aware about the benefits of using certified seed in onion.
Extension agencies should arrange state level demonstrations on use of certified
seed in onion to make the farmer fully aware of advantages of the certified seed.

c. No maintenance breeding for improved varieties

Since varieties when developed by the Universities / institutes do not pass
through maintenance breeding later, the varieties do not behave in different
characters in the same way as these were at the time of development. The
application of certification standards particularly for genetics purity therefore
becomes impossible. The Universities / Institutes should continue maintenance
breeding of their varieties for maintaining distinctiveness, uniformity and stability.

d. Most of the parameters of the varieties are influenced by agro climatic

conditions
In onion there are many characters like colour, shape, bolting, neck
thickness or doubles which are affected adversely by agro climatic conditions like
soil, temperature, rainfall, cultural practices etc. Practical application of certification
standards required to be seen at the time of certification where staff cannot have
proper judgment. The staff should, therefore, know the details of characters and
how and to what extent, they are influenced by adverse weather conditions. Based
on that the staff should assess the situation and apply their mind in certifying a
crop.

e. Staff with certification agencies are neither adequate nor they have
proper knowledge about the crop.
Onion is highly cross-pollinated crop and it requires through inspection or
check at different stages. If one stage is left it becomes difficult to meet the
requirements. For example if inspection is not managed at the time of bulb
selection, Similarly if isolation is not checked at the time of bolting it becomes a
frutile exercise later as roughing has no meaning at the time of flowering. This is
possible only when sufficient staff having good knowledge about onion is provided.
f. Unawareness of farmers about pre harvest and post harvest practices of
onion seed production.
The extension agencies as also staff certification agencies are supposed to
properly guide for production and post harvest practices for certified seed
production initially. Certification staffs presently do not guide. Presently since staff
themselves are not aware about pre harvest practices as also post harvest
practices, programmes many times fail as such farmers hesitate in going for
certified seed production. It is, therefore, necessary for certification staff to guide
the farmers initially.

g. Inadequate infrastructural facilities for storage of bulbs, cleaning,

grading and drying
Bulbs of rabi onion are required to be stored in ventilated godowns which are
not available. Seed requires is must which is mostly not available. Similarly for
enabling the seed producers to pack the seed in moisture proof containers for long
term storage, seeds are required to be dried to 6% moisture where dehumidified
driers are required. Such facilities are lacking at any places.

h. Certification standards are not realistic

Presently standards which have been fixed are not realistic. The standards
need to be fixed based on the type of material being developed by the institutes.
The effects of agro climatic conditions on different parameters need to be
considered. Isolation distances are not adequate particularly between white and red
varieties.

i. Non availability of adequate quality breeder / foundation seed of a

variety
Even if everyone is ready for taking up certified seed production, adequate
quality breeder seed foundation seed with the concerned institute is not available.
Because of this problem in fact many good varieties in onion have been lost before
going to farmers. The seed production programme, therefore, should be properly
planned right from production of breeder seed to certified seed so as to make
available quality seed in adequate quantities for improving production and quality.
j. Low price of seed available in the market
Many times onion seed price in the market are very low compared to quality
seed / certified seed. This is mainly because farmers collect seed from premature
bolters / takes up in situ method where though quality is poor quality is in
abundance, Govt. should, therefore give some.
 SEED PRODUCTION OF CUCURBITACEOUS VEGETABLES

Land Requirements: There are no land requirements as to previous crop, but the
land should be free of volunteer plants. Generally the soil should be well drained
and aerated.

Isolation Requirements: Most of the cucurbits are monoecious in character and a

few are dioecious. A number of hermaphrodite and andromonoecious cultivars
are also available in some crops. Pollination is largely done by insects. For pure
seed production and isolation distance all around seed field is necessary to
separate it from fields of other varieties, fields of the same variety not conforming
to varietal purity requirements for certification, from wild cucurbit species, and to
separate musk melon from long melon and vice versa, and pumpkin from summer
and winter squashes and vice versa as follows

Class Minimum distance (meters)

Foundation 1000

Certified 500

Flower structure in cucurbits

GENETIC PURITY AND SEED HEALTH STANDARDS FOR CUCURBITS

Factors Minimum permitted level(%)

FS CS

Open pollinated variety

Off-type 0.10 0.2

Objectional weed plant None None

Hybrids
Off-type in seed parent
0.01 0.05
Off-type in pollen parent
None 0.05

Pollen shedders in seed parent - 0.10

Seed borne diseases ***

Muskmelon * 0.1 0.20

Summer squash ** 0.1 0.5

Cucumber mosaic virus ,** Cucumber mosaic virus, watermelon mosaic virus
SEED CERTIFICATION STANDARDS IN INDIA FOR CUCURBITS

Factors
Minimum permitted level (%)

Foundation seed Certified seed

Pure seed (minimum) 98 95

Inert matter (maximum) 2 5

other crop seed (maximum) None None

Weed seed (maximum) None None

Other objectional varieties (only for

5/kg 10/kg
hybrids)

Germination (minimum) 60 60

Moisture for ordinary pack 7.0 7.0

(maximum)

Moisture for vapour proof pack 6.0 6.0

(maximum)

Seed production details in Cucurbitaceous vegetables

Particulars Bittergourd Snakegourd Ribbedgourd Ashgourd /

Pumpkin
Isolation Foundation seed 1000 m and certified seed 500 m
Season June - July and Feb – March
Varieties CO1, MDU1, CO1, CO2, CO1, CO2, CO1, CO2
Coimbatore PKM1, MDU1 PKM 1
long green &
long white
Seed rate / ha 2.5 2.5 2.5 2.5 / 1.0
female flower Spraying of Ethrel 200 - 250 ppm at two true leaf stage and
increased by after a week of 1st spray
Spacing (cm) Take pits of size 45x45x45 cm at 2.5x2.0 m distance
Fertilizers / 6:12:6 12:24:12 9:15:9 6:12:6
(NPK g/pit)
Physiological Change of fruit colour in any Complete Change of fruit
maturity part or 1/3 of fruit tip to drying of colour to
yellow to red fruits orange brown
in pumpkin and
ashy coating
and metallic
sound in
ashgourd
Processing Hand picking Hand picking BSS 4 wire 16/64 round
mesh sieve perforated
sieve
Fruit to seed 30 15-16 13-14 1.0-1.3
recovery (%)
Seed yield 120-150 220-250 200-250 120-150
(kg/ha)

Techniques of Hybrid Seed Production in cucurbits

i. Hand emasculation and hand pollination

This technique is frequently used for melon seed production. In this species,
andromonoecious lines are common and they must be emasculated and hand
pollinated if used as the female parent for producing hybrid seed. This method has
also been used for some watermelon and cucumber hybrids. This technique is
applicable for limited scale production, since lot of trained labour are required in
pinching, pollen collection and hand pollination.

ii. Hand emasculation and pollination by insect

The male flowers from female lines are pinched off day before of anthesis
regularly, which honeybees and other insects (voluntary) uses as a pollinating
agents. The male and female are grown in alternate rows. The fruit set on female
lines are of hybrid and harvested for seed extraction. The planting ratio varies
within the crops e.g. summer squash 3:1 and 4:1 in muskmelon and cucumber but
depend upon the population of bees in plot. This technique is also used in bottle
gourd, pumpkin, muskmelon, cucumber, summer squash and bitter gourd for
hybrid seed production.

iii. Use of genetic male sterility system

Genetic male sterility system has been utilized for commercial hybrid
production in muskmelon. The genetic male sterility in muskmelon is controlled by
single recessive gene (msms). For hybrid seen production, the male sterile line is
used as female parent. Since genetic male sterile line is maintained in heterozygous
forms, 50% fertile plants are to be removed at flowering. The other 50% having
non-dehiscent empty anther are retained in female rows. The female and male are
grown in 4:1 ratio. However, to maintain the good plant population in female rows
it is suggested that seed parent should be sown with double seed rate. It is also
advised that female line seedling should be raised in polythene bags and
transplanted at flower appearance in order to avoid the fertile plants in female
rows. The pollination is done by honey bees and 1 to 2 medium sizes hives are
good enough to ensure the good pollination and fruit set at female row.

The male sterile line is maintained in heterozygous form by crossing with

maintainer line under adequate isolation distance or under cover.

iv. Use of gynoecious sex form

The gynoecious sex form has been commercially exploited in hybrid seed
production of cucumber. For hybrid seed production female and male rows are
planted in 4:1 ratio. The female (seed parent) bear only female flowers and
pollination in done by insect (honeybee). To ensure the good fruit and seed
recovery, the sufficient population of honeybee 1 to 1½ colony of medium size has
to be kept at the boundary of seed production plot to boost the amount of crossing.
The parental lines i.e. male parent maintained by selfing (mixed pollination) and
rouge out undesirable plants before contamination take place. The female lines i.e.
gynoecious lines maintained by inducing the staminate flower through the sprays of
silver nitrate 200 ppm at two to four true leaf stage and then selfing is carried out.
It was observed that 10-11 male flowers appear per 100 nodes.

The performance of gynoecious lines is unstable under high temperature and

long photo period conditions because of their thermo-specific responses for
gynoecious stability. That is why the gynoecious cucumber did not receive much
attention in the tropical countries. However, few true breeding tropical gynoecious
lines in cucumber and muskmelon have been developed at IARI. As a result of
development of true breeding line, muskmelon hybrid Pusa Rasraj was developed.
These homozygous gynoecious lines are maintained by using GA 3 , 1500ppm or
silver nitrate 200-300 ppm or sodium thio sulphate 400 ppm to induce staminate
flowers at two and four true leaf stage. Homozygous lines are planted in strict field
isolation. The gynoecious lines are crossed with monoecious male parent to produce
F1 hybrid.

v. Hybrid seed production through chemical sex expression

The hybrid seed can also be produce in cucurbits by the application of

chemicals for attaining the sex of cucurbits. Specific chemicals are known to induce
femaleness and maleness as desired. The spraying of ethrel (2-choloro-ethyl-
phosphonic acid) 200-300 ppm at two and four true leaf stage and another at
flowering is useful for inducing the pistilate flower successively in first few nodes on
the female in bottle gourd, pumpkin and squash for F1 seed production. The row of
male parent is grown side by the side of female and natural cross pollination is
allowed. In the absence of insect, hand pollination is possible when two sexes are
separate. Four to five fruit set at initial nodes are sufficient for hybrid seed. The
complete suppression of male flowers in squash can be achieved by applying ethrel
at higher concentration (400-500 ppm) twice.

The other chemicals like GA3, (10-25 ppm) in cucumber, MH-(100 ppm),
ethephon (600 ppm) in squash induces female flowers.
 Seed Certification

It is a legally sanctioned system for quality control and seed multiplication

and production. It involves field inspection, pre and post control tests and seed
quality tests.

Purpose of seed certification

To maintain and make available to the farmers, through certification, high

quality seeds and propagating materials of notified kind and varieties. The seeds
are so grown as to ensure genetic identity and genetic purity.

Eligibility for certification of crop varieties

Seeds of only those varieties which are notified under section 5 of the Seeds
Act, 1966 shall be eligible for Certification.

Breeder seed is exempted from Certification. Foundation and Certified class

seeds come under Certification.

Breeder seed is produced by the plant breeder which is inspected by a

monitoring team consisting of the breeder, representative of seed certification
agency (DDA), representative of NSC (Deputy Manager) & nominee of crop co-
ordinator (s – 11). The crops shall be inspected at appropriate stage.

Phases of seed certification or Seed certification procedures

1. Receipt & Scrutiny of application

2. Verification of seed source

3. Field inspection

4. Post harvest supervision of seed crops

5. Seed sampling & testing

6. Labelling, tagging, sealing and grant of certificate.

1. Receipt & scrutiny of application

a. Application for registration

Any person, who wants to produce certified seed shall register his name with
the concerned Assistant Director (AD) of seed certification by remitting Rs. 25/- per
crop, per season. There are 3 seasons under certification viz., kharif (June-Sep),
Rabi (Oct. – Jan.) & Summer (Feb-May).

The applicant shall submit two copies of the application to the ADSC 10 days
before the commencement of the season or at least at the time of registration of
sowing report.

On receipt of the application, the ADSC will verify the time limit, variety
eligibility & its source, the class mentioned, remittance of fee etc.

The application, if accepted will be given an application no (e.g. Paddy / K /

01- 05-06, where Paddy refers the crop to be registered, K-the season, 01-the
application no & 05-06 -the financial year). The original application is retained and
the duplicate is returned to the applicant.

b. Sowing report: (Application for the registration of seed farm)

The seed producer who wants to produce certified seeds shall apply to the
ADS.C, in the prescribed sowing report form in quadriplicate with prescribed
certification fees along with other documents such as tags to establish the seed
source.

Class of seed Source of seed

1. Foundation class Breeder seed

2. Certified class Foundation seed

3. F. Class stage II Foundation class stage – I

4. C. Class stage II Certified class stage - I

Separate sowing reports are required for different crop varieties, different
classes, different stages and if the seed farm fields are separated by more than 50
metres.
 Separate sowing reports are also required if sowing or planting dates differ
by more than 7 days and if the seed farm area exceeds 25 acres.

The sowing report shall reach the concerned ADAS.C within 35 days from the
date of sowing or 15 days before flowering whichever is earlier. In the case of
transplanted crops, the sowing report shall be sent 15 days before flowering.

The producer shall clearly indicate on the reverse of sowing report, the exact
location of the seed farm in a rough sketch with direction, distances marked form a
permanent mark like mile stone, building, bridge, road, name of the farm if any,
crops grown on all four sides of the seed farm etc, to facilitate easy identification of
the seed farm by the seed certification officer.

The AD S.C, on receipt of the sowing report, scrutinizes & register the seed
farm by giving a S.C number for each sowing report. Then he will send one copy of
the sowing report to the S.C officer, one to the D.D.S.C & the third to the producer
after retaining the fourth copy.

2. Verification of seed source

During his first inspection of seed farm the S.C officer, will verify whether the
seed used to raise the seed crop is from an approved source.

3. Field Inspection

Objective

The objective in conducting field inspection is to verify the factors which can
cause irreversible damage to the genetic purity or seed health.

Inspection Authority

The seed certification officer authorized by the registering authority shall

attend to field inspections.

Crop stages for inspection

The number of field inspections and the stages of crop growth at which the
field inspections should be conducted vary from crop to crop. It depends upon
duration, and nature of pollination of the seed crop.
 If the crop is grown for hybrid seed production, the no. of field inspections
during the flowering stage should be more than in the case of self-pollinated /
cross/ often cross pollinated varieties.

In hybrid seed production and variety seed production of cross pollinated

crops, the inspection during flowering should be made without any prior notice of
the seed grower to judge the quality of operation undertaken by him to maintain
the genetic purity of the crop. But in the case of self-pollinated crops the seed
grower may be informed about the date of inspection.

In the former case if prior notice is given to the seed grower, it may not be
possible to detect the damage by the contaminants, whereas in the latter case prior
notice will lead to improvement of the quality of the seed production work and thus
the quality of seed.

The key points to be observed at each stage of inspection are

Stage of crop Key points to be observed at Inspection

Verification of seed source
Confirmation of acreage given in the report
Land requirement to keep check on genetic as well as
physical contamination and spread of disease inoculums.
I.Pre-flowering stage
Planting ratio
(Vegetative stage)
Border rows
Isolation distance
Guide the grower in identification of Off-types, pollen
shedder, diseased plants, shedding tassels etc.
Confirm the observation of plants inspection were
II. Flowering Stages: correct.
(May be II & III Confirm whether grower had continued thorough
inspections, When roguing, after the previous inspection.
5% of plants begin to Verify the removal & occurrence of Off-types, pollen
flower) shedders, shedding tassels, objectionable weed plants &
diseased plants.
 Confirm the correctness of observations, made in earlier
inspections
III. Inspection
Guide the grower on roguing, based on pods, earhead,
during post flowering
and pre-harvest seed & chaff characters such as colour, shape & size
stage
Explain to the grower when & how to harvest the crop &
process
Verify that male parent rows have been
harvested separately.
Ensure complete removal of off-types, other crops,
IV. Inspection during
harvest weeds & diseased plants etc.
(This is the last
Seal properly by the certification agency of the threshed
inspection conducted
on a seed crop) produce after initial leaning & drying.
Instruct the seed growers for safe storage &
transportation.

MINIMUM NUMBER OF FIELD INSPECTIONS REQUIRED

FOR DIFFERENT CROPS FOR CERTIFICATION

Minimum
Crop no. of Stages of crop
inspection

Paddy & Wheat 2 Flowering to harvest

Sorghum 4 Ist before flowering, II nd & IIIrd during

flowering, IVth prior to harvest.
Hybrid

Varieties 3 Ist before flowering, II nd during flowering

and IIIrd prior to harvest
Maize 4 Ist before flowering
Inbred lines, Single Rest during flowering
crosses, Other hybrids

Varieties 2 I st before flowering

IInd during flowering

Bajra 4 Ist before flowering

Hybrids II nd & IIIrd during flowering,
IVth prior to or during harvest

Ist before flowering

Varieties 3 IInd during 50% flowering
IIIrd prior to harvest

Green gram,Black 2 Ist before flowering

gram, Red gram
II nd at flowering & fruiting stage
Cowpea

Ground nut 2 Flowering to harvest

Sesame 3 Ist before flowering

(Gingelly) II nd during flowering
IIIrd from fruit maturity to harvest

Sunflower 2 Flowering to harvest

Rape & mustard 3 Ist before flowering

II nd from flowering to fruiting
IIIrd from fruit maturity to harvest

Soyabean 2 Flowering to harvest

Castor 2 Flowering to harvest

Cotton (Varieties) 2 Flowering to harvest

(Hybrids) 4 Ist before flowering
II nd & IIIrd during flowering
IVth during harvest
Brinjal, Tomato 3 Ist before flowering
Chilli, Bhendi IInd from lowering to fruiting
IIIrd during maturity

Carrot 3 Ist early (20-30 days after sowing),

IInd when lifted & re-planted,
IIIrd during flowering.

Cabbage 3 Ist before marketable stage

IInd when the heads have formed
IIIrd during flowering

Cauliflower 4 Ist before marketable stage

IInd during curd formation
IIIrd when most plants have formed curds
IV th during flowering

Onion 3 Ist during early vegetative stage

IInd during bulb formation
(seed to seed)
IIIrd during flowering

Field Counts

The purpose of field inspection is to find out field standards of various factors
in the seed farm. It is impossible to examine all the plants in the seed farm.
Hence, to assess the field standards of various factors random counting is followed.

The number of counts taken and the method employed in taking counts vary
from crop to crop. It is necessary to take a minimum of 5 counts upto 5 acres &
an additional count for every 5 acres or part thereof as given below:

Area of the field (in No. of counts to be

acres) taken

Upto 5 5

6-10 6
 11-15 7

16-20 8

21-25 9

Double Count

In any inspection, if the first set of counts shows that the seed crop does not
confirm to the prescribed standard for any factor, a second set of counts should be
taken for that factor. However, when the first set of counts shows a factor more
than twice the maximum permitted, it is not necessary to take a second count.

On completion of double count, assess the average for the two counts. It
should not exceed the minimum permissible limit.

NO. OF PLANTS FOR A COUNT

No. of plants
S.no. Crop / heads per Remarks
count

1. Soyabean, Jute, Lucerne, Mesta, 1000 plants Closely

Berseem planted crops

2. Beans, Cluster beans, Cowpea, Peas, 500 plants Medium

Green gram, Blackgram, Mustard,Niger, spaced crops
Sesame, Bengal gram, Safflower

3. Bhendi, Brinjal, Chilli, Castor, Cole 100 plants Wide spaced

crops, Cotton, Cucurbits, Maize, Potato, crops
Groundnut, Redgram, Tomato &
Sunflower

4. Bajra, Barley, Oats, Paddy, Wheat, 1000 heads Tillering crops

Ragi, Sorghum
Points to be observed before counting

1. All plants falling in each count must be examined for each factor

2. In hybrid seed field, the prescribed number of the field counts should be
taken in each parent separately.

Sources of contamination or factors to be observed

The contaminants are

1. Physical contaminants

2. Genetical contaminants.

 Physical contaminants are inseparable other crop plants, objectionable

weed plants and diseased plants.

 Genetical contaminants consist of off-types, pollen shedders and

shedding tassels.

a. Off Type

Plant that differs in morphological characters from the rest of the population
of a crop variety.

Off-type may belong to same spp. or different spp. of a given variety. Plants
of a different variety are also included under off-types.

Volunteer plants & mutants are also off-types.

b. Volunteer Plant

Volunteer plants are the plants of the same kind growing naturally from seed
that remains in the fields from a previous crop.

c. Pollen Shedders

In hybrid seed production involving male sterility, the plants of ‘B’ line
present in ‘A’ line are called Pollen shedders.
 Sometimes ‘A’ line tends to exhibit symptoms of fertile anthers in the ear
heads of either on the main tiller or side tiller and these are called Partials. These
partials are also counted as pollen shedders.

d. Shedding Tassels

These are plants which shed or shedding pollen in female parent rows. When
5 cm or more of the entire spike shed pollen they are also counted as Shedding
tassels.

e. Inseparable Crop Plants

These are plants of different crops which have seeds similar to seed crop.

Crop Inseparable crop plants

Wheat Barley, oats, gram, & Triticale

Barley Oats, gram, wheat & Triticale

Oats Barley, gram, wheat & Triticale

Triticale Wheat, barley, oats, gram & Rye

f. Objectionable Weed Plants

These are weeds

1. Whose seeds are difficult to be separated once mixed

2. Which are poisonous

3. Which have smothering effect on the main crop

4. Which are difficult to eradicate once established.

5. Difficult to separate the seeds. These seeds cause mechanical admixtures

Common name of
S.No Crop Botanical name
the weed

1. Paddy Wild rice Oryza sativa var fatua

2. Wheat Wild morning glory Convolvulus arvensis

3. Sunflower Wild sunflower Helianthus spp

 4. Bhendi Wild okra Abelmoschus spp

5. Rape, mustard Mexican prickly poppy Argemone mexicana

6. Lucerne Dodder Cuscuta spp

g. Designated Diseases

The diseases which may reduce the yield and quality of seeds are
termed as Designated diseases.

S.No Crop Name of the Casual organism

Disease
1. Wheat Loose smut Ustilago tritici
2. Sorghum Grain smut Sphacelotheca sorghii
Head smut
3. Pearl millet Ergot Claviceps microcephala
Grain smut Tolyposporium pencillariae
Downy mildew Sclerospora graminicola
4. Cowpea Anthracnose Colletotrichum
lindemuthianum
5. Green gram Halo blight Pseudomonas phasiolicola
6. Gingelly Leafspot Cercospora sesami
7. Sunflower Downy mildew Plasmopara halstedii
8. Brinjal Phomopsis blight Phomopsis vexans
9. Chilli Leaf blight Alternaria solani
Anthracnose Colletotrichum capsici
10. Tomato Early blight Alternaria solani
Leaf spot Stemphylium solani
Tobacco mosaic (TMV)
virus

Land Requirement

The field offered for certified seed production should not been grown in the
previous season with the same crop. If it was grown, the variety should be the
same. In that case, the field should be irrigated at least 3 weeks before sowing and
ploughed just prior to sowing, in order to destroy germinating seeds.

Isolation

Separation of seed fields from fields of other varieties of the same crop,
same variety fields not conforming to varietal purity requirements, and other
related species fields and fields affected by diseases to prevent genetic & disease
contamination.

The minimum distance to be maintained between the seed crop and the
contaminant is called Isolation distance.

Crop F.S (m) C.S (m)

Self pollinated crops
Cereals and Millets
Paddy 3 3
Wheat 3 3
Pulses
Green gram 10 5

Black gram 10 5

Soya bean 3 3
Bengal gram 10 5
Cowpea 10 5
Lab lab 10 5
Oil Seeds
Groundnut 3 3
Vegetables
Tomato 50 25
Cluster beans 10 5
French beans 10 5
Peas 10 5
lettuce 50 25
Potato 5 5
Often Cross Pollinated crops
Millet
Sorghum Variety 200 100
Sorghum hybrid 300 200
Pulses
Red gram 200 100
Oil Seeds
Sesame 100 50
Cotton (variety) 50 30
Vegetables
Brinjal 200 100
Chillies 400 200
Bhendi 400 200

Cross Pollinated Crops

Millets
Maize (varieties) 400 200

Inbred line 400 -

Single cross hybrid 400 -
Double cross hybrid - 200
Bajra variety 400 200
Bajra hybrid 1000 200
Sun hemp 200 1000
Castor 300 150
Sunflower variety 400 200
Sunflower hybrid 600 400
Cabbage 1600 1000
Beetroot 1600 1000
Radish 1600 1000
Cauliflower 1000 500
Onion 1000 800
 Carrot 400 200
Amaranthus 1000 500
Gourds

Inspection Report

The seed certification officer after taking field counts and comparing them
with the minimum field standards, the observations made on the seed farm field
should be reported in the prescribed proforma to

1. Deputy Director of S.C

2. To the Seed producer

3. AD, S.C

4. Retained with him.

Assessment of seed crop yield

It is necessary to avoid malpractices at the final stage during harvest

operation.

The seed certification officer is expected to fix the approximate seed yield.

L.F.R REPORT (Liable For Rejection Report)

If the seed crop fails to meet with any one factor as per the standards, L.F.R
report is prepared & the signature of the producer is obtained & sent to D.DSC within
24 hrs.

RE-Inspection

For the factors which can be removed without hampering the seed quality,
the producer can apply for re-inspection to the concerned D.D,S.C within 7 days
from the date of F.I rejection order. For re-inspection half of the inspection charge
is collected.
4. Post Harvest Supervision Of Seed Crop

The post harvest inspection of a seed crop covers the operations carried out
at the threshing floor, transport of the raw seed produce to the processing plant,
pre-celeaning, drying, cleaning, grading, seed treatment, bagging & post processing
storage of the seed lot.

Pre-requisites for processing

1. Processing report should accompany the seed lot

2. ODV test for paddy should be done at the time of sealing & issue of
processing report or before processing. If the result exceeds 1% the produce
may be rejected.

3. It should correlate with the estimated yield.

4. Seed should be processed only in approved processing unit.

5. Field run seed should be brought to the processing unit within 3 moths from
the date of final inspection. Processing & sampling should be done within 2
months in oil seed crops & 4 months for other crops from the date of receipt
in the processing unit. In cotton, the kapas from the passed lot should be
moved to the ginning factory within 5 days from the date of issue of
processing report. The ginning should be done within 3 months from the
date of final harvest inspection report. Ginned seeds should be moved to
seed processing unit within 5 days of ginning. Inspection and sampling
should be done within 3 months after ginning.

Intake of Raw Produce & Lot Identification

The seed certification officer in-charge of the seed processing plant may,
after verification of the above stated documents and total amount of seed accept
the produce for processing.

After verification he should issue a receipt to the seed grower. Each seed lot
has to be allocated a separate lot number for identification.
Processing of seed lot

1. It is done to remove chaff, stones, stem pieces, leaf parts, soil particles etc
from the raw seed lot.

2. Grading to bring out uniformity in the seed lot.

3. Seed treatment to protect it from storage pests & diseases.

Processing Inspection

1. The processing should be done in the presence of concerned seed

certification officer.

2. The recommended sieve size should be used for grading.

3. While processing of paddy, the work of perfect processing has to be

evaluated then & there.This is done by conducting a float test. Take 400
seeds from the processed seed & put into a tumbler of water. Count the
floating paddy seeds. Maximum float admissible is 5%. If the float seeds
exceed the limit, adjust the air flow or feeding to perfect the processing.

4. In maize, before shelling, the cobs should be examined for off-type and off -
coloured kernels. Individual cobs should be examined with reference to its
Varietal characters. The cobs of off-types and off-coloured kernels should be
rejected.

5. Seed Sorting in Cotton.

The ginned seeds will be evaluated for its quality. A maximum of 3% for the
following factors can be taken into account.

1. Immature seeds
2. Ill-filled seeds
3. Broken seeds
4. Stained seeds &
5. Over fuzzy seeds.
Groundnut Pod Verification

 In groundnut 4% of ill-filled pods can be allowed.

 After processing, the seeds may be treated, packed, weighed & sealed before
the SCO.

 The unit of packing may be equal to the seed rate of 1/2 or one acre or ha

5. Seed Sampling & Testing

During packaging S.C officer will draw samples according to ISTA Procedure
& send the sample to ADSC concerned within a day of sampling. The ADSC will
inturn send the sample to the STL within 3 days of receipt of the sample for testing
seed standards viz. physical purity, germination, moisture content & seed health as
prescribed. The STO will communicate the result to the ADSC concerned within 20
days.

On receipt of the analytical report, the ADSC will communicate the result to
the producer & SCO.

6. Labelling, tagging, sealing and grant of certificate

After receiving the seed analytical report, the SCO will get the tag from the
ADSC & affixes labels (producer’s label) and tags (Blue for C.S & White for F.S)
to the containers & sealed to prevent tampering and grant certificate fixing a
validity period for 9 months.

Tagging should be done within 60 days of testing.

Resampling & Reprocessing

When a seed lot does not meet the prescribed seed standards in initial test,
on request of the producer SCO may take resample.

If the difference in germination analysed & required is within 10, then

straight away re-sampling can be done. If it is > 10, reprocessing & resampling
may be done.

The producer should request the SCO concerned in writing within 10 days
from the receipt of the result. No charge is collected for resampling.
 When a seed lot, fails even after free sampling, reprocessing can be taken
upon with special permission from D.S.C. For such reprocessing a fee of Rs. 20/- Q
and lab charges of Rs. 10/- Q is collected.
 Seeds Act and Rules

Introduction

The seed is an important agricultural input and it plays vital role in increasing
production and productivity. There is a need to safeguard the farmers with the
supply of genetically pure and quality seeds. Any new variety produced by the
Scientist has to be multiplied many times to meet the needs of the farmers. In
order to ensure the availability of quality seeds, Government of India have enacted
Seeds act, 1966 and Seed rules, 1968. The seed (Control) order, 1983 was
promulgated under essential commodities act, 1955 in order to ensure the
production, marketing and equal distribution of the seeds.

Seeds Act, 1966

The object of Seed Act is to regulate the quality of certain notified kind /
varieties of seeds for sale and for matters connected therewith. The seed act
passed by the Indian Parliament in 1966 was designed to create a 'Climate' in
which the seeds man could operate effectively and to make good quality seed
available to cultivators. Seeds rule under the act were notified in September 1968
and the act was implemented entirely in October, 1969. This act extent to the
whole of India and it has 25 sections.

Seed legislation could broadly be divided into two groups

1. Sanctioning legislation

Sanctioning legislation authorizes formation of Advisory bodies, Seed

Certification Agencies, Seed Testing laboratories, Foundation and Certified Seed
Programmes, Recognition of Seed certification Agencies of Foreign countries
Appellate authorities etc.

2. Regulatory legislation

Regulatory Legislation controls the quality of seeds sold in the market

including suitable agencies for regulating the seed quality. On quality control basis,
the Seeds Act could conveniently be divided into the following:
I. Minimum limit and labeling of the notified kind / varieties of seed

a. Power to notify the kind / variety

b. Labeling provisions

c. Seed testing

d. Seed analyst

e. Seed inspectors

f. Penalty

g. General provisions

II. Seed Certification

III. Restriction of Import and Export of Seeds

I. Minimum limits and labeling

Quality control as envisaged in the Act is to be achieved through pre and

post marketing control, voluntary certification and compulsory labeling of the seeds
of notified kind / varieties.

(a) Power to notify the kind / varieties

New varieties evolved by the State Agricultural Universities and ICAR

institutes are notified and released /notified respectively under section 5 of the
seeds act in consultation with the central seed committee and its sub committees
constitute under section 3 and 3(5) of the Seeds Act. As on date more than 2500
varieties and 130 varieties were notified and denotified under this section. List of
varieties notified and denotified from 1969 to 2005 are compiled and made
available in the form of a book called catalogue of varieties notified and denotified
under section 5 of the Seeds Act. Functions of the Central Seed Committee and its
sub-committee are defined in Clauses 3 and 4 of part II of seed rule.

(b) Labeling provision

Minimum limits for germination, physical purity and genetic purity of varieties
/ hybrids for crops have been prescribed and notified for labeling seeds of notified
kind / varieties under section 6(a) of the Seeds Act. Size of the label, colour of the
label and content of the label were also notified under sub clause (b) of Section 6 of
Seeds Act. Colour of the label is opel green and size of the label is 10 cm x 15 cm
or proportionate thereof. Responsibility for making labeling content of mark or
label, manner of marking, false / misleading statement on label etc., are defined
under clause 7,8,9,10,11 and 12 of part V of seeds rule.

Section 7 of the act regulates the sale of notified kind or varieties. Accordingly no
person shall keep for sale, offer to sell, barter or otherwise supply any seed of any
notified kind or variety, after the dates recorded on the container mark or label as
the date unto which the seed may expected to retain the germination not less than
prescribed under clause (a) of section 6 of the Act.

(c) Seed Testing

There is a provision to set up a central seed laboratory and state seed

laboratory to discharge functions under section 4(1) and 4(2) of the Seed Act, In
the year 1968 there were 23 state seed testing laboratories in the country. At
present there are 86 Seed testing laboratories functioning in the country. During
1995-96 these laboratories tested about 5 lakh samples. Seed testing laboratories
have been assigned certain important functions under part III (5) of Seed Rule.

(d) Seed Analysts

State Government could appoint the Seed Analysts through notification in the
Official Gazette under Section 12 of the Seed Act defining his area and his
jurisdiction. Seed Analyst should posses certain minimum qualification as prescribed
under clause 20 part IX of Seed Rule.

(e) Seed Inspectors

Classes of seed

The State Government, under section 13 of the Act may appoint such a
person as it thinks fit, having prescribed qualification (Clause 22 part IX of Seed
Rule) through notification, as a Seed Inspector and define the areas within which he
shall exercise jurisdiction for enforcing the seed law. He will be treated as a public
servant within a meaning of section 21 of the I.P.C. (45 of 1860). He has power to
examine records, register document of the seed dealer. He will also exercise such
other powers as may be necessary for carrying out the purposes of this Act or rule
made there under. Duties of Seed inspectors are defined in clause 23 of part IX of
Seed rule. He can issue, stop sale order in case the seed in question contravenes
the provision of relevant Act and rules for which he can use form No.III. When he
seizes any record, register documents or any other material , he should inform a
magistrate and take his order for which he can use form No.IV.

(f) Penalty

If any person, contravenes any provision of the Act or Rule, or prevents a

seed inspector from taking sample under this Act or prevents a Seed Inspector from
exercising any other power conferred on him could be punished under section 19 of
the act with a fine of five hundred rupees for the first offence. In the event of such
person having been previously convicted of an offence under this section with
imprisonment for a term, may extend to six months or with fine, which may extent
to one thousand rupees or with both.

II. Seed certification

The object of the Seed Certification is to maintain and make available to the
public through certification high quality propagating material of notified kind /
varieties so grown and distributed as to ensure genetic identity and genetic purity.
The certified standards in force are Indian Minimum seed certification standards and
seed certification procedures form together for the seed certification regulations.
Seeds of only those varieties which are notified under section under Section 5 of
the seeds act shall be eligible for certification.

 Breeder seed

 Foundation seed

 Certified Seed

Breeder seed

 Breeder seed is a seed directly controlled by the breeder.

 Breeder seed should be genetically so pure as to guarantee that in the

subsequent generation.
  Breeder seed could not come under the perview of seed certification as it is
not meant for public sale.

 Breeder seed should be packed and supplied with breeder's golden yellow
stag as per the guideline given in Indian Minimum Seed Certification
standards. It is also the fact that no standard for breeder seed have been
prescribed.

Foundation seed

 Foundation class of seed and certified class of seed are to be certified by

the Certification Agencies as per the Indian Minimum Seed Certification
Standards.

 Section 8 of the Seeds Act provide state government or the Central

Government consultation with State Government may be notification in
official gazette, established certification agencies for the state to carry out
the functions entrusted to certification agency by or under this Act (Part IV,
clause 6, part VI clause 14 of Seeds Rule).

Certified seed

 Seed act section 9 provides any person desires of producing certified seed
shall register his name with concerned seed certification agency duly
remitting the prescribed fee in form No.1 for grant of certificate. Certificate
could be granted in form No.11 after meeting the requirement of
certification agency prescribed under Part VII clause 15,16 and 17 of Seed
rule.

 It should have the minimum genetical purity of 99%

 Certified seed may be the progeny of certified seed , provided this

reproduction does not exceed two generations beyond foundation seed and
provided that if certification agency determines the genetic and physical
purity, if not be significantly altered

 In case of highly self pollinated crops certification of one further generation

may be permitted
  Certified seed produced from certified seed ,shall be eligible for further
seed increase under certification, except in case of highly self pollinated
crops, where certification of one further generation may be permitted

 Certification tags issued once for certified seed not eligible for further seed
increase under certification

 For paddy and wheat, certified seed produced from certified seed is eligible
for certification by NSC up to two generations from foundation seed

Seed (Control) Order, 1983

III. Restriction of Export and Import of Seeds

There is a provision to restrict export and import of seeds of notified kinds or

varieties. The section 17 defines as under “No person shall for the purpose of
sowing or planting by any person (including himself) export or import or cause to
be exported or imported any seed of any notified kind or variety unless.

 It conforms to the minimum limits of germination and purity specified

for that seed under clause (a) of Section 6 and

 Its container bears in the prescribed manner the mark or label with the
correct particular thereof specified for that seed under clause (b) of
section 6.

Background of the case

The Ministry of civil supplies through an order dated 24.4.1983 had declared
the seed for sowing or planting materials of food crops, fruits, vegetables, cattle
fodder and jute to be essential commodities in exercise of power conferred by
Section 2(a) (viii) of Essential Commodities Act, 1955. It was followed by the issue
of Seed (control) order dated 30th December, 1983 by the Ministry of Agriculture,
Dept. of Agriculture and Co-operation in exercise of powers contained in section 3
of Essential Commodities Act, which deals with Central Governments power to
control, and regulate production, supply and distribution of essential commodities.
The Seed (control) order, 1983 had been notified as per Gazette notification, G.S.R
832(E) dated 30. 12.1983. The notification under reference holds good and remains
operative. Joint Secretary (Seeds), Government of India, Ministry of Agriculture,
Department of Agriculture and Cooperation has been appointed as Seed Controller
for implementation of seed (control) order.

Gist of the Seed (Control) order, 1983

Issue of License to dealers

All persons carrying on the business of selling, exporting and importing seeds
will be required to carry on the business in accordance with terms and conditions of
license granted to him for which dealer has to make an application in duplicate in
Form 'A' together with a fee of Rs.50/- for license to licensing authority unless the
State Government by notification exempts such class of dealers in such areas and
subject to such conditions as may be specified in the notification.

Based on such enquiry as it thinks fit for licensing authority may grant in
form 'B' or refuse in provisions of the Order. The refusal to grant license shall be
accompanied by clear recording of reasons for such refusal.

Renewal of License

A holder of license shall be eligible for renewal upon and applicable being
made in the prescribed form 'C' (in duplicate) together with a fee of rupees twenty
before the expiry of license or at the most within a month of date of expiry of
license for which additional fee of Rs.25/- is required to be paid.

Appointing of Licensing authority

The state government may appoint such number of persons as it thinks

necessary to be inspector and define the area of such Inspector’s jurisdiction
through notification in the official gazette.

Time limit for analysis of samples by Seed testing lab

Time limit for analysis of samples by seed testing lab and suspension /
cancellation of license may be done by Licensing authority after giving an
opportunity of being heard to the holder of license, suspend or cancel the license on
grounds of mis-representation of a material in particular or contravention in
provision of the order.

Suspension / Cancellation of license

The Licensing authority may after giving an opportunity of being held to the
holder of license, suspend or cancel the license on grounds of mis-representation of
material in particular or contravention in provision of the Order.

Appeal

The state government may specify authority for hearing the appeals against
suspension / cancellation under this order and the decision of such authority shall
be final. Any person aggrieved by an order of refusal to grant or amend or renew
the license for sale, export / import of seed may within 60 days from the date of
Order appeal to the designated authority in the manner prescribed in the Order.

Miscellaneous

The licensing authority may on receipt of request in writing together with

Rs.10/- can amend the license of such dealer. Every seed dealer are expected to
maintain such books, accounts and records to this business in order and submit
monthly return of his business for the preceding months in Form 'D' to the licensing
authority by 5th day of every month

The Seeds Act, 1966

(Act No.54 of 1966) [29th December, 1966]

An Act to provide for regulating the quality of certain seeds for sale, and for
matters connected therewith.

It is enacted by Parliament in the Seventeenth Year of the Republic of India as

follows:

Short Title, Extent and Commencement

1. (1)This Act may be called the Seeds Act, 1966.

 (2)It extends to the whole of India.

(3) It shall come into force on such date as the Central Government may, by
notification in the Official Gazette, appoint, and different dates may be
appointed for different provisions of this Act, and for different States or for
different areas thereof.

Definitions

2. In this Act, unless the context otherwise requires,

1. "Agriculture" includes horticulture;

2. "Central Seed Laboratory" means the Central Seed Laboratory
established or declared as such under sub-section (1) of section 4;
3. "Certification agency" means the certification agency established under
Section 8 or recognised under Section 18;
4. "Committee" means the Central Seed Committee constituted under
sub-section (1) of Section 3;
5. "Container" means a box, bottle, casket, tin, barrel, case, receptacle,
sack, bag, wrapper or other thing in which any article or thing is
placed or packed;
6. "Export" means taking out of India to a place outside India;
7. "Import" means bringing into India from a place outside India;
8. "Kind" means one or more related species or sub-species of crop
plants each individually or collectively known by one common name
such as cabbage, maize, paddy and wheat;
9. "notified kind or variety" , in relation to any seed, means any kind or
variety thereof notified under Section 5;
10."Prescribed" means prescribed by rules made under this act;
11."seed" means any of the following classes of seeds used for sowing or
planting-

I. seeds of food crops including edible oil seeds and seeds of fruits
and vegetables;
 II. cotton seeds;
III. seeds of cattle fodder;

and includes seedlings, and tubers, bulbs, rhizomes, roots, cuttings,

all types of grafts and other vegetatively propagated material, of food
crops or cattle fodder;

12."Seed Analyst" means a Seed Analyst appointed under section 12;

13."Seed Inspector" means a Seed Inspector appointed under section 13;
14."State Government", in relation to a Union territory, means the
administrator thereof;
15."State Seed Laboratory", in relation to any State, means the State
Seed Laboratory established or declared as such under sub-section (2)
of section 4 for that State; and
16."Variety" means a sub-division of a kind identifiable by growth, yield,
plant, fruit, seed, or other characteristic.

Central Seed Committee

3. (1) The Central Government shall, as soon as may be after the commencement
of this Act, constitute a Committee called the Central Seed Committee to advise the
Central Government and the State Governments on matters arising out of the
administration of this Act and to carry out the other functions assigned to it by or
under this Act.

2. The Committee shall consist of the following members, namely:-

i. a Chairman to be nominated by the Central Government;

ii. eight persons to be nominated by the Central
Government to represent such interests that Government
thinks fit, of whom not less than two persons shall be
representatives of growers of seed;
iii. One person to be nominated by the Government of each
of the States.
(3) The members of the Committee shall, unless their seats become vacant earlier
by resignation, death or otherwise, be entitled to hold office for two years and shall
be eligible for renomination.

(4) The Committee may, subject to the previous approval of the Central
Government, make bye-laws fixing the quorum and regulating its own procedure
and the conduct of all business to be transacted by it.

(5) The Committee may appoint one or more sub-committees, consisting wholly of
members of the Committee or wholly of other persons or partly of members of the
Committee and partly of other persons, as it thinks fit, for the purpose of
discharging such of its functions as may be delegated to such sub-committee or
sub-committees by the Committee.

(6) The functions of the Committee or any sub-committee thereof may be exercised
notwithstanding any vacancy therein.

(7) The Central Government shall appoint a person to be the secretary of the
Committee and shall provide the Committee with such clerical and other staff as the
Central Government considers necessary.

Central Seed Certification Board

"8A. ,(1) The Central Government shall, by notification in the Official Gazette,
establish
a Central Seed Certification Board (hereinafter referred to as the Board) to advise
the Central
Government and the State Governments on all matters relating to certification and
to co-ordinate
the functioning of the agencies established under section 8.

(2) The Board shall consist of the folJowing members, namely:-

(i) a Chairman, to be nominated by the Central Government;

 lii) four members, to be nominated by the Central Government from out of the
persons employed by th~ State Governments as 'Directors 'of Agriculture;

(iii) three members, to be nominated by the Central Government from out of

the persons employed by the Agricultural Universities as Directors of
Research;

(iv) thirteen persons, to be nominated by the Central Government to represent

such interests as that Government thinks fit, of whom not less than four
persons shall be representatives of seed producers or tradesmen.

(3) A member of the Board shall, unless his s~at becomes vacant earlier by
resignation or otherwise - be entitled to hold officefor two years from the date of
his nomination:

Provided that a person nominated under clause (if) or clause (iii) of sub-section (2)
shall hold office only for so long as he holds the appointment by virtue of which his
nomination was made.

Central Seed Laboratory and State Seed Laboratory

4. (1) The Central Government may, by notification in the Official Gazette, establish
a Central Seed Laboratory or declare any seed laboratory as the Central Seed
Laboratory to carry out the functions entrusted to the Central Seed Laboratory by
or under this Act.

(2) The State Government may, by notification in the Official Gazette, establish one
or more State Seed Laboratories or declare any seed laboratory as a State Seed
Laboratory where analysis of seeds of any notified kind or variety shall be carried
out by Seed Analysts under this Act in the prescribed manner.
Power to notify kinds or varieties of seeds

5. If the Central Government, after consultation with the Committee, is of opinion

that it is necessary or expedient to regulate the quality of seed of any kind or
variety to be sold for purposes of agriculture, it may, by notification in the Official
Gazette, declare such kind or variety to be a notified kind or variety for the
purposes of this Act and different kinds or varieties may be notified for different
States or for different areas thereof.

Power to specify minimum limits of germination and purity, etc.

6. The Central Government may, after consultation with the Committee and by
notification in the Official Gazette, specify-

a. the minimum limits of germination and purity with respect to any seed
of any notified kind or variety;
b. the mark or label to indicate that such seed conforms to the minimum
limits of germination and purity specified under clause (a) and the
particulars which such mark or label may contain.

Regulation of sale of seeds of notified kinds or varieties

7. No person shall, himself or by any other person on his behalf, carry on the
business of selling, keeping for sale, offering to sell, bartering or otherwise
supplying any seed of any notified kind or variety, unless-

a. such seed is identifiable as to its kind or variety;

b. such seed conforms to the minimum limits of germination and purity
specified under clause (a) of section 6;
c. the container of such seed bears in the prescribed manner, the mark
or label containing the correct particulars thereof, specified under
clause (b) of section 6; and
d. he complies with such other requirements as may be prescribed.
Certification agency

8. The State Government or the Central Government in consultation with the State
Government may, by notification in the Official Gazette, establish a certification
agency for the State to carry out the functions entrusted to the certification agency
by or under this Act.

Grant of certificate by certification agency

9. (1) Any person selling, keeping for sale, offering to sell, bartering or otherwise
supplying any seed of any notified kind or variety may, if he desires to have such
seed certified by the certification agency, apply to the certification agency for the
grant of a certificate for the purpose.

(2) Every application under sub-section (1) shall be made in such form, shall
contain such particulars and shall be accompanied by such fees as may be
prescribed.

(3) On receipt of any such application for the grant of a certificate, the certification
agency may, after such enquiry as it thinks fit and after satisfying itself that the
seed to which the application relates conforms to the minimum limits of
germination and purity specified for that seed under clause (a) of section 6, grant a
certificate in such form and on such conditions as may be prescribed.

Revocation of certificate

10. If the certification agency is satisfied, either on a reference made to it in this

behalf or otherwise, that-

a. the certificate granted by it under section 9 has been obtained by

misrepresentation as to an essential fact; or
b. the holder of the certificate has, without reasonable cause, failed to
comply with the conditions subject to which the certificate has been
granted or has contravened any of the provisions of this Act or the
rules made thereunder;
then, without prejudice to any other penalty to which the holder of the certificate
may be liable under this Act, the certification agency may, after giving the holder of
the certificate an opportunity of showing cause, revoke the certificate.

Appeal

11. (1) Any person aggrieved by a decision of a certification agency under section 9
or section 10, may, within thirty days from the date on which the decision is
communicated to him and on payment of such fees as may be prescribed, prefer an
appeal to such authority as may be specified by the State Government in this
behalf:

Provided that the appellate authority may entertain an appeal after the expiry of
the said period of thirty days if it is satisfied that the appellate was prevented by
sufficient cause from filing the appeal in time.

(2) On receipt of an appeal under sub-section (1), the appellate authority shall,
after giving the appellant an opportunity of being heard, dispose of the appeal as
expeditiously as possible.

(3) Every order of the appellate authority under this section shall be final.

Seed Analysts

12. The State Government may, by notification in the Official Gazette, appoint such
persons as it thinks fit, having the prescribed qualifications, to be Seed Analysts
and define the areas within which they shall exercise jurisdiction.

Seed Inspectors

13. (1) The State Government may, by notification in the Official Gazette, appoint
such persons as it thinks fit, having the prescribed qualifications, to be Seed
Inspectors and define the areas within which they shall exercise jurisdiction.
(2) Every Seed Inspector shall be deemed to be a public servant within the
meaning of section 21 of the Indian Penal Code (45 of 1860) and shall be officially
subordinate to such authority as the State Government may specify in this behalf.

Powers of Seed Inspector

14. (1) The Seed Inspector may-

a. take samples of any seed of any notified kind or variety from-

i. any person selling such seed; or

ii. any person who is in the course of conveying, delivering or
preparing to deliver such seed to a purchaser or a consignee; or
iii. a purchaser or a consignee after delivery of such seed to him;

b. send such sample for analysis to the Seed Analyst for the area within
which such sample has been taken;
c. enter and search at all reasonable times, with such assistance, if any,
as he considers necessary, any place in which he has reason to believe
that an offence under this Act has been or is being committed and
order in writing the person in possession of any seed in respect of
which the offence has been or is being committed, not to dispose of
any stock of such seed for a specific period not exceeding thirty days
or, unless the alleged offence is such that the defect may be removed
by the possessor of the seed, seize the stock of such seed;
d. examine any record, register, document or any other material object
found in any place mentioned in clause (c) and seize the same if he
has reason to believe that it may furnish evidence of the commission
of an offence punishable under this Act; and
e. exercise such other powers as may be necessary for carrying out the
purposes of this Act or any rule made thereunder.

(2) Where any sample of any seed of any notified kind or variety is taken under
clause (a) of sub-section (1), its cost, calculated at the rate at which such seed is
usually sold to the public, shall be paid on demand to the person from whom it is
taken.

(3) The power conferred by this section includes power to break-open any container
in which any seed of any notified kind or variety may be contained or to break-open
the door of any premises where any such seed may be kept for sale:

Provided that the power to break-open the door shall be exercised only after the
owner or any other person in occupation of the premises, if he is present therein,
refuses to open the door on being called upon to do so.

(4) Where the Seed Inspector takes any action under clause (a) of sub-section (1),
he shall, as far as possible, call not less than two persons to be present at the time
when such action is taken and take their signatures on a memorandum to be
prepared in the prescribed form and manner.

(5) The provisions of the Code of Criminal Procedure, 1898 (5 of 1898), shall, so far
as may be, apply to any search or seizure under this section as they apply to any
search or seizure made under the authority of a warrant issued under section 98 of
the said Code.

Procedure to be followed by Seed Inspectors

15. (1) Whenever a Seed Inspector intends to take sample of any seed of any
notified kind or variety for analysis, he shall-

a. give notice in writing, then and there, of such intention to the person
from whom he intends to take sample;
b. except in special cases provided by rules made under this Act, take
three representative samples in the prescribed manner and mark and
seal or fasten up each sample in such manner as its nature permits.

(2) When samples of any seed of any notified kind or variety are taken under sub-
section (1), the Seed Inspector shall-
 a. deliver one sample to the person from whom it has been taken;
b. send in the prescribed manner another sample for analysis to the Seed
Analyst for the area within which such sample has been taken; and
c. retain the remaining sample in the prescribed manner for production in
case any legal proceedings are taken or for analysis by the Central
Seed Laboratory under sub-section (2) of section 16, as the case may
be.

(3) If the person from whom the samples have been taken refuses to accept one of
the samples, the Seed Inspector shall send intimation to the Seed Analyst of such
refusal and thereupon the Seed Analyst receiving the sample for analysis shall
divide it into two parts and shall seal or fasten up one of those parts and shall
cause it, either upon receipt of the sample or when he delivers his report, to be
delivered to the Seed Inspector who shall retain it for production in case legal
proceedings are taken.

(4) Where a Seed Inspector takes any action under clause (c) of sub-section (1) of
section 14:

a. he shall use all despatch in ascertaining whether or not the seed

contravenes any of the provisions of section 7 and if it is ascertained
that the seed does not so contravene, forthwith revoke the order
passed under the said clause or, as the case may be, take such action
as may be necessary for the return of the stock of the seed seized;
b. if he seizes the stock of the seed, he shall, as soon as may be, inform
a magistrate and take his orders as to the custody thereof;
c. without prejudice to the institution of any prosecution, if the alleged
offence is such that the defect may be removed by the possessor of
the seed, he shall, on being satisfied that the defect has been so
removed, forthwith revoke the order passed under the said clause.
(5) Where as Seed Inspector seizes any record, register, document or any other
material object under clause (d) of sub-section (1) of section 14, he shall, as soon
as may be, inform a magistrate and take his orders as to the custody thereof.

Report of Seed Analyst

16.(1) The Seed Analyst shall, as soon as may be after the receipt of the sample
under sub-section (2) of section 15, analyse the sample at the State Seed
Laboratory and deliver, in such form as may be prescribed, one copy of the report
of the result of the analysis to the Seed Inspector and another copy thereof to the
person from whom the sample has been taken.

(2) After the institution of a prosecution under this Act, the accused vendor or the
complainant may, on payment of the prescribed fee, make an application to the
court for sending any of the samples mentioned in clause (a) or clause (c) of sub-
section (2) of section 15 to the Central Seed Laboratory for its report and on receipt
of the application, the court shall first ascertain that the mark and the seal or
fastening as provided in clause (b) of sub-section (1) of section 15 are intact and
may then despatch the sample under its own seal to the Central Seed Laboratory
which shall thereupon send its report to the court in the prescribed form within one
month from the date of receipt of the sample, specifying the result of the analysis.

(3) The report sent by the Central Seed Laboratory under sub-section (2) shall
supersede the report given by the Seed Analyst under sub-section (1).

(4) Where the report sent by the Central Seed Laboratory under sub-section (2) is
produced in any proceedings under Section 19, it shall not be necessary in such
proceedings to produce any sample or part thereof taken for analysis.

Restriction on export and import of seeds of notified kinds or varieties

17. No person shall, for the purpose of sowing or planting by any person (including
himself), export or import or cause to be exported or imported any seed of any
notified kind or variety, unless-
 a. it conforms to the minimum limits of germination and purity specified for that
seed under clause (a) of section 6; and
b. its container bears, in the prescribed manner, the mark or label with the
correct particulars thereof specified for that seed under clause (b) of section
6.

Recognition of seed certification agencies of foreign countries

18. The Central Govt. may, on the recommendation of the Committee and by
notification in the Official Gazette, recognise any seed certification agency
established in any foreign country, for the purposes of this Act.

Penalty

19. If any person-

a. contravenes any provision of this Act or any rule made thereunder; or

b. prevents a Seed Inspector from taking sample under this Act;or
c. prevents a Seed Inspector from exercising any other power conferred
on him by or under this Act;

he shall, on conviction, be punishable-

i. for the first offence with fine which may extend to five hundred
rupees, and
ii. in the event of such person having been previously convicted of an
offence under this section, with imprisonment for a term which may
extend to six months, or with fine which may extend to one thousand
rupees, or with both.

Forfeiture of property

20. When any person has been convicted under this Act for the contravention of
any of the provisions of this Act or the rules made thereunder, the seed in respect
of which the contravention has been committed may be forfeited to the
Government.

Offences by companies

21. (1) Where an offence under this Act has been committed by a company, every
person who at the time the offence was committed was in charge of, and was
responsible to the company for the conduct of the business of the company, as well
as the company, shall be deemed to be guilty of the offence and shall be liable to
be proceeded against and punished accordingly:

Provided that nothing contained in this sub-section shall render any such person
liable to any punishment under this Act if he proves that the offence was committed
without his knowledge and that he exercised all due diligence to prevent the
commission of such offence.

(2) Notwithstanding anything contained in sub-section (1), where an offence under

this Act has been committed by a company and it is proved that the offence has
been committed with the consent or connivance of, or is attributable to any neglect
on the part of, any director, manager, secretary or other officer of the company,
such director, manager, secretary or other officer shall also be deemed to be guilty
of that offence and shall be liable to be proceeded against and punished
accordingly.

Explanation. – For the purpose of this section,-

a. "company" means any body corporate and includes a firm or other

association of individuals; and
b. "director", in relation to a firm, means a partner in the firm.

Protection of action taken in good faith

22. No suit, prosecution or other legal proceeding shall lie against the Government
or any officer of the Government for anything which is in good faith done or
intended to be done under this Act.
Power to give directions

23. The Central Government may give such directions to any State Government as
may appear to the Central Government to be necessary for carrying into execution
in the State any of the provisions of this Act or of any rule made there under.

Exemption

24. Nothing in this Act shall apply to any seed of any notified kind or variety grown
by a person and sold or delivered by him on his own premises direct to another
person for being used by that person for the purpose of sowing or planting.

Power to make rules

25. (1) The Central Government may, by notification in the Official Gazette, make
rules to carry out the purpose of this Act.

(2) In particular and without prejudice to the generality of the fore-going power,
such rules may provide, for-

a. the functions of the Committee and the travelling and daily allowances
payable to members of the Committee and members of any sub-
committee appointed under sub-section (5) of section 3;
b. the functions of the Central Seed Laboratory;
c. the functions of a certification agency;
d. the manner of marking or labeling the container of seed of any notified
kind or variety under clause (c) of Section 7 and under clause (b) of
section 17;
e. the requirements which may be complied with by a person carrying on
the business referred to in section 7;
f. the form of application for the grant of a certificate under section 9,
the particulars it may contain, the fees which should accompany it, the
form of the certificate and the conditions subject to which the
certificate may be granted;
 g. the form and manner in which and the fee on payment of which an
appeal may be preferred under section 11 and the procedure to be
followed by the appellate authority in disposing of the appeal;
h. the qualifications and duties of Seed Analysts and Seed Inspectors;
i. the manner in which samples may be taken by the Seed Inspector, the
procedure for sending such samples to the Seed Analyst or the Central
Seed Laboratory and the manner of analyzing such samples;
j. the form of report of the result of the analysis under sub-section (1) or
sub-section (2) of section 16 and the fees payable in respect of such
report under the said sub-section (2);
k. the records to be maintained by a person carrying on the business
referred to in section 7 and the particulars which such records shall
contain; and
l. any other matter which is to be or may be prescribed.

(3) Every rule made under this Act shall be laid as soon as may be after it is made,
before each House of Parliament while it is in session for a total period of thirty
days which may be comprised in one session or in two successive sessions, and if,
before the expiry of the session in which it is so laid or the session immediately
following, both Houses agree in making any modification in the rule or both Houses
agree that the rule should not be made, that rule shall, thereafter have effect only
in such modified form or be of no effect, as the case may be; so however, that any
such modification or annulment shall be without prejudice to the validity of
anything previously done under that rule.
 New seed policy {1988}

The Government of India evolved a New seed policy implemented from

October 1, 1988.

The policy laid special emphasis on

- Import of high quality of seeds

- A time bound programme to modernize plant quarantine facilities

- Effective implementation of procedures for quarantine /post entry

quarantine and

- Incentives to encourage the domestic industry

- Import of quality seeds.

1. Bulk import of seeds of coarse cereals, pulses and oil seeds may replace (or)
displace the local productions.

2. Transfer of technology may not be actual one, because due to bulk import of
seeds or import of technology, instead we can import the germplasm of
superior variety if any and could be developed locally to meet the demand
(i.e.,) incorporate the advantages of exotic variety to the local types(or) even
direct multiplication's after adaptive trials.

3. As we have superior varieties of international standard (e.g.) Maize,

Sorghum, Bajra, or even in oil seeds like groundnut etc., the bulk import is
not necessiated. Instead we need varieties suitable to agroclimatic zones
besides higher yields.

4. Import of flower seeds could be encouraged in order to earn foreign

exchange through export of flowers and it can be imported under (OGL) open
general license. But there is a fear of introduction of new pest and diseases
as they are coming without post entry quarantine checkup.
Strengthening of quarantine

Since, 1st October 1988 only bulk import of seeds was under taken without
any progress either in the strengthening of quarantine facilities.

Threat of pest and disease

Introduction of new pest and disease would pose a new problem due to bulk
import due to lack of post entry quarantine. To avoid this threat, the imported
seeds should be subjected to testing and it should be done by one person from
ICAR. Entry of exotic variety without proper field testing may change the disease
pattern if that particular strain is becoming susceptible to existing pathogens.

(e.g.) Kernal burnt - which was not noticed in the previous years is now a major
disease on wheat after the introduction of Kalyansona.

Genetic erosion

It is another danger, due to introduction of similar strains there is a danger

of genetic uniformity and eliminates local diversified strains which leads to problem
of non-availability of improved strains if there is any outbreak of disease.

Incentives to domestic seed industry

Indigenous seed production / seed industry will be affected because of the

entry of multi nation diseases. Since the policy is allowing indiscriminate bulk
imports through private sectors at the same time the import duty on seeds has
been reduced to 15 per cent. Import duty on advanced machines and equipment
used in seed production or processing has also been reduced and interest on post
shipment credit has also been slashed down to help importers. Income tax rebate
and deduction are available to the taxpaying units on the revenue expenditure or in
house research and development. Incentives are also being provided to seeds
located in backward areas and growth centers.

Application of biotechnology in agriculture

The multination would prevent the III world countries in enjoying the full
benefit of biotechnology. The bulk import of seed indicates accepting the monopoly
rights and the limitation of potential bio-technology in agriculture.
Advantages of biotechnology in agriculture

Certain plants fertilize themselves through nitrogen fixation, which is one of

the most promising areas of genetic engineering. Bacterium on the roots of plants
like groundnut, and soyabean take nitrogen from the air and transform it into
nitrates. Scientists are studying the possibility of transforming the genes
responsible for nitrogen fixation in wheat, rice, and maize (in which nitrogen
fixation doses not occur). They feel new strains can be grown without expensive
chemical fertilizers.

Plant variety protection (PVP) and the Indian agriculture (Protection of

Plant Variety & farmers right Bill,2001)

The Intellectual Property Rights (IPRs) are generally being applicable to

industrial property only. The patent laws of India did not provide for IPRs on living
organisms including plant varieties. The question of plant variety protection has
been brought in to sharp focus by Agreement on Trade Related Aspects of
Intellectual Property Rights (TRIPS) which is a part of Agreement establishing World
Trade Organization (WTO). India is a signatory to TRIPS agreement, which casts an
obligation on member countries to provide for a system of plant variety protection
either through patents or through a sui generis legislation framework or a
combination thereof. Under these agreements, a legislative framework for plant
variety protection has to be provided by member countries within a specified time
period. While this has lent some urgency to the question of plant variety protection,
the question of plant variety rights, even independent of the obligations posed by
TRIP’s agreement, has been under active consideration in view of our strong
agricultural research system. The plant breeding programmes have become more
sophisticated and high input based. The extent of investment by the State on public
research, in evolving varieties of commercial significance, is coming down with
responsibility of evolving new varieties of crops of commercial significance being left
to the private sector commercial organisations. There is also a move on the part of
the international research institutions, who at one time played a pioneering role in
plant breeding and genetic work, to focus on pure or strategic research. In the
wake of the global economic liberalization, it is only expected that agriculture is
accorded the status of an industry and given all incentives and impetus, normally
required for a fast developing, competitive business. To meet our food demands, as
well as to exploit our export potential in agricultural commodities, development and
use of new plant varieties having specific agronomic nutritive or market preference
characteristics are essential. New varieties may be bred for higher yields, greater
resistance to biotic and abiotic stresses, longer shelf life, better consumer
preference, higher industrial value, low input requirements and so on. To meet
these demands the variety improvement activities based on conventional as well as
biotechnological methods requires heavy investments both in scientific, man power
and economic terms. It is therefore, understandable that the fruits of such intensive
efforts will have to be protected from misuse, and also ensuring an appropriate
incentive (reward) to the breeder.

The following are the plant variety protection steps:

1. Historical developments of plant variety protection

For over 60 years, different forms of protection of new plant varieties

through the system of Plant Breeders' Right (PBR’s) have been in existence in
industralised countries which essentially means that the holder of the PBR can
prevent others from producing propagating material of the protected variety and /
or marketing the same. In order to coordinate inter country implementation of PBR
a " Union Internationale Pour La Protection Des Obtention Vegetables" (UPOV) was
established by International Convention for Protection of New Varieties of plants
(the UPOV convention), which was signed in Paris in 1961. The convention entered
into force in 1968. It was revised in 1972, 1978 and 1991. The 1978 Act entered
into force in 1981. The 1991 act has not yet entered into force.

The purpose of UPOV convention is to ensure that the member States of the
Union acknowledge the achievements of breeder of new plant varieties by making
available to them exclusive property rights, on the basis of a set of uniform and
clearly defined principles. To be eligible for protection, varieties have to be (I)
distinct from existing known varieties (ii) sufficiently homogenous (uniform) (iii)
stable and (iv) new in the sense that they must not have commercialised prior to
certain dates established by reference to the date of the application for protection.

2. Scope of protection of plant varieties under UPOV convention

Both the 1978 and 1991 conventions set out a minimum scope of protection
offer to member states for the possibility of taking national circumstances into
account in their legislation. Under 1978 Act, the minimum scope of the Plant
Breeders' right requires that the holders' authorization for the production for
purposes of commercial marketing, the offering for sale and marketing of
propagating material of protected variety.

The 1991 Act contains more detailed provision defining the acts concerning
propagating material in relation to which holders' authorization is required.
Exceptionally, but only where the holder has no reasonable opportunity to exercise
his right in relation to the propagating material, his authorization may be required
in relation to any specified acts done with harvested material of the variety.

3. Duration of plant breeder's rights

Like all intellectual property rights, plant breeder’s rights are granted for a
limited period of time (15-20 years) at the end of which varieties protected by them
pass into public domain. The rights are also subject to controls, in the public
interest, against any possible abuse.

4. Exemptions

It is also important to note that authorization of the holder of plant breeders'

rights is not required for the use of his variety for research purpose, including its
use in the breeding of further new varieties.

From the inception of UPOV in 1961, farmers have been allowed to use their
own harvested material of protected varieties for the next production cycle on their
own farms. On farm saving is still a practice in UPOV countries. The 1991 UPOV
convention contains an "Optional exception" which provides that it is unto the
national government to decide whether to permit farmers to use the seed of a PBR
protected variety for propagation purposes on their own holdings or not.
5. Sovereign rights on biological resources

Another major development, which has taken place along with India signing
the World Trade Agreement, is global Biodiversity Convention. India is a signatory
to this convention, which became operational on December 29, 1993. Among other
things it reaffirms that "the states have sovereign rights over their own biological
resources" and that states are responsible for conserving their biological diversity
and for using their biological resources in a sustainable manner".

6. Suggestions for a SUI system of plant variety protection

The proposal of 1991 UPOV convention which extents plant breeders rights to
the harvested material, is not appropriate for our country. The frame work for plant
variety protection has to be evolved in a manner that prevents situations where
repeated imports of improved varieties are not required so as to avoid dependence
on foreign sources of supply.

While, finalizing legislation on PVP, the government needs to strike a balance

between its commitment under WTO, growth of the seed sector and their interests
of the farmers, which through a difficult task, is not impossible to achieve.

7. Seed Industry Development in Post PVP period

In the post PVP period, we anticipate fairly high investment in seed research
from private sector and healthy competition with public sector in crop breeding and
seed production and distribution. However, public sector institutions will continue to
play major role in developing varieties of wheat rice, chick pea, pigeon pea,
mungbeans, urdbeans, groundnut, sugarcane, jute, potato and millets. The
continued improvement of these crops is most vital for our food security system.
The public sector will have to continue to develop varieties for rainfed, salt affected,
hilly and low lying flood prone regions. In export potential of food grains and other
agricultural commodities, breeding for quality of produce will have to be given
priority. We may also tailor varieties suited to the needs of the importing countries.
Since there is growing concern about the use of chemical pesticides in crop
production, the present research programme of breeding for resistance against the
pests and diseases will have to be strengthened further. Strategic research on
breeding for research against pests and diseases will be priority areas of
research of a public institution. We anticipate that the material generated from
these research programmes will be made available to the private sector.

Seed industry both in public and private sector is likely to develop at a fast
rate after the legislation on plant variety protection is enacted. The recent
experience shows that contribution of both public and private sector in Seed
industry development is complimentary. While private sector seed companies are
concentrating on hybrids of millets, oil seeds, cotton and vegetables, the public
sector seed corporations are engaged in seed production and distribution of self-
pollinated crops. It has also been observed that due to competition among the seed
companies, the farmers have been benefited not only in respect of stability in prices
of hybrid seeds but also better quality of seeds. It is expected that with
programmatic policy planning, faster growth of both public and private sector in
seed research and development will be ensured so that they can play important role
in improving the incomes and standards of living of our farmers.
 Lecture: 25
LIPASES AND PHOSPHOLIPASES
Lipids constitute one of the four major classes of compounds that are found in living
systems. The lipids of metabolic significance include triacylglycerol, phospholipids and
the products of lipid metabolism such as free fatty acids and glycerol.
Lipases
 Triacylglycerols or triglycerides undergo hydrolysis by lipases to form glycerol and
fatty acids, which undergo further oxidation generating energy.
 Lipases have been reported to be present in dry seeds of some species, e.g.
castor bean, Scots pine and Douglas fir but at a low level, or absent in others e.g.
apple.
 In most cases of seeds, following imbibitions, there appears to be a rise in lipase
activity but whether this increase is due to the de novo synthesis of the enzyme or
activation of existing lipases has not been determined.
 A decline in lipase activity is always associated with decline in acylglycerol
reserves.
 In castor bean, as in many other fat-storing seeds, free fatty acids do not
accumulate, but are rapidly degraded and converted to carbohydrate within the
endosperm.
 In other seeds such as germinating seeds of oil palm (Elaeis guineensis), a
different pattern of fat mobilization can be observed.
 The products of lipid catabolism are transported via specialized structures called
haustorium through its vascular system.
 Lipases are generally non-specific and can hydrolyse a wide variety of
triacylglycerols
 They initiate digestion by hydrolyzing triacylglycerols to form free fatty acids and
1,2-diacylglycerols.
 Complete hydrolysis of triacylglycerols produces glycerol and fatty acids.
 Lipase hydrolyses easily the terminal fatty acids to produce 2-monoacyl glycerol as
major

Phospholipases
 Phospholipases are the hydrolytic enzymes acting on phospholipids and
splitting into different products.
  There are four types of phospholipases known as phospholipase A1,

phospholipase A2 or B1, phospholipase C and phospholipase D.

Phospholipase A
 Phospholipase A is present in large amounts in snake venom and human
pancreas.
 It is also designated as phospholipase A1.
 It catalyses the hydrolysis of the fatty acids in the 2 or -position of the
phospholipids.
 Though this enzyme attacks on glycerophosphatides, it is fairly specific for
phosphatidyl choline (lecithin).
 The enzyme is relatively stable to heat (below pH 7.0).
 The product of the hydrolysis, a lysolecithin, (monoacylphosphoryl choline)
has a powerful hemolytic activity.

Phospholipase B (A2)
 It is otherwise termed as lysophospholipase and widely distributed in nature
often in association with phospholipase A.
 Phospholipase B is also designated as phospholipase A2 since it acts on
the lysolecithin (the product obtained from phospholipid by the action of
phospholipase A1).
  The action of this enzyme following that of phospholipase A yields
glycerophosphorylcholine as the final product.

Phospholipase C
 Phospholipase C is mostly found in the plant kingdom but it may also be
present in some animal tissues and venoms.
 It catalyses the liberation of a 1,2-diacylglycerol and phosphorylcholine from
phosphatidylcholine.
 Phosphorylcholine is also liberated from sphingomyelin by this enzyme.
Phospholipase D
 Phospholipase D, an enzyme described mainly in plants catalyses the
hydrolysis of choline from phosphatidylcholine leaving phosphatidic acid.
 Lecture: 26
OXIDATION OF FATTY ACIDS

Fatty acids obtained by hydrolysis of fats undergo different oxidative

pathways designated as alpha (), beta () and omega () pathways.
-oxidation
 -Oxidation of fatty acids has been found in certain tissues especially in brain
tissue of mammals and plant systems.
 It does not require CoA intermediates and no high-energy phosphates are
generated.
 This type of oxidation results in the removal of one carbon at a time from the
carboxyl end of the fatty acid.
 The physiological role of -oxidation in plants is not yet fully established but it has
been suggested that it may be involved in the degradation of long chain fatty acids
as observed in many animal tissues.
 -Oxidation is clearly the main source of the odd-carbon fatty acids and their
derivatives that occur in some plant lipids.
 In this process, sequential removal of one carbon at a time from free fatty acids of
chain length ranging from C13 to C18 occur.

-Oxidation
 -Oxidation is normally a very minor pathway brought about by hydroxylase
enzymes involving cytochrome P-450 in the endoplasmic reticulum.
 Fatty acids with oxygen function (alcoholic or carboxyl) at the methyl terminal end (
-end) are formed by -oxidation and frequently occur as constituents of cutin and
suberin.
 The requirements for the oxygenase-mediated conversion of a -methyl fatty acyl
CoA into a -hydroxymethyl fatty acyl CoA are molecular oxygen, reduced pyridine
nucleotide and a non-heme iron protein in higher plants.
-Oxidation of fatty acids
In 1904, Franz Knoop made a critical contribution to the elucidation of the
mechanism of fatty acid oxidation and demonstrated that most of the fatty acids are
degraded by oxidation at the -carbon.
 -Oxidation of fatty acids takes place in mitochondria.
 Fatty acids are activated before they enter into mitochondria for oxidation.
Activation of fatty acids
 Fatty acids are converted into active intermediate in a reaction with ATP and
coenzyme A.
 A thioester linkage between the carboxyl group of a fatty acid and the sulfhydryl
group of coenzyme A is formed with the hydrolysis of ATP.
 This activation reaction takes place on the outer mitochondrial membrane
catalysed by acyl CoA synthetase.
 Several acyl CoA synthetases each specific for fatty acids of different chain length
are present in the membrane of mitochondria.
Penetration of long chain fatty acids into mitochondria
 Long chain acyl-CoA molecules do not readily get into the inner mitochondrial
membrane and are carried across the inner membrane by conjugating with carnitine (
-hydroxy -trimethyl ammonium butyrate), a zwitterionic compound formed from lysine.
 Activation of lower fatty acids and their oxidation within the mitochondria occur
independently of carnitine, but long-chain acyl CoA will become oxidised unless they
form acylcarnitines.
 The acyl CoA combines with carnitine in the presence of carnitine acyltransferase
I, which is bound to the outer mitochondrial membrane.
 Acylcarnitine is transported in, coupled with the transport out of one molecule of
carnitine.
 The acylcarnitine then reacts with coenzyme A catalyzed by carnitine palmitoyl
transferase II, located on the inside of the inner membrane.
 Acyl CoA is reformed in the mitochondrial matrix and carnitine is liberated.
Oxidation
A saturated acyl CoA is oxidised by a recurring sequence of four reactions

 Oxidation in presence of FAD, hydration, oxidation in presence of NAD+, and

thiolysis by CoASH.
 In -oxidation, 2 carbons are cleaved at a time from acyl CoA molecules, starting
from the carboxyl end.
 The chain is broken between the -and -carbon atoms.
 The two-carbon units formed are acetyl CoA.
i) The first reaction in -oxidation of acyl CoA is the formation of trans 2- enoyl CoA or
, -unsaturated acyl CoA in presence of acyl-CoA dehydrogenase and the coenzyme,
FAD.
ii) The next step is the hydration of the double bond between C-2 and C-3 by enoyl
CoA hydratase with the formation of -hydroxy acyl CoA.
iii) In the third step, the -hydroxy acyl CoA is dehydrogenated in the presence of

-hydroxy acyl CoA dehydrogenase and NAD+ forming -ketoacyl CoA.

iv) In the last step of -oxidation, -ketoacyl CoA reacts with coenzyme A in the
presence of the enzyme, thiolase.
The products of this reaction are acetyl CoA and an acyl CoA containing two carbons
less than the original acyl CoA molecule that underwent oxidation.
By the above steps of -oxidation fatty acids are completely degraded to
acetyl CoA units. The acetyl CoA formed from fatty acids can be oxidised to carbon
dioxide and water via citric acid cycle.
Energetics of  oxidation
The energetics or the energy conserved in terms of ATP by oxidation of a
molecule of palmitic acid is given below:
 Palmitic acid (16 carbons) undergoes -oxidation forming eight molecules of acetyl
CoA by undergoing seven -oxidation spirals.
 When one cycle of -oxidation takes place, one molecule of FADH2, one molecule

of NADH and one molecule of acetyl CoA are produced.

 Electrons from these reducing equivalents (FADH2 and NADH) are transported

through the respiratory chain in mitochondria with simultaneous regeneration of

high-energy phosphate bonds.
 Mitochondrial oxidation of FADH2 eventually results in the net formation of about

1.5 ATP.
 Likewise, oxidation of electrons from NADH yields 2.5 molecules of ATP. Hence, a
total of four ATP molecules are formed per cycle and ten molecules of ATP are
formed through Krebs’s cycle from each molecule of acetyl CoA.
 8 Acetyl CoA through TCA cycle yield (8x10) = 80 ATP
7 -oxidation spiral reactions yield (7x4) = 28 ATP
--------------
Total 108 ATP
-------------
ATP utilized in the initial step = 2 ATP
Hence, complete oxidation of palmitic acid yields 106 ATP.

Oxidation of monounsaturated fatty acids

 Oxidation of monounsaturated fatty acids follows many of the reactions of saturated
fatty acids except the requirement of two additional enzymes, an isomerase and a
novel reductase.
 Reactions of monounsaturated fatty acid are explained by considering the
oxidation of a C-16 unsaturated fatty acid, palmitoleic acid, having a single double bond
between C-9 and C-10 .
 Palmitoleic acid is activated and transported across the inner mitochondrial
membrane in the same way as saturated fatty acids.
 Palmitoleoyl CoA undergoes three cycles of degradation as in  oxidation. But the

cis 3 decenoyl CoA formed after the third cycle does not serve as a substrate for acyl
CoA dehydrogenase.
 The presence of a double bond between C-3 and C-4 prevents the formation of
another double bond between C-2 and C-3.
 An isomerase converts the cis double bond into a trans double bond and shifts the
position of double bond between C-2 and C-3.
 The subsequent or follow up reactions are those of the  oxidation pathway in which

the trans 2 decenoyl CoA is a regular substrate.

Oxidation of polyunsaturated fatty acids

The oxidation of a polyunsaturated fatty acid, linoleic acid, with cis-9 and cis-12
double bonds, is considered.

 The cis-3 double bond formed after three rounds of -oxidation is converted into a
trans double bond by the isomerase.
 This permits one more round of -oxidation.
 The acyl CoA produced by four rounds of -oxidation of linoleic acid contains a cis-
4 double bond, which undergoes dehydrogenation by acyl CoA dehydrogenase

yielding trans 2, cis-4 dienoyl intermediate.

 This intermediate is not a substrate for the next enzyme in the -oxidation pathway.

 This intermediate is converted into a trans 3 enoyl CoA to the trans 2 form, an
intermediate generally found in -oxidation pathway and results in complete oxidation of
the fatty acid.
 Lecture: 27
Fatty acid and triacyl glycerol biosynthesis
Biosynthesis of fatty acids
 It was thought that fatty acid biosynthesis occurred by reversal of the β-oxidation
pathway.
 On the contrary, it occurs by a separate pathway that differs from β-oxidation in
several ways.
i. Synthesis takes place in the cytosol, in contrast with degradation or oxidation, which
occurs in the mitochondrial matrix.
ii. Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl group of an
acyl carrier protein (ACP) whereas intermediates in fatty acid breakdown are bonded
to coenzyme A.
iii. The enzymes of fatty acid synthesis in animals are joined in a single polypeptide
chain called fatty acid synthase. In contrast, the degradative enzymes do not seem to
be associated. Plants employ separate enzymes to carry out the biosynthetic reactions.
iv. The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid

oxidation are NAD+ and FAD.

Pathway for the movement of acetyl-CoA units from within the
mitochondrion to the cytoplasm for use in lipid and cholesterol biosynthesis.
The following seven steps are involved in fatty acid biosynthesis.

Formation of malonyl CoA

The synthesis of malonyl CoA from acetyl CoA is catalyzed by acetyl CoA
carboxylase having biotin as prosthetic group. The production of malonyl CoA is the
initial and controlling step in fatty acid synthesis. In this reaction, bicarbonate serves
as a source of CO2. The reaction takes place in two steps, namely carboxylation of

biotin involving ATP and transfer of the carboxyl group to acetyl CoA resulting in
malonyl CoA.

 Acetyl CoA carboxylase plays a key role in regulating fatty acid metabolism and
the same is inactivated by phosphorylation.
 ii) Formation acetyl and malonyl ACP
Acetyl transacylase and malonyl transacylase catalyze the formation of
acetyl ACP and malonyl ACP respectively. Acetyl transacylase can transfer acetyl as
well acyl groups whereas malonyl transacylase is highly specific.
Acetyl
transacylase
Acetyl CoA + ACP ----------------→ acetyl - ACP + COASH

Malonyl
transacylase
Malonyl CoA + ACP ---------------→ Malonyl - ACP + COASH

iii) Formation of acetoacetyl - ACP (β -ketoacyl ACP)

 Acetyl ACP condenses with malonyl ACP to form acetoacetyl ACP.
 Carbondioxide is eliminated from malonyl ACP.
iv) Reduction of β-ketoacyl ACP to β-hydroxyl acyl ACP.
 The β- keto group in acetoacetyl ACP is reduced by NADPH- dependent β-ketoacyl
reductase.
v) Formation of unsaturated acyl ACP.
The β-hydroxyl group combines with the hydrogen atom attached to the γ-carbon
and a water molecule is removed to form α, β-unsaturated acyl ACP.
vi) Formation of Acyl ACP
 The unsaturated acyl ACP is converted in the next step to a saturated acyl ACP by the
enzyme α,β-unsaturated acyl ACP reductase using NADPH as the coenzyme.
 The resultant product contains two carbon atoms more than the starting material.
 Addition of subsequent acetyl units through malonyl ACP leads to the formation of 16-
carbon palmitate.
Stoichiometry of fatty acid synthesis
The stoichiometry of the synthesis of palmitate is given below:

Acetyl CoA + 7 malonyl CoA + 14 NADPH + 20 H+ --------------→

Palmitate + 7 CO2 + 14 NADP+ + 8 CoASH + 6 H2O

 The equation for the synthesis of the malonyl CoA used in the above reaction is
7 Acetyl CoA + 7 CO2 + 7 ATP --------→ 7 malonyl CoA + 7ADP

+ 7 Pi + 14 H+
The overall stoichiometry for the synthesis of palmitate is

8 Acetyl CoA + 7 ATP + 14 NADPH + 6H+ -------→ Palmitate +

14 NADP + 8 CoASH + 6 H2O + 7 ADP + 7 Pi

Fatty acid synthesis and degradation are reciprocally regulated so that both are
not simultaneously active.
Elongation of fatty acids or synthesis of long chain fatty acids
 Elongation by the fatty acid synthase complex stops upon formation of palmitate (16
C).
 Further elongation and the formation of double bonds are carried out by other enzyme
systems.
 The major product of fatty acid biosynthesis is the 16-carbon fatty acid, palmitate.
 Additional enzymes are required to synthesise longer chain fatty acids.
 Chain elongation reactions occur both in mitochondria and in microsomes.
Microsomes are small membrane-enclosed vesicles derived from the endoplasmic
reticulum of cells.
 Mitochondria and microsomes carry out chain elongation by adding two-carbon units to
fatty acids.
 The microsomal system has great physiological significance in that it provides the long
chain fatty acids (18-24C) required for the myelination of nerve cells in animal
system.
 Chain elongation occurs by a cycle of condensation, reduction, dehydration
followed by another reduction that parallels cytosolic fatty acid biosynthesis.
 The more active elongation system adds two carbons to palmitoyl-CoA to make it
steroyl CoA.
 The mechanism of elongation is identical with that known in the synthesis of palmitate
except the enzyme systems and the acyl carrier protein.
Biosynthesis of unsaturated fatty acids
 Palmitate and stearate serve as precursors of the two most common monounsaturated

fatty acids, palmitoleate, 16:1, (Δ 9) and oleate, 18:1 (Δ 9) respectively.

 Each of these fatty acids has a single double bond between C-9 and C-10.
 The double bond is introduced into the fatty acid chain by an oxidative reaction
catalysed by fatty acyl-CoA desaturase, which is NADPH-dependent enzyme.

 The unsaturated fatty acids, linoleate, 18:2 (Δ9,12) and α-linolenate, 18:3 (Δ9,12,15)
cannot be synthesised by mammals; but plants can synthesise both.
 The desaturases responsible for synthesis of both the above fatty acids are present in
endoplasmic reticulum of plants.
 The plant desaturases oxidise phosphatidylcholine-bound oleate and produce
polyunsaturated fatty acids and do not directly add double bonds to the fatty acids.
 Once ingested, the linoleate are readily converted to other polyunsaturated fatty acids
like γ-linolenate, arachidonic acid etc. in animals and human beings.

Biosynthesis of triacylglycerols
  Triacylglycerols are not synthesised by reversal of lipolysis.
 They are synthesisd by a different mechanism in which both glycerol and
fatty acids are activated by ATP before they are incorporated into
acylglycerols.

i) Activation of glycerol
 Glycerol kinase catalyses the activation of glycerol to glycerol
3-phosphate.
 If glycerol kinase is found in low quantity or absent, glycerol 3-phosphate
will be formed from dihydroxyacetone phosphate obtained from glycolysis
and this reaction is catalysed by the enzyme glycerol 3-phosphate
dehydrogenase.
ii) Activation of fatty acids
 Fatty acids are activated to acyl CoA by the enzyme acyl CoA
synthetase, utilizing ATP and CoASH.
 Two molecules of acyl CoA combine with glycerol 3-phosphate to form
1,2-diacylglycerol phosphate.
 Formation of 1,2-diacyl glycerol phosphate takes place in two stages,
catalysed by glycerol 3-phosphate acyl transferase and then by
1-acyl glycerol 3- phosphate acyl transferase.
 The phosphate group is removed from 1,2-diacyl glycerol phosphate by
phosphatidate phosphatase to form 1,2-diacyl glycerol.
 Triacylglycerols are finally formed by esterification of one or more
molecule of acyl CoA with the diacylglycerol.
Alternative pathway for triacylglycerol biosynthesis
 In this pathway, dihydroxyacetone phosphate from glycolysis is reduced
by NADPH, acylated and converted to lysophosphatidate.
This pathway accounts for less than 10% of total triacylglycerol synthesis.
 Lecture: 28
TRANSAMINATION, DEAMINATION AND DECARBOXYLATION

 Protein metabolism is a key physiological process in all forms of life.

 Proteins are converted to amino acids and then catabolised.
 The complete hydrolysis of a polypeptide requires mixture of peptidases because
individual peptidases do not cleave all peptide bonds.
 Both exopeptidases and endopeptidases are required for complete conversion
of protein to amino acids.
Amino acid metabolism
 The amino acids not only function as energy metabolites but also used as
precursors of many physiologically important compounds such as heme, bioactive
amines, small peptides, nucleotides and nucleotide coenzymes.
 In normal human beings about 90% of the energy requirement is met by oxidation
of carbohydrates and fats. The remaining 10% comes from oxidation of the carbon
skeleton of amino acids.
 Since the 20 common protein amino acids are distinctive in terms of their carbon
skeletons, amino acids require unique degradative pathway.
 The degradation of the carbon skeletons of 20 amino acids converges to just
seven metabolic intermediates namely.
i. Pyruvate
ii. Acetyl CoA
iii. Acetoacetyl CoA
iv. -Ketoglutarate
v. Succinyl CoA
vi. Fumarate
vii. Oxaloacetate
 Pyruvate, -ketoglutarate, succinyl CoA, fumarate and oxaloacetate can serve as
precursors for glucose synthesis through gluconeogenesis.Amino acids giving rise
to these intermediates are termed as glucogenic.
 Those amino acids degraded to yield acetyl CoA or acetoacetate are termed
ketogenic since these compounds are used to synthesize ketone bodies.
 Some amino acids are both glucogenic and ketogenic (For example,
phenylalanine, tyrosine, tryptophan and threonine.
Catabolism of amino acids
The important reaction commonly employed in the breakdown of an amino
acid is always the removal of its -amino group. The product ammonia is excreted
after conversion to urea or other products and the carbon skeleton is degraded to
CO2 releasing energy. The important reaction involved in the deamination of amino

acids is
i. Transamination
ii. Oxidative deamination
iii. Non oxidative deamination
Transamination
 Most amino acids are deaminated by transamination reaction catalysed by
aminotransferases or transaminases.
 The -amino group present in an amino acid is transferred to an -keto acid to
yield a new amino acid and the -keto acid of the original amino acid.
 The predominant amino group acceptor is -keto glutarate. Glutamate's amino
group is then transferred to oxaloacetate in a second transamination reaction yielding
aspartate.
Glutamate + oxaloacetate ---------- -ketoglutarate + aspartate
pyridoxal phosphate

 Pyridoxal phosphate, the coenzyme of pyridoxine (vitamin B6) plays an important

role in these reactions.
 Amino transferase reactions occur in two stages.
o Pyridoxal phosphate is covalently attached to the amino transferases via a
Schiff's base linkage formed between the aldehyde group of pyridoxal phosphate
and the epsilon amino group of lysine residue of the enzyme. Pyridoxal phosphate
is converted to pyridoxamine phosphate.
o In the second stage, the amino group attached to pyridoxamine phosphate is
transferred to a different keto acid to yield a new amino acid and releases pyridoxal
phosphate
Oxidative deamination
 Transamination does not result in net deamination, since one amino acid is
replaced by another amino acid.
 The function of transamination is to funnel the amino nitrogen into one or a
few amino acids.
 For glutamate to play a role in the net conversion of amino groups to ammonia, a
mechanism for glutamate deamination is needed so that -ketoglutarate can be
regenerated for further transamination.
 The generation is accomplished by the oxidative deamination of glutamate by
glutamate dehydrogenase.
 Glutamate is oxidatively deaminated in the mitochondrion by glutamate

dehydrogenase. NAD+ or NADP+ functions as the coenzyme.

 Oxidation is thought to occur with the transfer of a hydride ion from glutamate's 

carbon to NAD(P)+ to form -iminoglutarate, which is then hydrolysed to 

-ketoglutarate and ammonia.
 The ammonia produced is then converted to urea in mammals
Two non-specific amino acid oxidases namely, L-amino acid and D-amino acid
oxidases catalyse the oxidation of L and D-amino acids utilizing FAD as their
coenzymes.
Amino acid + FAD + H2O ------------ -Keto acid + NH3 + FADH2

Non-oxidative deamination
 Amino acids such as serine and histidine are deaminated non-oxidatively
The other reactions involved in the catabolism of amino acids are decarboxylation,
transulfuration, desulfuration, dehydration etc.

Decarboxylation
 The decarboxylation process is important since the products of decarboxylation
reactions give rise to physiologically active amines.
 The enzymes, amino acid decarboxylases are pyridoxal phosphate-
dependent enzymes.
 Pyridoxal phosphate forms a Schiff's base with the amino acid so as to stabilise
the -carbanion formed by the cleavage of bond between carboxyl and -carbon
atom.
 The physiologically active amines epinephrine, nor-epinephrine, dopamine,
serotonin, -amino butyrate and histamine are formed through decarboxylation of
the corresponding precursor amino acids.
 Lecture 29
Ammonia assimilating enzymes, GDH, GS and GOGAT

Biosynthesis of ammonia
Ammonia is produced from the catabolic pathways of amino acids. Some
of the ammonia that is generated is recycled and used in a variety of
biosynthetic processes. The excess ammonia is excreted directly or converted
to uric acid or urea for excretion depending on the organism.

 Many aquatic organisms simply excrete ammonia as NH4+ into the

surrounding medium.
 Most terrestrial vertebrates convert the ammonia into urea (humans, other
mammals and adult amphibians) or uric acid (birds, reptiles).
 In plants ammonia is also derived from nitrate absorbed from the soil.
Nitrate is first converted to nitrite and then to ammonia.
 The major route for the assimilation of ammonia into organic nitrogen
is the result of the collaborative activity of glutamine synthetase (GS)
and glutamate synthase (also called as Glutamine oxoglutarate
aminotransferase or GOGAT).
 Ammonia is fixed with the help of glutamine synthetase which catalyses
the joining of ammonia to glutamic acid.
 The enzyme GOGAT is dependent either on NADPH (bacteria, roots and
developing seeds but not in leaves) or ferredoxin (leaves, legume nodules,
roots and legume seeds) to transfer the amino nitrogen from glutamine to
oxoglutarate.
 The net reaction is the production of one molecule of glutamate from one

molecule of oxoglutarate and one molecule of NH4+

 An additional enzyme glutamate dehydrogenase(GDH) is widely

distributed but is not significantly involved in ammonia assimilation because
of high Km value.
 All the 20 protein amino acids are synthesised by plants and
microorganisms.
 Human beings are able to synthesise only 10 amino acids, which are
called as non-essential amino acids.
 The synthesis of non essential amino acids require only one or two step
reactions whereas the synthesis of essential amino acids require multi
step reactions.
 The synthesis of 20 amino acids is grouped into families where the
precursor compounds are same for one family .
 Lecture: 31
SECONDARY METABOLITES - OCCURRENCE, CLASSIFICATION AND FUNCTIONS OF
PHENOLICS

Secondary metabolites
 Organic compounds produced by the plants which have no direct role in the growth and
development are called as secondary metabolites.
 There are about 100,000 secondary compounds that are produced by the plants and the
structures of more than 15000 alkaloids, 30000 terpenes, several thousand phenyl
propanoids, 1000 flavoniods, 500 quinones, 700 polyacetylenes and 800 non-protein amino
acids have already been characterised.
 These secondary compounds produced by plants are grouped into five major groups.
1. Phenolics
2. Terpenoids
3. Alkaloids
4. Special nitrogen metabolites
5. Cuticular compounds
Phenolics
 Phenolics are a group of compounds characterized by at least one aromatic ring bearing
one or more hydroxyl groups.
 Most of the thousands of phenolics known to date are of plant origin.
 These phenolic compounds are biosynthesised through shikimate pathway.
Shikimate pathway
 Shikimate pathway is an important pathway in plants through which many secondary plant
products are synthesised.
 The key starting materials are phosphoenolpyruvate (PEP) and erythrose 4P derived
from glycolysis and pentose phosphate pathways, respectively.
 These two compounds condense to produce a six carbon cyclic compound with one carbon
(COOH) side chain namely shikimate.
 Then shikimate is phosphorylated and condensed with another molecule of PEP to produce
a cyclic compound containing a three carbon and one carbon side chains.
 This is finally converted to aromatic amino acids phenylalanine and tyrosine.
 These amino acids are deaminated followed by hydroxylation at different carbon atoms in
the aromatic ring to form cinnamic acid derivatives.
 These cinnamic acid derivatives are utilised for the synthesis of different phenolic
compounds.
Functions of phenolics
 Phenolics are of great importance as cell wall components.
 They form part of cell wall structures such as lignins, cutins and suberins, which
provide mechanical support and function as barriers against microbial attack.
 The flavonoids and anthocyanins contribute to flower and fruit colours. This is important
for attracting insects and animals to the plant for pollination and seed dispersal.
 Phenolics also play a defensive role in plants by protecting against predators.
 Simple phenolic acids, polyphenolics like tannins and phenolic resins at the plant surface
are effective feeding deterrents.
 Phenolics are accumulated as post-infectional low molecular compounds called
phytoalexins as a result of microbial attack.
 Among the phenolic phytoalexins, hydroxycoumarins and hydroxycinnamate conjugates
contribute to disease resistance mechanism in plants.
 Phenolic compounds also produce allelopathic effect. A well known compound from
Juglans species is juglone which is highly toxic for a wide range of plants. It occurs in the
plant as a non-toxic glucoside and is made active by deglucosylation and oxidation after
leaching from the leaves into the soil.
 Phenolics also function as signal molecules in the interaction between nitrogen fixing
bacteria and leguminous plants.
 These plants exude flavonoids which act selectively in Rhizobia as inducers of
nodulation gene transcription.
 Salicylic acid is strongly implicated as a signal molecule which induces active defense
responses in several plant species against many types of pathogens.
 Recently, it has been shown that phenolic compounds function as effective antioxidants.
 Polyphenolics are important in foodstuffs, wines and herbal teas because of their
astringent taste.
 Plants rich in polyphenolics were used as tanning agents in leather industries.
 Phenolic pigments (anthocyanins, flavones etc) of fruits are most widespread food colours
occurring in fruit juices, wines and jams.
 Anthocyanins have considerable potential in the food industry as safe and effective food
additives.