2

SYLLABUS
THEORY:
 Introduction and application of biotechnology in agriculture and
forestry.

 Various techniques of plant tissue culture such as cell culture,

micro propagation, meristem culture, anther culture, embryo
culture, soma clonal variation, protoplast isolation and fusion,
cryo-preservation and their applications.

 Restriction enzymes and introduction to various blotting

techniques such as Southern, Northern and Western blotting.

 Genetic Engineering and recombinant DNA technology

 Vectors for gene transfer.

 Direct and Indirect methods of gene cloning and transgenic plants.

RFLP, PCR, construction of genomic library and DNA cloning.

REFERENCE BOOKS:

1. Introduction to Plant Biotechnology by H S Chawla, Oxford &

IBH Publishing Co. Pvt. Limited, New Delhi, ( Price Rs. 240/-)
2. Plant Biotechnology – Laboratory manual of Plant Biotechnology
by H S Chawla ( Price = Rs. 160/-)
3. Basic Biotechnology, Bu’lock, J D and Kristiansen, B, ( 1987),
Academic Press, London.
4. Biotechnology by Singh, B D ( 1998), Kalyani Publishers,
Ludhiana (Price: Rs. 185/-)
5. Plant Tissue Culture: Theory and Practice by Bhojwani, S S and
Razdan, M K (1983), Elsevier, Amsterdam
6. Elements of Biotechnology, Gupta P K
 3

INDEX

____________________________________
SR. NO. NAME OF CHAPTER PAGE NO.
___________________________________
1. INTRODUCTION TO BIOTECHNOLOGY

2. TISSUE CULTURE : THE BASICS

3. TISSUE CULTURE : THE TECHNOLOGY

4. TISSUE CULTURE FOR MICROPROPAGATION

5. TISSUE CULTURE FOR CROP IMPROVEMENT

6. GERMPLASM CONSERVATION AND CRYOPRESERVATION

BIOTECHNOLOGY

7. INTRODUCTION / HISTORY OF BIOTECHNOLOGY

8
8. BASIC TECHNIQUES IN MOLECULAR BIOLOGY

9.. FUNDAMENTALS OF GENE CLONING

10. LIBRARY CONSTRUCTION

11. MOLECULAR MARKERS

* * * * * *
 4

FIGURE INDEX

SR. Description CH Chapter Pag

1 APT e
ER No.
No.
1 Shoot tip culture 4 Microprpagation 20
2 Single Node culture 4 Microprpagation 21
3 Axillary bud method for 4 Microprpagation 21
micropropagation
4 Plant regeneration via organogenesis 4 Microprpagation 22

5 Clonal Propagation 4 Microprpagation 23

6 Anther culture & Microspore culture 5 TC for crop 26
Improvement
7 Protoplast isolation 5 TC for crop 33
Improvement
8 Protoplast fusion 5 TC for crop 35
Improvement
9 Cryopreservation of shoot tip 6 Cryopreservation 39
10 Electrophoresis 8 Basic technique in 45
Molecular biology
11 Southern Blot 8 Basic technique in 46
Molecular biology
12 Hybridization in Southern blotting 8 Basic technique in 47
Molecular biology
13 Comparison of Southern/ Northern/ 8 Basic technique in 48
Western blot Molecular biology
14 Gene cloning 9 Gene cloning 51
15 Restriction digestion 9 Gene cloning 55
16 Comparison of advantages and 9 Gene cloning 57
disadvantages of vectors
17 Gene gun- Particle delivery system 9 Gene cloning 58
18 Agrobacterium mediated gene 9 Gene cloning 59
transfer
19 Genomic library construction 10 Library construction 61
20 Processing of RNA in Eukaryote 10 Library construction 62
21 cDNA library construction 10 Library construction 63
22 Comparison of genomic and cDNA 10 Library construction 64
library
23 PCR protocol 11 Molecular markers 66
24 RFLP 11 Molecular markers 69
 5

CHAPTER - 1
GENERAL INTRODUCTION TO
PLANT BIOTECHNOLOGY
Biotechnology can be simply defined as “A very broad field, which encompasses
manipulations of living organisms themselves or the products that they make or the
processes they carry out." They combine the use of many branches of life science such
microbiology, biochemistry, plant breeding, genetics, tissue culture and many more. It involves
the basic knowledge of Genetics.

Biotechnology, based on molecular biology and genetic engineering, offers manifold

beneficial applications in the areas of agriculture, food and dairy, environmental control,
veterinary and aqua culture apart from industry, health-care and energy production.
Biotechnology has opened new vistas in agriculture.

ADVANTAGES OF BIOTECHNOLOGY:
1. The new techniques are relatively fast,
2. They are highly specific
3. They are energy and resource efficient.

The new economic policy of globalisation in India is going to give tremendous boost to
agricultural production using Biotechnological tools providing necessary competitive edge in
crop productivity with custom-built quality.

MAIN FEATURES OF BIOTECHNOLOGY:

1. Consist two basic techniques: tissue culture and genetic engineering.
2. Bypass sexual process in development of new crop cultivars.
3. Overcomes the barriers of cross incompatibility in distant crosses.
4. It helps in development of transgenic plants.
5. Rapid method of crop improvement. Tetraploidy plants can be obtained in one step
through protoplast fusion.

Bio-technology refers to the application of various biological organisms for

processes for mass production of useful substances/products for industry, medicine
and agriculture. e.g Penicillium and Streptomyces fungi used for mass production of
antibiotics such as penicillin and streptomycin.

BRANCHES OF BIOTECHNOLOGY

1. Plant Biotechnology: Combination of tissue culture and genetic engineering techniques.

Development of transgenic plants which are resistant to biotic and abiotic stress.
 6

Development of improved high yielding varieties, Use of embryo rescue technique,

synthetic seeds, cryopreservation etc.
2. Animal Biotechnology: To develop transgenic animals for increased milk and meat
production and vaccine production.
3. Medical Biotechnology: For large-scale production of various drugs and hormones. e.g.
vaccines for chicken pox, polio, hepatitis etc.
4. Industrial Biotechnology: Commercial production of various organic substances such
as acetic acid, citric acid- Penicillin-Streptomycin, enzymes, proteins etc.
5. Environmental Biotechnology: Detoxification of Industrial waste and effluents,
treatment of sewage water, microbes used as pollution-sensors.

There are many bottlenecks to conventional breeding as it is random crossing, selection is

tedious, and you have to wait for long time for expression of phenotypes and more time to evolve
new varieties. Biotechnology provides more precision and speed to plant breeding.
Biotechnology has proven useful for following genetic markers in plant breeding. For instance
plant varieties can be crossed by conventional means, and, by analyzing a few cells of the newly
sprouted plant, one can predict some of the expected properties of the progeny, by looking at the
presence or absence of certain genes.

Transgenic Vs Conventional Breeding

Sr. Particulars Transgenic Conventional

No. Breeding Breeding
1 Sexual process Bypassed Involved

2 Methods Tissue culture and Hybridization

Genetic Engineering
3 Transfer of genes from Possible Not possible
microbes / animals
4 Frequency of desired plants Very low Adequate

5 Technical skill required Very High Moderate

6 Expenditure Very High Low

7 Time required fro new variety 3 – 4 years 12 – 15 years

8 Improvement by polygenic Not possible Possible

traits
9 Facility Well equipped lab Field facilities

Before we learn more about plant biotechnology, let us know about some definitions.
 7

[Link]: Biotechnology, in the simplest and broadest sense, is a series of

enabling technologies which involve the manipulation of living organisms or their sub-
cellular components to provide useful products, processes, or services.
[Link] MANIPULATION: Identify a gene from another species which controls a trait of
interest and / or modify an existing gene (create a new allele)
[Link] : A piece of DNA that controls the expression of a trait.
4. RECOMBINANT DNA TECHNOLOGY: The technology used in the isolation or
synthesis and joining together of unlike pieces of DNA so that biologically active
recombinant DNA molecules can be manufactured in the lab. These recombinant DNA
molecules can then be introduced into bacteria, yeasts, or other cells where they can
replicate and function (code for protein synthesis). Also known as genetic engineering.
[Link] DNA: New combinations/arrangements of DNA constructed in the
laboratory by inserting a foreign DNA.
[Link] ENZYMES: They are molecular scissors which cut the DNA at a
specific site; isolated from bacteria where they are used as Bacterial defense against
viruses.
[Link] VECTOR: Genetic element into which genes can be recombined and
replicated.
[Link] : An organism that has a new genetically engineered DNA sequence
found in every one of its cells. Genetically engineered organisms are transgenic. These
two terms are used interchangeably.
9. CHROMOSOME: The structural component in the cell that carries the genes; composed
of DNA and proteins.
10. CLONAL PROPAGATION: Asexual reproduction of plants that are considered to be
physiologically and/or genetically uniform and to have originated from a single individual
or explant.
11. CLONE: (1) A population of recombinant DNA molecules all carrying the same
inserted sequence. (2) A population of cells derived from a single cell by mitoses. (3) A
population of plants derived from a single individual by vegetative propagation. (4) To use
recombinant DNA techniques to insert a particular gene or other DNA sequence into a
vector molecule. (5) To propagate a clone.
12. IN VITRO PROPAGATION: Propagation of plants in a controlled, artificial
environment using plastic or glass vessels, aseptic techniques, and a defined growth
medium.
13. TOTIPOTENCY: The ability of undifferentiated plant tissues to differentiate into
functional plants when cultured in vitro.
14. HYBRIDIZATION: (1) The pairing of complementary DNA or RNA strands to give
stable DNA-DNA or DNA-RNA duplexes. The efficiency of hybridization is a tes of
sequence homology. (2) Production of offspring, or hybrids, from genetically dissimilar
parents. The process can be used to produce hybrid plants (by cross-breeding two different
varieties) or hybridomas (hybrid cells formed by fusing tow unlike cells, used in producing
monoclonal antibodies).

The study of plant biotechnology is very important as it leads to the development of

many novel plants having many new traits which can attribute disease or insect resistance or
better quality or system to fight against biotic and abiotic stress.
 8

The study of plant biotechnology is divided in two major areas :

A) The use of tissue culture techniques for crop improvement and micropropagation
B) The use of gene manipulation technique using biotechnology tools.

Following techniques could be used for plant biotechnology

TISSUE CULTURE TECHNIQUES:

FOR MICROPROPAGATION
A) Cell culture and Callus culture
B) Meristem culture and Shoot tip culture
C) Node culture
D) Axillary bud culture
E) Organ culture

FOR CROP IMPROVEMENT

1) Anther and microspore culture
2) Somaclonal variation
3) Embryo / ovule culture
4) Somatic hybridization ( Protoplast isolation and fusion )

GENE MANIPULATION TECHNIQUES:

1. Gene identification and isolation
2. Gene cloning
3. Gene transfer
4. Expression
For gene transfer there are several techniques:

1) Gene transfer with vectors:

1. Through vectors such as plasmid, viruses, bacteriophage etc.

2) Gene transfer and plant transformation without vectors:

1. Micro injection.
2. Particle bombardment.
3. Direct up take.

We will discuss all these techniques in subsequent chapters.

 9

CHAPTER - 2

T
TIISSSSU
UEEC
CUUL
LTTU
URRE
E :: T
THHE
EBBA
ASSIIC
CSS
HISTORY OF TISSUE CULTURE:

Haberlandt ( 1902 ) : First attempt of Plant tissue culture.

Hannig ( 1904 ) : Embryo cultures of selected crucifers.

Robbins ( 1922 ) : In vitro culture of root tips.

Laibach ( 1925 ): Use of embryo culture in interspecific crosses of Linum

White P R ( 1934 ): Successful culture of tomato roots ( He maintained them

upto 1968)

Gautheret ( 1939 ) :Continuous growing cultures of carrot

Gautheret, Nobecourt, White ( 1939 ) : Founders of development of TC. Used dividing –

meristem cells ( undifferentiated cells ). Successful establishment of continuously grown
callus culture.

Skoog ( 1944 ): In vitro adventitious shoot formation in tobacco.

Muir [Link]. ( 1954 ) : First plant from single cell

Miller ( 1955 ) : Discovery that kinetin is essential for cell division.

Maheshwari and Rangaswami ( 1958 ): Regeneration of somatic embryo in vitro from

the nucleus of Citrus ovules.

Gautheret ( 1959 ) : Publication of first handbook on plant tissue culture

Kanta ( 1960 ) : First successful test tube fertilization in Papaver

Murashige and Skoog (1962 ): Nutrition medium for plant tissue culture.

Guha and Maheshwari ( 1964 ): Production of first haploid plant from pollen grains of
Datura.
Power ( 1970 ): First achievement of protoplast fusion

Takebe ( 1971 ): Regeneration of first plant from protoplast.

 10

Plant tissue culture techniques are also employed in agriculture to a great deal. These
methods have many applications in agriculture: It is used for Generation of variability,
development of haploids, embryo rescue for interspecific hybridization and somatic
hybridization. It is used for selection for disease resistance, selection for salinity and metal
toxicity resistance and drought resistance, Micro-propagation and preservation of germplasm.
Many companies also have commercialized these methods and plant tissue culture raised plants
are available in the market for the farmers.

Definitions:
Plant tissue culture : Growth of living plant tissues in a suitable culture medium (in vitro) is
known as plant tissue culture.
Culture medium : It is a nutrient medium, which contains all essentials micro and macro
nutrients, sugar, vitamins, hormones etc. which allows the growth of the cells to grow invitro.
The pH of medium should be 5.5.
Cell autonomy: Any cell, which is isolated, separated from plant and which continue to grow is
called cell autonomy. It was given by Schwan in 1839.
Differentiation: It is the development process by which cells are formed then the tissues and
then organs are formed. Finally the whole plant is produced. This process is known as
differentiation.
De-differentiation: The conversion of differentiated tissue, back to undifferentiated form is
known as dedifferentiation ( Callus formation from root/leaf/stem/ tissue).
Re-differentiation: When differentiated tissue is converted into undifferentiated form and this is
reconverted to differentiated form is known as re-differentiation.
Organogenesis: The process of initiation and development of a structure that shows natural
organ form and/or function.
Plant regeneration: Laboratory technique for forming a new plant from a clump of plant cells.
The process of recovering plantlets from in vitro cultures through organogenic or embryogenic
development, either by adventitious or de novo origination.
Undifferentiated cells: Strictly, the cells that are determined but not yet expressing cell
specialization. They may also be referred as pro-determined cells.
Subculture: The process, by which tissue or explant is first subdivided, then transferred into
fresh culture medium.
Somatic cell hybridization: The fusion of plant protoplasts derived from somatic cells that
differ genetically.
Somaclonal variation: Variation that occurs and accumulates in cultures of cells and tissues; it
may be either genetic or epigenetic in basis. When the rate of variation exceeds the normal
mutation rate of that species or genotype in vivo, the variation may be said to arise as a result of
growth or manipulation in vitro, and may be the result of growth in unorganized states.
Protoplast: A membrane-bound cell formed after the cell wall is removed from a microbial or
plant cell by the action of pectinase and cellulase.
Protoplast fusion: A technique for producing somatic hybrids between two sources of
protoplasts by treating with agents such as polyethylene glycol (PEG) and Ca=2 ions to induce
 11

cell membrane fusion. The two protoplast sources may be from the same or highly divergent
species.
Meristem: A localized group of actively dividing cells, from which permanent tissue systems
(root, shoot, leaf, flower) are derived. The main categories of meristems are (i) apical meristems,
in root and shoot tips; (ii) lateral meristems (vascular and cork cambiums); and (iii) intercalary
meristems, in the nodal region and at the base of certain leaves.
In vitro propagation: Propagation of plants in a controlled, artificial environment using plastic
or glass vessels, aseptic techniques, and a defined growth medium.
Haploid: (1) Characterizing a cell in which there is half the usual number of chromosomes, or
only one chromosome set. Sex or gametic cells are haploid. (2) Such a cell or organism.
Friability: The tendency for cultured plants cells to separate easily from one another.
Explant: Tissue taken from its original site (plant or seed) and transferred to an artificial
medium to establish a cell tissue culture system.
Embryogenesis: The process of initiation and development of embryos or embryo-like
structures from somatic cells (more specifically: somatic embryogenesis).
Electroporation: Application of an electrical current across a membrane (as in a protoplast),
inducing: (i) temporary pores and permitting uptake of molecules, organelles, etc., or (ii) fusion
of neighboring membranes.
Cybrid: The variable cell resulting from the fusion of a cytoplast with a whole cell, thus
creating a cytoplasmic hybrid.
Batch culture: A suspension culture in which cells grow in a finite volume of nutrient medium.
Cultures commonly exhibit five growth phases: lag, exponential, linear, deceleration, and
stationary phases.
Androgenesis: Development of plants from the male gametophyte by the culture of anthers or
microspores.
Morphogenesis: The formation of complex structures / organs is called morphogenesis. It is the
next level of differentiation whereby increasingly complex structures such as roots, stem, leaves
are formed.
TYPES OF PLANT TISSUE CULTURE :

Cell culture: Regeneration of a plant from a single cell in nutrient medium. Its purpose is to
obtain genetic variants at cellular level and production of secondary metabolites.

Seed culture : It is very successful in Orchids. This is because seeds are small and sometime do
not germinate. They have limited food reserve. With this method, the in vitro germination
is quick.
Callus culture: Growth of unorganized mass of cells as tissue is called callus. Differentiated
tissues such as roots, stem, leaf, flower etc. is used as starting material for callus induction.
Purpose: Creation of genetic variants, virus free plants, as a source of suspension and
protoplast culture and production of secondary metabolites.
Suspension culture : culture of cells or cell aggregates in moving liquid in form of
suspension.
 12

Protoplast culture: Cells without cell wall is known as protoplast. Purpose: Somatic
hybridization, creation of cybrids, transplantation of direct up take of nucleic acid / DNA
and creation of genetic variants.
Organ culture: Culture of excised piece of organ in vitro. Different names are given depending
on source ( Meristem, shoot tip, root culture, ovule culture, anther, endosperm culture,
nucleus culture etc.) Purpose: Regeneration from an organ, which has separate identity
such as anther, ovule, embryo and bud, adventitious root formation and production of
mutants.
Meristem culture: Culture of apical dome or meristem with / without one leaf primordial is
known as meristem culture. Purpose: Recovery of virus free stocks, vegetative
propagation, germplasm conservation in vitro. Regeneration of a plant from tissue of an
actively dividing organ like stem tip, root tip or vegetative bud ( Shoot tip culture, single
nod culture etc.)
Anther and pollen culture : Production of haploids to obtain homozygous plants, as a
starting point for mutant induction, creation of all male plants, to breed at lower ploidy
level.

Ovule / Embryo / Ovary culture: It is explained by Hannig as sterile isolation and growth
of immature embryo. There are two techniques: Mature and immature embryo. When the
immature embryos are used, it is called embryo rescue technique. Purpose: Prevention of
embryo abortion in wide crosses –Interspecies and intergeneric and distant hybrids is
successful (Overcoming incompability to prevent early flower fall), achievement of test
tube fertilization, multiplication of plants where seed germination is a problem (seed
dormancy), production of haploids, shortening breeding cycle and in vitro clonal
propagation.
Somatic embryo culture: Development of somatic embryo from cell, callus or explants, for
clonal propagation and pathogen free plants.

BASIC REQUIREMENTS OF TISSUE CULTURE

1. ASEPTIC CONDITIONS: Tissue Culture laboratory should have aseptic condition. It
should be well sterilized against pathogens. Ex-plant and glassware should be properly
sterilized.
2. CONTROL OF TEMPERATURE: Air conditioning of Tissue Culture laboratory is
essential. Temperature between 18-25 0 C. is maintained.
3. PROPER CULTURE MEDIUM : Culture media developed by Murashige and Skoog
(1962) and Gamborg et. al. (1968) are used with some modifications.
4. SUB CULTURING: Transfer of tissue or callus from old culture media to fresh culture
media is called sub-culturing. This is essential to maintain good health of the callus or
tissues.

IMPORTANT STEPS OF TISSUE CULTURE TECHNIQUE:

1. Isolation of tissues: Tissues for regeneration can be isolated with the help of sterilized /
blade from any plant part. Isolated tissues are sterilized to grow on culture medium. Tissues
should be insect and disease free.
 13

2. callus formation and Regeneration: Tissues proliferate on the culture medium and
give rise to mass of unorganized cell called callus. Callus is of two types: friable and
compact. Friable callus can be easily manipulated for suspension culture.
3. Embryogenesis: Formation of somatic embryo from callus.
4. Organogenesis: The process of differentiation of shoots and roots from the somatic embryo.
Complete plant develops directly from the somatic bud. Plants obtained are transferred after some
time to pot culture from the culture medium. Soil should be sterilized before transplantation.

The Protocol of tissue culture:

1. The selection of Explant material
2. Sterilization of Explant
3. The selection of specific media and its composition
4. Sterilization of media
5. Inoculation of explant material
6. Incubation ( Culture growth )
7. Culture multiplication ( Establishment of culture )
8. Sub-culturing
9. Transfer of Complete plant to soil / pot
10. Acclimatization to the natural environment

APPLICATIONS OF TISSUE CULTURE

1. Generation of variability :
a. Gametoclonal variation -Anther or ovule culture
b. Somaclonal variation -Callus cultures of somatic explant e.g Meristem
c. Protoclonal variation -Regenerated from callus cultures of protoplast .
2. Development of haploids: Wheat , Rice
3. Embryo rescue: For interspecific hybridization for distant hybridization.
4. Somatic hybridization: Fusion of cells takes place through protoplast. Isolation of
protoplast is done from two different species and fused. Later selection of hybrid cells is
done. Culturing of hybrid cells will lead to regeneration of plants from hybrid tissues.
5. Selection for disease resistance: Pathogen is included in culture medium. Resistant
genotypes do not support growth of pathogenic fungi. It is done for potato wilt and root rot
resistance. Virus resistance has been achieved in tomato, tobacco and alfalfa.
6. Selection for salinity and metal-toxicity: Only resistant or tolerant cells will
survive.
7. Selection for drought resistance: High proline content is an indicator of water
stress tolerance. e.g. tomato and sorghum.
8. Micropropagation: Biggest advantage to industry for rapid mass multiplication of
plants. Regeneration of meristematic cells (undifferentiated cells) is also possible. With this
method, multiplication of superior hybrids on a large scale is done.
9. Preservation of Germplasm : Cells or tissues can be preserved in liquid nitrogen
for long term storage. Cells are treated with dimethyl sulphoxide to protect them against
freezing injury.
****
 14

CHAPTER - 3

TISSUE CULTURE : THE TECHNOLOGY

Let us try to learn the technology of tissue culture step-by-step in details.
In the tissue culture techniques, the selection and processing of various tissues play very
important role for the success.

The Protocol of Tissue culture:

 The selection of Explant material
 Sterilization of Explant
 The selection of specific media and its composition
 Sterilization of media
 Inoculation of explant material
 Incubation ( Culture growth )
 Culture multiplication ( Establishment of culture )
 Sub-culturing
 Transfer of Complete plant to soil / pot
 Acclamatization to the natural environment

SELECTION AND PROCESSING OF TISSUES:

CELL CULTURE: It contributes to understanding inter-relationship and complementary

influence on multi-cellular organisms. Haberlandt was the pioneer worker to give the importance
of selection of tissues.
Advantages of individual cells vs. intact organ / whole plants
1. We can study the pathways of metabolism
2. It permits quick administration and withdrawal of diverse chemicals/substances, which
will make targeted mutant selection very easy.
3. It also helps in detecting cytogenetical and metabolic variation depending on stage of
growth and cultural conditions ( spatial heterogeneity ).
4. Cell line can be used to produce high yielding cultures and cultures with superior
agronomic traits.
ISOLATION OF SINGLE CELLS:
The single cells can be isolated either from plant organs or from cultured tissues.

FROM PLANT ORGANS: Single cells can be obtained from leaf tissues because of
homogenous population of cells. This gives large scale and defined cell cultures. There are two
methods of isolation of single cells : Mechanical Method and Enzymatic Method

Mechanical method : The isolation is done from mesophyll cells from mature leaves of
monocot, dicot and grasses which are active in photosynthesis and respiration. The procedure
 15

involves mild maceration of 10 gm of leaves in 40 ml of grinding medium (20 µM Sucrose, 10

µM MgCl2, 20 µM Tris- HCl buffer, pH 7.8 ) with pestle and mortar. Then it is passed through 2
layers of muslin cloth and later washed by centrifugation at low speed in same medium. Large-
scale production of individual cells is also possible with this method.

Enzymatic Method: Takebe (1968) treated tobacco leaf tissues with pectinase enzyme and
obtained large number of metabolically active cells. Potassium dextran sulphate improved yield
of cells. This method is convenient and yield is more and damage is minimum. Osmotic
protection is provided to the cells while macerozyme degrade the middle lamella and cell wall of
parenchymatous tissues. This method is not suitable to cereals ( Hordeum vulgare, Zea mays)
because mesophyll cells are elongated with a no. of inter-locking constriction which prevent their
isolation.

FROM CULTURED TISSUES: Freshly cut pieces of surface sterilized plant organs are placed
in a solidified nutrient medium consisting of auxins and cytokinins to initiate cultures. Explant
on such medium exhibit callusing at the cut ends; gradually extends to entire surface of tissue.
Callus is separated from an explant and transferred to fresh medium. Repeated subculture on
agar medium improves viability of callus, which is a pre-requisite for raising a fine cell
suspension in a liquid medium. The pieces of callus are transferred on a continuously agitated
liquid medium on a shaker. The movement of culture medium exerts mild pressure on small
pieces of tissue breaking them in to free cells, which favors gaseous exchange and uniform
distribution of cells.
GROWTH AND SUB-CULTURE OF SUSPENSION CULTURE:

There are four stages of growth of the cells.

1. Lag phase: It is an adjustment phase in which the cells adjust to the new environment
and starts getting the nutrients.
2. Log phase: It is the phase of the growth and multiplication of cells. It is also called
exponential phase as the number of cells increase in log numbers.
3. Stationary Phase: The cell density becomes static because of exhaustion of nutrients,
depletion of oxygen and increase in the concentration of toxic compounds.
 16

Timing of subculture is very important and it is primarily dependent on

a) Initial cell density
b) Duration of lag phase
c) Growth rate of cell line.
The normal incubation time of stock culture is 21 – 28 days. Subculture time in actively growing
cells is 6 – 9 days.

FACTORS AFFECTING TISSUE CULTURE: There are several factors, which affect the
success of tissue culture. They can be broadly divided in chemical factors and physical factors.
They are summarized below:

(1) MEDIA: One of the most important factor governing the growth and morphogenesis of
plant in the culture is the composition of the medium.
(a) Mineral salts: The MS medium is high in nitrate, potassium and ammonium, while B5
medium ( for soybean cell culture) is rich in potassium and nitrate levels. Gamborg
medium is rich in mineral salts. Iron is essential for growth and morphogenesis.
( b) Carbon and Energy source: Sucrose is the carbon source in most of the medium.
However, glucose and fructose can substitute sucrose. Sugar induces flower production.
(c) Vitamins: Pyridoxine, nicotinic acid and biotin are required for growth while riboflavin
inhibits the growth. Similarly, folic acid, PABA and thiamine increase growth. Ascorbic
acid only increases the growth when levels of thiamine were suboptimal.
(d) Auxins and Cytokinins: They are growth regulators. Both are added to the medium to
obtain morphogenesis although no universal ration of auxin: cytokinins exist. It will vary
from crop to crop.
(e) Other organic compounds: Green plants should be able to synthesize all amino acids
required for protein synthesis. Adenine sulfate can enhance growth and shoot formation.
Meso-inositol is involved in the synthesis of phospholipids, cell wall pectin and
cytoplasmic membrane systems.
(f) pH: The pH of the medium should be optimum. ( 5.5 for MS medium). The pH affects the
uptake of nutrients, ions and solidification of medium. Higher the pH, harder will be the
medium. Low pH keeps it in liquid form. The pH drops during autoclaving to a tune of
0.3, so slightly higher pH is adjusted before autoclaving.
(2) THE EXPLANT: An explant is a piece of tissue or organ, which is removed from
the plant for the purpose of culturing. There are three factors which should be
considered for the selection of explant 1) The size of explant, 2) the source of
explant and 3) physiological age of explant.
( a ) Explant size: As a general rule, the size of the explant should be bit big because small
explant have low survival rate. The no. of shoots produced from the explant from a shoot-tip
explant may also be influenced by the size of explant. In Chrysanthemum, the small shoot tip
( 0.2 – 0.5 mm) and shoot meristem ( 0.1 -0.2 mm) produced only a single shoot while large
explant ( 0.5 -1.55 mm) produced multiple shoots.
( b ) Source of Explant: The explant should be healthy and free from disease. It can be
from leaves, bulb scales, petals, anthers etc.
 17

( c ) Physiological age: The physiological age of explant also influence the type and extent
of morphogenesis. Young juvenile tissues have higher degree of morphogenic competence as
compared to older tissues.
( d ) Genotype: The genotype of the plant chosen for propagation may influence the
response in culture. Within a species, some genotype appears to propagate easily while other
fails to respond. Genes control the differences in hormonal and nutritional requirement for
differentiation.
( e ) Season: Morphogenetic ability seems to be limited to periods of vegetative growth.
Seasonal variation in morphogenic response has been found in Lilium if the explant is
obtained in spring or summer. Seasonal variation is also seen in production of haploid plant
from anthers in Tobacco.
( C ) LIGHT: Light plays an important role in inducing organogenesis. It may be divided in
a) Photo period b) wave length and c) Light intensity.
a) PHOTO PERIOD: Photo period affects the bud formation. Dark period is
necessary to induce root and embryo formation. Sunny days and longer period
decrease bud formation.
b) WAVE LENGTH: Specific wavelength is to be chosen for specific period
for the success of tissue culture.
c) LIGHT INTENSITY: It has been observed that optimum light intensity for
plant tissue culture may differ for each type of plant. Optimum light for
cultured tissue in Stage- I and Stage – II is around 1000 (1K) lux while for
Stage – III, it is between 3000 – 10,000 lux. Higher light intensities improved
survival of transferred plants.
( D ) TEMPERATURE: The optimum temperature range is 24 – 28 0 C for growth of organ
depending on species. Plant tissue culture is affected by alternating day / night temperature
cycle.

( E) GAS PHASE: Variability in tissue culture response may be, in part, due to influence of
gas phase of culture. Ethylene has a wide range of stimulatory and inhibitory effects. The
action of ethylene is antagonistic to morphogenesis while positive effects on callus
formation. CO2 also decrease the growth rate.
Ethanol and acetylene: Inhibits photosynthesis, organogenesis, embryogenesis
Ethylene: Inhibits morphogenesis but promote callus formation
Acetylaldehyde: Inhibits morphogenesis but promote callus formation
Carbon dioxide: Increase in photosynthesis
Oxygen: Increased respiration
Use of cotton/ wool plug is good for easy exchange of gas. Non-absorbent cotton is a
good material.
( F ) POLARIITY: Inhibits morphogenesis but promote callus formation Polarity effects
in plants and tissue culture is a well documented phenomenon. Orientation of explant on medium
influences the organogenesis. Polarity effects may be caused by chemical gradient in the plant or
anatomical difference between the explant tissues.
( G ) SUBCULTURE: The extended sub-culturing of callus or cell suspension leads to
decrease in morphogenetic potential and variability from culture. Variation may result from
segregation chimeras, change in chromosome number, their structures, genetic mutation and
adaptation.
****
 18

CHAPTER - 4

TISSUE CULTURE FOR MICROPROPAGATION

The applications of tissue culture techniques are enormous. The commercial applications
for micropropagation have attracted the industry. While the use of tissue culture techniques for
crop improvement has been popular with the scientists.
The commercial use of micro-propagation can yield 1,000,000 (one million) plants in six
month time. This has attracted the commercial firms to go for massive use of tissue culture.

There are three major stages of micropropagation:

1) Stage – I : Initiation of sterile culture of Explant ( Establishment )
2) Stage – II : Proliferation / multiplication of shoots from Explant ( Proliferation )
3) Stage – III : Transfer of shoots to rooting medium followed by planting into soil
( Rooting and Hardening )

Following events occur during the plant from the explant material. Plant regeneration can
be either from tissue sections (Adventitious origin ) or from cell or callus ( de novo origin ).

Selection of Explant

Surface sterilization

Establishment in growth medium

Transfer to proliferation medium

Shoot or Embryoid formation

Transfer of shoots to rooting medium

Transfer of shoots to sterilized soil

Acclimatization

Transfer to natural Soil environment

 19

ADVANTAGES OF MIROPROPAGATION:
A) Only small amount of tissue is needed.
B) It helps in bulking up of rapidly new cultivars that would take, otherwise, many years.
C) Speedy International Exchange of material. This helps in maintaining the sterility of
material and reduces the quarantine period.
D) In vitro stocks are proliferated any time of the year. You can have year-round nursery
for ornamental / fruit / tree spp.
E) Production of disease free plants ( Virus free ).
F) Seed production on large scale ( Axillary bud proliferation)
G) Germplasm storage is also done. The meristems are genetically stable so it is an
excellent material for production of virus free plants.
H) Artificial seeds can be produced from immature embryos. The embryos are
encapsulated with hydrogel such as alginate, seed gums of guar or tamarind, plant
exudates of Acacia, or microbial products like dextran and xanthans. They are analog
to true seed, produced by somatic embryogenesis. (Advantages: Well protected, rigid
enough for handling)

PROBLEMS ASSOCIATED WITH MICROPROPAGATION:

 It has extensive requirement of sophisticated instruments and trained man power.
 Although, precautions are taken, still, chances of contamination. There are high
losses in very short time.
 Genetic stability of the culture may be pronounced. The plants produced by shoot
tip culture are stable but adventitious shoot and callus cultures are unstable.
 During repeated cycles in vitro shoot multiplication, cultures show water-soaked,
almost translucent leaves. This declines in the rate of growth and eventually they
may die. This phenomenon is known as VITRIFICATION.

TISSUE CULTURE TECHNIQUES:

FOR MICROPROPAGATION
F) Cell culture and Callus culture
G) Meristem culture and Shoot tip culture
H) Node culture
I) Axillary bud culture
J) Organ culture

FOR CROP IMPROVEMENT

5) Anther and microspore culture
6) Somaclonal variation
7) Embryo / ovule culture
8) Somatic hybridization ( Protoplast isolation and fusion )

(1) CELL CULTURE: Regeneration of a plant from a single cell in nutrient medium. Its
purpose is to obtain genetic variants at cellular level and production of secondary
metabolites.
 20

TYPES OF CELL CULTURE:

There are two types of cell cultures: Batch culture and Continuous culture

BATCH CULTURE:
Take 20 -75 ml of medium in 100 -250 ml flask, inoculate the cell culture and incubate
the flasks. There are many problems associated with the batch cultures.

Problems:
1. The growth declines after 3-4 cell generation signaling the stationary phase.
2. No steady growth pattern. There is constant change in the pattern of the cell growth.
3. Uniform cell types are not available. You may find all types of cells.
4. Enzyme concentration and metabolites may not be fixed.
5. Constant sub culturing is required to get uniform cells.
6. Maintenance of sterility is a problem.

CONTINUOUS CULTURE: The advantage of this method is that large-scale cultures are
grown under steady state. The fresh medium is added and cells mass and used medium is drained
at a constant interval.
There are two types of continuous cultures:
a) Closed type: Here the addition of medium is balanced with the outflow.
b) Open type: Equal addition and withdrawal is done. This allows indefinite maintenance of
cultures at a constant and sub-maximal growth rate. Two types of open continuous
culture are available: Chemostat and Turbidostat
Chemostat: Cell growth is maintained by constant inflow of fresh medium consisting of N, P of
glucose at a concentration so as to be growth limiting. All other constituents are present at higher
concentrations so that desired growth rate is maintained by controlling addition / removal of
medium / cells.
Turbidostat: The input of the medium is intermittent, as it is mainly required to control the rise
in the turbidity due to cell growth. The turbidity is pre-selected on the biomass density in culture.

ADVANTAGES OF CONTINUOUS CULTURES:

1. Ease of maintaining sterility over a long period of time.
2. Less detrimental effects during mechanical failures.
3. Fair degree of automation is possible.
4. There is Versatility with regard to growth conditions such as temperature, aeration,
stirring speed, illumination, nutrient and growth regulator levels etc.

APPLICATIONS OF CELL CULTURE:

1. Embryogenic cell suspension offers the possibility of large-scale clonal propagation.

2. Embryo can be made dormant. Somatic embryos from cell suspensions can be used
for long-term storage in germplasm banks.
3. Embryogenic cell suspension cultures are good for theoretical / practical applications.
Selection scheme can be employed for soma clonal variation against biotic / abiotic
stress and genetic variability can be created.
 21

4. Production of important chemicals: Somatic embryo of celery has the same flavor as
that of natural mature plant. Similarly somatic embryo of cacao has the same lipids as
cacao butter.
5. Production of secondary metabolites: The secondary metabolites such as the
chemicals of fragrance, flavor, natural sweeteners, anti-microbials, pharmaceuticals
etc. can be produced on large scale using this method. The major functions of
secondary metabolites include warding off predators; attract the pollinators and
disease resistance.
6. The protoplast isolation and fusion can be done for somatic hybridization and
creating genetic variability.

PRODUCTION OF SECONDARY METABOLITES:

The secondary metabolites are very important products of plants. Although, they are
produced in micro-quantity in the plant, considering their industrial and therapeutic importance
large scale production is essential. They can also be produced in bulk by cell suspension cultures.
There are many advantages of cell cultured secondary metabolites over the plant produced one.

ADVANTAGES OF SECONDARY METABOLITE PRODUCTION

CELL CULTURED Vs NATURAL PLANT

1. No environmental effects including pest, disease and seasonal effects.

2. They are produced more accurately to market demand under controlled conditions.
3. Any cell of a plant could be multiplied to yield specific metabolites.
4. It can give consistent quality
5. Their production can be automated, regulated and their productivity can be improved.
6. Less labor and production cost is involved.
7. This method is best for long life-cycle plant like Papaver, which is a source of Thebaine.
8. New route of synthesis could be worked out from mutants. Even novel products can be
synthesized which are generally not produced by the plants.

LIMITATIONS OF CELL PRODUCED SECONDARY METABOLITES:

A) If natural product is produced in differentiated cell in a particular organ, then there is

very little production in cell line cultures.
B) The production of secondary metabolites is low as compared to whole plant.
C) Cell cultures are genetically unstable. The mutation leads to reduced productions.
D) Vigorous stirring is required to avoid clumping which leads to damage to cells.
E) It is costly because it requires high sugar concentrations in the medium.
F) If there is contamination / infection in the medium, production is low.
G) The economics is to be worked out (Cost / kg in Rs ).

GROWTH MEASUREMENTS IN CELL CULTURE:

There are several methods, which can be employed for the growth measurement in cell
cultures.

1. Cell Numbers: This is an indispensable growth parameter for suspension cultures.

 22

Cell aggregates ( One volume )

12% chromium trioxide ( Four volume )

Heated at 70 0 C

Cells stained and plasmolysed

Aggregates partially macerated

Shrinkage of protoplasts

Cells are Counted

2. Fresh Weight : This is also a good method for growth measurement.

A) Collection of cells on pre-weighed nylon membrane fabric filters supported on a funnel.
B) The cells are washed with water to remove the medium.
C) They are drained under vacuum.
D) The dried cells are weighed.

Limitation: Large samples are required for accurate result.

3. Dry weight / Packed Cell Volume ( PCV ) : This is a method of choice in

many laboratories.
Appropriate volume of suspension

Centrifuge at 2000 X for 5 minute

Pellet volume or packed cell volume is noted.

( PCV is expressed as ml pellet / ml of culture)

Supernatant is discarded

Pellet is washed

Pellet is dried overnight at 80 0 C

Cooled and reweighed for dry weight

 23

SYNCHRONIZATION OF SUSPENSION CULTURE:

The cells are never of the same age and physiological conditions in the suspension culture
which leads to uneven growth. Following methods can be employed to synchronize the
suspension cultures.
a. Cold treatment at 4 0 C
b. Starvation
c. Use of inhibitors, which temporarily block the chain of events.
d. Treatment with 0.02 % Colchicine.

FACTORS AFFECTING THE SUCCESS OF SHOOT TIP CULTURE:

1. The size of explant plays very important role. Bigger the size of explant, more is the
success.
2. Season during which explants are obtained also affects the success. Explants
dissected at the end of dormancy yields good result.
3. Actively growing tips are best as they have strong growth potential and low virus
concentrations.
BU D C U LTU R E:

Buds contain quiescent / active meristems. Most vascular plants have indeterminate mode
of growth. The bud culture is divided in two.

a) Single Node culture ( Potato, peas, roses, tomato, cucumber, eucalyptus etc.)
b) Axillary bud culture: ( Strawberry, gerbera )

The hormone- cytokinins plays important role as high cytokinins stops the apical
dominance and allows Axillary buds to develop. The ratio of cytokinins to auxin is kept at 10 : 1.

( 2 ) MERISTEM AND SHOOT TIP CULTURE: The use of meristem is very important
 24

in production of virus-free plants. Morel and Martin ( 1952 ) used meristem culture for in vitro
virus eradication of Dahlia. Morel is pioneer for shoot tip culture of orchids. Shoot tip culture
is more successful in herbaceous plants because of weak apical dominance and strong root
generating capacities as compared to woody plants. Shoots of all Angiosperms and
Gymnosperms can be multiplied using this technique.

AXILLARY BUD METHOD FOR MICROPROPAGATION

 25

PLANT REGENERATION VIA ORGANOGENESIS

******
 26

CHAPTER - 5

T
TIIS
SSSU
UEEC
CUUL
LTTU
URRE
E FFO
ORRC
CRRO
OPP IIM
MPPR
ROOV
VEEM
MNNT
T

The applications of tissue culture have found its way, widely in micropropagation. But
the possibility of its use in crop improvement and its integration in biotechnological tools has
opened new chapters in molecular biology work. There are several methods, which can be used
or integrated in the crop improvement program.
FOR CROP IMPROVEMENT

1. Anther and microspore culture

2. Somaclonal variation
3. Embryo / ovule culture
4. Somatic hybridization ( Protoplast isolation and fusion)
( 1 ) ANTHER CULTURE: It is a simple, quick and efficient method.
Anther and Pollen Culture : Production of haploids to obtain homozygous plants, as
a starting point for mutant induction, creation of all male plants, to breed at lower ploidy level.
Androgenic method: The basic principle of this method is to stop the development of pollen
cell whose fate is normally a gamete (sexual cell) and to force its development directly into the
plant. The haploids can be obtained either by culture of excised anther or culture of isolated
pollen (microspore).
Young flower buds with immature anthers in which microspores are
confined within anther sac at appropriate stage of pollen development

Surface sterilized and rinsed with water

Calyx is removed from flower bud

Corolla slit is opened and stamens are removed

It is placed on a sterile petri dish

One of the anthers crushed in aceto-carmine to test the pollen stage

Anther is gently separated from filament and intact uninjured anther is inoculated on
medium ( injured anthers are discarded which leads to callus development)

10-20 anthers on solid or 50 anthers in liquid medium (10 ml)

After 3- weeks, 3-5 cm plantlets are removed and transferred to another medium for
further development

Transferred to soil or pot

 27

FACTORS AFFECTING ANTHER CULTURE:

A) The Genotypes : Anther culturing ability is genetically controlled so simple media should
be tested for different cultivars.
B) Physiological status of donor cell at the time of excision of anther play important role.
C) Stage of Pollen is also very important.
D) Pre-treatment of anther with cold / heat or etherel has positive effect.
E) The composition of culture media is the key factor.
F) Pre-treatment of anthers / flower buds at low temperature ( 3 -10 0 C ) for 2 -30 days
stimulates embryogenesis.

APPLICATIONS / IMPORTANCE OF ANTHER CULTURE:

1. It is useful in production and utilization of homozygous lines.
2. Release of new varieties through F1 double haploid system.
3. In mutagenesis of haploid and selection of mutants resistant to disease.
4. Inheritance study becomes easy in polyploid species using anther culture technique.
5. Transfer of desired alien genes.
6. It is useful in diocious plant for producing more male plants.
7. Fusion of haploid protoplast is possible.
8. Large nos. of haploid plants can be produced.

HAPLOID PRODUCTION THROUGH BALBOSUM METHOD:

Haploid: Those plants, which possess a gametophytic chromosome (single set) in their
sporophytes. It can be explained in a way that a cell in which there is half the usual number of
chromosomes, or only one chromosome set. Here the gametic cells are haploid.
Generally, the frequency of haploid formation is only 0.001 to 0.01 %. Spontaneous haploids can
be produced by process of apomixes or parthenogenesis (Embryo development from unfertilized
eggs). Haploids can be induced artificially through in vivo and in vitro methods.
In vivo methods of artificial haploid Production:
1. Gynogegesis
2. Androgenesis
3. Genome elimination by distant hybridization ( Bulbosum method)
4. Chemical treatment
5. Temperature shock
6. Irradiation effects
In vitro methods of artificial haploid Production:
1. Anther culture method
2. Pollen culture
3. Ovary / ovule / embryo culture method

MONOPLOIDS: Possess half the number of chromosome from diploid.: Maize , Barley.
POLYPLOID: Half the no. of gametophytic chromosome from polyploidy: Potato, Wheat
ANDROGENESIS: The haploid production through anther culture is called androgenesis.
 28

GYNOGENESIS: The production of haploid from male ovule culture where the female gamete
is triggered to sporophytic development.
Genome elimination by distant hybridization by Bulbosum method:
Kasha and Kao developed this method in 1970. In some cases of distant hybridization,
chromosomes of one species are gradually eliminated from the zygote Since, they do not have
the two parental genomes in full, this does not prevent embryo development but the resulting
embryo and the F1 plant obtained from them are not true interspecific hybrids.
Generally, the chromosome from one genome are successively eliminated due to mitotic
irregularities and in extreme cases, chromosomes from only one genome may remain in the
embryo. Such embryo is consequently haploid. e.g. Hordeum bulbosum x H. vulgare. IN this
method, embryo is to be rescued before it degenerates. This method is also applied in potato also.

MICROSPORE CULTURE: It can be defined as the production of haploid plants through

in vitro culture of male gametophytic calls i.e. microspore or immature pollen.

Anther culture has main disadvantage that plants not only originate from pollen but also
from various other parts of anther especially in dicot plants which result in plant population with
various ploidy level. In Microspore culture this limitation is overcome.

ADVANTAGES OF MICROSPORE CULTURE:

 Uncontrolled effects o f anther wall and tissue are eliminated and other factors can
be regulated.
 The sequence o f androgenesis can be observed from single cell.
 Microspores are ideal for uptake, transformat ion and mutagenic studies.
 Higher yields can be obtained with this method.
 29

( 2 ) SOMACLONAL VARIATION:
DEFINITION: Larkin and Scowcroft (1981) defined it as the variability generated by the use of
tissue culture cycle is termed as Somaclonal variation.
Somaclonal variation: Variation that occurs and accumulates in cultures of cells and tissues; it
may be either genetic or epigenetic in basis. When the rate of variation exceeds the normal
mutation rate of that species or genotype in vivo, the variation may be said to arise as a result of
growth or manipulation in vitro, and may be the result of growth in unorganized states.
Generally it is expected that tissue culture raised plants should be the exact copies of parent
plants but in practice, there are variation which is due to change in chromosome nos. and
structure either due to genetic disorder (mutation) or cultural conditions.
Nomenclature:
The nomenclature is given in the following way.
Plants generated through tissue culture as R or R0 ----- R1, R2 R3, R4……..
OR
SC1 ( = R0) ----- SC1, SC2, SC3, SC4, ….
( In both cases , they are self fertilized progeny )
SCHEME FOR OBTAINING SOMACLONAL VARIATIONS: The Somaclonal
variation can be obtained by two methods:
1. Without in vitro selection pressure or
2. With in vitro selection pressure
WITHOUT in vitro SELECTION PRESSURE:

EXPALNT  EXPLANT DERIVED CALLUS  SHOOT GENERATION

 PLANT FORMATION TRANSFER TO FIELD
 SCREENING FOR DESIRABLE TRAITS
 AGRONOMIC TRIALS
WITH in vitro SELECTION PRESSURE:
Explant Pathogen

Explant derived callus Purified culture filtrate

Multiplication of callus Toxin isolation

Determination of lethal concentration

Small pieces of callus on toxic medium for generation of toerant callus

Regeneration of plant

In vivo testing against toxin / pathogen

Disease resistant plant

 30

FACTORS AFFECTING SOMACLONAL VARIATIONS:

1. Genotype: The genotype of the variety affects the production of variation.

2. Explant source: The source of explant also affects the Somaclonal variations. As in
case of Geranium, root and petiole cuttings give Somaclonal variations while stem
cuttings does not give the variation. Similarly in case of sugarcane, the stem cuttings
give good result.
3. Duration of cell culture: The variation increase with the cell culturing duration in the
medium.
4. Culture conditions: The incorporation of 2,4 D, NAA, BAP give better result.

FACTORS FOR in vitro SELECTION:

A) Selection of propagules: The variation can be obtained fro the explants of plant
suspension / protoplast or callus.
B) Selective agent: The agent causing Somaclonal variation could be an analogue,
herbicide or any toxin from any pathogen.
C) Selection of techniques: The concentration of toxin should be sub-lethal ( 70 -90 %)
and the time interval should be sufficient to cause variation and the plant should not
die.
D) Regeneration of Plants: Sometime, the capacity is lost due to reduced level of auxin
and light intensity.
E) In vivo testing: Conditions for disease development should be provided.
F) Agronomic analysis: The analysis of agronomic parameters should be also
considered.
G) Resistance stability: This is a very important factor as many times, the resistance is
lost in sub-culturing. So the stability is given due consideration in selection.

APPLICATIONS OF SOMACLONAL VARIATIONS:

i) Novel variants: Improved scented Geranium variety ( Velvet rose ), Pure thorn-
less black berries, PUSA Jaikisan variety of mustard, Citronella BIO-13. These all
above mentioned cases are produced through Somaclonal variation method.
ii) Somaclones and Disease resistance: Eye spot diseases in Sugarcane, Potato (late
blight), Tobacco (Phytophthora), ,Tomato Fusarium wilt, maize and rice
( Helminthosporium leaf spot, Rapeseed ( Phoma )
iii) Somaclones and abiotic stresses: Proline increase cold hardening, freezing
tolerance in wheat, salt, Aluminum herbicide resistance.
iv) Salt tolerance: Rice, wheat, Brassica and tobacco
v) Aluminum tolerance: Alfalfa, carrot, sorghum and tomato
vi) Drought resistance: Sorghum R-III
vii) Herbicide resistance : Glyphosate, sulfonyl urea in tobacco and soybean
viii) Insect resistance: Aphid resistance in Wheat
ix) Seed quality: Alien genes introduced into the variety to improve the quality.
 31

BASIS OF SOMACLONAL VARIATION:

1. Karyotype change
2. Change in chromosome structure
3. Single gene mutation
4. Cytoplasmic genetic changes
5. Mitotic crossing over
6. Gene amplification and nuclear changes
7. Transpossable elements

DISADVANTAGES OF SOMACLONAL VARIATIONS:

1. It is uncontrollable and unpredictable

2. Variation is cultivar dependent
3. Variation is not always stable and heritable.
4. Not all the changes are novel.
5. Variable frequency of Somaclonal variation

( 3 ) EMBRYO / OVULE / OVARY CULTURE:

IN VITRO POLLINATION AND EMBRYO CULTURE:

Barriers to production of distant hybrids:

The difficulties encountered in production of interspecific hybrid may be grouped into
three broad classes.
(i) Failure of zygote formation. ( Pre-fertilization or pre-zygotic barriers )
(ii) Failure of zygote development ( Post fertilization barriers ).
(iii) Failure of hybrid seedlings development.

(i) Pre fertilization / pre-zygotic barriers :

In this case there will be no zygote formation. It may arise due to following.

(a) Inability of pollen to germinate on foreign stigma.

(b) Failure of pollen tube to reach ovule due to excessive length of style.
(c) Failure of pollen tube to reach base of style before ovary abscises because of slow
growth of pollen tube.
(ii) Post fertilization barrier: In this case fertilization takes place and zygote is produced but
the development of zygote is blocked at various Stages due to various reasons.

(a) Some species contain lethal genes, which cause death of the zygote, the lethal genes do
not have any effect on the species, carrying them but affect interspecific hybrid.
(b) In some cases death of embryo occurs due to genetic imbalance between the two spp.
(c) In some cases chromosome elimination of one parent during development of embryo.

This does not prevent embryo development, but the resulting embryo or FI plant is not
true hybrid.
 32

(d) In few cases embryo development may be blocked by an incompatibility between

cytoplasm of the species used as female parent and genome of the parent used as
male parent.
( e) In most of the cases endosperm develop poorly, due to which seeds are shrunken
such seeds do not germinate and this condition is known as endosperm abortion.
(iii) Failure of hybrid seedling development:

Some distant hybrid dies during seedling development or even after initiation of
flowering.
The pre zygotic barriers can be overcome by in vitro pollination where as post zygotic
barriers can be overcome by embryo rescue or ovary culture techniques.
I In vitro pollination/fertilization: Pollination performed in the test tube on callus medium is
known as in vitro pollination and seed development after such pollination is known test tube
fertilization. The test tube fertilization was first used by Kanta et.a1. 1962 in New Delhi.

.Application of in vitro pollination:

( I ) By in vitro pollination hybrid plants have been obtained in the following interspecific
crosses.
(a) Gossypium hirsutum X G. arborium (b) Nicotiana tabacum X N. rustica
(c)Zea mays X Z. Mexicana
( 2 ) Haploid plants can be also be obtained through in vitro pollination e.g. when Mimulus luteas
is pollinated in vitro with pollen of Torenia fournieri, unfertilized egg develops
parthenogenetically into haploid plant.

(II) EMBRYO AND OVULE CULTURE (EMBRYO RESCUE TECHNIQUE) :

(a) Embryo culture: In vitro culture of hybrid embryos useful in cross combination where
fertilization occurs and embryo begins to develop but degenerates prior to full maturity due to (i)
Inability of endospern1 to carry out its normal role in supplying nutrients to developing embryos.
(ii) The maternal tissue is antagonistic to the development of embryo. In other words embryo
culture is useful when the embryo has the ability to mature but is prevented from doing on where
the shriveled seeds are obtained which do not germinate.

APPLICATION OF EMBRYO I OVULE CULTURE TECHNIQUES :-

1) Obtaining rare hybrids. The most important use of embryo / ovule culture is raising rare
hybrid through embryo rescue. Laibach in 1925 cultured hybrid embryo between Unum
austriacum X L. perenne and obtained hybrid plant through this embryo rescue technique.

Through embryo culture technique (embryo rescue technique) fertile hybrid have been
obtained in various incompatible intra-specific and intra-generic crosses.
(i) Lycopersicum esculentum X L. peruvianum.
(it) Lycopersicum esculentum X L. chifense
(ill) Lycopersicum esculentum X Solanum lycopersiodes
(iv) Trificum X Agilopsis
(v) Hordium X secule ..
 33

Through ovule culture technique, hybrids have been obtained in the following
incompatible crosses.

(i) A belmoschus esculentus X A. ficalneus

(ii) Brassica oleracea X B. compestris
(iii) Glycine max X G. tomentelly
(iv) Nicotiana rustica X N. tabacum
( 2 ) Haploid production :-
In certain interspecific / intergeneric crosses due to chromosome elimination of one
parent during first few divisions of embryogenesis haploid embryo / plant is formed. This
haploid embryo generally aborts. By rescuing this haploid embryo, haploid plant can be
produced.

( 3 ) Shortening the breeding cycle :-

In some plants the life cycle is long due to long dormant period of seeds by growing
embryo, this period may be reduced or shortened. Rosa normally takes a whole year to come
into flowering through embryo culture it has been possible to take two generation in one year in
malus seed takes 9 months to germinate but by embryo culture within 4 weeks seedling are
produced in orchids ovules are cultured to reduce duration.
( 4 ) Rapid seed viability test :-
Germination of excised embryo is more rapid method for testing viability of seeds.

( 5 ) Propagation of rare plants :-

Embryo culture technique is also useful for propagation of plant where seed germination
does not occur seeds of Musa balbisiana a wild relative of banana do not germinate in nature
were seedling can be readily obtained by culturing their embryos.

( 4 ) PROTOPLAST ISOLATION AND FUSION : It is also known as

Somatic Hybridization.

Protoplast: A membrane-bound cell with all the components of cell, formed after the cell wall
is removed from a microbial or plant cell by the action of pectinase and cellulase is called
protoplast. Hanstein (1880) gave this term.
The protoplast is unusual as the outer plasma membrane is fully exposed which is the
only barrier between internal and external environment. Cocking (1960) first used the enzyme to
release protoplast.
USEFULNESS OF PROTOPLAST: It is used for the cell fusion or somatic hybridization.
Since, it can take up the foreign DNA or cell organelle or bacteria or virus through plasma
membrane; it has gained importance in crop improvement program.
PROTOPLAST ISOLATION:
There are two popular methods for isolation of protoplast from plant cell.
1. Mechanical Method
2. Enzymatic Method
 34

MECHNICAL METHOD OF PROTOPLAST ISOLATION: Highly vacuolated cells of

storage tissue, such as onion bulb scales, radish root or beetroots are used. The cells are
plasmolysed in iso-osmotic solution, which will lead to withdrawal of contents to centre. The
tissue are dissected and de-plasmolysed to release preformed protoplast.
DISADVANTAGES OF MECHANICAL METHOD:
1. Restricted to certain tissue having large vacuolated cells only.
2. Yield of protoplast is very low.
3. Method is tedious and laborious.
4. Viability of protoplast is low because of presence of substances of damaged
cells in the protoplast.
ENZYMATIC METHOD OF PROTOPLAST ISOLATION:
Takebe (1968) first used commercial enzymes to produce protoplasts. There is a mixture
of enzymes such as cellulose, hemicellulase and pectinase (degrades middle lamella) is used. The
commercial pectinase, which is also known as macerozyme (macerase) is produced from
Rhizopus.
Cellulase  Onozuka R-10
Driselase  Both cellulolytic and pectinolytic
Macerozyme  Macerase from Rhizopus
Other enzymes such as helicase, colonase, cellulolysin, glusulase, zymolase, pectolyase etc.
FACTORS AFFECTING ENZYME ACTION:
1. Physiological state of tissue and cell material : The source for protoplast isolation
could be leaves, petioles, shoot / root apex, fruit hypocotyls, stem, embryo, callus,
mesophyll cells etc. The best source is mesophyll cells because they ( cells ) are loosely
arranged.
2. Type of enzymes: Since varieties of enzymes are available, different combination of
enzymes can be used depending upon the composition of tissue material. The type of
enzyme will play a major role.
3. pH : The ideal pH for carrying out enzyme reaction is 4.7 to 6.0. There will be adverse
effect of extreme low / high pH.
4. Temperature: The enzyme reaction is to be carried out at specific optimum temperature.
The best temperature range is 25 -30 0 C.
5. Duration of Enzyme treatment: The reaction time also plays a big role. The ideal time
range is 30 minutes to 20 hrs.
During the enzyme treatment, the protoplast needs to be stabilized. The osmoticum is added
to prevent the bursting of protoplast.
OSMOTICUM: Non-ionic substance: Soluble carbohydrates. Mannitol at 0.3 -0.7 M-best
Ionic substance: KCl, CaCl2, MgSO4
The PURIFICATION OF PROTOPLAST is done using filtration, centrifugation and washing.
 35

VIABILITY STUDY OF PROTOPLAST:

The viability and intactness of protoplast can be done using following methods.
1. Phase contrast microscopy
2. Staining of protoplast with FDA ( 5.0 % Flourescein di-acetate) which accumulates
inside plasma lamella.
3. It can also be stained with 0.01 % Phenosafranine. The viable cells remain unstained.
Calcoflour white ( CFW ) or Evan’s blue ( 0.025 %) can also be used.
4. Photosynthetic studies can also determine the viability of protoplast.
5. The oxygen uptake study reveals the viability too.
6. The variation of protoplast size with osmotic change, can also determine the viable
protoplast. The dead or fragmented will not respond to osmotic change.

PROTOPLAST CULTURING:
1. Agar culture method:
2. Liquid culture: This is preferred in earlier stages of growth. This allows easy
dilution and transfer; osmotic pressure and density of cells can be reduced.
3. Liquid droplet method.
4. Hanging droplet method
5. Feeder layer method
6. Co-culturing method.

PROTOPLAST FUSION ( SOMATIC HYBRIDIZATION ):

The protoplast fusion is useful in somatic cell genetics and crop improvement. The
biggest advantage of this method is that the species cross barrier is broken. The plants, which are
incompatible, can be used for crossing purpose. It can also be called somatic hybridization..
Somatic cell hybridization: The fusion of plant protoplasts derived from somatic cells that
differ genetically. It is the method of hybrid production through fusion of somatic protoplast
under in vitro conditions and subsequent development of heterocaryon to a hybrid plant.
Protoplast fusion: A technique for producing somatic hybrids between two sources of
protoplasts by treating with agents such as polyethylene glycol (PEG) and Ca+2 ions to induce
cell membrane fusion. The two protoplast sources may be from the same or highly divergent
species.
 36

Cybrid (Somatic hybrid) : It is nuclear genome of one parent but cytoplasmic genes
(Plastome) from both the parents in the fused hybrid. It can be explained as the creation of
variable cell resulting from the fusion of a cytoplast with a whole cell, thus creating a
cytoplasmic hybrid.
Protoplast fusion is possible between taxonomically distant plant spp. beyond the limits
of sexual crossability. This creates cells with new genetic, nuclear or cytoplasmic constitution.
There are differences between sexual and somatic hybridization.
No. Sexual Hybridization Somatic hybridization
1 It is in vivo process It is in vitro process.
2 It is applicable to sexually compatible crops It is applicable to sexually incompatible
crops
3 It involves the fusion of male and female It is a fusion of diploid protoplasts
gametes which are haploid
4 Fusion product is diploid and formed embryo Fusion product is tetraploid heterocaryon
from callus and hybrid plants are generated
5 Hybrid contain cytoplasm of female parent only Hybrid contains cytoplasm of both parents.
6 Useful in plant breeding Cybrid or somatic hybrid is used for
transfer of cytoplasm

PROTOPLAST FUSION;
The protoplast fusion can be carried out by two methods.
1. Spontaneous fusion : This is possible with callus culture. But the problem is that it does
not regenerate into whole plant.
2. Induced Fusion: This is doe not using a fusing agent (Fusogen : The agent used for
fusion of two cells / protoplast is called fusogen). There are several different types of
agents used for this purpose.
a) NaNO3 : It has very low frequency of fusion ( Power, 1970)
b) Ca ions at high pH of 10.5 can also be used.
c) Poly Ethylene Glycol ( PEG ): Most successful  Kao and Michayluk (1974)
d) Electro-fusion : The electrical impulses are given for the fusion which bring two
protoplast together due to reduction in surface tension . The method is also called
ELECTROPORATION.
Advantages of PEG:
1. It is reproducible. 2. It has high frequency of fusion.
3. It has very low cytotoxic effects. 4. Formation of bi-nucleate heterokaryons.
5. It is non-specific i.e. Inter-specific, inter-generic or inter-kingdom spp. can be fused.
 37

THE MECHANISM OF PROTOPLAST FUSION:

The fusion of protoplast occurs due to the agglutination or adhesion of two protoplasts
due to reduction in the surface tension. Later the plasma membrane fusion takes place at
localized site. The genetic material exchange takes place and there is formation of heterocaryon.
The question arises that how do we identify and select the hybrid cell? There are several
methods, which can be employed.

1. Chlorophyll deficiency complementation. 2. Auxotroph complementation.

3. Complementation of resistance markers. 4. Use of metabolic inhibitors and
5. Use of visual characteristics.
SCHEMATIC DIAGRAM OF PROTOPLAST FUSION
 38

APPLICATION OF SOMATIC HYBRIDIZATION ( PROTOPLAST FUSION ) :

a) Production of novel interspecific / intergeneric crosses those are difficult to hybridize.
As we have seen earlier that crossability barrier is removed in this method.
b) This method is used for somatic hybridization for gene transfer such as genes for
disease resistance, abiotic stress resistance, quality characters like erucic acid, nicotin
content etc. and cytoplasmic male sterility ( CMS ).
c) It can be very well employed for production of auto-tetraploids.
d) Sexually sterile ( haploid, triploid etc) plant can be fused to produce fertile diploid /
polyploids.
e) Hybridization is possible between juvenile phased plants.
f) Production of heterozygous lines within a single spp. ( Potato / root tubers is normally
vegetatively propagated ).
g) Somatic cell fusion is useful for study of cytoplasmic genes.
h) It is also possible to create unique nuclear – cytoplasmic combination.
PROBLEMS OF PROTOPLAST FUSION:
1) Protocol for plant regeneration from protoplast to actual plant should be available.]
2) Efficient selection method selection of fused cell should be available.
3) In taxonomically apart cell, the end products are unbalanced because they are not
natural products of their life cycle.
4) There is development of chimeric callus (two nuclei are replicating separately).
5) Regeneration products are often variable due to Somaclonal variation. There are
chances of elimination, translocation and segregation.
6) The biggest limitation of this method is that the gene transfers are totally random. The
fused cell may not have the desired genes in which we may be interested.
7) Stability of fused cell is also a problem.
8) Back crossing is very essential for variety.

*****
 39

CHAPTER - 6

GERMPLASM STORAGE & CRYOPRESERVATION

It has been a common practice to store the seeds under a drying place ( 5.8 % water) with
low humidity and – 18 0 C. But there are problems associated with the normal storage of seeds.

PROBLEMS OF SEED STORAGE :

1. Some plants do not produce fertile seeds.

2. Some seeds remain viable for only limited time. Later the seeds loose the germination
capacity.
3. Some seeds are heterozygous so they are suitable for maintaining the true-to- type
genotype.
4. Some seeds deteriorate very rapidly.

Because of these problems, new and novel techniques for seed / germplasm storage have
been developed. This is an in vitro conservation. There are many advantages of in vitro storage
of germplasm.

GERMPLASM CONSERVATION: It is the technique of germplasm conservation (storage

of cells, tissues, embryo or seeds) by ultra low temperature in liquid nitrogen at – 196 0 C OR
Conservation, storage and maintenance of germplasm in in vitro by tissue culture technique in
aseptic conditions in freezing conditions at – 196 0 C in liquid N2.
ADVANTAGES OF in vitro CONSERVATION:

A) Plant species which are in danger of being extinct can be preserved. There are many
plants which are about to vanish from this earth because of man-made and
environmental factors, which can be saved.
B) In vitro conservation saves the storage space, time, labor and land requirement.
C) Sterile plants can be maintained in laboratory conditions.
D) Reduction in growth decreases the number of necessary sub-culturing.
E) If a sterile culture is obtained, sub-culture in vitro sis the only safe way.
There are two methods of Germplasm storage

1. CRYOPRESERVATION and 2) SLOW GROWTH METHOD

( 1 ) CRYOPRESERVATION: The word cryo has been coined from a Greek word
Kryos meaning frost. The same principle is applied here too. The preservation is done in
freezing conditions.
 40

Following methods are utilized

(1) On a solid CO2 at – 79 0 C
(2) Low temperature Deep freeze at – 80 0 C
(3) In vapor phase of N2 at – 150 0 C or
(4) In liquid Nitrogen at – 196 0 C which is used for plant materials.

The basic mechanism of low temperature storage is very simple as at freezing

temperatures, the metabolic activities are reduced and biological deterioration stops. However,
two aspects should be critically considered.

1. We should know degree of freeze tolerance of a particular plant species.

2. The formation of ice crystals with in the cell causes the damage to the cell. You
have to minimize the formation of crystals inside the cell.

OBJECTIVES OF CRYOPRESERVATION:
1. Conservation of Somaclonal and gametoclonal variation
2. Maintenance of recalcitrant seeds.
3. Conservation of cell lines producing medicines.
4. Storage of pollens for enhancing longetivity.
5. Conservation of rare germplasm arising through somatic hybridization or genetic
manipulation.
6. Delaying the process of ageing
7. Storage of meristem culture for micro propagation micro grafting and disease free
plants.
8. Conservation of plant material from endangered species.
9. Establishment of germplasm bank
10. Exchange of germplasm and information at International level.

STEPS OF CRYOPRESERVATION:

1. Raising sterile tissue culture plant in in vitro conditions.

2. Addition of cryo-protectants and pre-treatment with certain chemicals.
3. Freezing the material at a specific temperature.
4. Storage for a specific period
5. Thawing (The process of bringing back from freezing temperature to normal room
temperature) – This is done when we want to re-utilize the storage material.
6. Determination of survival (This is very important as some material may die at this
temperature) by specific methods.
7. Plant growth and regeneration.
Raising sterile plant culture:
All plant material can not survive at – 196 0 C. Small, richly cytoplasmic meristematic
cells survive better. Cell suspensions and organized structures can be preserved with this method.

Addition of Cryo-protectants: Glycerol, glycols, acetamide, sucrose, mannose, proline and

DMSO ( Di-methyl sulfoxide ) is used . However, DMSO at 5 -10% is the best.
 41

ADVANTAGES OF DMSO: It has low molecular weight. It is easily miscible. It is non-toxic

and easily permeable and washable. These properties make it best cryo-protectants.

BASICS OF CRYOPRESERVATION:
Freezing of plant cells involves the conservation of some or all of their liquid water to
ice, while thawing is a reversal of this transition.
In plant tissue, water can be divided into:
1. Extra cellular water
2. Intra-cellular : Bound water and Free water
Free water is available for freezing, while bound water is with macro molecular
constituents, in structural and functional role. Freezing of plant cells involves both free and
bound water, freezing of bound water to be move harmful to survival.

CRYOPRESERVATION OF SHOOT TIPS

Freezing: There are three methods of freezing

1) Rapid freezing : Vials are plunged into liquid nitrogen ( - 300 – 10000 C /
minute). Generally the shoot tips and embryo can be preserved.
2) Slow freezing: Gradual increase of 0.1 to 100 C / minute up to 1000 C is done
in liquid nitrogen. It is good for meristems of peas and potatoes.
3) Step-wise freezing : Advantages of both the methods are obtained.

Thawing: The thawing is done in warm water at 35 – 45 0 C. Shaking is done continuously till
all the ice crystals goes away.
 42

Determination of Survival : Realistic tests, staining method of FDA, TTC ( tri phenyl
tetrazolium chloride) , Evan’s blue is done.

Plant growth and regeneration: Washing out of cryo-protectants and growing them in the
culture media is done.

ADVANTAGES OF CRYOPRESERVATION:
1. To ensure the availability of useful germplasm for use in future.
2. Some crop species do not produce seeds and can not be preserved by
conventional method which can be preserved.
3. Vegetatively propagated plants possess high degree of heterogeneity.
Propagation by seeds leads to segregation and loss of germplasm; such species
can be preserved.
4. Plants do not produce viable seeds and take long time to reproductive maturity
can be regenerated in short time.
5. Some plants need to manifest by vegetative form only i.e. tuber, root cutting
etc. can be preserved.
6. We can preserve the plant species, which produce recalcitrant (toxin) and loose
the viability of seeds when it is dried at certain water content or exposed to low
temperature.

LIMITATIONS / PROBLEMS OF CELL CULTURE PRESERVATION

1) Sub culturing over an extended period of time may cause undesirable

changes.
2) Chromosomal aberration.
3) Decline in morphogenetic potentials.
4) Loss of biochemical characteristics.
5) Accumulation of point mutations.

SLOW GROWTH METHOD:

There are several key factors, which can be applied for this method.

1. Bonsai type
2. Non-freezing by temperature ( 1 – 4 0 C),
3. Low oxygen supply,
4. Use of Growth retarding Hormones such as – CCC and ABA.
5. Osmotic Inhibitors ( 3-6 % Mannitol)

****
 43

CHAPTER - 7
INTRODUCTION / HISTORY OF BIOTECHNOLOGY

A. Goals of biotechnology
1. To understand more about the processes of inheritance and gene expression
2. To provide better understanding & treatment of various diseases, particularly genetic
disorders.
3. To generate economic benefits, including improved plants and animals for agriculture
and efficient production of valuable biological molecules e.g. Vitamin A fortified
engineered rice

Recombinant DNA—Transfer of genetic material between organisms of the same or different

species, also termed genetic engineering.

DNA recombination occurs naturally

1. Sexual reproduction
2. Bacterial transformation
3. Viral transfer of DNA
Recombinant DNA technology is used to isolate and study genes
1. A gene is located on a chromosome map
2. A DNA library of that organism is produced using Restriction enzymes, plasmid
DNA and bacteria
3. The gene of interest is isolated ("cloned") from the library
a. Complementary base pairing rules allow design of synthetic DNA probes
b. Plasmid DNA isolation, restriction digestion, and electrophoresis are used to
purify the gene away from its bacterial host
4. Multiple copies of the gene of interest are produced for study using polymerase chain
reaction

HISTORY:

Year Name of Scientist Contribution

1953 Watson and Crick Structure of DNA

1962 Jacob and Monad Lactose Operon

1968 Messelson and Yuan Term Restriction enzyme coined

1970 Smith and Nathan Discovery of first RE Hind III

 44

Year Name of Scientist Contribution

1972 Berg [Link]. First recombinant DNA molecule

1972 Mertz and Davis Joining two DNA with enzyme ligase

1972 Temin Discovery of reverse transcriptase

( RNA to DNA )

1973 H. Boyer and S Cohen Creation of hybrid plasmid

1974 Zaenen and Larebeke Discovery of Ti plasmid

1977 Chilton Successful integration of Ti plasmid

in plant
1977 Maxam and Gilbert Gene sequencing method

1977 Sharp and Roberts Discovery of split genes

1980 Eli Lilly and co. Commercial production of insulin

from bacteria
1980 RFLP technique was developed

1982 Krens Incorporation of DNA by protoplast

1983 Kary Mullis Polymerase Chain Reaction ( PCR)

1984 De Block and Horsch Transformation of Tobacco with

Agrobacterium

1986 Powell and Abel TMV resistant plant in tobcacco and

tomato by cDNA of coat protein

1987 Barton Isolation of Bt gene

1990 Human Genome mapping launched

1990 Welsh and McClelland and RAPD was discovered

William

1991 Fodor DNA Micro Array system discovered

1995 Vos DNA finger printing by AFLP

1997 Blattner Sequencing E. coli genome

2001 Wenter [Link]. Human genome sequencing completed

 45

Benefits of Biotechnology
Improve outcome of genetic diseases
• Reduced Land Use
• Reduced Pesticide and fertilizer Use
• Less environmental pollution
• More and better production of crops in terms of yield and quality

Agricultural applications
1. Disease resistant plants,
2. Nitrogen fixing genes,
3. Salt tolerance,
4. Bioremediation,
5. BT producing plants (cotton, tobacco, tomato corn etc.),
6. Roundup resistant plants ( Herbicide resistant ),
7. Nutritionally altered plants - lower fat, different amino acids;
8. Polygalacturonidase inactivated tomato  prevent softening of tomato, Flavr Savr ,
Vitamin A supplemented rice- Golden Rice (1999)
9. Plants as vaccine factories.

Biotechnology Improves Traits or Special Features

• Potential to cure genetic diseases of animals and human being
• Improve animal and plant traits
• Improve resistance to disease and pests
• Improve nutrition and quality of crops
• Expression of proteins in target cells or organisms
• Increase yields of crops
• Increase the shelf life of fruits
• Transgenic Food

*****
 46

CHAPTER - 8
BASIC TECHNIQUES IN MOLECULAR BIOLOGY

BASIC TECHNIQUES IN MOLECULAR BIOLOGY:

To explain the processes of gene cloning and gene manipulation, following techniques
are essential.
( 1 ) AGAROSE GEL ELECTROPHORESIS:

( a ) AGAROSE GEL ELECTROPHORESIS: It is a standard method for separation ,

identification and purification of DNA or RNA upto 20 kb.

ADVANTAGES:
1. It is a simple and rapid method.
2. It is capable of resolving DNA fragments, which can not be separated by other
methods.
3. Location of DNA can be determined.
4. DNA can be recovered back and used for cloning.
Agarose is linear polymer obtained from sea weed which forms a gel ( 0.4 to 1.0 % )

HOW IT WORKS: When an electric field is applied, DNA which is negatively charged
migrates towards anode. The rate of migration depends on
a) Molecular size of DNA
b) Agarose concentration
c) Conformation of DNA
d) Composition of buffer
Larger molecules migrate slowly than smaller molecules.

Type of Buffers: There are several different type of buffers employed in the agarose gel
electrophoresis depending upon type of work. TAE (Tris Acetate Buffer ), TBE ( Tris Borate
EDTA buffer ), TE ( Tris EDTA buffer) , TPE ( Tris Phosphate buffer ) etc.

Staining Reagent : Ethidium bromide ( Etbr) is used for staining DNA molecule. The dye Etbr
is an intercalating dye which goes inside the bases of DNA molecule in the chain. It is toxic and
mutagenic at high concentrations. It fluoresce and gives orange color under ultra violet ( UV)
light.
( b ) PULSE FIELD GEL ELECTROPHORESIS ( PFGE) : It can detect higher MB DNA (
100 – 1000 MB DNA ). Here we change intermittently the direction of electric field ( 120 0 every
90 seconds for 18 – 24 hrs). This was developed by Schwartz and Cantor in 1984.

( c ) POLYACRYLAMIDE GEL ELECTROPHORESIS ( PAGE ) :

( d ) ISO ELECTRIC FOCUSSING ( IEF ) :

 47

( 2 ) BLOTTING TECHNIQUES: Blotting describes the immobilization of sample

either nucleic acid or proteins on to a solid support generally nylon or nitro-cellulose membrane.
The blotted samples ( either DNA or RNA) on a nitro-cellulose or nylon membrane are used as
target for hybridization with labeled probes such as DNA/ RNA sequence or antibody.

There are several techniques used in blotting

1. Southern Blotting: This is used for DNA samples.
2. Northern Blotting: It is used for blotting RNA samples.
3. Western Blotting : Here the proteins are blotted.
SOUTHERN BLOTTING TECHNIQUE:

Southern developed the method in 1975. He demonstrated that DNA restricted fragments
obtained in agarose gel can be transferred to solid support ( nitro cellulose ) and detected as band
after hybridizing with complementary radio labeled probes.

Purified DNA digested with Restriction Enzyme

Agarose gel Electrophoresis
Agarose gel with DNA fragments
1. Depurination treatment
2. Neutralizing solution
Agarose gel equilibrated
Transfer of DNA fragments on Nitrocellulose
/ nylon membrane by capillary action
DNA transferred to nitro cellulose membrane
Fixation of DNA to membrane
( Baking at 80 0 C / UV cross-linking )
Membrane with DNA fragments

Addition of labeled probe

Hybridization of DNA with labeled probe

Detection by auto radiography on X ray films

Detection of hybridized fragments

 48

SCHEMATIC REPRESENTATION OF SOUTHERN HYBRIDIZATION

Southern Blot : Gel electrophoresis followed by transfer to a membrane

DNA Cleaved with a restriction enzyme

DNA Cleaved with a
restriction enzyme

Smear of restriction fragments

Hybridization of DNA on membrane to specific probe

 49

Southern Blot

PERFECT CONDITIONS ARE NECESSARY FOR HYBRIDIZATION

Factors affecting hybridization:
1. Bound nucleic acid is embedded and in accessible to probe.
2. The hybridization buffer affects the speed of reaction.
3. Dextran sulfate act as volume extruder to increase the rate and extent of
hybridization.
4. Dried milk, heparin and SDS depress non-specific binding of probe.
5. Formamide decrease the melting temperature of DNA.

NORTHERN HYBRIDIZATION: It is opposite to Southern hybridization. AS in southern

hybridization, DNA is bound on the membrane, in Northern, RNA is bound on the nitro cellulose
membrane.

DEFINITION: The separation of RNA by gel electrophoresis followed by hybridization with

radio-labelled RNA / DNA probe. The procedure is same as that of Southern hybridization.

How ever, there are differences also.

[Link] is blotted. RNA is not denatured as is done in Southern hybridization.

[Link] from nitro cellulose, diazotized cellulose ( DBM) and O-amino phenyl thio ether
( APT) cellulose can also be used.

SOUTHERN AND NORTHERN BLOTTING REQUIRE PURIFIED NUCLEIC ACID

AND IT IS TIME CONSUMING AND EXPENSIVE METHOD.

WESTERN BLOTTING: It is a method of protein blotting. It can be defined as blotting of

electrophoresed protein bands from a SDS- PAGE on to a membrane ( nylon / nitrocellulose) and
their detection with fluorescent antibody probe and visualization the color under UV light.

SDS-PAGE separates protein on the basis of size and charge of protein. The gel is over-layered
with a nitro cellulose membrane and electric field is applied. The protein migrates from gel to
membrane and become bound. Later, they are incubated with casein ( this will block all the
 50

unoccupied sites with casein on membrane). Radio labeled antibody against the protein is added
and the mixture is incubated. The radio labeled antibody will bind to the homologous protein
125
bands which are visualized and detected either using I or autoradiography on X ray film or
ELISA method is done for visual detection of color.

Western blot is similar in concept to northern, except use PAGE

AUTORADIOGRAPHY: It is the process of photographic image formation, caused by

exposure of X ray film to emission from radio isotopes.

DOT-BLOT ANALYSIS: If only detection is to be done, Dot-blot is used. Here, the purified
DNA/RNA is blotted on nitrocellulose and hybridization with probe is done and it is detected.

APPLICATIONS:

1. Rapid detection of specific sequences of DNA / RNA.

2. Determination of relative amount of DNA / RNA in a complex mixture. It can be done
using densitometer.
3. The polymorphism can be detected in different cultivars or varieties of any crop.
4. The genes can be located or identified from different clones.
 51

The procedure for these three blots is summarized below:

The important properties of the three are shown below:

*****
 52

CHAPTER - 9
FUNDAMENTALS OF GENE CLONING
Gene cloning: The isolation and amplification of an individual gene sequence by insertion of
that sequence into a bacterium using a vehicle where it can be replicated.
There are numerous examples of gene cloning in various systems.

NO SYSTEM EXAMPLE
1. Prokaryote to Prokaryote Transfer of nif gene from Rhizobium to E.
coli
2. Prokaryote to Eukaryote Transfer of Bt toxin gene from Bacillus
thuringiensis to tobacco, tomato, cotton etc.
3. Eukaryote to Prokaryote Insulin producing gene from pancreas of pig
or any animal to E. coli.
4. Eukaryote to Eukaryote Disease / insect resistance gene from
resistant variety to susceptible variety

The above-mentioned examples suggest that any gene of our interest from any organism
can be transferred in a precise manner-using gene cloning method.
Before we look into the strategies of gene cloning, let us try to understand some terminology.
1. GENE MANIPULATION: Identify a gene from another species, which controls a
trait of interest and / or modify an existing gene (create a new allele).
2. RECOMBINANT DNA: New combinations/arrangements of DNA constructed in the
laboratory by inserting a foreign DNA.
3. RECOMBINANT DNA TECHNOLOGY: The technology used in the isolation or
synthesis and joining together of unlike pieces of DNA so that biologically active
recombinant DNA molecules can be manufactured in the lab. These recombinant DNA
molecules can then be introduced into bacteria, yeasts, or other cells where they can
replicate and function (code for protein synthesis). Also known as genetic engineering.
4. CLONING VECTOR: Genetic element into which genes can be recombined and
replicated.
5. TRANSGENIC : An organism that has a new genetically engineered DNA sequence
found in every one of its cells. Genetically engineered organisms are transgenic. These
two terms are used interchangeably.

GENE MANIPULATION TECHNIQUES:

1. Gene identification and isolation
2. Gene cloning
3. Gene transfer
4. Expression
 53

For gene transfer there are several techniques:

1) Gene transfer with vectors:

1. Through vectors such as plasmid, viruses, bacteriophage etc.
2) Gene transfers and plant transformation without vectors:
1. Micro injection. 2. Particle bombardment.
3. Direct up take.
We will discuss first the gene cloning technology using vectors.

BASIC EVENTS OF GENE CLONING: There are several steps of gene cloning.

a) Isolation of gene of interest after getting pure form of DNA from a cell.
b) Incorporation of gene of interest ( fragment of DNA ) to be cloned into a vector.
c) The vector ( r-vector ) is introduced into a host cell by transformation.
d) Cells that have acquired r-DNA are detected and selected using appropriate method.
e) r-DNA is multiplied within host cell to produce no. of identical copies of cloned gene.

COMPONENTS OF GENE CLONING: There are several components of gene cloning.

1. Restriction enzymes: They are used for cutting and joining the DNA molecules.
2. Vectors: They are used for inserting gene of interest in a cloning vehicle.
 54

3. DNA fragments: Gee libraries where gene of interest is cloned and preserved in a
clone in a vector.
4. Selection of clone f transformed cells that have acquired recombinant DNA.

( 1 ) RESTRICTION ENZYMES:
Let us try to understand basic terminology of restriction and modification.
Restriction: Identification of incoming DNA to the cell (Bacteria) and its destruction by
cleaving into pieces if it is recognized as foreign DNA.
Modification: Protection of the cell’s own DNA by methylation of certain bases so that host
DNA is not cleaved.

RESTRICTION ENZYMES: They are molecular scissors which cut the DNA at a specific site;
isolated from bacteria where they are used as Bacterial defense against viruses.
Messelson and Yuan in 1968 gave the term Restriction Enzyme. Later, Smith and
Nathan in 1973 gave the nomenclature and classified these enzymes.
There are both three or four letter word in which, the source of the organisms from where
it was isolated and their number in Roman letter to suggest the order of discovery is mentioned.
1. Hae : Haemophilus aegypticus
2. Hin : Haemophilus influenza
3. Hinf : Haemophilus influenza serotype f
If more than one enzyme is isolated from single origin, then they are denoted Roman
numericals ( I, II, III etc.) e.g. EcoRI, HindIII, SalI etc.

There are three classes of restriction enzymes ( Yuan, 1981):

1. Type – I 2. Type – II and 3. Type - III

TYPE – I : They are complex enzymes which act as endonuclease and methylase
function. They require ATP, Mg +2. They are single, multi-functional enzyme that recognizes 15
bp in length (methylated at adenosine) and cleavage site is 1000 bp away. They show specificity
for recognition but not for cleavage. The biggest disadvantage is that they produce heterogeneous
fragments. Generally, they are not used in gene cloning techniques.
e.g. EcoK, EcoB etc.

TYPE – II : They are the most common restriction enzymes used in gene cloning.
They are simple enzyme having single polypeptide. They have separate methylase and
endonuclease activity. The recognition and cutting site is the same one. They generally recognize
six nucleotide (hexa nucleotide). Some also recognize 4 , 5 or 8 bp too.

PvuI : Proteus vulgaris ………CGATCG……..

PvuII : Proteus vulgaris ............CAGCTG..........
EcoRI : E. Coli .............GAATTC.........
They are stable and require only Mg +2 . The recognition sequence have an axis of
rotational symmetry ( Palindromic sequence)

Palindromic sequence: The sequence read the same in either direction on opposite strand.
“Madam, in Eden I’m Adam”
“ Limdi game gadi mali” or “ Jare Bawa Bareja”
 55

EcoRI 5’ – G A A T T C - 3’
3’ - C T T A A G - 5’

The restriction enzymes produce two types of cuts. Both these cuts have their importance
genetic engineering work.
1. Staggered ( sticky) or cohesive end
2. Blunt end

1. Staggered end: The examples are EcoRI, PstI, HindIII, SalI etc. There are two types of
products. One is 5’….G OH 3’ and another is 3’ ….CTTAAP 5’.

2. Blunt Ends: The examples are :

Hae III : 5’ G G C C …3’

3’ C C G G …. 5’

AluI 5’ A G C T … 3’ Arthobacter luteus

3’ T C G A ….5’

Please note that 5’ end always carry phosphorus group while 3’ end carry hydroxyl
group. This property allows them to form complementary base pairs with any other DNA
molecule of any origin.

ISOSCHIZOMERS: It can be defined as two enzymes having same recognition site e.g.
HindIII and HsuI. Both have the same recognition site
….AAGCTT….
…. TTCGAA….
 There are some enzymes who recognize the same site but the cleavage point is different.
e.g. SmaI and XmaI

SmaI 5’ C C C G G G …3’
3’ G G G C C C … 5’

XmaI 5’ C C C G G G …3’
3’ G G G C C C … 5’
 Some restriction enzyme having different recognition sites, produce same sticky ends( It
means that they generate identical overlapping terminal)

BamHI GGATCC
BglII A G A T C T (This means that cleaved fragments by any of
Sau3A GATC these enzymes can be joined).
 56

STAR ACTIVITY: The enzymes change the specificity of recognition and cutting due to
change in reaction conditions such as pH, concentration of NaCl etc.

TYPE – III: There are two subunits. One is for recognition and modification another
for nuclease activity. They require ATP, Mg. The problem is that the recognition sites are
asymmetric, non-palindromic. The single strand ends produced by Type –III differ from each
other and cannot recombine at random. They lack ATPase activity. Since cleaved products are
not uniform, they are not very useful in gene cloning.
e.g. HgaI, MboII, FokI etc.

DNA LIGASE ENZYMES : It can be defined as those enzymes which joins DNA
fragments. It is the final step of construction of rDNA molecule. The process is called ligation.
e.g. T DNA ligase from E. coli
Here the vector DNA and foreign DNA cut with the same restriction enzyme are mixed
together, which forms complementary base pairs. Ligase acts on the DNA at the 5’ phosphorus
group and forms a phosphodiester bond.
DNA MODIFYING ENZYMES: There are a several other enzymes which are very
important in gene cloning and are used at specific place. Few of them are discussed below.

1. Kinase : They transfer the phosphate group from γ32 P to a 5’ terminal of DNA /
RNA. The example is bacteriophage T4 kinase.
2. Alkaline Phosphatase: It removes the 5’ end phosphorus group from the DNA and
modify them.
3. DNA Polymerase: DNA polymerase I is responsible for the synthesis of nucleic acid
using single stranded DNA as template.
4. Terminal transferase: These enzymes can add oligonucleotides tails to the 3’ end of
DNA duplex. Here the homo-polymer extension can be done.

5’ GAATTC ..3’ + Enzyme and ATP  5’GAATTCAAAAAAA…3’

(oligo d (A) tail)

5. S1 Nuclease: It converts cohesive ends of DNA to blunt / flush ends or trimming

single stranded ends. It is used when annealing of two incompatible end require
overlapping ends to be removed.
6. Lambda ( λ ) exonuclease: It is used for 5’ end modification. It removes nucleotides
from 5’ ends of DNA.
7. Exonuclease III : It is used for 3’ end modification.
8. Bal 31 nuclease: This enzyme simultaneously degrade both ends. DNA ends are
shortened. It produces blunt ends. They have highly specific activity.

9. Linkers: Linkers are the synthetic oligonucleotides which self-associate to form

dsDNA with recognition sequence for restriction enzyme. These can be ligated to
blunt ends and cut with specific restriction enzyme to give cohesive end.
10. Adapters: They are chemically synthesized DNA with preformed cohesive ends.
 57

VECTORS:
VECTOR: It can be broadly defined as genetic element into which genes can be recombined
and replicated. A random DNA segment or specific gene is linked into small replicating, circular
DNA molecule which is called vector. The vectors are used to form rDNA which can be
propagated in a suitable host and multiplied in large numbers.
The largest numbers of vectors exists for E. coli.

TYPES OF VECTORS:
1. Plasmids 5. Bacterial Artificial
2. Cosmids chromosome ( BAC)
3. Phagemids. 6. Transpossons.
4. Yeast Artificial chromosome - 7. Plant virus vector
YAC 8. Insect virus vector

IDEAL PROPERTIES OF VECTORS:

1. It should contain replicon to enable it to replicate.
2. It should have several marker genes, which help to differentiate transformed cells such as
antibiotic resistance, toxin production etc.
3. It should have unique cutting site within a marker gene leading to inactivation and
identification.
4. It should contain suitable gene control elements such as promoter, terminator, and
ribosome binding sites. etc.
 58

( 1 ) PLASMIDS: The properties of plasmids are as follows:

a. Double stranded, closed, circular DNA which are extra chromosomal units.
b. They carry out self-replication and are inherited independently.
c. They are found in variety of bacteria
d. The examples are pBR – 322, pUC, pACYc, colE1 etc.
e. The size of the DNA is in the range of 1 – 200kb.
f. Copy no. are from 7–500. If they have relaxed replication control = then > 1000.

pBR – 322 : 15 – 20
pUC - : 7 – 500
pACYc : 10 - 12
colE1 : 15 – 20
There are three general classes of plasmids.
1. Virulence plasmids. – Toxin gene producing
2. Drug resistance plasmid – Antibiotic resistance
3. Fertility plasmids – plasmids required for bacterial conjugation
The plasmid vectors are added with certain features.
1. The size of the vector is reduced to minimum so that larger fragments can be
accommodated. The vector should not be bigger than > 15 kb size.
2. It should contain the origin of replication.
3. It should be added with selectable markers.
4. Introduction of synthetic cloning sites such as poly-linkers is done inside the marker
gene.
5. There is incorporation of Axillary sequence for visual detection of r-clone by histo-
chemical tests.
pBR 322 : It was artificially developed by Boliviar and Rodriguez in 1977. The size
is 4.36kb. It is double stranded having a copy no. of 15 – 20 in E. coli. It has ampicillin
resistance (ampR) and tetracycline resistance ( tetR) genes. The gene coding for β-
lactamase is introduced which modifies the ampicillin resistance gene and inactivates to
make it susceptible to ampicillin. It has been completely sequenced. There are many sites
for restriction enzymes on this plasmid.
pUC : Plasmid University of California : It is 2.7 kb having ampicillin resistance (
ampR) and lacZ genes with many sites for restriction. There are pUC 18, pUC 19 etc.
plasmids.
EMBL : European Molecular Biology Laboratory

Ti plasmid: It has been obtained from Agrobacterium tumefaciens. The Ti plasmid is

a tumor inducing plasmid which cause gall formation in dicot plants. It is invades a plant
tissue and cause cancerous gall formation ( crown gall). A part of Ti plasmid is integrated
into the plant chromosomal DNA. It has been widely used in genetic engineering and
producing transgenic plant. The example of Bt gene incorporation in Ti plasmid is well
known. Similarly, Ri plasmid which are obtained from Agrobacterium rhizogenes which
cause hairy root disease in plants.
( 2 ) COSMIDS : It can be defined as the plasmid vectors that contain a bacteriophage cos
site, which directs insertion of DNA into the phage heads.
 59

Advantages of cosmids:
A. Large DNA upto 45kb can be inserted.
B. DNA can be introduced into the host using bacteriophage derived by in vitro
packaging.
Disadvantages of cosmids:
1. They are difficult to store in bacterial host in glycerol stock solution.
2. In vitro packaging is needed to maintain cosmids inside the viral heads.

( 3 ) BACTERIOPHAGE VECTORS: Bacteriophages are viruses that infect bacteria.

They are also called phages. They have either double stranded DNA( T2, T4, T6 etc.) and
single stranded ( φ x174, M13 etc. ) . There are DNA phages such as T2, T4, T6, λ, φ
x174 and RNA phages such as MS2. The bacteriophage cloning vectors based on λ are λgt10,
λgt11, EMBL3, EMBL4 etc.
( 4 ) PHAGEMIDS: They are plasmid vectors having both bacterial as well as phage origin
of replication site. e.g. pBluescript-II KS.

( 5 ) YEAST ARTIFICIAL CHROMOSOME ( YAC ): They are artificially derived

vectors from the yeast. The biggest advantage is that it can accommodate large piece of DNA
inside them. They can accommodate as large as 1000 – 2000 kb fragment e.g. pYAC4, while
bacterial artificial chromosome ( BAC ) can accommodate around 50 – 300 kb fragment e.g.
pBeloBAC11. This property is exploited in genomic library construction.

( 6 ) SHUTTLE VECTORS: These are the plasmids which are capable of propagation and
transferring – shuttling genes between prokaryote (E. coli) and eukaryote (yeast). The
fundamental requirement is that both should have unique ori site for each type of cell and
different markers for both.

( 7 ) BINARY VECTORS: It can replicate in E. coli or Agrobacterium tumefaciens;

Agrobacterium can cause these vectors to insert into a plant’s chromosome.

Selecting the Vector

Vector Advantages Disadvantages
Can carry foreign DNA Must remain small in
Plasmid into bacteria and yeast size to increase uptake
and avoid damage

Can carry large Technically more

Cosmid
DNA fragments difficult to construct than
recombinant plasmid

Virus Can reach plant and Disabled virus may

human cells regain disease-
disease-
causing properties
 60

GENE TRANSFER AND PLANT TRANSFORATION

WITHOUT VECTORS
The gene can be transferred to any host system without the use of vectors also. The
transfer of gene and plant transformation can also be done using following techniques.
1. Micro injection. 2. Particle bombardment. 3. Direct up take.

1. PARTICLE BOMBARDMENT METHOD:

Foreign DNA is delivered in to plant cells through high velocity metal particles.
a. PLANT METERIAL TO BE USED: Regenerable tissue or organs are required for
bombardment. Generally meristem or immature embryo is used
b. METAL PARTICLES: Gold particles being dense can penetrate in to deeper cell
layers than tungsten.
c. TECHNIQUE OF DELIVERING DNA: Bombardment is done with particle gun.
1. Gun powder driven device-Require more DNA.
2. Helium particle inflow gene more common.

Minute particles (1.0-1.5 micro meter) of gold coated with the DNA of interest. The DNA
coated particles are accelerated with such force that they should penetrate the outer cell wall
of target tissues. Some DNA enters in the nuclei and integrated with the DNA of host
resulting in transformation.

d. IDENTIFCATION AND REGENERATION OF TRANSFORMED TISSUE:

With the help of polymerase chain reaction (PCR) technique transformed tissues are
identified and regenerated in culture medium into whole plant.
 61

2. DIRECT DNA TRANSFER: It involves electroporation with Poly ethyl glycol

(PEG) with calcium phosphate. A suspension of protoplast with desired DNA is prepared.
Then high voltage current is applied through the protoplast DNA suspension. Electric current
leads to formation of small temporary holes in the membrane of protoplast through which the
DNA can pass. After entry in to the cell the foreign DNA gets incorporated with host genes
resulting genetic transformation. This method used only in those crops regeneration is
possible from protoplast e.g Rice.

3. BY MICRO INJECTION : Plasmid DNA is delivered in host cells by mechanical

means with the help of microscopic needles. In this method regeneration from protoplast is
the basic requirement.
 62

Agrobacterium Mediated gene transfer: The Ti plasmid of Agrobacterium can

be used for the plant transformation. The gene of interest can be inserted into the Ti plasmid,
which is introduced into the plant cell and expression takes place.

*****
 63

CHAPTER - 10

LIBRARY CONSTRUCTION

L IB R A R Y C O N ST R U C T IO N : A collection of DNA fragments (small to large) that

represents either the entire genomic DNA sequence of a particular species (Genomic) or the
entire array of coding sequences (cDNA) of a particular cell type, organ), etc…….
The methods used for cloning genes are essentially the same for other cloning methods i.e.
restriction digestion, ligation, etc. In other words, a DNA library is a comprehensive collection of
cloned DNA of the organism of interest.
Libraries are constructed using either a viral or plasmid vector and are generally housed
and propagated in a population of bacterial cells (E. coli). The challenge of a molecular biologist
is to employ all kinds of different methods (radio-labelling, Southern blotting, plasmid-prep,
microbiology, etc.) to pluck out from a library a clone or series of clones corresponding to their
gene of interest.

Why make a library?

There are several reasons why we would want to make a library.
1. To identify the gene coding sequence
2. For expression cloning and production of the protein
3. As a probe for identifying its chromosomal location
4. For the development of an assay to measure its gene expression.

There are two types of library being constructed.

1. Genomic library :– derived from whole genome of an organism.

2. Complimentary DNA (cDNA) library :–derived from purified mRNA
sequence where in splicing of RNA has taken place and only functional RNA
is used.
GENOMIC LIBRARY: Large pieces of randomly cleaved DNA (10 kb-350kb in length)
derived from genomic DNA cloned into an appropriate vector or lambda phage.
Five types of vectors used to clone genomic fragments:
1) Lambda Phage – 10-25 kb
2) Cosmids – ~40 kb
3) P1 Phage - ~100-150 kb
4) BAC Clones – ~100-350 kb
5) YAC Clones – ~250-1000 kb

How do we Randomly Generate a Library?

 Fragments for a genomic library to isolate single-copy genes are produced by partial
digestion of genomic DNA with restriction enzymes. Helps to ensure that there is no
systematic exclusion of sequences from a cloned library.
 64

 Generally purified genomic DNA is obtained from organ tissue (liver is commonly used)
or other source and cut with a frequent restriction enzyme cutter.
 Most common is Sau3A, which recognizes 4-bp GATC that is compatible (can anneal to
the overhangs produce but other enzymes).
 DNA is partially cut to produce a wide variety of sizes (10 –350 kb) and cloned into the
restriction sites of an appropriate vector (i.e. lambda phage, P1 phage, BAC vector).

Genomic libraries are very important to locate the gene of interest on a very large
chromosome of plant. The exact location is not known so if we construct a genomic library and
screen the clones for a particular gene, it will be very useful for gene cloning purpose.
 65

STEPS OF GENOMIC LIBRARY CONSTRUCTION:

1. Isolation of Pure DNA: Different methods can be used and purity can be checked.
2. Partial digestion of DNA: It is digested with such a restriction enzyme, which can
produce 15 -20 kb fragments. e.g. Sau3A which can produce 9 -25 kb fragments. The
remaining genome is uncut.
3. Cloning the fragments in Vector: The cloning is done in lambda phage because of its high
cloning and packaging efficiency. A part of the lambda, which is non-essential is cut and
foreign DNA is inserted. It is called Replacement vector e.g. EMBL3, lambda DASH,
lambda FIX etc.
4. Ligation of fragment to vector: The non-essential region of lambda genome is removed
with same RE used for genomic DNA cutting and vector arms are purified. They are
mixed and annealed together> Proper conditions are chosen for producing r-clones.
5. Packaging: The resulting recombinant lambda phage after the ligation, is packaged by
using packaging extract containing head and tail proteins and some enzymes.
6. Transformation of bacterial cells: The bacterial cell E. coli is transformed with the
recombinant phage, multiplied and tested for particular trait. Identification and analysis
of cloned genes are carried out using several tests.

a. Colony / plaque hybridization

b. Immunological detection.
c. Southern blot analysis and
d. Detection of nucleic acid sequences.

cDNA Library: Many times, another type of library is constructed which is called cDNA
library. In this type, the functional RNA is used.
 66

The procedure of producing cDNA library is shown in the following figure:

STEPS OF CREATION OF cDNA LIBRARY:

Tissue

Extract mRNA

Copy into ssDNA using Reverse transcriptase and dNTPs

Formation of cDNA/RNA hybrid

Treatment of hybrid molecule with RNase

Treatment with DNA polymerase to produce dsDNA

Insert into plasmid vector

Transform bacteria with recombinant plasmid

Selection of transformed bacterial clone containing gene of interest

 67

Two Types of DNA Libraries

Commercial and Public Sources of Genomic and cDNA Libraries

Companies : Stratagene, Invitrogen, Novagen, Amplicon Express, QBiogene, Origene,
Research Genetics, Edge, Incyte Genomics, Genome Systems many of new entries in market.

DIFFERENCES BETWEEN GENOMIC AND cDNA LIBRARY

Genomic Library cDNA Library

Source Done for DNA only Done for mRNA

Intron & exons both are No introns are present

present

DNA sequences All sequences from organism: Transcribed sequences: for

for general cellular functions specific cellular functions

Production Restriction digestion & cDNA produced and Insertion of

insertion of fragments into cDNA into plasmids
plasmids
Screening DNA or mRNA probe mRNA probe

Used to Study genes, introns Study transcribed genes

and regulatory sequences

****
 68

CHAPTER - 11
MOLECULAR MARKERS
For plant genotyping and DNA finger printing, there are several molecular markers used
in plant biotechnology, which detect the polymorphisms at the DNA level.

1. PCR based Techniques: Randomly amplified polymorphic DNA ( RAPD ), Micro-

satellites or simple Sequence Repeat Polymorphism ( SSRP), Amplified Fragment
Length polymorphism (AFLP), Arbitrarily Primed –PCR ( AP-PCR)

2. Non- PCR based Techniques: Restricted Fragment Length Polymorphism (RFLP)

POLYMERASE CHAIN REACTION ( PCR ) : It can be defined as the in vitro method of
DNA replication ( amplification ). It is also called Peoples’ Choice Reaction. Kary Mullis
discovered it in 1983. He was awarded Nobel Prize in Chemistry in 1993. He demonstrated that
oligonucleotides primers could be used to amplify segments of DNA / cDNA. It is a
extraordinarily powerful and versatile technique.

Primers: A pair of short single-stranded oligonucleotides those are identical to the 5'-ends of the
sense and anti-sense strands that will be amplified.

SALIENT FEATURES OF PCR

1. It is a semi-conservative DNA replication by DNA polymerase.
2. It does the selective amplification of chosen region of DNA.
3. The important point is that the DNA molecule is not cloned in plasmid or any vector.
4. The only requirement is the knowledge of sequence of border region of chosen DNA so
that short oligonucleotides can bind.
5. Amplification is carried out in vitro by a thermo-stable heat resistant DNA polymerase
from a organism called Thermus aquaticus. The enzyme is known as Taq DNA
polymerase.
Components of PCR

1) Primers: A pair of short single-stranded oligonucleotides that are identical to the 5'-ends
of the sense and anti-sense strands that will be amplified.
2) Taq DNA polymerase. Taq is a DNA polymerase that was isolated from the bacterium
Thermus aquaticus, which normally lives in hot springs in temperatures close to
100° C. The enzymes from this beast, including Taq, have evolved to be stable at
high temperatures, which means the enzyme is stable under the extreme
temperature conditions of PCR.
3) Template DNA: This is the DNA from which you amplify your fragment of interest. It can
be from any source, including ancient DNA from fossils!
4) dNTPs: Just like in all other DNA sequencing reactions, the nucleotide building blocks
must be present.
STEPS OF PCR
0
a. Denaturation of a template DNA duplex at 94 C by heating ( Separation of two strands ).
 69

[Link] ( joining ) of oligonucleotides primer to the target sequence of separated DNA at

55 - 65 0 C.
c. DNA synthesis by Taq DNA polymerase from the 3’ OH end of each primer by at 72 0 C in
presence of dNTPs ( dATPs, dTTPs, dCTPs, dGTPs ).
 70

TYPES OF PCR:
1. INVERSE PCR ( iPCR ): The PCR which magnifies the sequences, outside the
boundaries of known sequences.
2. RT – PCR ( Reverse Transcriptase - PCR ): mRNA is converted to cDNA using reverse
transcriptase and then PCR is carried out.
3. RAPID AMPLIFICATION OF cDNA END ( RACE ):
4. QUANTITATIVE TRT-PCR: You will be able to quantify the cDNA using this
method.
5. ANCHORED PCR
6. ASSYMETRIC PCR: Partially for sequencing of DNA.
7. PCR FOR SITE DIRECTED MUTAGENESIS
8. MULTIPLEX PCR
9. BOOSTER PCR

GENERAL APPLICATIONS:
( a ) Diagnosis of genetic disorders.
( b ) The genetic identification of samples such as hair, sperm, blood stain involved
in criminal cases, maternal/paternal disputes, rape cases etc.
( c ) Analysis of homologous genes in evolutionary biology.

APPLICATIONS OF PCR IN PLANT BIOTECHNOLOGY:

1. Generation of specific sequence of cloned dsDNA as molecular probe.
2. Generation of probes for cDNA.
3. Generation of cDNA libraries from small amount of RNA.
4. Sequencing of DNA
5. Sexing of the embryo
6. DNA finger printing in criminal and court cases in forensic sciences.
7. Identification of genetic mutations in prenatal diagnosis in babies and in plant.
8. PCR detection of a gene transferred into a plant genome.
9. In the study of population biology and evolutionary studies.
10. Used as a molecular marker technique in RAPD (Randomly amplified
polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism).

ADVANTAGES OF PCR:
A. Large amount of amplification from small amount of DNA is possible. Nanogram
amounts of DNA can be generated from a single template molecule.
B. Technique is quick and simple.
C. Technique is very sensitive.
D. The biggest advantage is that the DNA need not be in pure form.
 71

PROBLEMS OF PCR:

1. Nucleotide sequence of boundary region must be known.

2. As PCR is very sensitive technique, it may generate false signals due to previous
contamination.
There are several differences between PCR and gene cloning

PCR Gene cloning

Final Result Selective amplification of Selective amplification of
DNA of our choice DNA of our choice
Manipulation Manipulation is done in vitro Manipulation is done in vivo
Selection of specific First step Last step
segment of DNA
Concentration of Need only nanogram quantity Needed in microgram quantity
starting material
Biological reagent DNA, Taq DNA polymerase. Restriction enzyme, vector,
requirement dNTPs bacterial cell, markers for
detection
Automation Yes No
Labor intensive No Yes
Error probability Less More
Applications Much more Many
Cost Less More
Users’ skill Not required. Has been Required
automated
Time taken for end Hardly 2 – 3 hrs. 2 – 3 days
result

RFLP :- Restriction Fragment Length Polymorphisms

Some of the techniques developed for DNA manipulation are used to detect DNA
variations known as restriction fragment length polymorphisms (RFLPs) where use of cloned
fragments of chromosomal DNA as genetic markers, usually termed as "RFLP mapping" with
RFLP denoting' Restriction Fragment Length Polymorphism is done.

In this technique, DNA is digested with a restriction enzyme. Homologous restriction

fragments of DNA, which differs in size or length can be used as genetic markers to follow
chromosome segments through genetic crosses. A large piece of DNA is reduced to a series of
smaller fragments of defined size by digestion with restriction enzymes. The fragments produced
will thus be specific for each target DNA restriction enzyme combination and can be used as a
"finger print" specific for a given target DNA or for the organism containing that DNA.
 72

Advantages of RFLP markers

1. RFLP markers can be directly screened at DNA level and therefore behave in a
codominant manner.
2. The phenomenon of dominance/recessivity does not operate.
3. Allelic variation is much greater than that for morphological markers.
4. These markers are phenotype neutral.
5. These markers are free of epistatic interactions of environmental factors.

Applications of RFLP

1. Abundant natural variation in DNA sequences provides unlimited number of RFLPs. This
is helpful in construction of saturated linkage maps.
2. RFLPs' closely linked to qualitative / quantitative traits, can be used for tagging purposes.
3. RFLPs can be used in directed or compressed (in time and space) breeding activities.
4. RFLPs can be used in strain and varietal identification.
5. RFLPs can be employed in germplasm cataloguing.
6. Screening human DNA for the presence of potentially deleterious genes.
7. Providing evidence to establish the innocence of, or a probability of the guilt of, a crime
suspect by DNA "fingerprinting" in forensic science.

Steps of RFLP :

1. Isolation and purification of high molecular weight plant DNA.

2. Cut the DNA with Restriction enzymes producing DNA fragments of precisely defined
length. Separate them by electrophoresis, with the smaller fragments migrating farther
than the larger fragments.

3. Vacuum blotting of DNA from agarose gel and transferring them on nitrocellulose
membrane. Preparation of plasmid DNA, purification and in vitro labelling and
separation of labeled DNA probe.

4. Southern hybridization using the radio labeled (or fluorescent) probe with the fragments
on the nitrocellulose. One or more of the fragments can be visualized with a "probe" (a
molecule of single-stranded DNA that is complementary to a run of nucleotides in one or
more of the restriction fragments.).

5. Autoradiography on X ray film. If probes encounter a complementary sequence of

nucleotides in a test sample of DNA, they bind to it by Watson-Crick base pairing and
thus identify it.
 73

RAPD (Randomly Amplified Polymorphic DNA) :

“It is PCR based amplification of random DNA segments with single primer
which detect polymorphism in the absence of specific nucleotide sequence
information.”. Here single short oligonucleotides primers are arbitrarily selected to
amplify a set of DNA aegments distributed randomly through out the genome.

RAPD STEPS…

1. DNA extraction
2. DNA amplification by PCR using random primers
3. Amplified products separated by gel electrophoresis
4. Visualization of markers on the gel & gel photography
 74

COMPARISON OF RAPD AND RFLP

CHARACTERISTICS RAPD RFLP

Principle DNA amplification Restriction Digestion

Detection DNA staining with Etbr Southern Blotting on X

ray films
DNA quantity required 5 – 25 nanogram 5.0 microgram

Purity of the DNA required Crude can also work Relatively pure DNA

Primer requirement Random primer No primer

Probe requirement None Set of specific probe

Use of radio-isotopes None Yes

Part of genome surveyed Whole genome Generally low copy

coding region
Types of probe requirement Random 9 -10 mer Species specific cDNA

Dominance Dominant/ Null Co-dominant

Automation Easy Difficult

Reliability Intermediate High

Recurring cost High High

APPLICATIONS OF RAPD:
1. Construction of genomic maps. It has been done for Arabidopsis, Helianthus, pine
etc.

2. Mapping of traits: Used for indirect selection in segregating populations during plant
breeding programs. It is used for tagging genes of economic value.

3. Analysis of genetic structure of populations.

4. Finger printing of individuals

5. Targeting markers to specific regions of genome.

6. Used for identification of somatic hybrids

7. Used and recommended for evaluation of characteristics of genetic resources.

 75

LIMITATIONS OF RAPD:

A. RAPD polymorphisms are inherited as dominant-recessive characters

B. RAPD markers are short primers so even a mistake in even a single nucleotide can
prevent the amplification.

C. RAPD is sensitive to changes in PCR conditions resulting in changes to some of the

amplified fragments.

Amplified Length Fragment Polymorphism ( AFLP ) :

“It is a method for PCR amplification of restriction digests of genomic DNA

following the ligation of oligonucleotides.” It is a combination of RFLP and RAPD
methods. It is applicable universally and is highly reproducible. It is based on PCR
amplification of genomic restriction fragments generated by specific restriction enzymes
and oligonucleotide adapters of few nucleotide bases.

AFLP STEPS…
1. DNA extraction
2. DNA digestion with restriction enzymes
3. Ligation of oligonucleotides
4. Primer annealing
5. DNA amplification using PCR
6. Separation of amplified DNA on agarose gel
7. Visualization of markers on the gel & gel photography

ADVANTAGES OF AFLP:

1. Extremely sensitive technique

2. It has high reproducibility making it superior to RAPD
3. It has wide scale applications
4. It discriminates heterozygote from homozygote when a gel scanner is used.

*****