BT403

AGRICULTURAL BIOTECHNOLOGY

HANDOUTS

VIRTUAL UNIVERSITY OF PAKISTAN

 1. Agriculture Biotechnology Introduction

Biotechnology: it is use of biological processes, organisms, or systems. It is defined as a set of

tools that uses living organisms (or parts of organisms) to make or modify a product, improve
plants, trees or animals, or develop microorganisms for specific uses.

Benefits of Biotechnology

It is used for all these three purposes

HEAL THE WORLD

Biotechnology heals the world by creating more precise tools for disease detection and by
reducing rates of infectious disease.

FUEL THE WORLD

It fuels the world by using biofuels, improving manufacturing process efficiency and by
reducing the use of and reliance on petrochemicals

FEED THE WORLD

It feeds the world by generating higher crop yields, using biotech crops and by improving food
and crop oil content.

Agriculture Biotechnology is collection of scientific techniques to improve plants, animals

and microorganisms, to manipulate the genetic makeup, for the production or processing of
 agricultural products, to Increase agricultural productivity, to enhance breeders’ ability to make
improvements in crops and livestock and to enable improvements that are not possible with
traditional crossing of related species alone.

Agricultural biotechnology is the term used in crop and livestock improvement through
biotechnology tools. This monograph will focus only on agricultural crop biotechnology.
Biotechnology encompasses a number of tools and elements of conventional breeding
techniques, bioinformatics, microbiology, molecular genetics, biochemistry, plant physiology,
and molecular biology.

2. History of agriculture biotechnology

A brief history

 It is practiced for so long about 8-10,000 years ago to improve organisms by: Selection
and breeding
 Selection: it is selection and saving of the best looking plants and seeds, the selection
features are: Faster growth, higher yields, pest and disease resistance, larger seeds, or
sweeter fruits
 Breeding: it is done by artificially mating, cross-pollination, and desirable
characteristics from different parent plants could be combined in the offspring.
Superior plants are selected and breed them to create new and improved varieties of
different crops.
 In 1990 -The first food product of biotechnology (an enzyme used in cheese production
and a yeast used for baking) appeared on the market.
 In1995, farmers have been growing genetically engineered (GE) crops.
 In 2003, 7 million farmers in 18 countries more than 85 percent of them resource-poor
farmers in the developing world were planting biotech crops.

Why is agricultural biotechnology important?

In a world where 7 Billion people, living mostly in rural areas, go hungry every day, Food demand
is set to double in the next thirty years and arable land is limited, Advances in agriculture are critical
if we are to reduce hunger and promote growth and development in a socially acceptable and
environmentally sustainable way.
 Benefits of agriculture biotechnology
Agricultural biotechnology has been used to protect crops from disastrous diseases. Biotech crops
can make farming more profitable by increasing crop quality and may in some cases increase
yields. Biotech crops may provide enhanced quality traits such as increased levels of beta-carotene
in rice to aid in reducing vitamin A deficiencies. Agriculture biotechnology produces herbicide-
tolerant crops.The tools of agricultural biotechnology have been invaluable for researchers in
helping to understand the basic biology of living organisms. Biotechnology has helped to make
both insect pest control and weed management safer and easier while safeguarding crops against
diseases.

3. Applications of agriculture biotechnology I

Genetic engineering:

Genetic engineering is also called genetic modification, is the direct manipulation of an

organism's genes using biotechnology. All crops improved with transferred DNA to date
have been developed to aid farmers to increase productivity by reducing crop damage from
weeds, diseases or insects. It inserts fragments of DNA into chromosomes of cells, uses
tissue culture to regenerate the cells into a whole organism with Different genetic
composition from the original cells. This is also known as rDNA technology that produces
transgenic organisms.

Molecular markers: Scientists can use molecular markers to select plants or animals that
possess a desirable gene, even in the absence of a visible trait. Thus, breeding is more precise
and efficient.

Types of Molecular markers:

There are three types of molecular markers:

Morphological markers:

Morphological markers are those traits that are scored visually, or morphological markers are
those genetic markers whose inheritance can be followed with the naked eye.

Biochemical molecular markers:A molecular marker is a molecule contained within a

sample taken from an organism (Biochemical molecules). The first biochemical
molecular markers used were the protein based markers. One of the earliest protein based
markers to be used was Isozyme. These are different forms of an enzyme exhibiting the
same catalytic activity but differing in charge and electrophoretic mobility.

DNA based markers:

The sequence of nucleotides in DNA of an individual is unique and thus determines its identity.
The ultimate difference between individuals lies in the nucleotide sequence of their DNA. These
can be used to diagnose the presence of the gene without having to wait for gene effect to be
seen.

4. Applications of agriculture biotechnology II

Molecular diagnostics: Molecular diagnostics are methods to detect genes or gene products
that are very precise and specific. Molecular diagnostics are used in agriculture to more
accurately diagnose crop/livestock diseases.

Vaccines;

Biotechnology-derived vaccines are used in livestock and humans. They may be cheaper,
better and/or safer than traditional vaccines. They are also stable at room temperature, and do
not need refrigerated storage. Biotechnology-derived vaccines are used in livestock and
humans. They are cheaper, better, safer than traditional vaccines, stable at room temperature
and do not need refrigerated storage

Tissue culture

Tissue culture is the regeneration of plants in the laboratory from disease-free plant parts.
This technique allows for the reproduction of disease-free planting material for crops.
Examples of crops produced using tissue culture include citrus, pineapples, avocados,
mangoes, bananas, coffee and papaya.

Flowers

Gene identification and transfer techniques used to improve the color, smell, size and other
features of flowers. It is used to make improvements to other common ornamental plants, in
particular, shrubs and trees. Some of these changes are similar to those made to crops, such
 as enhancing the cold resistance of a breed of tropical plant, so it can be grown in northern
gardens.

Nutrient Supplementation

In an effort to improve human health, nutrient supplementation is needed, particularly in

underdeveloped countries, scientists are creating genetically altered foods that contain nutrients
known to help fight disease or malnourishment. An example of this is Golden Rice, which contains
beta-carotene, the precursor for Vitamin A production in our bodies.

Pest resistant crops

Pest resistant GM crops (primarily cotton and maize) are genetically modified so they are toxic to
certain insects. They are often called Bt crops. The introduced genes were originally identified in a
bacterial species called Bacillus thuringiensis.

5. Tissue culture

Introduction: Tissue culture is the term used for “the process of growing cells artificially in the
laboratory” Tissue culture involves both plant and animal cells. Tissue culture produces clones,
in which all product cells have the same genotype (unless affected by mutation during culture).
 Brief history: Tissue culture had its origins at the beginning of the 20th century with the work
of 1- Gottleib Haberlandt (plants) and 2-Alexis Carrel (animals).

The first commercial use of plant clonal propagation on artificial media was in the germination
and growth of orchid plants, in the 1920’s

In the 1950’s and 60’s there was a great deal of research, but it was only after the development
of a reliable artificial medium (Murashige & Skoog, 1962) that plant tissue culture really ‘took
off’ commercially

Critical requirements:

Tissue culture of both plant and animal has several critical requirements.

1. Appropriate tissue (some tissues culture better than others)

2. A suitable growth medium containing energy sources and inorganic salts to supply cell
growth needs.

3. This can be liquid or semisolid

4. Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and
animal tissue and can over run a culture

5. Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well
defined and the growth substances are provided in serum from the cell types of interest
 6. Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste
metabolites

Steps of tissue culturing

1. selection of explant:

Explants are small pieces of plant parts or tissues that are aseptically cut and used to initiate a
culture in a nutrient medium. Explants can be taken from different parts of a plant such as
shoots, leaves, stems, flowers, roots, and from many types of mature cells provided they are
able to de-differentiate into totipotent cells.

2. Establishment of the explant

Establishment of the explant in a culture medium The medium sustains the plant cells and
encourages cell division. It can be solid or liquid

3. Multiplication

The explant gives rise to a callus (a mass of loosely arranged cells) which is
manipulated by varying sugar concentrations and the auxin (low): cytokinin
(high) ratios to form multiple shoots.The callus may be subdivided a number of times Dividing
shoots.Warmth and good light are essential
4. Root formation

The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios.
Promote root formation.Ready to transfer to soil

6. Growth Regulators
Growth

Growth is an irreversible change in Mass, i.e. increase in size, volume and weight of any part
of plant’s body. It means quantitative increase in plant body e.g. Cell division Cell
enlargement.

Development

Development is an irreversible change in state. It means the qualitative change in plant body
e.g. Seed Seedling Vegetative maturation Flowering. Growth is a continuous process
Development is phase to phase process.
Plant growth regulating compounds

 Plant growth regulating compounds are:

 Natural and Synthetic

 Natural- found naturally in plants

 Synthetic- human made

 Both groups regulate or influence:

 Cell division

 Cell differentiation

 Root and shoot growth

 Senescence (plant aging)

Plant growth regulators

Plant Growth regulators (PGR) refers to natural or synthetic substances influence the growth
and development.

Classification of PGR On the Basis of Origin

Natural hormone: natural hormones are produced by some tissues in the plant they are also
called endogenous hormones. e.g. IAA (Indol acetic acid)

Synthetic hormone: synthetic hormones are produced artificially and similar to natural
hormone in physiological activity. They are also clso called Exogenous hormones. e.g. 2,4- D,
NAA (Naphthalene acetic acid).

Growth promoting hormones/Growth promoter: they increase the growth of plant. e.g.
Auxins. Gibberellins, Cytokinins etc.

Growth inhibiting hormones/Growth retardant: they inhibit the growth of plant. e.g. ABA,
Ethylene.

Auxins

It is derived from the Greek word "auxein" means- "to grow/increase". Auxins may be defined
as growth promoting substances which promote growth along the vertical axis when applied
in low concentration to the shoot of the plant. Auxins are synthesized in the stem and root
apices and transported through the plant axis. They occur universally in all plants as Active
growth = Auxin production.

Role of Auxins: Auxins stimulate cell elongation and influence a host of other developmental
responses, they are involved in root initiation, vascular differentiation, tropic responses, apical
dominance and in development of auxiliary buds, flowers and fruits

Auxins in plant tissue culture are used to induce callus from explants , and cause root and shoot
morphogenesis and parthenocarpy

Gibberellin: They have a regulatory function and are produced in the shoot apex primarily
in the leaf primordial (leaf bud) and root system.

Role of gibberellins: They are used to stimulates stem growth dramatically, they also
stimulate cell division, Cell elongation (or both) and controls enzyme secretions, they
are involved in overcoming dormancy in seeds and buds also used commercially in
increasing fruit size of seedless grapes and Stimulating seed germination & seedling
growth

Cytokinins :

Roles of cytokinins are : Promotion of cell division also found in all tissues with
considerable cell division. Ex: embryos (seeds) and germinating seeds, young developing
fruits roots supply cytokinins upward to the shoots. They also interact with auxins to
influence differentiation of tissues (may be used to stimulate bud formation).

Effect of cytokinins
 Ethylene

It is a growth retardant. Ethylene promotes ripening , it is gaseous hormone, it is produced

in the actively growing meristems of the plant, in senescing ripening or ageing fruits, in
senescing (ageing or dying) flowers, in germinating seeds and in certain plant tissues as a
response to bending, wounding or bruising. Ethylene as a gas, diffuses readily throughout the
plant. It may promote leaf senescing and abscission (leaf fall). Increases female flowers in
cucumbers (economically - will increase fruit production). It is also responsible for de greening
of oranges, lemons and grapefruit – ethylene gas breaks down chlorophyll and lets colors show
through.

Abscissic acid

It is also a growth retardant which induces stomata closing, also involved in nhibition of bud
growth and shoot formation. It is widespread in plant body – moves readily through plant.
ABA appears to be synthesized by the leaves, it Interacts with other hormones in the plant,
counteracting the growth - promoting the effects of auxins & gibberellins. It is also involved
with leaf and fruit abscission (fall), onset of dormancy in seeds and onset of dormancy (rest
period) in perennial flowers and shrubs. ABA is effective in inducing closure of stomata in
leaves, indicating a role in the stress physiology in plants. (ex: increases in ABA following
water, heat and high salinity stress to the plant)

7. Sterile Techniques
Sterile techniques are used to clean equipment and for surface sterilization of explants.

Clean Equipment
It is used for successful tissue culture requires the maintenance of a sterile environment. All
tissue culture work is done in a laminar flow hood. The laminar flow hood filters air with a
dust filter and a high-efficiency particulate air (HEPA) filter. It is important to keep the hood
clean, which can be done by wiping it with 70% alcohol. The instruments used should also be
dipped in 70% ethanol and sterilized using flame or glass beads. Hands should be disinfected
with ethanol before handling cultures in order to avoid contamination.

It is imperative to maintain axenic conditions throughout the life of cultures: from explant to
the production of whole plants. Entire experiments have been lost because of an episode of
fungal or bacterial contamination at any stage of culture. Especially problematic are fungal
contaminants that are propagated by spores that might blow into a hood from an
environmental source. Therefore, it is important to work away from the unsterile edge of a
laminar flow hood.

Surface Sterilization of Explants

Plant tissues inherently have various bacteria and fungi on their surfaces. It is important that
the explant be devoid of any surface contaminants prior to tissue culture since contaminants
can grow in the culture medium, rendering the culture non sterile. In addition, they compete
with the plant tissue for nutrition, thus depriving the plant tissue of nutrients. Bacteria and
especially fungi can rapidly overtake plant tissues and kill them. The surface sterilants are
chosen for an experiment typically depend on the type of explant and also plant species.
Explants are commonly surface-sterilized using sodium hypochlorite (household bleach),
ethanol, and fungicides when using field-grown tissues. The time of sterilization is dependent
on the type of tissue; for example, leaf tissue will require a shorter sterilization time than will
seeds with a tough seed coat. Wetting agents such as Tween added to the sterilant can
improve surface contact with the tissue.

Although surface contamination can be eliminated by sterilization, it is very difficult to

remove contaminants that are present inside the explant that may show up at a later stage in
culture. This internal contamination can be controlled to a certain extent by frequent transfer
to fresh medium or by the use of a low concentration of antibiotics in the medium.
Overexposing tissues to decontaminating chemicals can also kill tissues, so there is a
balancing act between sterilizing explants and killing the explants themselves.

Culture Conditions and Vessels

Cultures are grown in walk-in growth rooms or growth chambers. Humidity, light, and
temperature have to be controlled for proper growth of cultures. A 16-h light photoperiod is
optimal for tissue cultures, and a temperature of 22–258C is used in most laboratories. A
light intensity of 25–50mol.m-2s-1 PAR (photosynthetically active radiation) is typical for
 tissue cultures and is supplied by cool white fluorescent lamps. A relative humidity of 50–
60% is maintained in the growth chambers. Some cultures are also incubated in the dark.
Cultures can be grown in various kinds of vessels such as petri plates, test tubes, “Magenta
boxes,” bottles, and flasks

8. Basic Steps of plant tissue culture

Plant tissue culture

• Plant tissue culture is a technique of growing plant cells, tissues, organs, seeds or other
plant parts in a sterile environment on a nutrient medium

Basic techniques of plant tissue culture are

 Culture vessels
 Culture medium
 Sterilization
 Inoculation
 Incubation
 Induction of callus
 Morphogenesis = Organogenesis Embryogenesis
 Hardening
Culture vessels: Cultures can be grown in various kinds of vessels such as petri plates, test
tubes, “Magenta boxes,” bottles, and flasks.
Culture medium: The important media used for all purpose experiment are Murashige and
Skoog medium (MS medium). The culture medium is closed with cotton plug/ or aluminium
foil sheet. The pH of the medium is adjusted to 5.8 (acidic range).

Composition of culture medium:

• Nutrient Medium
• Medium depends upon the type of plant tissue or cell used for culture
• Generally nutrient consist of
• inorganic salts (both micro & macro elements) a carbon source (usually sucrose)
• Vitamins (eg. nicotinic acid, thiamine, pyridoxine
• Amino acids (eg. arginine)
• Growth regulators (eg. auxins)
• An optimum pH (5.7) is also vary important
Sterilization

Sterilization Methods are used in Tissue Culture Laboratory. All the materials, e.g., vessels,
instruments, medium, plant material, etc., used in culture work must be freed from microbes
therefore, they are sterilized.

Sterilization techniques

• sterilization is achieved by one of the following approaches:

• dry heat treatment
• flame sterilization
• autoclaving
• filter sterilization
• wiping with 70% ethanol
• surface sterilization.
Inoculation

Transfer of explant (root, stem, leaf, etc.) on to a culture medium is called inoculation. The
inoculation is carried out under aseptic condition for which an apparatus called laminar air
flow chamber is used. Flamed and cooled forceps are used for transfer of plant materials to
different culture media kept in glasswares.

Incubation

The culture medium with the inoculum is incubated at 26 - 28oC with the light intensity at
2000 to 4000 lux (unit of intensity of light) and allowing photoperiod of 16 hour of light and
8 hours of darkness.
 Induction of callus: Due to activity of auxins and cytokinins, the explant is induced to form
callus. The callus is an unorganized mass of undifferentiated tissue. The mechanism of callus
formation is that auxin induce cell elongation and cytokinin induces cell division as a result of
which masses of cells are formed.

 Morphogenesis
Formation of new organs from the callus under the influence of auxin and cytokinin is called
morphogenesis.

Roots and shoots are differentiated from the callus.

Such embryos are called somatic embryos result in the formation of young plantlet.

 Types of morphogenesis:
Organogenesis

Embryogenesis

 Organogenesis: it is formation of new organs such as shoot and root is known as

organogenesis. The development of shoot from the callus is called caulogenesis and
formation of root is called rhizogenesis respectively.
 Embryogenesis
Formation of embryos (ie. bipolar structure having shoot and root) from the callus is called
embryogenesis.

These embryos arise from somatic callus tissue and are called somatic embryos or embryoids
or somaclonal embryos.

 Hardening
Exposing the plantlets to the natural environment in a stepwise manner is known as hardening.

Finally the plantlets are gradually transferred to the soil.

9. Types of tissue culture, callus culture

Types of tissue culture
 These are the types of in vitro cultures
 Callus Cultures
 Cell Suspension Culture
  Anther/Microspore Culture
 Protoplast Culture
 Embryo Culture
 Meristem Culture
Types of in vitro cultures

Callus Culture

It is an unorganized mass of thin-walled parenchyma cells which is involved in formation of wound

callus is observed in all groups of living organisms. In Plant tissue culture, callus is produced by
cultivation of plant tissue on nutrient media under in vitro conditions. Presence of growth hormones in
the culture media promotes callus formation and proliferation

Role of Callus

In vitro callus provides totipotent cells for plant regeneration via organogenesis or somatic
embryogenesis. Callus is used as a target tissue for genetic transformation. Callus formation is
initiated for plant regeneration of other transformed tissues

Dispersal of friable callus into single cells is used for the initiating cell suspension cultures
Initiation and maintenance of callus cultures

(1) Selection of suitable parent material

(2) Choice of explant and method of isolation

(3) Culture medium and conditions required

(4) Optimization of culture conditions

Selection of suitable parent material

Parent plant must be healthy and free from decay or disease

Mother plant be actively growing and not be about to enter a period of dormancy

Choice of explant and method of isolation

 Any part of mother plant; Plant organs or specific plant tissue or plant cells
 Explant must contain living cells
 Younger tissue is more callus responsive due to the presence of large no. of actively
dividing cells
 Explants isolated in sterile conditions
 Ensure proper sterilization methods for a particular explant
Culture medium and conditions required

 Culture the sterilized explants on suitable autoclaved culture medium

 Incubate cultures at 22-24 ± 2°C in light or dark
 Most callus will be initiated from the cut surfaces within 3-8 wks.
Optimization of culture conditions

 Consult literature to know previous callus initiation attempts for species under
consideration
  For pioneering callus culture attempt, modify medium previously used for a related
species
 Start with one of the defined media and manipulate hormone concentrations
 Set up a growth Latin Square of 25 culture plates with 5 each of auxin and cytokinin
conc.
 Callus for this growth trial should be uniform and of large (20 mg) size.
Callus growth measurement

 Subculture vigorous growing callus (2-5 mm dia.)

 Slow growing callus plus explant transferred to fresh medium
 Callus growth assessment via fresh and dry wets.
 Plot a growth curve.

10. Cell Suspension Cultures

Cell suspension cultures are rapidly dividing suspensions of cells grown in liquid medium; they
grow more rapidly than callus cultures and more ready to culture manipulations. They are
comprised of cell aggregates and dispersed single cells

Techniques of cell culture

Initiation of cell suspension culture

Fragments of undifferentiated callus 2 - 3 g / 100 mL

Liquid medium

↓ aeration --------

Subculture

↓ agitation --------

Suspension cell cultures

Initiation of Cell Suspensions:

Initiation and establishment time depends on plant species and growth medium. Dicots are
easier to establish in suspensions than monocot. It is initiated by agitating a fragment of callus
in a vol. of liquid medium on a shaker, there are three procedures used for cell dispersion:

1. Initiate from friable callus

2. non-friable callus

3. Callus treated with cell wall degrading enzymes

Initiation from Friable Callus

It is most commonly used starting material, easily fragmented during agitation in liquid medium,
achieved by:

 Callus passaged on a 7d cycle for 2-3 wks.

 Ratio of auxin/cytokinins altered, increase auxin concentration. Use 2-3 g of friable
callus per 100 ml of liquid medium.
 Low levels of callus tissue fail to replicate.
 Subculture the cells to fresh medium at a ratio of 1:1. Filter the actively dividing cell
suspensions to remove large callus aggregates to get fine suspensions
Initiation from non-friable callus

First repeatedly subculture or transfer callus fragments to semi-solid medium until friable,
initiate and establish cell suspensions

• Initiation from callus treated with enzymes

 Pectinase
 Cellulase
(breaks down middle lamella of cell wall and separates plant cells)

Maintenance of Cell Suspensions

• Varieties of culture vessels are available, suitable ones allow large surface area to
maximize gas exchange. E.g. 20 ml culture medium + 100 ml flask, 50-100 ml in a 250
ml flask, flasks enclosed with sterilized/dried aluminium foil caps. Shaker speed should be
100-120 rpm with optimum incubation conditions

Small batch culture

• Culture volume : in a small fixed volume : <100 ML

Growth Characteristics of Cell Suspensions

 Plant cell suspensions consist of cells with diverse morphology and state of
aggregation. Two morphological types of cells can be distinguished:

 Cell aggregates made up of small cells

 Large and elongated single cells

 Proportion of the cell type depends on the passage of culture and nature of auxin

Monitoring the Growth of Culture

To monitor the growth following should be checked:

 Cell number
 PCV
 Fresh wt. and Dry wt.
 Cell Viability
 Medium conductivity & pH
 Use two methods simultaneously till stationary phase

Growth Curve of Suspension Cultures

 Plotted for fresh, dry wts. and PCV.

 Gives the length of culture cycle
 Used to decide the subculture interval of suspensions
 Lag Phase
 Exponential Phase
 Linear Phase
 Decelerating Phase
 Stationary Phase
Uses of Cell Suspensions

You can study various factors and compounds affecting growth and differentiation, cell
division, rapid preparation of protoplasts, large scale production of commercial plants via
somatic embryogenesis and commercial production of secondary metabolites.

11. Anther/Microspore Culture

Introduction

Anthers or pollens can be cultured on a suitable medium containing sucrose (usually 2%), iron,
vitamins, hormones etc. The hormonal component of the medium is important for initiation of
growth. Usually to the culture medium auxin, cytokinin etc. are added either singly or in
various combinations. Low concentration of auxin stimulates callus formation. In a medium
supplemented with auxin embryoid formation usually occurs at a faster rate as observed in
 anther culture of Datura. Anthers cultured on a medium containing coconut milk or kinetin
develop embryoids which later form haploid plantlets. Callus is formed from pollen grains on
a medium supplemented with yeast extract or casein hydrolysate.

Factors Affecting Anther and Pollen Culture:

1. Activated charcoal:

It has a stimulatory effect on embryogenesis and this has been observed in anther cultures of
potato, rye, tobacco, etc. This may be due to removal of inhibitory substances from agar by
activated charcoal. Charcoal may absorb the degradation product (5-2-furfural) of sucrose.
Anther cultures of Petunia and Nicotiana indicate that activated charcoal removes both
exogenous and endogenous growth hormones from culture medium.

Temperature:

Temperature has significant effect on pollen embryoid development. In Datura embryoids are
not formed if cultures are maintained at 20°C or below. In Nicotiana tabacum optimal
temperature for embryoid growth is 25°C.

Pre-treatment of anthers at 3—10°C for 2—30 days stimulates embryogenesis. Wenzel (’77)
observed that buds of Secale cerale pretreated at 6°C for 6—10 days develop embryoids. In N.
tabacum if the buds are pre- treated at 5°C for 72 hours than 58% anthers produce embryoids.
Sometimes pre-treatment at high temperature helps embryoid formation. In Brassica
campestris pre-treatment of anthers at 35°C for 24 hours helps embryoid formation.

Centrifugation of the anthers at 3—5°C for approximately 30 minutes helps embryoid

formation

Stage of the anther:

• Particular stage of the anther at the time of culture is important. Usually anthers just before
or immediately after pollen mitosis are most suitable for culture.

• Suitable stages of anthers for culture are pre-mitotic, mitotic and post-mitotic.

a. Pre-mitotic stage:
Anthers at this stage have microspores which have just completed the first meiotic division
and the pollens are immature, uninucleate and starch-free.

Anthers of Hordeum vulgare and Hyocyamus at this stage are suitable for culture. According
to Nitsch (’72) and Sunderland (’71) anthers with uninucleate pollens are suitable for culture.

b. Mitotic stage:
 In some plants, anthers at first pollen division stage are most suitable for culture, as observed
in Nicotiana tabacum and Datura innoxia.

c. Post-mitotic stage:

Early bi-cellular stage of pollen development is most suitable for culture in Atropa belladonna
and Nicotiana sp. etc. Mature anthers are usually unsuitable for culture, but in Brassica oleracea
mature anthers are the proper stage for anther culture. Anthers of proper stage are chosen by
selecting flower buds of definite length under fixed environmental conditions.

Photoperiod and light intensity:

Higher number of embryoids are formed when anthers are taken from plant grown under short
days and high light intensities.

Flowering time:

Anthers taken from flowers at the beginning of the flowering period of the plant are most
suitable for culture.

Endogenous auxin:

Embryogenic pollens are found near the tapetum within the anthers. The tapetum may release
some substance which initiates embryogenic development in pollens. This is observed in
Hyoscyamus niger by Raghavan (’78).

Age of the plant:

Usually anthers from younger plants are more suitable for culture.

Methods of Anther culture

1. Selected plants are cultivated until they reach flower bud stage.

2. In some cases flower buds are chilled few days prior to culture.

3. Flower buds of proper size and developmental stage are taken and surfaces sterilized with
alcohol or hypochlorite solution for 10—20 minutes. Buds are rinsed several times in sterile
double distilled water.

4. The anthers are carefully excised from flower buds using force and dissecting needle.
Filaments must be removed prior to culture; otherwise callus may be formed at the cut ends.

5.Anthers may be cultured either on agar-solidified culture medium or placed on a filter paper
bridge over a liquid medium.
 6. Anthers are cultured at 25°G in presence or absence of light. Light is essential after plantlets
are formed. Continuous illumination from cool white fluorescent lamp of 300 lux is satisfactory.

7. After a period of 4—5 weeks in culture plantlets are formed. From a single anther many
plantlets are formed

8. These plantlets are carefully separated quite early and cultured on a fresh root-inducing
medium containing 0.5% agar and all other components in half-strength to that of the anther
culture medium.

9. After formation of proper root system they are transplanted to pots. These pots are preferably
kept in humid condition for few days

12. Protoplast Culture

Protoplasts

Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane
and all other cellular components. They represent the functional plant cells but for the lack of
the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and
this process is referred to as somatic hybridization (or protoplast fusion). Cybridization is the
phenomenon of fusion of a normal protoplast with an enucleated (without nucleus) protoplast
that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).

Protoplast isolation: It refers to the separation of protoplast from plant tissue, it is important to
isolate viable and uninjured protoplast as gently and as quickly as possible, it involves two
methods:

a. Mechanical

b. Enzymatic

Mechanical method:
 Tissue is immersed in 1.0 M sucrose until protoplasm shrunk away from their enclosing cell
wall (Plasmolysis). Plasmolysed tissue is cut with a sharp knife at such a thickness that only
cell walls are cut Plasmolysed cell. Undamaged protoplast in strips are released by osmotic
swelling when placed in a low concentration of sucrose solution. Problem encountered: some
cells release uncut complete protoplast while the rest produces broken dead protoplasts

Enzymatic method:

it refers to the use of enzymes to dissolve the cell wall for releasing protoplasts. It involves two
methods:

I. Direct method (One step only)

II. Sequential method (Two step method)

 Direct method :
 Incubation of leaf segments overnight in enzyme solution
 Mixture is filtered and centrifuged
 Protoplast forms pellet
 Then washed with sorbitol and re-centrifuged
 Clean protoplasts float
 They are pipetted out

Sequential method

Two enzyme mixtures(mixture A and mixture B) are used one after the other. Leaf segments
with mixture A (Macerozyme in manifold at pH 5.8) are vacuumed infiltrated for 5 mins,
transferred to a water bath at 25°C and subjected to slow shaking. The enzyme mixture is
then replaced by fresh ‘enzyme mixture A’ and leaf segments are incubated for another hour.
he mixture is filtered using nylon mesh and centrifuged for 1 min, washed 3 times with 13%
mannitol . Cells are then incubated with ‘enzyme mixture B’ (Cellulase in mannitol solution
at pH 5.4) for above 90 mins at 30°C. The mixture is centrifuged for 1 min so that protoplast
form a pellet and clean 3 times with sorbitol

Purification of protoplast

Protoplasts are purified by removing: Undigested material (debris),Bursts protoplasts , and

enzymes. Debris are removed by filtering the preparation through a nylon mesh . Enzymes
are removed by centrifugation whereby the protoplasts settle to the bottom of the tube and
the supernatant removed with the help of a pipette. Intact protoplasts are separated from
broken protoplasts through centrifugation and removed by a pipette as they are collected at
the top of tube

Protoplast Culture

Isolated protoplast can be cultured in an appropriate medium to reform cell wall and
generate callus. Optimal culture conditions are:

 Optimal density to the culture.

 Optimal auxin to cytokinin ratio, glucose and sucrose.
 Maintain osmoprotectant in the medium
 Temperature: 20-28°C pH: 5.5-5.9 0.25% Casein hydrolysate BAP and NAA

Culture of protoplasts

Protoplasts cultured in suitable nutrient media first generate a new cell wall. The formation
of a complete cell with a wall is followed by an increase in size, number of cell organelles,
and induction of cell division. The first cell division may occur within 2 to 7 days of culture.
It results in small clumps of cell, also known as micro colony, within 1 to 3 weeks. From
such clumps, there are two routes to generate a complete plant (depending on the species).
Plants are regenerated through organogenesis from callus masses. The micro calli can be
made to develop into somatic embryos, which are then converted into whole plant through
germination
 Importance of Protoplast Culture

The protoplast in culture can be regenerated into a whole plant. Hybrids can be developed
from protoplast fusion. It is easy to perform single cell cloning with protoplasts. Genetic
transformations can be achieved through genetic engineering of protoplast DNA. Protoplasts
are excellent materials for ultra-structural studies.

Isolation of cell organelles and chromosomes is easy from protoplasts. Protoplasts are useful
for membrane studies (transport and uptake processes).Isolation of mutants from protoplast
cultures is easy.

13. Embryo Culture

What is embryo?

A seed plant embryo is part of a seed, consisting of precursor tissues for the leaves, stem and
root as well as one or more cotyledons. The young sporophyte of a seed plant usually
comprising a rudimentary plant with plumule, radicle, and cotyledons

What is Embryo Culture?

The embryo of different developmental stages, formed within the female gametophyte through
sexual process, can be isolated aseptically from the bulk of maternal tissues of ovule, seed or
capsule and cultured in vitro under aseptic and controlled physical conditions in glass vials
containing nutrient solid or liquid medium to grow directly into plantlet

Culturing method

The general method of embryo culture follows the following steps.

1. Pluck healthy and mature fruits from the field and wash thoroughly in running water for
about an hour.

2. Surface sterilize with 0.01% Tween-20 for 15 min, rinse seeds several times with distilled
water and finally treat with 0.01% HgCl2 solution for 10-15 min.

3. Finally rinse it for six times with sterile distilled water.

 4. Incubate the cultures at 22-25°C under a 16 h photoperiod of 2000 lux luminous
intensity.

5. After two weeks of inoculation the embryo begins to swell on callus proliferation
medium. Distinct callus growth is observed after 4 weeks.

6. After 8 weeks of inoculation transfer the callus on shoot regeneration medium. Within 4
weeks of transfer into second medium the callus turns green and produces soft spongy
tissue. Some of these tissues are differentiated into embryoids.

7. The embryoids produce cluster of budlets when subcultured onto shoot regeneration
medium.

8. The budlets grow into shoots and produce 2-3 leaf appendages within 12 weeks.

9. Thereafter, they are separated into individual shoots and then subcultured into a fresh
medium of the same composition until shoots develop.

Types of Embryo Culture

There are two types of embryo culture:

1. Mature Embryo Culture

Mature embryos are isolated from ripe seeds and cultured in vitro. Mature embryo cultures are
carried out in the following conditions

Conditions

1. When the embryos remain dormant for long periods.

2. Low survival of embryos in vivo.

3. To avoid inhibition in the seed for germination.

4. For converting sterile seeds to viable seedlings.

 2. Embryo Rescue

Embryo rescue involves the culture of immature embryos to rescue them from unripe or
hybrid seeds which fail to germinate. This approach is very useful to avoid embryo abortion
and produce a viable plant. Wild hybridization involving crossing of two different species of
plants from the same genus or different genera often results in failure. This is mainly because
the normal development of zygote and seed is hindered due to genetic barriers.

Applications of Embryo Culture:

Embryo culture can be used in:

 Prevention of Embryo Abortion

 Overcoming Seed Dormancy
 Shortening of Breeding Cycle
 Production of Haploids
 Overcoming Seed Sterility
 Clonal Propagation

14. Meristem/shoot tip culture

Meristem

A meristem is the tissue in most plants containing undifferentiated cells (meristematic cells),
found in zones of the plant where growth can take place

What is Meristem Culture?

Meristem culture is the in vitro culture of a generally shiny special dome-like structure mea-
suring less than 0.1 mm in length and only one or two pairs of the youngest leaf primordia,
most often excised from the shoot apex.

Principle

The excised shoot tips and meristem can be cultured aseptically on agar solidified simple
nutrient medium or on paper bridges dipping into liquid medium and under the appropriate
 condition will grow out directly into a small leafy shoot or multiple shoots. Alternatively the
meristem may form a small callus at its cut case on which a large number of shoot primordia
will develop. These shoot primordia grow out into multiple shoots. Once the shoots have been
grown directly from the excised shoot tip or meristem, they can be propagated further by nodal
cuttings. This process involves separating the shoot into small segments each containing one
node. The axillary bud on each segment will grow out in culture to form yet another shoot.

Protocol

 Remove the young twigs from a healthy plant. Cut the tip (1 cm) portion of the twig.
 Surface sterilize the shoot apices by incubation in a sodium hypochlorite solution (1%
available chlorine) for 10 minutes. The ex- plants are thoroughly rinsed 4 times in ster-
ile distilled water.
 Transfer each explants to a sterilized petri dish.
 Remove the outer leaves from each shoot apices with a pair of jeweler’s forceps. This
lessens the possibility of cutting into the softer underlying tissues.
 After the removal of all outer leaves, the apex is exposed. Cut off the ultimate apex
with the help of scalpel and transfer only those less than 1 mm in length to the surface
of the agar medium or to the surface of filter-paper Bridge. Flame the neck of the
culture tube before and after the transfer of the excised tips. Binocular dissecting mi-
croscope can be used for cutting the true meristem or shoot tip perfectly.
 Incubate the culture under 16hrs light at 25°C
 As soon as the growing single leafy shoot or multiple shoots obtained from single shoot
tip or meristem, develop root, transfer them to hormone free medium
 The plantlets formed by this way are later transferred to pots containing compost and
kept under greenhouse conditions
Importance of Shoot Tip/Meristem Culture

The uses of shoot tips and meristem in tissue culture are very varied and include mainly:

1. Virus eradication,
2. Micro-propagation and
3. Storage of genetic resources.
Virus Eradication

This technique is also valuable for the maintenance of carefully defined stocks of specific vari-
eties and cultivars in disease Free State. The size of the meristem explant is critical for virus
eradication.

Micro Propagation

A sexual or vegetative propagation of whole plants using tissue culture techniques is referred
to as micro-propagation. Shoot tip or meristem culture of many plant species can successfully
be used for micro-propagation.

Storage of Genetic Resources

Many plants produce seeds that are highly heterozygous in nature or that is recalcitrant. Such
seeds are not accepted for storing genetic resources. So, the meristem from such plants can be
stored in vitro.

15. Regeneration Methods of Plants in Culture

Plant regeneration

The process of growing an entire plant from a single cell or group of cells. Regeneration is
possible because plant cells can be made totipotent using hormones. Differentiated tissue:
stems, leaves, roots, etc. Undifferentiated (embryonic) cells are totipotent: can become a whole
new plant by differentiating into a whole new plant.
Plant regeneration

The main objective in plant cultures is to regenerate a plant or plant organ from the callus
culture. The regeneration of plant or plant organs only taken place by the expression of cellular
totipotancy of the callus tissues. In agriculture biotechnology, tissue culture is most important
for the regeneration of transgenic plants from single transformed cells

Organogenesis

Organogenesis is the formation of organs: either shoot or root. In vitro organogenesis depends
on the balance of auxin and cytokinin and the ability of the tissue to respond to phytohormones
during culture. It takes place in three phases.

In the first phase the cells become competent;

Next, they dedifferentiate.

In the third phase, morphogenesis proceeds independently of the exogenous phytohormone.

Indirect Organogenesis

Formation of organs indirectly via a callus phase is termed indirect organogenesis. Induction
of plants using this technique does not ensure clonal fidelity, but it could be an ideal system
for selecting somaclonal variants of desired characters and also for mass multiplication.
Induction of plants via a callus phase has been used for the production of transgenic plants in
which

the callus is transformed and plants regenerated or

the initial explant is transformed and callus and then shoots are developed from the explant.
 Direct Organogenesis

The production of direct buds or shoots from a tissue with no intervening callus stage is termed
direct organogenesis. Plants have been propagated by direct organogenesis for improved
multiplication rates, production of transgenic plants, and—most importantly—for clonal
propagation. Typically, indirect organogenesis is more important for transgenic plant
production.

16. Somatic Embryogenesis

Introduction

Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a

single somatic cell or group of somatic cells. Somatic embryos are formed from plant cells that
are not normally involved in the development of embryos, i.e. ordinary plant tissue.

Somatic embryoid formation

It may be formed from:

 Vegetative cells of a mature plant

 Reproductive cells other than zygote or
 Cotyledons, hypocotyl or young plantlets
Hypocotyl, Cotyledon

The hypocotyl (short for "hypocotyledonous stem"meaning "below seed leaf") is the stem of a
germinating seedling, found below the cotyledons (seed leaves) and above the radicle (root).It
is the part of the stem of an embryo plant, beneath the stalks of the seed leaves or cotyledons
 and directly above the root. An embryonic leaf in seed-bearing plants, one or more of which
are the first leaves to appear from a germinating seed.

A cotyledon is a significant part of the embryo within the seed of a plant, and is defined as "the
embryonic leaf in seed-bearing plants, one or more of which are the first to appear from a
germinating seed."

Steps
of
somatic embryogenesis

The steps which are involved in somatic embryogenesis are:

 Initiation of embryogenic culture

 Prolifertion of embryogenic culture
 Pre-maturation of somatic embryos
 Maturation of somatic embryos
 Plant development
Types of Somatic Embryogenesis

There are two types of somatic embryogenesis

• Direct somatic embryogenesis

• Indirect somatic embryogenesis

Direct somatic embryogenesis

In this type of embryogenesis, the embryos initiate directly from explants in the absence of
callus formation.
Indirect Embryogenesis

In this type of embrogenesis, the embryos are developed through cell proliferation i.e., callus
formation. The cells from which embryos arise are called as ‘Induced embryogenic determined
cells’ (IEDC). Here growth regulators with specific cultural conditions are required for initiation
of callus and then redetermination of those cells into the embryo development.

Advantages:

Advantages of somatic embryogenesis are:

 Higher propagation rate

 Suitable for suspension culture
  Artificial seed production
 Somaclonal variation
 Germplasm conservation
 Labour savings

Disadvantages

 Disadvantages of somatic embryogenesis are:

 Low frequency embryo production
 Incomplete embryo production
 May create unwanted genetic variation
 Inability to generate large number of normal , free living plantlet
 Plantlets are weaker
 Respone tissue specific
Factors influencing somatic embryogenesis

The factors that influence somatic hybridization are:

1. Auxin

2. Cytokinin

3. Nitrogen

4. Activated charcoal

5. Age of culture

1. Auxin

In medium having relatively high concentration of auxin embryonal budding or embryonal

clumps have been observed. For cell differentiation the medium should contain auxin and
reduced nitrogen.Subsequent development takes place in medium with no auxin or low
concentration of auxin and reduced nitrogen. In some plants first and second stages occur in
the first medium and plantlet development takes place in the second medium.

2. Cytokinin

• The role of cytokinin in embryogenesis is not clear. Embyogenesis in carrot cell suspension
is stimulated by addition of zeatin in medium lacking auxin but inhibited by the addition
of kinetin. Inhibitory effect of exogenous cytokinin may be due to an increase in
endogenous cytokinin in growing embryoids. The role of cytokinin in embryogenesis is
not clear. Embyogenesis in carrot cell suspension is stimulated by addition of zeatin in
medium lacking auxin but inhibited by the addition of kinetin. Inhibitory effect of
exogenous cytokinin may be due to an increase in endogenous cytokinin in growing
embryoids

3. Nitrogen

The ratio of nitrogen to auxin is an important factor controlling embryogenesis. Embryo

development can be initiated on White’s medium with low nitrogen content only in absence of
auxin. At low nitrogen concentration organic nitrogen is more suitable than inorganic
nitrogen.Substances used as a source of nitrogen are potassium nitrate, ammonium chloride,
glutamine, glutamic acid, alanine, urea etc.

4. Activated charcoal

Presence of activated charcoal in the medium helps embryogenesis in several cases. Activated
charcoal may adsorb the inhibitory substances present in the medium.

5. Age of the culture

Embryogenesis usually occurs in short-term cultures. With older cultures this ability decreases
and ultimately it is completely lost. This may be due to either the inability to synthesise some
embryogenetic substances or changes in the ploidy level which may lead to loss of
morphogenetic potential.
 17.Application of plant cell Culture in crop improvement
Introduction

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part
of the group of technologies called plant biotechnology. Tissue culture has been exploited to
create genetic variability from which crop plants can be improved, to improve the state of
health of the planted material and to increase the number of desirable germplasms available
to the plant breeder.

Germplasm are living genetic resources such as seeds or tissues that are maintained for the
purpose of plant breeding, preservation, and other research uses. Tissue culture protocols are
available for most crop species, although continued optimization is still required for many
crops, especially cereals and woody plants. Tissue culture techniques, in combination with
molecular techniques, have been successfully used to incorporate specific traits through gene
transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and
embryos have been used to create new genetic variation in the breeding lines, often via
haploid production.

Haploid is the term used when a cell has half the usual number of chromosomes. Cell culture
has also produced somaclonal and gametoclonal variants with crop improvement potential.

Somaclonal variation is the variation seen in plants that have been produced by plant tissue
culture. Chromosomal rearrangements are an important source of this variation

Gametoclonal variation has been defined as the variation among plants regenerated from
gametic cells in culture. The culture of single cells and meristems can be effectively used to
eradicate pathogens from planting material and thereby dramatically improve the yield of
established cultivars. Large scale micro propagation laboratories are providing millions of
plants for the commercial ornamental market and the agricultural, clonally propagated crop
 market. With selected laboratory material typically taking one or two decades to reach the
commercial market through plant breeding, this technology can be expected to have an ever
increasing impact on crop improvement as we approach the new decade.

Applications

 The applications of various tissue culture approaches to crop improvement are

following:
 Breeding & biotechnology
 Wide hybridization
 Haploidy
 Somaclonal variation
 Micropropagation
 Synthetic seed
 Pathogen eradiction
 Germplasm preservation

18.Plant Breeding and Biotechnology

Plant breeding can be conveniently separated into two activities: manipulating genetic
variability and plant evaluation. Historically, selection of plants was made by simply
harvesting the seeds from those plants that performed best in the field. In spite of the general
lack of integration of most plant biotechnology and plant breeding programs, field trials of
transgenic plants have recently become much more common. More than 50 different plant
species have already been genetically modified, either by vector dependent (e.g.
Agrobacterium) or vector independent (e.g. biolistic, micro-injection and liposome) methods.
In almost all cases, some type of tissue culture technology has been used to recover the
modified cells or tissues. In fact, tissue culture techniques have played a major role in the
development of plant genetic engineering. Tissue culture will continue to play a key role in
the genetic engineering process for the predictable future, especially in efficient gene transfer
and transgenic plant recovery.
 19. Wide Hybridization
Definition

Hybridization between individuals from different species, belonging to same genus or different genera,
is termed as distant or wide hybridization. A critical requirement for crop improvement is the
introduction of new genetic material into the cultivated lines of interest, whether via single genes,
through genetic engineering, or multiple genes, through conventional hybridization or tissue culture
techniques. During fertilization in angiosperms, pollen grains must reach the stigma of the host plant,
germinate and produce a pollen tube. The pollen tube must penetrate the stigma and style and reach
the ovule. The discharge of sperm within the female gametophyte triggers syngamy and the two sperm
nuclei must then fuse with their respective partners. The egg nucleus and fusion nucleus then form a
developing embryo and the nutritional endosperm, respectively. This process can be blocked at any
number of stages, resulting in a functional barrier to hybridization and the blockage of gene transfer
between the two plants.

Barrier for crossing:

There are two barriers for crossing

Pre-Zygotic

Post-Zygotic

Pre –zygotic Barriers

Post- zygote barriers are those which occur prior to fertilization, they are due to the genic
differences in different species. There is failure of pollen germination, slow growth of pollen
tube, inability of the pollen to reach the ovary and arrest of pollen tube in style ovary and ovule.

Post- Zygotic barriers

Post- zygote barriers are those which occur after fertilization, hybrid in viability and weakness leading
to chromosome elimination, lethality and embryo abortion, hybrid sterility and hybrid breakdown with
weak or sterile individuals in F2 owing to recombination of genes complements of the parental species.

Techniques to overcome isolation barriers

Pre-zygotic barriers can be overcome by: In-vitro fertilization, Protoplast fusion, Embryo culture, Use
of growth hormones (IAA, NAA) and adopting bridging species technique.

In vitro Fertilization

IVF has been used to facilitate both interspecific and intergeneric crosses, to overcome physiological
based self-incompatibility and to produce hybrids. A wide range of plant species has been recovered
through IVF via pollination of pistils and self and cross-pollination of ovules. This range includes
agricultural crops, such as tobacco, clover, com, rice, cole, canola, poppy and cotton. The use of delayed
pollination, distant hydridization, pollination with abortive or irradiated pollen, and physical and
chemical treatment of the host ovary have been used to induce haploidy.

Protoplast Fusion

Protoplast fusion has often been suggested as a means of developing unique hybrid plants which cannot
be produced by conventional sexual hybridization. Protoplasts can be produced from many plants,
including most crop species. However, while any two plant protoplasts can be fused by chemical or
physical means, production of unique somatic hybrid plants is limited by the ability to regenerate the
fused product and sterility in the interspecific hybrids rather than the production of protoplasts. Perhaps
the best example of the use of protoplasts to improve crop production is that of Nicotiana, where the
somatic hybrid products of a chemical fusion of protoplasts have been used to modify the alkaloid and
disease-resistant traits of commercial tobacco cultivars.

Protoplast fusion should focus on four areas:

Agriculturally important traits

Achieving combinations that can only be accomplished by protoplast fusion

A somatic hybrids integrated into a conventional breeding programme and

The extension of protoplast regeneration to a wider range of crop species

Embryo Culture

The most common reason for post-zygotic failure of wide hybridization is embryo abortion due to poor
endosperm development. Embryo culture has been successful in overcoming this major barrier as well
as solving the problems of low seed set, seed dormancy, slow seed germination, inducing embryo
growth in the absence of a symbiotic partner, and the production of monoploids of barley. Interspecific
and intergeneric hybrids of a number of agriculturally important crops have been successfully produced,
including cotton, barley, tomato, rice, jute, Hordeum X Secale,Triticum x Secale,Tripsacumx lea and
some Brassicas.

Embryo Culture

The most common reason for post-zygotic failure of wide hybridization is embryo abortion due to poor
endosperm development. Embryo culture has been successful in overcoming this major barrier as well
as solving the problems of low seed set, seed dormancy, slow seed germination, inducing embryo
growth in the absence of a symbiotic partner, and the production of monoploids of barley. The most
common reason for post-zygotic failure of wide hybridization is embryo abortion due to poor
endosperm development. Embryo culture has been successful in overcoming this major barrier as well
as solving the problems of low seed set, seed dormancy, slow seed germination, inducing embryo
growth in the absence of a symbiotic partner, and the production of monoploids of barley.

Bridging species technique

When direct cross between tow species with the same or different ploidy levels are difficult to
accomplish a third specie (Bridge specie) is used to make such crosses possible

Pre-zygotic barriers

Pre- zygote barriers can be overcome by: Tissue culture techniques, Back crossing, Doubling of
chromosomes and Embryo rescue.

Backcrossing

Backcrossing is a crossing of a hybrid with one of its parents or an individual genetically similar to its
parent, in order to achieve offspring with a genetic identity which is closer to that of the parent.It is
used in horticulture, animal breeding and in production of gene knockout organisms.

Doubling of chromosomes
 Artificial production of doubled haploids is important in plant breeding. Haploid cells are produced
from pollen or egg cells or from other cells of the gametophyte, then by induced or
spontaneous chromosome doubling, a doubled haploid cell is produced, which can be grown into
a doubled haploid plant.

Embryo rescue

The term “embryo rescue” refers to a number of in vitro techniques whose purpose is to
promote the development of an immature or weak embryo into a viable plant. Embryo
rescue has been widely used for producing plants from hybridizations in which failure of
endosperm to properly develop causes embryo abortion. Embryo rescue is one of the earliest
and successful forms of in-vitro culture techniques that is used to assist in the development of
plant embryos that might not survive to become viable plants

20. Haploids
Introduction

A haploid is a cell or organism that has a single set of chromosomes that are not paired. The haploid
gamete is normally produced during plant cell division. During fertilization, these cells normally
merge with other similar haploid cells. A haploid cell only has half the number of chromosomes
as are present in diploids.

Applications of Haploid Plants are:

• In Vitro production of haploids can solve some problems in genetic studies

• The following points highlight the top applications of haploid plants

Development of Pure Homozygous Line

In plant breeding, it is very much essential to get the pure homozygous line which is generally
obtained through selfing for 6-7 generations. But by the use of anther/pollen culture it can be
reduced to few months or a year. These genetically pure homozygous lines are used for breeding
as well as genetic research purpose. This technique is also helpful for breeding of these plants
which have more elongated juvenile phase.

Selection of Mutants Resistance to Diseases

Selection of mutants with resistance to disease is of prime importance in crop improvement.

Haploids provide a relatively easier system for the induction of mutations. Some examples of using
anther culture technique in mutant successfully are tobacco mutants resistant to black shank
disease and wheat lines resistant to scab.

Transfer of Desired Alien Gene

Chromosomal instability in haploids makes them potential tools for introduction of alien
chromosomes on genes during wider crossing programs.

Induction of Mutagenesis

Haploid cell cultures are useful material for induction of mutations and to study the effect of
mutation. This method can overcome the masking effect of presence of dominant gene. The
screening method for detection of mutational effect Is also easier in this technique.

Induction of Genetic Variability

The pollen/microspore is easy explant for production of genetically variable types by introducing
the different foreign genes through different transformation procedure. These transformed or
transgenic haploids can be used further in breeding program.

Development of Aneuploids

Haploids have been used in the production of aneu plaids like monosomies in wheat, trisomies in
potato. In tobacco nullisomics were derived from haploids obtained from monosomies which could
not produce nullisomics on selfing. Nullisomic is a genetic condition involving the lack of both
the normal chromosomal pairs for a species (2n-2).

21.Somaclonal Variation

Somaclonal variations

The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal
variations. These are genetic variations in plants that have been produced by plant tissue culture
and can be detected as genetic or phenotypic traits.

Basic features of somaclonal variations

Variations in number and structure of chromosomes are commonly observed. Regenerated plants
with altered chromosomal changes often show changes in leaf shape and colour, growth rate and
habit, and sexual fertility. It is generally heritable mutations and persists in plant population even
after plantation into the field.

Mechanism of Somaclonal Variations

 Genetic (Heritable Variations)

 Pre-existing variations in the somatic cells of explant
 Caused by mutations and other DNA changes
 Occur at high frequency
 Epigenetic (Non-heritable Variations)
 Variations generated during tissue culture
 Caused by temporary phenotypic changes
 Occur at low frequency
Causes of Somaclonal Variations are:

1. Physiological Cause
 2. Genetic Cause

3. Biochemical Cause

1. Physiological Cause

Physiological causes are: Exposure of culture to plant growth regulators and Culture conditions.

2. Genetic Cause

• Genetic causes are: Change in chromosome number: Aneuploidy, gain or loss of 1 or

more chromosomes, Polyploidy, gain or loss of an entire genome, Translocation, arms of
chromosomes switched, inversion, piece of chromosome inverted.

• Change in chromosome structure: Deletion, Inversion, Duplication, Translocation,

• Gene Mutation: Transition, Traversions, Insertion and Deletion

• Plasmagene Mutation : It is a self-replicating extra nuclear determiner of hereditary

characteristics.

• Transposable element activation : Transposable elements (TEs), also known as

"jumping genes" or transposons, are sequences of DNA that move (or jump) from one
location in the genome to another.

• DNA sequence : Change in DNA and Detection of altered fragment size by using
Restriction enzyme

• Change in Protein : Loss or gain in protein band and Alteration in level of specific
protein

• Methylation of DNA: Methylation inactivates transcription process.

Biochemical Cause

It is the Lack of photosynthetic ability due to alteration in carbon metabolism e.g. biosynthesis of
starch via carotenoid pathway and Nitrogen metabolism processes.
 • Advantages of Somaclonal Variations are: help in crop improvement, creation of
additional genetic variations, increased and improved production of secondary
metabolites, selection of plants resistant to various toxins, herbicides, high salt
concentration and mineral toxicity, suitable for breeding of tree species.

Applications of Somaclonal Variations

Production of agronomically useful plants: As a result of somaclonal variations, several novel

variants of existing crops have been developed, e.g., pure thorn-less blackberries, somaclonal
variations are useful and improved morphological characters in different crops.

Resistance to diseases: Somaclonal variations have largely contributed towards the development
of disease resistance in many crops e.g. rice, wheat, maize, sugarcane, tobacco, apple, tomato.

• Resistance to abiotic stresses: It has been possible to develop biochemical mutants with
abiotic stress resistance.

• i. Freezing tolerance e.g. wheat.

• ii. Salt tolerance e.g., rice, maize, tobacco.

• iii. Aluminium tolerance e.g., carrot, sorghum, tomato.

Resistance to herbicides

Certain somaclonal variants with herbicide resistance have been developed. E.g.

i. Tobacco resistant to glyphosate, sulfonylurea and picloram.

• ii. Carrot resistant to glyphosate.

• iii. Lotus resistant to 2, 4-dichlorophenoxy acetic acid

Improved seed quality

A new variety of Lathyrus sativa (grass Pea) seeds with a low content of neurotoxin has been
developed through somaclonal variations. Neurotoxins are an extensive class of exogenous
chemical neurological insults that can adversely affect function in both developing and mature
nervous tissue.

22.Micropropagation
Micropropagation is the practice of rapidly multiplying reserve plant material to produce a large
number of progeny plants, using modern methods of plant tissue culture. Micropropagation is used to
multiply noble plants, such as those that have been genetically modified or raised through
conventional plant breeding methods. It is also used to provide a sufficient number of seedlings to
plant from a common plant that does not produce seeds, or does not respond well to vegetative
reproduction.

Micropropagation Technique

Micropropagation is a complicated process and comprises mainly 4 stages (I, II, III and IV). But
initial step 0 is also necessary.

Step 0: This is the initial step in micro-propagation, and involves the selection and growth of
common plants for about 3 months under controlled conditions.

Stage I – Establishment: In this stage, the initiation and establishment of the culture is
achieved in a suitable medium. The selection of appropriate explants is important. The most
 commonly used explants are organs, shoot tips and axillary shoots. The explant selected is
surface sterilized and washed prior to use.

Stage II – Multiplication: At this stage, the main activity of Micro propagation occurs in a
defined culture medium. Phase II mainly involves the multiplication of shoots or the rapid
formation of explant embryos.

Stage III – Rooting: This stage involves the transfer of shoots to a medium for rapid
development in shoots. Sometimes sprouts are planted directly on the ground to develop
roots. In vitro rooting of shoots is preferred while simultaneously handling a large number of
species.

Stage IV – Acclimatization: This stage involves the establishment of seedlings in the soil.
This is done by transferring seedlings from stage III of the laboratory to the environment of
the greenhouse. For some plant species, stage III is omitted, and un rooted shoots of stage II
are planted in pots or in a suitable compost mix.

Factors Affecting Micro-propagation: there is need of optimization of several factors is

necessary for success in clonal propagation in vitro (micro-propagation).

Genotype of the plant: Selection of the correct genotype of plant species (by screening) is
necessary to improve micropropagation. In general, plants with a vigorous germination and
branching ability are more suitable for micro-propagation.

Physiological status of explants: The explants (plant materials) of the most recently produced
parts of plants are more effective than those of the older regions. A good knowledge of the
process of natural propagation of donor plants, with special reference to the stage of growth and
seasonal influence, will be useful in the selection of explants.

Culture media: Conventional plant tissue culture media are suitable for micropropagation
during stage I and stage II. However, for stage III, certain modifications are essential. The
addition of growth regulators (auxins and cytokinins) and alterations in mineral composition is
essential. This depends largely on the type of crop.
Light: The photosynthetic pigment in cultured tissues absorbs light and, therefore, influences
micropropagation. Variations in daytime lighting also effect micropropagation. In general, a
lighting of 16 hours of day and 8 hours of night is suitable for the proliferation of shoots.

Temperature: The majority of micropropagation culture necessitates optimum temperature

around 25 °C. However, there are some exceptions.

Micropropagation applications are:

 Suitable alternative to traditional methods

 High prevalence of plants
 The production of disease-free plants
 Seed production in some crops
 Cost effective process
 Automated micro propagation
 Very small size explants can be used
 Only practical method of multiplying genetically modified cells or cells after
protoplast fusion.
 Ease in keeping, packing and transport of material multiplied by micropropagation.

23.Synthetic Seed
Synthetic seed: Many fruit crops are difficult to multiply by conventional propagation methods
and improve through traditional breeding programs. Among the innovative techniques of micro
propagation, the concept of somatic embryogenesis with synthetic seed production or artificial
seed technology is very promising. Synthetic seed is referred to as encapsulated somatic embryos,
which functionally mimic seeds and can develop into seedling under suitable conditions. Synthetic
seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell aggregates, or any
other tissue that can be used for sowing as a seed and that possess the ability to convert into a plant
under in vitro or ex vitro conditions and that retain this potential also after storage. In simple words
synthetic seed contains an embryo produced by somatic embryogenesis enclosed within an
artificial medium that supplies nutrients and is encased in an artificial seed covering.
Why Synthetic Seeds:

In some of the horticultural crops seeds propagation is not successful due to; heterozygosity of
seeds particularly in cross pollinated crops, minute seed size e.g.; orchids, presence of reduced
endosperm, some seeds require mycorrhizal fungi association for germination e.g.: orchids, no
seeds are formed.

Characteristics of Synthetic Seeds:

i. High volume. Large scale propagation method

ii. Maintains genetic uniformity of plants

iii. Direct delivery of propagules to the field, thus eliminating transplants

iv. Lower cost per plantlet

v. Rapid multiplication of plants

Advantages of Synthetic Seeds are:

i. Ease of handling while in storage

ii. Easy to transport

iii. Has potential for long term storage without losing viability

iv. Maintains the clonal nature of the resulting plants

v. Serves as a channel for new plant lines produced through biotechnological advances to be
delivered directly to the green house or field

vi. Allows economical mass propagation of elite plant varieties

24.Germplasm conservation
Germplasm: A germplasm is a collection of genetic resources for an organism. Germplasm is a
living tissues from which new plants can be grown. It can be a seed or another plant part-a leaf,a
piece of stem,pollen or even just a few cells that can be turned into the whole plant. For plants, the
germplasm may be stored as a seed, stem, Callus, Whole plant in nurseries.

Germplasm conservation: Plant germplasm is genetic source material in the form of Seeds,
Cultured cells Callus, Pollens. The in-situ /ex-situ preservation of this material is known as
“Germplasm conservation”. Germplasm provide the raw material (genes) which the breeder uses
to develop commercial crop varieties.

What is the need of Preservation: Preservation/Conservation of plant biodiversity is an important

issue. Storage of Economically important, endangered, rare species and make them available when
needed. The conventional methods of storage failed to prevent losses caused due to various
reasons.

Methods of Germplasm conservation are:

1. In-situ Preservation

2. Ex-situ Preservation

In-situ Preservation: it is preservation of the germplasm in their natural habitat, conservation of

domesticated and cultivated species in the farm or in the surroundings. However, there is a heavy
loss or decline of species, populations and ecosystem composition, which can lead to a loss of
biodiversity, due to habitat destruction and the transformations of these natural environments;
therefore, in situ methods alone are insufficient for saving endangered species.

Ex-situ preservation: 1. It is used to maintain the biological material outside their natural
habitats, for storage in seed banks, field gene collections, in vitro collections and botanical gardens.
Ex situ conservation is a viable way for saving plants from extinction, and in some cases, it is the
only possible strategy to conserve certain species. In vitro conservation is especially important for
vegetatively propagated and for non-orthodox seed plant species. Non-Orthodox seeds are seeds
which do not survive drying and/or freezing during ex-situ conservation.

Approaches for the in vitro conservation of germplasm:

 • Cryopreservation (freeze-preservation)

• Cold storage

• Low-pressure and low-oxygen storage

• Cryopreservation: Cryopreservation (Greek, krayos-frost) literally means preservation in

the frozen state. The principle involved in cryopreservation is to bring the plant cell and
tissue cultures to a zero metabolism or non-dividing state by reducing the temperature in
the presence of cryoprotectants In this case the cells are preserved in the frozen state. The
germplasm is stored at a very low temperature using

• Solid carbon dioxide (at -790C)

• Using low temperature deep freezers (at -800C)

• Using vapour nitrogen (at- 1500C)

• Liquid nitrogen (at-1960C).

Cold Storage: Cold storage is a slow growth germplasm conservation method. It conserves the
germplasm at a low and non-freezing temperature (1- 9°C). The growth of the plant material is
slowed down in cold storage in contrast to complete stoppage in cryopreservation. Thus it prevents
cryogenic injuries. Long term cold storage is simple, cost effective. It yields germplasm with good
survival rate. Virus free strawberry plants could be preserved at 10°C for about 6 years. Several
grape plants have been stored for over 15 years by using a cold storage at temperature around 9°C
and transferring them in the fresh medium every year.

Low pressure and low oxygen storage: In low- pressure storage, the atmospheric pressure
surrounding the plant material is reduced. In the low oxygen storage, the oxygen concentration is
reduced. The lowered partial pressure reduces the in vitro growth of plants.In the low-oxygen
storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg
reduces plant tissue growth. Due to the reduced availability of 02, and reduced production of CO2,
the photosynthetic activity is reduced. It inhibits the plant tissue growth and dimension. This
method has also helped in increasing the shelf life of many fruits, vegetables and flowers. The
germplasm conservation through the conventional methods has several limitations such as short-
lived seeds, seed dormancy, seed-borne diseases, and high inputs of cost and labour. The
techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storages help
us to overcome these problems.

Applications or significance of germplasm conservation are: The conservation of germplasm

involves the preservation of the genetic diversity of a particular plant or genetic stock. It can be
used at any time in future.It is important to conserve the endangered plants or else some of the
valuable genetic traits present in the existing and primitive plants will be lost. Main crops produce
recalcitrant or short lived seeds. Similarly, in case of clonal crops seeds are not the best material
to conserve due to their genetic heterogeneity and unknown worth. Their genes need to be
conserved. The roots and tubers loose viability rapidly. Their storage requires large space, low
temperature and is expensive. In addition, materials modified by genetic engineering may some,
times be unstable. Such materials are needed to be conserved intact for future use.

25.Genetic Markers in Plant Breeding

Genetic marker: A genetic marker is a gene or DNA sequence with a known location on a
chromosome. It can be used to identify individuals or species. It can be described as a variation
(which may arise due to mutation or alteration in the genomic loci) that can be observed. Any
phenotypic difference controlled by genes, that can be used for studying recombination processes
or selection of a more or less closely associated target gene Anything in the genome that is variable
and can be used to compare individuals. Detectable allelic variation on a chromosome it can be a
phenotype, can also be a unique detectable sequence of DNA. They are points of variation that can
be used to identify individuals or species, or may be used to associate an inherited disease with a
gene through genetic linkage with nearby but possibly unidentified or uncharacterised genes.
Examples include single nucleotide polymorphisms (SNPs) and minisatellites.

Polymorphism: In biology and zoology is the occurrence of two or more clearly different morphs
or forms, also referred to as alternative phenotypes, in the population of a species.
Single nucleotide polymorphisms (SNPs): A single-nucleotide polymorphism, often abbreviated
to SNP. A variation in a single nucleotide that occurs at a specific position in the genome, where
each variation is present to some appreciable degree within a population. For example, at a specific
base position in the human genome, the C nucleotide may appear in most individuals, but in a
minority of individuals, the position is occupied by an A. This means that there is an SNP at this
specific position, and the two possible nucleotide variations – C or A – are said to be alleles for
this position. As SNPs are thought to play a major role in the induction of phenotypic variation in
plants, so it is very important to identify the functional SNPs regarding crop improvements. SNPs
also can identify the genomic diversity of species to demonstrate the speciation and evolution, and
associate genomic variations with phenotypic traits

Types of genetic markers are:

Molecular markers

Morphological markers

26.Molecular Markers
Molecular markers

It is a sequence of DNA or protein that can be screened to reveal key attributes of its state or
composition and thus used to reveal genetic variation, also known as “Genetic Marker”.
Genetic markers are the sequences of DNA which have been traced to specific location on the
chromosomes and associated with particular traits.

Classification of molecular markers:

Molecular Markers are classified as:

1. Protein Based Markers/ Biochemical Markers

2. DNA Based Markers

 Protein Based Markers/ Biochemical Markers: Plant Breeding Markers related to the
variations in protein and amino acid banding pattern.

Isozyme markers: Multiple forms of the same enzyme coded by the different genes

Allozyme : one enzyme, one locus; two or more alleles in a population

Advantages: advantages of molecular markers are:

They are simple, inexpensive, electrophoretically resolvable, and detectable, does not require
DNA extraction or the availability of sequence information, primers or probes, quick and easy
to use, codominant markers that have high reproducibility.

Disadvantages of molecular markers are: they are relatively low abundance and low level
of polymorphism, can be affected by environmental conditions, they may change depending
on the type of tissue used for the analysis.

Applications of molecular markers are:

They are used for detection of the gene introgression (gene movement) and recombination, for
comparative mapping, for determination of the genetic diversity and phylogenetic
relationships.

27. DNA based markers

DNA Markers

A gene or other fragment of DNA whose location in the genome is known is called DNA marker.
It refers to any unique DNA sequence which can be used in DNA hybridization, PCR or restriction
mapping experiments to identify that sequence. It can be identified by a range of molecular
techniques such as RFLPs, RAPDs, AFLP, SNPs, SCARs, microsatellites etc.

Advantages of DNA markers:

Advantages of DNA markers are presented below.

 They are highly polymorphic.

 They have simple inheritance (often co-dominant).
 They abundantly occur throughout the genome.
 They are easy and fast to detect.
 They exhibit minimum pleiotropic effect.
 Their detection is not dependent on the developmental stage of the organism.

Properties of DNA Marker: An ideal DNA marker should have some properties or characteristics

Polymorphism:

• Markers should exhibit high level of polymorphism.

• In other words, there should be variability in the markers.

• It should demonstrate measurable differences in expression between trait types and/or gene
of interest.

Co-Dominant:

• Marker should be co-dominant.

• It means, there should be absence of intra-locus interaction.

• It helps in identification of heterozygotes from homozygotes.

Multi-Allelic:

• The marker should be multi-allelic.

• It useful in getting more variability/ polymorphism for a character.

• A multiallelic site is a specific locus in a genome that contains three or more observed
alleles, again counting the reference as one, and therefore allowing for two or more variant
alleles.

Different flower colors due to allelic variation in multiple genes

No Epistasis:

• There should be absence of epistasis.

• It makes Identification of all phenotypes (homo- and heterozygotes) easy.

• Epistasis: the interaction of genes that are not alleles, in particular the suppression of the
effect of one such gene by another.

Neutral:
 • The marker should be neutral.

• The substitution of alleles at the marker locus should not alter the phenotype of an
individual.

• This property is found in almost all the DNA markers

No Effect of Environment: Markers should be insensitive to environment. This property is also

found in almost all the DNA markers.

Applications of DNA Marker in Crop Improvement are:

• i. DNA markers are useful in the assessment of genetic diversity in germplasm, cultivars
and advanced breeding material.

• ii. DNA markers can be used for constructing genetic linkage maps.

• iii. DNA markers are useful in identification of new useful alleles in the germplasm and
wild species of crop plants.

• iv. DNA markers are used in the marker assisted or marker aided selection. MAS has
several advantages over straight selection.

• v. DNA markers are useful in the study of crop evolution.

28. Morphological markers

Morphology:

Morphology is a branch of biology dealing with the study of the form and structure of organisms
and their specific structural features.

Plant Morphology:

Plant morphology or phyto morphology is the study of the physical form and external structure of
plants. This is usually considered distinct from plant anatomy, which is the study of the internal
structure of plants, especially at the microscopic level. Plant morphology is useful in the visual
identification of plants.

Morphological features of plants:

 Plants are characterized by following features:

 Roots
 Shoots
 Stem
 Leaves
 Flowers
 Fruits
Morphological Markers

They are visually characterized phenotypic characters. e.g.

• Flower colour ,seed shape , growth habit, pigmentation

• It involves Germplasm characterization and indirect selection

Advantages

Some advantages of morphological markers are:

• They are inexpensive to score and ready to experiments in natural populations

Disadvantages

Some disadvantages of morphological markers are:

• visible polymorphisms relatively rare

 • Most genetic variation not so easily observed (Variants are ambiguous)

• Genetic basis of variation can be complex, and is not necessarily easy to determine.

Limitations: some limitations of morphological marks are enlisted here:

• They do not represent the genome adequately, they give o stable inheritance( Need
repeated measures)

• They generally express late into the development of an organism. Hence their detection
is dependent on the development stage of the organism.

• They usually exhibit dominance, sometimes they exhibit deleterious effects. They
exhibit pleiotropy. They exhibit epistasis. They exhibit less polymorphism. They are
highly influenced by the environmental factors.

29. Transformation
Transformation

In molecular biology, transformation is the genetic alteration of a cell resulting from the direct
uptake and incorporation of exogenous genetic material from its surroundings through the cell
membrane. Transformation occurs most commonly in bacteria and in some species occurs
naturally. Transformation can also be effected by artificial means. Bacteria that are capable of
being transformed, whether naturally or artificially, are called competent

Competent cells
Competent Cells are more likely to incorporate foreign DNA if their cell walls are altered so that
DNA can pass through more easily. Such cells are said to be competent

Types of competence (there is two types of competence):

i. Natural competence

ii. Artificial competence

 i. Natural competence: Bacteria are able to take up DNA from their environment by
three ways; conjugation, transformation, and transduction. In transformation the DNA
is directly entered to the cell. Uptake of transforming DNA requires the recipient cells
to be in a specialized physiological state called competent state.

Artificial competence:

• It is a laboratory procedure by which cells are made permeable to DNA, with conditions
that do not normally occur in nature. This procedure is comparatively easy and simple, and
can be used in the genetic engineering of bacteria but in general transformation efficiency
is low. There are two main methods for the preparation of competent cells They are
Calcium chloride method and Electroporation. Transformation may also be used to
describe the insertion of new genetic material into nonbacterial cells including animal and
plant cells

History:

Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. Griffith
discovered that a harmless strain of Streptococcus pneumoniae could be made virulent after being
exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle"
from the heat-killed strain was responsible for making the harmless strain virulent.

In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin
MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and
using just this DNA was able to make a harmless strain virulent. They called this uptake and
incorporation of DNA by bacteria "transformation." Transformation using electroporation was
developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the
number of bacterial strains that could be transformed. Transformation of animal and plant cells
was also investigated with the first transgenic mouse being created by injecting a gene for a rat
growth hormone into a mouse embryo in 1982. In 1907 a bacterium that caused plant tumors,
Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was
found to be a DNA plasmid called the Ti plasmid. By removing the genes in the plasmid that
caused the cancer and adding in novel genes researchers were able to infect plants with A.
tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants. Not all
plant cells are susceptible to infection by A. tumefaciens so other methods were developed
including electroporation and micro-injection. Particle bombardment was made possible with the
invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in 1990.

30. Mechanisms of Transformation

Bacterial Transformation:

Bacterial transformation may be referred to as a stable genetic change brought about by the uptake
of naked DNA (DNA without associated cells or proteins) and competence refers to the state of
being able to take up exogenous DNA from the environment.

Two forms of competence exist:

1. Natural and

2. Artificial.

1. Natural competence: About 1% of bacterial species are capable of naturally taking up

DNA under laboratory conditions; many more are able to take it up in their natural
environments. Such bacteria carry sets of genes that provide the protein machinery to bring
DNA across the cell membrane(s). DNA material can be transferred between different
strains of bacteria, in a process called horizontal gene transfer.
2. Artificial competence: Artificial competence is induced by laboratory procedures and
involves making the cell passively permeable to DNA by exposing it to conditions that do
not normally occur in nature. Calcium chloride transformation is a method of promoting
competence. Chilling cells in the presence of divalent cations such as Ca 2+ (in CaCl2)
prepares the cell membrane to become permeable to plasmid DNA. The cells are incubated
on ice with the DNA and then briefly heat shocked (e.g., 42°C for 30–120 seconds) thus
allowing the DNA to enter the cells. This method works very well for circular plasmid
DNA. An excellent preparation of competent cells will give ~10 8 colonies per microgram
of plasmid. A poor preparation will be about 10 4/μg or less. Good, non-commercial
preparations should give 105 to 106 transformants per microgram of plasmid. The method,
however, usually does not work well for linear DNA, such as fragments of chromosomal
DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA.
 Interestingly, cells that are naturally competent are usually transformed more efficiently
with linear DNA than with plasmid DNA.

Electroporation: Electroporation is another method of promoting competence. In the method

the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell
membrane through which the plasmid DNA enters. This method is ready to the uptake of large
plasmid DNA. After the electric shock the holes are rapidly closed by the cell's membrane-repair
mechanisms. The efficiency with which a competent culture can take up exogenous DNA and
express its genes is known as transformation efficiency.

31. Bacterial transformation & selection

Bacteria can take up foreign DNA in a process called transformation. Transformation is a key
step in DNA cloning. It occurs after restriction digest and ligation and transfers newly made
plasmids to bacteria. After transformation, bacteria are selected on antibiotic plates. Bacteria
with a plasmid are antibiotic-resistant, and each one will form a colony. Colonies with the right
plasmid can be grown to make large cultures of identical bacteria, which are used to produce
plasmid or make protein

DNA cloning

Transformation and selection of bacteria are key steps in DNA cloning. DNA cloning is the
process of making many copies of a specific piece of DNA, such as a gene. The copies are
often made in bacteria. In a typical cloning experiment, researchers first insert a piece of DNA,
such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction
enzymes and DNA ligase and is called a ligation. After a ligation, the next step is to transfer
the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection
and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.

Steps of bacterial transformation and selection

Steps

Specially prepared bacteria are mixed with DNA (e.g., from a ligation). The bacteria are given
a heat shock, which causes some of them to take up a plasmid. Plasmids used in cloning
contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate
to select for ones that took up a plasmid. Bacteria without a plasmid die. Each bacterium with
a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony.
Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction
digest).

A colony containing the right plasmid is grown in bulk and used for plasmid or protein
production.
Why do we need to check colonies?

The bacteria that make colonies should all contain a plasmid (which provides antibiotic resistance).
However, it’s not necessarily the case that all of the plasmid-containing colonies will have the
same plasmid.

How does that work?

When we cut and paste DNA, it's often possible for side products to form, in addition to the
plasmid we intend to build. For instance, when we try to insert a gene into a plasmid using a
particular restriction enzyme, we may get some cases where the plasmid closes back up
(without taking in the gene), and other cases where the gene goes in backwards.

Why does it matter if a gene goes into a plasmid backwards?

In some cases, it doesn't. However, if we want to express the gene in bacteria to make a protein,
the gene must point in the right direction relative to the promoter, or control sequence that
drives gene expression. If the gene were backwards, the wrong strand of DNA would be
transcribed and no protein would be made. Because of these possibilities, it's important to
collect plasmid DNA from each colony and check to see if it matches the plasmid we were
 trying to build. Restriction digests, PCR, and DNA sequencing are commonly used to analyze
plasmid DNA from bacterial colonies.

32. Transformation in Plants

Gene Transfer is introduction of foreign genetic material, either DNA or RNA artificially or
naturally into a cell. It is often also referred to as transformation and is one of the foundations of
molecular biology. It is now possible to introduce and express DNA stably in nearly 150 different
plant species. To achieve genetic transformation in plants, we need the construction of a vector
(genetic vehicle)which transports the genes of interest, flanked by the necessary controlling
sequences i.e. promoter, Terminator, Selectable marker and other genes that deliver the DNA into
the host plant (Ex. vir genes of Agrobacterium)

Plant transformation

Plant transformation is a scientific approach whereby DNA from any organism is inserted into
the genome of a species of interest. The inserted DNA is called a “transgene”, and the resulting
plant is said to be “transgenic”. Transgenic plants are plants derived from cells in which genes
(often of nonplant origin) have been stably introduced by transformation to give the plant a
new and useful trait. Transgenic plants can be obtained after transformation of single cells and
the subsequent regeneration into complete, fertile plants by tissue culture protocols.
Transformed plant cells can be identified by their ability to grow on selective media containing
an antibiotic or a herbicide as transformation vectors contain selection genes conferring such
properties. Novel functions are expressed in transformed plant cells if the coding regions are
surrounded by promoter and terminator regions that are recognized by the plant transcription
machinery. The most preferred methods for plant transformation use either the particle gun or
the natural transformation system of Agrobacterium tumefaciens, as they can cope with cells
present in whole plants or tissues. Agrobacterium tumefaciens can be disarmed by deletion of
the onc‐genes that are naturally present between the 25‐bp repeats of the T‐DNA. Any gene
introduced between these repeats is translocated into plant cells by Agrobacterium
tumefaciens. Transformed cells with a single copy of the transgene usually show higher and
more stable expression than multicopy lines, in which expression may suffer from
posttranscriptional gene silencing. (T)‐DNA integration in plant cells occurs at random sites in
the genome by nonhomologous end‐joining and related backup pathways.

Targeted integration of transgenes can be accomplished in plant cells by using either a site‐specific
recombination system (e.g. Cre‐lox) or by homology‐directed integration in combination with a
site‐specific nuclease (e.g. a zinc‐finger nuclease). Agrobacterium tumefaciens‐mediated
transformation can be applied not only in dicotyledonous plants, but also in monocots such as
cereals and in yeasts and fungi. The T‐DNA can be used as a mutagen causing insertion mutations.
Libraries of Arabidopsis thaliana T‐DNA transformants are in use now in mutant screens to
identify insertion mutations in genes of interest (reverse genetics).

Vector-Mediated Gene Transfer:

Agrobacterium-Mediated Gene Transfer

Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium. It is rod shaped and

motile, and belongs to the bacterial family of Rhizobiaceae. A. tumefaciens is a phytopathogen,
and is treated as the nature’s most effective plant genetic engineer.

Some workers consider this bacterium as the natural expert of inter-kingdom gene transfer. In
fact, the major credit for the development of plant transformation techniques goes to the natural
unique capability of A. tumefaciens. Thus, this bacterium is the most beloved by plant
biotechnologists.
species of Agrobacterium:

i. A. tumefaciens that induces crown gall disease.

ii. A. rhizogenes that induces hairy root disease.

Crown Gall Disease and Ti Plasmid

Almost 100 years ago (1907), Smith and Townsend postulated that a bacterium was the
causative agent of crown gall tumors, although its importance was recognized much later. As
A. tumefaciens infects wounded or damaged plant tissues, in induces the formation of a plant
tumor called crown gall. The entry of the bacterium into the plant tissues is facilitated by the
release of certain phenolic compounds (acetosyringone, hydroxyacetosyringone) by the
wounded sites. Crown gall symptoms include round, wart-like growths 2 inches or larger in
diameter that appear at or just above the soil line, or on lower branches and stems. Plants with
several galls may be unable to move water and nutrients up the trunk and become weakened,
stunted and unproductive. Young plants can be killed by developing gall tissue. Crown gall
formation occurs when the bacterium releases its Ti plasmid (tumor- inducing plasmid) into
the plant cell cytoplasm. A fragment (segment) of Ti plasmid, referred to as T-DNA, is actually
transferred from the bacterium into the host where it gets integrated into the plant cell
chromosome (i.e. host genome). Thus, crown gall disease is a naturally evolved genetic
engineering process.

The T-DNA carries genes that code for proteins involved in the biosynthesis of growth
hormones (auxin and cytokinin) and novel plant metabolites namely opines, amino acid
derivatives and agropines, sugar derivatives The growth hormones cause plant cells to
 proliferate and form the gall while opines and agropines are utilized by A. tumefaciens as
sources of carbon and energy. As such, opines and agropines are not normally part of the plant
metabolism (neither produced nor metabolised). Thus, A. tumefaciens genetically transforms
plant cells and creates a biosynthetic machinery to produce nutrients for its own use.

As the bacteria multiply and continue infection, grown gall develops which is a visible mass
of the accumulated bacteria and plant material. Crown gall formation is the consequence of
the transfer, integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens
in the infected plant. The genetic transformation leads to the formation of crown gall tumors,
which interfere with the normal growth of the plant. Several dicotyledonous plants (dicots) are
affected by crown gall disease e.g. grapes, roses, stone-fruit trees.

33. Virus Mediated Gene Transfer

Viral transformation

Viral transformation is the change in growth, phenotype, or indefinite reproduction of cells

caused by the introduction of inheritable material. Through this process, a virus causes harmful
transformations of an in vivo cell or cell culture. Viral transformation can occur both naturally
and medically. Natural transformations can include viral cancers, such as human
papillomavirus (HPV) and T-cell Leukemia virus type I. Hepatitis B and C are also the result
of natural viral transformation of the host cells. Viral transformation can also be induced for
use in medical treatments. Cells that have been virally transformed can be differentiated from
untransformed cells through a variety of growth, surface, and intracellular observations.

Transduction

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral
vector.

Viral transformation (transduction)

Package the desired genetic material into a suitable plant virus and allow this modified virus
to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to
produce transformant cells. However genomes of most plant viruses consist of single stranded
RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a
form of transfection and not a real transformation, since the inserted genes never reach the
 nucleus of the cell and do not integrate into the host genome. The progeny of the infected
plants is virus free and also free of the inserted gene.

Plant Viruses as Vectors

Plant viruses are considered as efficient gene transfer agents as they can infect the intact plants
and amplify the transferred genes through viral genome replication. Viruses are natural vectors
for genetic engineering. They can introduce the desirable gene(s) into almost all the plant cells
since the viral infections are mostly systemic.

Plant viruses are non-integrative vectors:

The plant viruses do not integrate into the host genome in contrast to the vectors based on T-
DNA of A. tumefaciens which are integrative. The viral genomes are suitably modified by
introducing desired foreign genes. These recombinant viruses are transferred, multiplied and
expressed in plant cells. They spread systemically within the host plant where the new genetic
material is expressed.

Criteria for a plant virus vector

An ideal plant virus for its effective use in gene transfer is expected to posses the following
characteristics. The virus must be capable of spreading from cell to cell through
plasmodesmata. The viral genome should be able to replicate in the absence of viral coat
protein and spread from cell to cell. This is desirable since the insertion of foreign DNA will
make the viral genome too big to be packed. The recombinant viral genome must elicit little
or no disease symptoms in the infected plants. The virus should have a broad host range. The
virus with DNA genome is preferred since the genetic manipulations involve plant DNA.
The three groups of viruses — caulimoviruses, Gemini viruses and RNA viruses that are used
as vectors for gene transfer in plants are briefly described.

Caulimoviruses as Vectors:

The Caulimoviruses contain circular double- stranded DNA, and are spherical in shape.
Caulimoviruses are widely distributed and are responsible for a number of economically
important diseases in various crops. The caulimovirus group has around 15 viruses and among
these cauliflower mosaic virus (CaMV) is the most important for gene transfer. The other
caulimoviruses include carnation etched virus, dahlia mosaic virus, mirabilis mosaic virus and
strawberry vein banding virus.

Cauliflower mosaic virus (CaMV): CaMV infects many plants (e.g. members of Cruciferae,
Datura) and can be easily transmitted, even mechanically. Another attractive feature of CaMV
is that the infection is systemic, and large quantities of viruses are found in infected cells. A
diagrammatic view of the CaMV genetic map is depicted in Figure. The genome of CaMV
consists of a 8 kb (8024 bp) relaxed but tightly packed circular DNA with six major and two
minor coding regions. The genes II and VII are not essential for viral infection.
 •

Use of CaMV in gene transfer:

For appropriate transmission of CaMV, the foreign DNA must be encapsulated in viral protein.
Further, the newly inserted foreign DNA must not interfere with the native assembly of the
virus. CaMV genome does not contain any non-coding regions wherein foreign DNA can be
inserted. It is fortunate that two genes namely gene II and gene VII have no essential functions
for the virus. It is therefore possible to replace one of them and insert the desired foreign gene.
Gene II of CaMV has been successfully replaced with a bacterial gene encoding dihydrofolate
reductase that provides resistance to methotrexate. When the chimeric CaMV was transmitted
to turnip plants, they were systemically infected and the plants developed resistance to
methotrexate.

Limitations of CaMV as a vector

 CaMV vector has a limited capacity for insertion of foreign genes. Infective capacity of CaMV
is lost if more than a few hundred nucleotides are introduced. Helper viruses cannot be used
since the foreign DNA gets expelled and wild-type viruses are produced.

34. Virus Mediated Gene Transfer 2

Gemini Viruses as Vectors

The Gemini viruses are so named because they have geminate (Gemini literally means
heavenly twins) morphological particles i.e. twin and paired capsid structures. These viruses
are characterized by possessing one or two single-stranded circular DNAs (ss DNA). On
replications, ss DNA forms an intermediate double-stranded DNA. The Gemini viruses can
infect a wide range of crop plants (monocotyledons and dicotyledons) which attract plant
biotechnologists to employ these viruses for gene transfer. Curly top virus (CTV) and maize
streak virus (MSV) and bean golden mosaic virus (BGMV) are among the important Gemini
viruses. It has been observed that a large number of replicative forms of a Gemini virus genome
accumulate inside the nuclei of infected cells. The single-stranded genomic DNA replicates
in the nucleus to form a double-stranded intermediate. Gemini virus vectors can be used to
deliver, amplify and express foreign genes in several plants/ explants (protoplasts, cultured
cells). However, the serious drawback in employing Gemini viruses as vectors is that it is very
difficult to introduce purified viral DNA into the plants. An alternate arrangement is to take
the help of Agrobacterium and carry out gene transfer.

RNA Plant Viruses as Vectors

There are mainly two type’s single-stranded RNA viruses:

Mono-partite viruses:

These viruses are usually large and contain undivided genomes for all the genetic information
e.g. tobacco mosaic virus (TMV).

Multipartite viruses:

The genome in these viruses is divided into small RNAs which may be in the same particle or
different particles, e.g. brome mosaic virus (BMV). HMV contains four RNAs divided
between three particles. Plant RNA viruses, in general, are characterized by high level of gene
expression, good efficiency to infect cells and spread to different tissues. But the major
limitation to use them as vectors is the difficulty of joining RNA molecules in vitro.
Use of cDNA for gene transfer:

Complementary DNA (cDNA) copies of RNA viruses are prepared in vitro. The cDNA so
generated can be used as a vector for gene transfer in plants. This approach is tedious and slow
or complicated and therefore inefficient. However, some success has been reported. A gene
sequence encoding chloramphenicol resistance (enzyme- chloramphenicol acetyltransferase)
has been inserted into brome mosaic virus genome. This gene expression, however, has been
confined to protoplasts.

Limitations of Viral Vectors in Gene Transfer

The ultimate objective of gene transfer is to transmit the desired genes to subsequent
generations. With virus vectors, this is not possible unless the virus is seed-transmitted.
However, in case of vegetatively propagated plants, transmission of desired traits can be done
e.g. potatoes. Even in these plants, there is always a risk for the transferred gene to be lost
anytime. For the reasons referred above, plant biotechnologists prefer to insert the desired
genes of interest into a plant chromosome.

35. Vectorless or direct gene transfer

In the direct gene transfer methods, the foreign gene of interest is delivered into the host
plant cell without the help of a vector. The methods used for direct gene transfer in plants are

1. Chemical method
2. Physical method
 Chemical mediated gene transfer
1. Chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake
into plant protoplasts.
2. Calcium phosphate is also used to transfer DNA into cultured cells.
Liposome mediated gene transfer or Lipofection

Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids.
Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse
with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus.
Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant
cells.

Silicon carbide method

In this method, fibres of organic material like silicon carbide are used for gene transfer.
These fibres, when mixed with plasmid DNA and plant tissue or cells, help in penetration of
the foreign DNA into the plant tissue.

Microinjection

where the DNA is directly injected into plant protoplasts or cells (specifically into the
nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or
micropipette. This method of gene transfer is used to introduce DNA into large cells such as
oocytes, eggs, and the cells of early embryo.
Electroporation

Involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient

(temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA. The
cells are placed in a solution containing DNA and subjected to electrical shocks to cause
holes in the membranes. The foreign DNA fragments enter through the holes into the
cytoplasm and then to nucleus.

Particle gun/Particle bombardment

In this method, the foreign DNA containing the genes to be transferred is coated onto the
surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target
tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The
microprojectile bombardment method was initially named as biolistics by its inventor
Sanford (1988). Two types of plant tissue are commonly used for particle bombardment-
Primary explants and the proliferating embryonic tissues.

Transformation

This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The
transformation frequency (the fraction of cell population that can be transferred) is very good
in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-
50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation
efficiency refers to the number of transformants per microgram of added DNA. The CaCl2
breaks the cell wall at certain regions and binds the DNA to the cell surface.
 Conjuction

It is a natural microbial recombination process and is used as a method for gene transfer. In
conjuction, two live bacteria come together and the single stranded DNA is transferred via
cytoplasmic bridges from the donor bacteria to the recipient bacteria.

Selection of transformed cells from untransformed cells

The selection of transformed plant cells from untransformed cells is an important step in the
plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is introduced
into the plant along with the transgene followed by the selection of an appropriate selection
medium (containing the antibiotic). The segregation and stability of the transgene integration
and expression in the subsequent generations can be studied by genetic and molecular
analyses (Northern, Southern, Western blot, PCR).

36. Electroporation
Electroporation or electro-permeabilization is the process of applying electrical field to a
living cell for a brief duration of time in order to create microscopic pores in the plasma
membrane called electro-pores. This technique is used for transferring the recombinant DNA
molecule into wide range of hosts starting from bacteria to plant (plant protoplasts) and ani-
mal cells

Principle:

The phospholipid molecules of the plasma membrane are not static. When we apply electric
field to them their kinetic energy increases resulting in the increase in the membrane per-
meability at certain points. This is exactly where we see the formation of electro-pores. The
recombinant DNA can pass through these transient pores before they close.

Procedure

In this process cells are mixed with the recombinant DNA and the mixture is placed in a
small chamber with electrodes connected to a specialized power supply. Then a brief electric
impulse is discharged across the electrodes, which makes pores (holes) in the plasma
membrane. These pores remain for some time and are again resealed themselves.
Recombinant DNA enters the cell which are removed and plated in fresh selective medium.
The process of selection is then applied to isolate cells carrying recombinant DNA.
Advantages of electroporation:

1. This technique is simple, convenient and rapid, besides being cost-effective.

2. The transformed cells are at the same physiological state after electroporation.
3. Efficiency of transformation can be improved by optimising the electrical field strength,
and addition of spermidine.
Limitations of electroporation:

1. Under normal conditions, the amount of DNA delivered into plant cells is very low.
2. Efficiency of electroporation is highly variable depending on the plant material and the
treatment conditions.
3. Regeneration of plants is not very easy, particularly when protoplasts are used.

37. Particle Bombardment (Biolistics)

Biolistics is a method where cells are physically impregnated with nucleic acids or other
biological molecules. A biolistic particle delivery system is a device for plant transformation
where cells are bombarded with heavy metal particles coated with DNA/RNA. This technique
was invented by John Stanford in 1984 for introduction of DNA into cells by physical means
to avoid the host-range restrictions of Agrobacterium.Agrobacterium-mediated geneti
transformation system works well for dicotyledonous plants but has
low efficiency for monocots. Biolistic particle delivery system provides an effective and
versatile way to transform almost all type of cells. It has been proven to be a successful
alternative for creating transgenic organisms inprokaryotes, mammalian and plant species.

In this process, construct having gene of interest is coated on the surface oftiny particles of
gold or tungsten (0.6 – 1 mm in size). Prior to coating, DNA is precipitated with calcium
chloride, spermidine and polyethylene glycol. These coated microparticles are loaded on to the
macrocarrier and accelerated to high speed by using pressurized helium gas. Plant cell
suspensions, callus cultures, or tissues could be used as the target of thesemicroprojectiles. As
the microprojectiles penetrate the plant cell walls and membranes to enter the cells, coated
DNA is released from its surface and incorporated into the plant’s genome. In biolistics, use
of binary vectors with T-DNA border sequences is not required. This method is especially
important for monocots, for which efficiency of other transformation methods is not
satisfactory. A wide range of tissues such as apical and floral meristems, embryos, seedlings,
leaves, cultured cells and floral tissues could be used as target in this method.
A number of parameters should be carefully considered before using particle bombardment.

These can be classified under three categories

Physical parameters

Nature, chemical and physical properties of the metal particles utilized to carry the foreign
DNA. The nature and preparation of DNA,binding of DNA on the particles and target tissues.

Environmental parameters

Variables such as temperature, photoperiod and humidity of donor plants, explants, and
bombarded tissues affect physiology of tissues and influence receptiveness of the target tissue.

Biological parameters

Choice and nature of explants, pre- and post bombardment culture conditions, osmotic pre-
and post-treatment of explants.

Factors affecting bombardment

Several attempts are made to study the various factors, and optimize the system of particle
bombardment for its most efficient use.

Some of the important parameters are described.

Nature of micro particles:

Inert metals such as tungsten, gold and platinum are used as micro particles to carry DNA.

These particles with relatively higher mass will have a better chance to move fast when
bombarded and penetrate the tissues.

Nature of tissues/cells:

The target cells that are capable of undergoing division are suitable for transformation.

Some more details on the choice of plant material used in bombardment are already given.

Amount of DNA

The transformation may be low when too little DNA is used. On the other hand, too much
DNA may result is high copy number and rearrangement of transgenes. Therefore, the quantity
of DNA used should be balanced. Recently, some workers have started using the chemical
aminosiloxane to coat the micro particles with low quantities of DNA adequate enough to
achieve high efficiency of transformation.

Advantages of particle bombardment over Agrobacterium-mediated DNA transfer

This system is species independent and can been used successfully for a wide range of
organisms. Many species which are recalcitrant to other direct transfer methods or are not ready
to Agrobacterium-mediated transformation have been transformed by this technique.
Introduced DNA does not need sequences necessary for T-DNA replication and transfer as
complex interaction between bacterium and plant tissue does not take place. Transformation
of organelle DNA (mitochondria and chloroplasts) has also been achieved by this method.
Multiple genes can be introduced in a single plant. Particles can be coated with
DNA/RNA/siRNA/large fragments of nucleic acids.

Limitations of particle bombardment method:

 Limited regeneration capacity of tissue being bombarded

 Efficiency of stable integration of DNA.
 Insertion of multiple copies of the gene
 Integration of rearranged and/or truncated DNA sequences
 Damage to the cellular tissue.
 Specialized and expensive equipment’s are required
 38. Particle Bombardment (Biolistics) 2
Gene Delivery System:

This system has many names and also Known as Particle Bombardment, Biolistics,
Microprojectile bombardment, Particle acceleration, Particle inflow gun, Gene gun. A gene
gun or a biolistic particle delivery system, originally designed for plant transformation, is a
device for delivering exogenous DNA (transgenes) to cells. Using a gene gun directly shoots
a piece of DNA into the recipient plant tissue.

Tungsten or gold beads are coated in the gene of interest and fired through a stopping screen,
accelerated by Helium, into the plant tissue. The particles pass through the plant cells,
leaving the DNA inside.

Advantage:

This method can be use to transform all plant species.

No binary vector is required.

Transformation protocol is relatively simple.

Disadvantage:

Difficulty in obtaining single copy transgenic events.

High cost of the equipment and microcarriers.

Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.).

Transfer DNA is not protected.

39. Microinjection
The process of using a fine glass micropipette to manually inject transgene at microscopic or
borderline macroscopic level is known as microinjection. The transgene, in the form of
plasmids, cosmids, phage, YACs, or PCR products, can be circular or linear and need not be
physically linked for injection

Microinjection involves direct mechanical introduction of DNA intothe nucleus or cytoplasm

using a glass micro capillary injection pipette. The protoplasts are immobilized in low melting
agar, while working under a microscope, using a holding pipette and suction force. DNA is
then directly injected into the cytoplasm or the nucleus.

The injected cells are then cultured invitro and regenerated into plants. Successful examples of
this process has been shown in rapeseed, tobacco and various other plants. Stable transformants
can be achieved through this method but it requires technical expertise and is a time consuming
process.

Also, microinjection has achieved only limited success in plant transformation due to the thick
cell walls of plants and a lack of availability of a single-cell-to-plant regeneration system in
most plant species.

In this technique a traditional compound microscope (around 200X magnification) or an

inverted microscope (around 200x magnification) or a dissecting stereomicroscope (around
40-50x)is used.

Under the microscope target cell is positioned and cell membrane and nuclear envelope are
penetrated with the help of two micromanipulators. One micromanipulator holds the pipette
and another holds the micro capillary needle.

There are two types of microinjection systems; constant flow system and pulsed flow system

In the constant flow system the amount of sample injected is determined by the duration for
which needle remains in the cell. The constant flow system is relatively simple and inexpensive
but outdated.

The pulsed flow system has greater control over the volume of substance delivered, needle
placement and movement and has better precision. This technique results in less damage to the
receiving cell, however, the components of this system are quite expensive.
 40. Chemical Gene Transfer Methods
There are two types of chemical methods used for plant transformation

1. Polyethylene glycol (PEG)-mediated transfer

2. DEAE Dextran-Mediated transfer
Polyethylene glycol (PEG)-mediated transfer

Polyethylene glycol (PEG), in the presence of divalent cations (using Ca 2+), destabilizes the
plasma membrane of protoplasts and renders it permeable to naked DNA.

In this way, the DNA enters nucleus of the protoplasts and gets integrated with the genome.

The procedure involves the isolation of protoplasts and their suspension, addition of plasmid
DNA, followed by a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol and
calcium nitrate solution. As this mixture is incubated, protoplasts get transformed.

Advantages of PEG-mediated transformation:

 A large number of protoplasts can be simultaneously transformed.

 This technique can be successfully used for a wide range of plant species.
Limitations of PEG-mediated transformation:

 The DNA is susceptible for degradation and rearrangement.

 Random integration of foreign DNA into genome may result in undesirable traits.
 Regeneration of plants from transformed protoplasts is a difficult task.
(DEAE) Dextran Mediated transfer

This method was initially reported by Vaheri and Pagano in 1965 for enhancing the viral
infectivity of cell but later adapted as a method for plasmid DNA transfer. Diethyl aminoethyl
dextran (DEAE-dextran) is a soluble poly cationic carbohydrate that promotes interactions
between DNA and endo cytotic machinery of the cell.

In this method, the negatively charged DNA and positively charged DEAE – dextran form
aggregates through electrostatic interaction and form apolyplex. A slight excess of DEAE –
dextran in mixture results in net positive charge in the DEAE – dextran/ DNA complex formed.
These complexes, when added to the cells, bind to the negatively charged plasma membrane and
get internalized through endocytosis. Complexed DNA delivery with DEAE-dextran can be
improved by osmotic shock using DMSO or glycerol.
Several parameters such as number of cells, polymer concentration, transfected DNA
concentration and duration of transfection should be optimized for a given cell line

Advantages

This method is Simple and inexpensive, more sensitive and it can be applied to a wide range
of cell types and Can be used for transient transfection.

Disadvantages

DEAE is a toxic to cells at high concentrations. Transfection efficiency varies with cell type.
It can only be used for transient transfection but not for stable transfection. Typically
produces less than 10% delivery in primary cells.

41. Plastid transformation

Plastid
Plastids are the major organelle of plant and algal cells. These are the site of manufacture and
storage of important chemical compounds. It has circular, dsDNA copies and it replicates
autonomously of the cell. Plastids are thought to have been originated from endosymbiotic
bacteria and plastid genes show maternal inheritance.

Derived from proplastids in meristem

Plastids Have diverse functions

• Chloroplasts – green plastids – for photosynthesis

• Chromoplasts – coloured plastids – for pigment synthesis and storage
• Gerontoplasts – control dismantling of photosynthetic apparatus during senescence
• Leucoplasts – colourless plastids – monoterpene synthesis
• Leucoplasts include amyloplasts (starch), elaioplasts (fats), proteinoplasts (proteins) and
tannosomes (tannins)

Comparison of the nuclear and plastid genomes of angiosperm

 Why plastid is used for transformation?

Due to the High protein expression levels and absence of epigenetic effects. Plastids have
uniparental inheritance is commercially favored and easy transgene stacking in operons. Since
plastids are maternally inherited, they aren’t transmitted by pollen these are biologically safe.

Difficulty in delivering foreign DNA through double membrane of the plastid. The enormous
copy number (polyploidy) of the plastid genome. The desired genetic modification must be in
each copy of plastid genome in each cell. Failure to achieve homoplasmy results in rapid
somatic segregation and genetic instability. Repeated rounds of selection and regeneration are
required.

Chloroplast transformation requirements

For chloroplast transformation chloroplast specific expression vector is needed and a
method for DNA delivery through a double membrane of the chloroplast. An efficient
selection for the transplastome is required.
DNA delivery into plastids

Successful methods include biolistic and polyethylene glycol-mediated transfer. Biolistic is

preferred as it is less time-consuming and demanding. Integration of foreign DNA into plastid
genome occurs via homologous recombination. Homologous recombination operates in
plastids at a high efficiency.

Transformation of the chloroplast genome by bombarding tobacco leaves with micro

projectiles coated with DNA. Following bombardment, leaf discs are placed onto antibiotic
containing medium (panel A). Transgenic plants are regenerated from the transformed tissue
that is able to develop green chloroplasts (panel B).
 42. Methods of chloroplast transformation
The following points highlight the top five methods of chloroplast transformation in higher
plants.

The methods are:

1. Vectors for Plastid Transformation

2. Engineering for High-Level Protein Production

3. Biolistic Transfer

4. PEG Mediated Transformation

5. Galistan Expansion Femtosyringe Method.

Vectors for Plastid Transformation:

Vectors used for plastid transformation utilize left (LTR) and right (RTR) targeting regions to
direct inserting of transgene into the plastid region. Some of the commonly used plasmid
transformation vectors are plasmid repeat vector (pRV) and vector pRB94 and pRB95. Expres-
sion vector for chloroplast transformation contains two-open reading frame under the control
of chloroplast-specific promoter and termination signal.
 Presence of homologous sequence facilitate two recombination events which consequently
responsible for the insertion of marker gene and genome of interest into the LRT and RTR
regions of the plastid. Some of the well-known plastid transformation vectors are plasmid
repeat vector, and other is pRB95. Some of the expression system facilitated the expression of
ribosome binding site region inserted at intergenic regions allowing production of target
protein from polycistronic mRNA transcript

Engineering for High-Level Protein Production:

Strategies for production of high amount of recombinant proteins have been adopted like
utilization of strong promoter, and stable mRNA transcript determined by 5′ untranslated
(UTR) and 3′ untranslated (UTR) of the transgene. Considering these facts several plastid
expression vectors are designed to contain 5′ regulatory region PL casette and 3′ regulatory
region (T casette), strong sigma 70-type PEP promoter of the rRNA operon promoter (prnn).

The 3′ UTR regulatory sequence of mRNA include RNA stem loop structure, which acts as a
inefficient transcription terminator. Most 3′ UTR T casettes are derived from PbSA, rbcL and
rps 16 genes. The 5′ UTR (PL casette) region play important role in translation efficiency. In
addition, careful optimization of transgene codons, despite its prokaryotic nature of expression
system, resulted in high protein production.

Biolistic Transfer: Biolystic is one of the efficient approaches for plastid transformation.

Effective penetration and high transfer frequency are some of the plus points of biolystic
method. There are number of bacteria and viruses are known to infect chloroplast. It’s
envelop is made up of double membrane and actually considered that chloroplast
transformation was considered to be virtually impossible.

However, invention of biolistic gene gun technology paved the pathway of direct delivery of
target gene into the living cell. It is fortunate that DNA is deposited in a chloroplast and
successfully integrated into the chloroplast genome.

PEG Mediated Transformation:

Polyethylene glycol is widely used in transformation work. Despite its efficiency, PEG
mediated transformation is far behind than the biolistic approach. Foreign DNA is taken by
protoplast in presence of PEG and transported by unknown process from cytoplasm into the
chloroplast and finally integrated into the genome.

Galistan Expansion Femtosyringe Method:

The existing microinjection method in which recipent cells damaged by the release of
cellular contents into the needle after injection have raised fresh look into the designing of
novel approach for chloroplast transformation of wide range of species. A novel galinstan
femtosyringe method designed for chloroplast transformation involves microinjection of for-
eign DNA into chloroplast

The heat induced expansion of a liquid metal within a glass syringe forces the foreign DNA
through a minute capillary top with a diameter of approximately 0.1 µm. The liquid metals
employed in the specialized femtosyringe are generally galinstan, an alloy of gallium, indium
and tin.

43. Molecular biology of Chloroplast

Transformation
Stable transformation system depends on integration of the transforming DNA into the plastid
genome by homologous recombination. Sequence to be introduced into the plastid genome
must flanked on both side by region of homology with the chloroplast genome. Primary
transplastomic event results hetroplasmic cells. Hetroplasmy is unstable so it will resolve into
homoplasmy.
 Marker removal

• Recombination between directly repeated sequences excises the intervening DNA

sequence and one copy of the direct repeat.
 • The breakage and joining of DNA strands involved in recombination can be mediated by
the native homologous recombination machinery present in plastids.
Case Study – Lactuca sativa
1. Protoplast isolation

• Lettuce seeds were sterilized and sown on MS medium with 2% sucrose

• Shoot tips from leaves obtained were transferred to MS medium with 3% sucrose
• The leaves were cut into pieces and incubated in PG solution, followed by enzyme solution
consisting of 1% cellulase and 0.25% macerozyme
• Protoplast suspension was filtered through nylon mesh
• Protoplasts were collected at surface after centrifugation at 70g for 8min
2.Transformation and culture
• 10μl transforming DNA and 0.6ml PEG solution was added to protoplast suspension
and incubated at 25ºC for 10min
• Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a density of 3.6 X
104 protoplasts per ml
• The suspension was plated onto Petri dishes and cultured at 25ºC in the dark
• Selection was initiated on the 7th day by fresh medium containing spectinomycin
dihydrochloride

• 100% of spectinomycin-resistant lettuce cell lines were true plastid transformants

• A limitation was the high frequency of polyploid cell lines
 Analyses
• PCR – specific primers were used to assess the presence of aadA gene in resistant cell lines
• Immunoblot analysis – using HRP-conjugated secondary antibodies
• Southern and Northern blots were performed to look for target genes and their transcripts

Production of human therapeutic proteins

Why lettuce is favoured over tobacco?
• Most of the plant is leaf tissue and this tissue contains the greatest number of plastids per
cell
• Unlike tobacco, lettuce has no toxic alkaloids that need to be removed - low purification
and downstream processing costs
• Lettuce is a relevant human foodstuff that can be consumed without cooking
 44. Pesticide
Pesticides are substances that are meant to control pests, including weeds. Any substance or
mixture of substances intended for preventing, destroying, or controlling any pest, including
vectors of human or animal disease, unwanted species of plants or animals, causing harm
during or otherwise interfering with the production, processing, storage, transport, or
marketing of food, agricultural commodities, wood and wood products or animal feedstuffs.
Substances that may be administered to animals for the control of insects, arachnids, or other
pests in or on their bodies. The term includes substances intended for use as a plant growth
regulator, defoliant, desiccant, or agent for thinning fruit or preventing the premature fall of
fruit. Also used as substances applied to crops either before or after harvest to protect the
commodity from deterioration during storage and transport

Classification

Pesticides can be classified by target organism.

• Algaecides are used for killing and/or slowing the growth of algae.
• Antimicrobials control germs and microbes such as bacteria and viruses.
• Biopesticides are made of living things, come from living things, or they are found in
nature.
• Desiccants are used to dry up living plant tissues.
• Defoliants cause plants to drop their leaves.

• Disinfectants control germs and microbes such as bacteria and viruses.

• Fungicides are used to control fungal problems like molds, mildew, and rust.
• Herbicides kill or inhibit the growth of unwanted plants, aka weeds.
• Illegal and Counterfeit Pesticides are imported or sold illegally.
 • Insecticides are used to control insects.
• Insect Growth Regulators disrupt the growth and reproduction of insects.
• Miticides control mites that feed on plants and animals. Mites are not insects, exactly.
• Molluscicides are designed to control slugs, snails and other molluscs.
• Mothballs are insecticides used to kill fabric pests by fumigation in sealed containers.
• Natural and Biological Pesticides control pests using things found in nature, or man-made
versions of things found in nature.
• Ovicides are used to control eggs of insects and mites.
• Pheromones are biologically active chemicals used to attract insects or disrupt their mating
behavior. The ratio of chemicals in the mixture is often species-specific.
• Repellents are designed to repel unwanted pests, often by taste or smell.
• Rodenticides are used to kills rodents like mice, rats, and gophers.

45. Uses of pesticides

Pesticides are used to control organisms that are considered to be harmful, for example, they
are used to kill mosquitoes that can transmit potentially deadly diseases like West Nile virus,
yellow fever, and malaria. They can also kill bees, wasps or ants that can cause allergic
reactions. Insecticides can protect animals from illnesses that can be caused by parasites such
as fleas Pesticides can prevent sickness in humans that could be caused by moldy food or
diseased produce. Herbicides can be used to clear roadside weeds, trees, and brush. They can
also kill invasive weeds that may cause environmental damage. Herbicides are commonly
applied in ponds and lakes to control algae and plants such as water grasses that can interfere
with activities like swimming and fishing and cause the water to look or smell unpleasant.
Uncontrolled pests such as termites and mold can damage structures such as houses. Pesticides
are used in grocery stores and food storage facilities to manage rodents and insects that infest
food such as grain. Each use of a pesticide carries some associated risk. Proper pesticide use
decreases these associated risks to a level deemed acceptable by pesticide regulatory agencies.
DDT, sprayed on the walls of houses, is an organochlorine that has been used to fight malaria
since the 1950s. Recent policy statements by the World Health Organization have given
stronger support to this approach.

However, DDT and other organochlorine pesticides have been banned in most countries
worldwide because of their persistence in the environment and human toxicity. DDT use is not
always effective, as resistance to DDT was identified in Africa as early as 1955, and by 1972
nineteen species of mosquito worldwide were resistant to DDT

Dichlorodiphenyltrichloroethane, commonly known as DDT, is a colorless, tasteless, and almost

odorless crystalline chemical compound, an organochlorine, originally developed as an
insecticide, and ultimately becoming infamous for its environmental impacts. First synthesized in
1874, DDT's insecticidal action was discovered by the Swiss chemist Paul Hermann Müller in
1939. DDT was used in the second half of World War II to control malaria and typhus among
civilians and troops.

Benefits

Pesticides can save farmers' money by preventing crop losses to insects and other pests; in the
U.S., farmers get an estimated fourfold return on money they spend on pesticides. One study
found that not using pesticides reduced crop yields by about 10%.

Another study, conducted in 1999, found that a ban on pesticides in the United States may
result in a rise of food prices, loss of jobs, and an increase in world hunger. There are two
levels of benefits for pesticide use, primary and secondary. Primary benefits are direct gains
from the use of pesticides and secondary benefits are effects that are more long-term.

Primary benefits

• Controlling pests and plant disease vectors

• Improved crop/livestock yields
• Improved crop/livestock quality
• Invasive species controlled

Controlling human/livestock disease vectors and nuisance organisms

• Human lives saved and disease reduced. Diseases controlled include malaria,with millions
of lives having been saved or enhanced with the use of DDT alone.
• Animal lives saved and disease reduced
• Controlling organisms that harm other human activities and structures
• Drivers view unobstructed
• Tree/brush/leaf hazards prevented
• Wooden structures protected

46. Effects of pesticides

Health effects
Pesticides may cause acute and delayed health effects in people who are exposed. Pesticide
exposure can cause a variety of adverse health effects, ranging from simple irritation of the
skin and eyes to more severe effects such as affecting the nervous system, mimicking hormones
causing reproductive problems, and also causing cancer.

A 2007 systematic review found that "most studies on non-Hodgkin lymphoma and leukemia
showed positive associations with pesticide exposure" and thus concluded that cosmetic use of
pesticides should be decreased. Non-Hodgkin lymphoma is a cancer that starts in white blood cells
called lymphocytes, which are part of the body's immune system. leukemia involves the production of
abnormal white blood cells -- the cells responsible for fighting infection. There is substantial
evidence of associations between organophosphate insecticide exposures and neurobehavioral
alterations. Limited evidence also exists for other negative outcomes from pesticide exposure
including neurological, birth defects, and fetal death.

Environmental effects

Widespread use of pesticides in agriculture has experts worried due to their long-term
environmental damage. Some pesticides can stick around for years, posing a very real threat
to the ecological system and hence human health. Excessive and careless use of pesticides can
contaminate water sources and soil, make fruits and vegetables less nutritious, and reduce
biodiversity. Some pesticides have also been linked to the dramatic reduction in the number of
bees across the world, posing a huge threat to agriculture and food security, given that bees
pollinate more than 70% of all crops.

Economics

In one study, the human health and environmental costs due to pesticides in the United States
was estimated to be $9.6 billion: offset by about $40 billion in increased agricultural
production.

Additional costs include the registration process and the cost of purchasing pesticides: which
are typically borne by agrichemical companies and farmers respectively. The registration
process can take several years to complete (there are 70 different types of field test) and can
cost $50–70 million for a single pesticide.

At the beginning of the 21st century, the United States spent approximately $10 billion on
pesticides annually.

47. Effects of pesticides on human health

• Experts broadly classify the effects of pesticides as topical or systemic.
• Topical reactions are usually limited to areas of the body that have come in direct contact
with a pesticide.
• Inflammation of the skin (dermatitis) such as a rash or blisters is usually the most common
topical symptom.
• Other topical reactions may include sneezing, wheezing, and coughing, usually triggered
by petroleum distillates that many pesticides contain.
• Severe pesticide poisoning can cause seizures, change in heart rate, and sometimes even
coma and death
Short-Term Effects of Pesticides

• Short-term pesticide poisoning or acute toxicity from pesticides is usually the result of a
single and brief exposure to a pesticide.
• This kind of poisoning can happen due to exposure via the skin, inhalation, through the
eyes, or orally.
• Symptoms of acute toxicity can become apparent instantly or take as long as 48 hours
• Short-term effects of pesticides can manifest as:
• Nausea and vomiting
• Diarrhea
• Loss of consciousness
• Seizures
• Coughing and sore throat
• Extreme weakness
Long-Term Effects Of Pesticides

• While continual, low-dose exposure to pesticides don’t usually show immediate effects,
they cause serious harm to human health in the long term.

• Repeated exposure to pesticides, even in small doses, has been linked to a number of
diseases such as cancer, Parkinson’s, Alzheimer’s, sterility, and developmental disorders.
• Chronic exposure to pesticides can also lead to genetic changes and serious nerve disorders.
• Some studies have even linked pesticides to asthma, ADHD, depression, and anxiety
• Some pesticides contain chemicals that may be endocrine disruptors. These types of
pesticides can be especially damaging because they interfere with our hormones and
hormonal balance.
• Over a period of time, even low concentrations of these chemicals can cause obesity,
diabetes, thyroid tumors, decreased fertility, uterus abnormalities, and early puberty.
• Lastly, pesticides are also known to cause neurological issues such as loss of memory and
coordination, visual impairment, mood instability, and reduced motor skills
Effects Of Pesticides On Pregnant Women

• Exposure to pesticides and pesticide residue can lower fertility in women.

• A Harvard study found that women who ate more than two servings of fruits or vegetables
with high pesticide residue each day were 18% less likely to become pregnant and 26%
less likely to have a live birth compared to women with lower exposure

Effects Of Pesticides On Kids

Children are especially susceptible to harmful effects of pesticides. They can easily become
exposed to pesticides (via inhalation or skin contact) in schools, daycare, playgrounds,
hospitals, and any other public areas, no matter how careful you are. Kids’ bodies are smaller
and still growing, they take more breaths per minute, and they also eat and drink more relative
to their weight – all factors that make them more likely to absorb pesticides and residue. Their
little kidneys and liver also cannot eliminate pesticides from their bloodstream as effectively
as an adult’s.
48. Effects of pesticides on environment
 Although each pesticide is meant to kill a certain pest, a very large percentage of pesticides
reach a destination other than their target. Pesticides easily contaminate the air, ground and
water when they run off from fields, escape storage tanks, are not discarded properly, and
especially when they are sprayed aerially.

Water

Pesticides can be found in rain, ground water, streams, rivers, lakes and oceans. There are four
major ways that pesticides can reach the water:

• it can drift outside of the area of where it was sprayed,

• it can leach through the soil,
• it can be carried as runoff,
• or it may be spilled accidentally.

Soil

The use of pesticides decreases the general biodiversity in the soil. Soil quality is higher
without chemicals and this allows for higher water retention, necessary for plants to grow.

Plants

Nitrogen fixation, which is necessary for the growth of many large plants, is hindered by
pesticides that can be found in soil. This can lead to a large decline in crop yields. Application
of pesticides to crops that are in bloom can kill honeybees, which act as pollinators. This also
decreases crop pollination and reproduction.

Animals

Animals may be poisoned by pesticide residues that remain on food after spraying.

An application of pesticides in an area can eliminate food sources that certain types of animals
need, causing the animals to relocate, change their diet, or starve.

Poisoning from pesticides can even make its way up the food chain; for example, birds can be
harmed when they eat insects and worms that have consumed pesticides.

Birds

Birds are being harmed by pesticide use. Rachel Carson’s book Silent Spring discusses the
loss of several bird species due to accumulation of pesticides in their tissues. Types of
 fungicides used in farming are only slightly toxic to birds and mammals, but may kill off
earthworms, which can in turn reduce populations of the birds and mammals that feed on them

Aquatic Life

Fish and other aquatic biota may be harmed by pesticide-contaminated water. Application of
herbicides to bodies of water can cause plants to die, diminishing the water’s oxygen and
suffocating the fish. Repeated exposure of some pesticides can cause physiological and
behavioural changes in fish that reduce populations, such as abandonment of nests, decreased
immunity to disease, and increased failure to avoid predators. Additionally, as some pesticides
come in granular form, birds and other wildlife may eat the granules, mistaking them for grains
of food. A few granules of a pesticide are enough to kill a small bird. Herbicides may also
endanger bird populations by reducing their habitat.

Negative effects of pesticides

• Are carried on the wind

• Leaves residue on produce
• Remains inside produce and animals through bio accumulation
• Runs off into open water which contaminates public water supply as well as fish and other
seafood.
• Pesticides can enter the body through the skin, eyes, mouth, and nose.
• Fetuses can suffer from exposure and develop behavioral problems as well as growth issues
• Babies can develop lower cognitive scores and fewer nerve cells and can have a lower birth
rate as well as being born prematurely.
• Toxins from pesticides can stay in the body and build up in the liver
• There are many possible reactions that include fatigue, skin irritations, nausea, vomiting,
breathing problems, brain disorders, blood disorders, liver and kidney damage,
reproductive damage, cancer, and in some cases, death.
 49. Signs and symptoms by pesticide poisoning

How do pesticides enter our bodies?

Pesticides can enter your body during mixing, applying, or clean-up operations. There are
generally three ways a chemical or material can enter the body through the skin (dermal)
through the lungs (inhalation) by mouth (ingestion).

Dermal (absorption through skin or eyes)

In most work situations, absorption through the skin is the most common route of pesticide
exposure. People can be exposed to a splash or mist when mixing, loading or applying the
pesticide. Skin contact can also occur when you touch a piece of equipment, protective
clothing, or surface that has pesticide residue on it. Pesticides can also be absorbed through
your eyes. In addition, pesticides, can cause injuries to the eye itself.

Inhalation (through the lungs)

Inhalation may occur when working near powders, airborne droplets (mists) or vapors. The
hazard from low-pressure applications is fairly low because most of the droplets are too large
to remain in the air. Applying a pesticide with high pressure, ultra-low volume, or fogging
equipment can increase the hazard because the droplets are smaller and they can be carried in
the air for considerable distances. Pesticides with a high inhalation hazard will be labelled with
directions to use a respirator.

Ingestion (by mouth)

While ingestion (by mouth) is a less common way to be exposed, it can result in the most
severe poisonings. There are numerous reports of people accidentally drinking a pesticide that
has been put into an unlabeled bottle or beverage cup/container (including soft drink cans or
bottles). Workers who handle pesticides may also unintentionally ingest the substance when
eating or smoking if they have not washed their hands first.

Types of pesticide poisoning:

There are two types of pesticide poisoning:

Acute poisoning

This happens when someone has been exposed to a high dose of pesticide. This could occur
when the pesticide is being mixed, for example, or if a hose breaks drenching the person or
bystanders with liquid pesticide solution. Another example might be accidental ingestion of a
pesticide, such as a child swallowing the chemical.
 Chronic poisoning

This results from a person being exposed to a small amount of pesticide on many occasions
over a long period of time. Chronic poisoning may happen when the operator repeatedly uses
pesticide improperly, especially if they do not wear protective clothing and equipment or wears
protective clothing which is not clean or is worn out, like wearing cracked or torn gloves.

Symptoms of pesticide poisoning

Symptoms of mild poisoning includes headache, sweating, diarrhea irritation of nose

and throat eye irritation, nausea, fatigue, changes of mood, skin irritation, insomnia,
loss of appetite, thirst, weakness, restlessness, dizziness, sore joints and nervousness.

Symptoms of severe poisoning

Symptoms of severe poisoning includes vomiting, convulsions, loss of reflexes,

unconsciousness, inability to breathe, fever muscle twitching, thirst, constriction of eye
pupils (eye pupils become small), increased rate of breathing

50. Treatment for Pesticide Poisoning

When pesticides are released into the air, we breathe them in through our nose and mouth.
Once in the lungs, the pesticides quickly enter the blood and spread poison through the whole
body. Because some pesticides have no smell, it is often hard to know if they are in the air.
The most common forms of air-borne pesticides are fumigants, aerosols, foggers, smoke
bombs, pest strips, sprays, and residues from spraying. You can also inhale pesticide dust in a
storage area, when it is being used in an enclosed area, such as a greenhouse, or when it is
being transported to the fields. Pesticide dust in the air can travel miles to pollute an area far
from where it was used. It is easy for pesticide dust to get into houses.

If you think you have breathed in pesticides, get away from the pesticides right away! Do not
wait until you feel worse.

Treatment

If you or someone else breathes in pesticides: Get the person away from the area where she
breathed in the poison, especially if it is an enclosed area. Get fresh air. Loosen clothing to
make breathing easier. Sit with head and shoulders raised.

If the person is unconscious, lay her on her side and watch her to make sure there is nothing
blocking her breathing. If the person is not breathing, quickly do mouth-to-mouth breathing.
Seek medical help. Take the pesticide label or name of the pesticide with you.
 51. Treatment for Pesticide Poisoning 2
Like other toxic chemicals, pesticides can poison people in different ways:

They can poison through the skin and eyes, through the mouth (by swallowing) or through the
air (by breathing). Each kind of poisoning needs a different kind of treatment.

When pesticides get on the skin

Most pesticide poisonings are from pesticides being absorbed through the skin. This can
happen when they spill while being moved, when they splash during mixing, during spraying,
or when you touch crops that have just been sprayed. Pesticides can also get on your skin
through your clothes, or when you wash clothes with pesticides on them.

Rashes and irritation are the first signs of poisoning through the skin. Because skin problems
may be caused by other things, such as a reaction to plants, insect bites, infections, or allergies,
it can be hard to know if the problem is caused by pesticides.
Talk to other workers to find out if the crop you are working with causes this kind of reaction.
If you work with pesticides and get any unexpected skin rashes, it is safest to treat them as if
they are caused by pesticides.

Treatment

If you or someone else gets pesticides on the body:

Quickly remove any clothing the pesticides spilled onto. Wash the pesticides off the skin as
soon as possible with soap and cool water. If it got into the eye, rinse the eye with clean water
for 15 minutes.

If the skin is burned from pesticides:

Rinse well with cool water. Do not remove anything stuck to the burn. Do not apply lotions,
fats, or butter. Do not break blisters. Do not remove loose skin. Cover the area with a sterile
dressing, if available.

When pesticides are swallowed

People can swallow pesticides by eating, drinking, or smoking cigarettes in the fields while
working with pesticides, or by drinking water polluted with pesticides.

Children can drink or eat pesticides, especially if pesticides are stored in containers also used
to hold food, or left in the open or low to the ground.

Treatment

When someone swallow’s pesticides:

If the person is unconscious, lay her on her side and make sure she is breathing. If the person
is not breathing, quickly do mouth-to-mouth breathing. Mouth-to-mouth breathing can also
expose you to the pesticide, so cover your mouth with a pocket mask, a piece of cloth, or thick
plastic wrap with a hole cut in the middle, before you start mouth-to-mouth breathing. Find the
pesticide package and read the label right away. The label will tell you if you should make the
person vomit up the poison or not. If the person can drink, give her lots of clean water.

Seek medical help. If it is available, always take the pesticide label or name with you.

Do not vomit if the label says not to. Never vomit after swallowing a pesticide that contains
gasoline, kerosene, xylene, or other petroleum-based liquids. This will make the problem
worse. Never make the person vomit or drink if she is unconscious, confused, or shaking badly.
If you are sure vomiting is OK, give the person: a glass of very salty water or 2 tablespoons of
pounded strong-tasting edible plant (such as celery, basil, or another local herb) followed by 1
 or 2 glasses of warm water. Keep the person moving around. This can help her vomit sooner.
After vomiting, activated or powdered charcoal can help absorb any poison still in the stomach

52. Approaches to pesticide poisoning

When effectively applied, pesticides can kill or control pests, including weeds, insects, fungi,
bacteria, and rodents. Chemical pest control has contributed to dramatic increases in yields for
most major fruit and vegetable crops

Pesticide poisoning

A pesticide poisoning occurs when chemicals intended to control a pest affect non-target
organisms such as humans, wildlife, or bees

Types of pesticide poisoning.

Humans may be harmed by pesticides in two ways: they may be poisoned or injured.
Pesticide poisoning is caused by pesticides that harm internal organs or other systems inside
the body. Pesticide-related injuries usually are caused by pesticides that are external irritants.

Hazard

Hazard is the risk of harmful effects from pesticides. Hazard depends on both the toxicity of the
pesticide and your exposure.
Hazard = Toxicity x Exposure
Exposure

When a pesticide contacts a surface or organism, that contact is called a pesticide exposure.
For humans, a pesticide exposure means getting pesticides in or on the body. The toxic effect
of a pesticide exposure depends on how much pesticide is involved and how long it remains
there.
Types of Exposures

 Pesticides contact your body in four main ways:

 Oral exposure (when you swallow a pesticide),
  Inhalation exposure (when you breathe in a
 pesticide),
 Ocular – (through the eyes), or
 Dermal (through the skin)
Strategies to exposure/poison

 Diagnosis
 Prevention
 Treatment

53. Herbicides
A herbicide is a chemical substance used to control or manipulate undesirable vegetation,
especially weeds. Herbicides are extensively used in gardening, farming, and landscape turf
management. Herbicides tend to have wide-ranging effects on non-target species (other than
those the pesticide is meant to control or kill). Herbicides, also commonly known as
weedkillers, are chemical substances used to control unwanted plants. Modern herbicides are
often synthetic mimics of natural plant hormones which interfere with growth of the target
plants. The term organic herbicide has come to mean herbicides intended for organic farming.
Some plants also produce their own natural herbicides, such as the genus Juglans (walnuts), or
the tree of heaven; such action of natural herbicides, and other related chemical interactions, is
called allelopathy.
Herbicides are classified into two categories

selective and non-selective.

Selective herbicides kill specific unwanted plants while leaving desirable vegetation relatively
unharmed.
Non-selective herbicides (total weed killers) kill all or most plant species.

Methods of application

A herbicide can be applied directly to the plant, applied to the soil, or sprayed onto the foliage.
Herbicides are applied before, during, or after crop planting in row-crop farming to maximize
crop production by diminishing the development of unwanted plants. Herbicides are also
applied in ponds and lakes to control aquatic plants, in forests to prepare logged areas for
replanting, and to golf courses, lawns, parks, and other areas to clear out unwanted vegetation.
Herbicide Application time
Herbicides generally are applied at different times, depending upon the emergence time of the
weeds and upon the type of fruit plants. Herbicides that are applied at specific times include
the following:

Preplant herbicides are used before the crop is planted to control germinating weed seeds, and
are usually mixed into the top 2 to 3 inches of soil. No preplant herbicides are labeled for fruit
plants.
Preemergence herbicides are used after the crop has been planted, but before the weeds or crop
emerges. Restrictions on the age of plants to be treated must be followed.
Postemergence herbicides are used after the crop and/or weeds have emerged from the soil
surface and are growing. The most common of these is Round-Up®, which can be purchased
without a pesticide license.
Herbicides usually are more effective when temperatures before application have favored
uniform germination and rapid weed growth. Rapidly growing weeds are easiest to kill. High
temperatures at the time of application also tend to increase the activity of the herbicide but
also increase the possibility of crop injury. Moderate temperatures between 70 and 85°F are
the most favorable for spraying. Wind can also be a factor in herbicide application. It can cause
improper distribution over the weeds, reducing herbicide effectiveness while increasing the
danger of drift onto desirable plants. Fewer problems occur if sprays are used when the wind
velocity is low and the wind is blowing away from desirable plants.

Maximum Yield explains Herbicide

Besides selective and non-selective classifications, a herbicide can also be categorized

according to three other characteristics:

Persistence - How long the herbicide remains potent

Mechanism of action that how does it works. Means of uptake - How the plants will absorb it
(e.g., through the roots, aboveground foliage, etc.)
Mode of action

A herbicide’s effectiveness is strongly influenced by its toxic mode of action and the
application method. Herbicides can act by inhibiting a plant’s amino acid production, growth,
photosynthesis, cell division, or by mimicking natural auxin hormones to cause deformities.
Most modern herbicides are synthetic mimics of a natural plant’s hormones that obstruct the
target plant’s growth. Some plants such as the tree of heaven and juglans (walnuts) produce
their own natural herbicide. Organic herbicides are useful and are commonly used in organic
gardens, but they are less effective and more costly than synthetic herbicides because they
based on natural materials. For difficult cases, a combination of several herbicides is
recommended when dealing with herbicide resistance.
 First herbicides

Although research into chemical herbicides began in the early 20th century, the first major
breakthrough was the result of research conducted in both the UK and the US during the
Second World War into the potential use of herbicides in war. The first modern herbicide, 2,4-
D, was first discovered and synthesized by W. G. Templeman at Imperial Chemical Industries.
In 1940, he showed that "Growth substances applied appropriately would kill certain broad-
leaved weeds in cereals without harming the crops." By 1941, his team succeeded in
synthesizing the chemical. In the same year, Pokorny in the US achieved this as well.

54. Types of Herbicides

The different types of herbicides are all designed to kill plant tissue. However, they
accomplish it by two basic methods. They are known as Contact Herbicides and Systemic
Herbicides.
Contact herbicides: Contact is a word that means the chemical in that specific type of herbicide
will kill the parts of the plant it contacts. For broadleaf weeds this means it will kill the above
ground leafy part of the plants. It will not directly kill the below ground plants parts, such as
roots, bulbs, tubers, or rhizomes. Contact herbicides are popular because they work quickly by
killing the tissue in as fast as one day. Some herbicides will combine contact with systemic
chemicals for a faster effect. New Round Up Weed and Grass Killer has combined both for faster
control. For some plants, killing the above ground portions will not be enough to wipe out the
plant completely. Most plants will regrow plant tissue and the herbicide will need to be
reapplied. However, each time the plant has to use energy to start growth again will weaken the
plant and eventually kill it.
Systemic Herbicide

For systemic types of herbicides, the word "Systemic" means the plant absorbs through the
leaves or stems and transports it internally throughout the plant. The chemical travels with the
sap so it usually doesn't have the quick "knockdown" effect. The greatest benefit of a systemic
type of herbicide is that it will kill the entire plant, roots and all. The speed of chemical
movement in the plant is largely dependent on soil and air temperature.
A chemical sprayed in early spring may take a couple weeks longer to work than the same
chemical sprayed in mid-summer. The speed of kill is also dependent on the "mode of action"
of the chemical (how the chemical works inside the plant).
Five types of herbicides:

Broad spectrum - these work on a wide variety of plants.

Selective - these work on a narrow range of plants.
Contact - these kill plant tissue at or near the point of contact with it (they do not spread around
the plant). Therefore, they require even coverage in their application.
 Systemic - these move through the plant tissues via the plant's circulation system, and these
can be injected into the plant.
Residual - these can be applied to the soil in order to kill weeds by root uptake. They remain
active in the ground for a certain length of time, and can control germinating seedlings.

55. Effect of Herbicides on People

Herbicides are poisonous chemicals that are used to kill unwanted plants, and are considered
to be a type of pesticide. They are frequently used around the home and farm and represent a
serious health hazard to adults, but they are especially hazardous to children and pets.
Types

Herbicides are commonly found as liquids or powders, and are sometimes premixed into
fertilizer products.
Herbicides are classified according to the types of plants that they affect.
Broad-spectrum herbicides will kill any plant on which they are applied, while selective
herbicides are designed to target only certain types of plants.
Contact herbicides affect only the part of a plant that the chemical touches, while systemic
herbicides are designed to be drawn up into the plant through its roots or absorbed through its
leaves and stems.
Systemic herbicides kill the entire plant. Although many modern herbicides are less toxic than
their predecessors, they are still poisons and should always be handled with caution.
Skin Irritations and Allergic Reactions

According to the University of Missouri, herbicides are designed to be toxic to plants but in
general are not highly toxic to mammals. Skin irritations are some of the most common effects
when a person comes into contact with herbicides, and are most likely to happen on exposed
areas, such as the hands and forearms. Some chemicals may burn the skin and should be
washed off immediately with cold water. The Department of Veterans Affairs confirms that
chloracne, a form of acne, is associated with exposure to Agent Orange. It can be mild or
severe, and last up to several years. In severe cases, the skin may thicken and flake off. During
the Vietnam War, the U.S. military sprayed millions of gallons of the herbicide Agent Orange
between 1961 and 1971, in an attempt to defoliate trees in the jungle and deprive the enemy of
food and cover.
Cancers

The Department of Veterans Affairs acknowledges that the herbicide Agent Orange is
responsible for a wide range of health problems in Vietnam veterans, including several types
of cancer. The National Academy of Sciences concluded that there was a positive correlation
between the incidence of Hodgkin’s disease, a cancer of the lymph system, and exposure to
the herbicide Agent Orange. The VA also notes that non-Hodgkin’s lymphoma is also
associated with exposure to the defoliant. Herbicides are also suspected as causes of prostate
cancer, cancers of the lungs and bronchial tubes, and cancers of the larynx and trachea.
Nervous System Disorders

Some herbicides can cause nervous system disorders, such as peripheral neuropathy.
The early symptoms of this disease include numbness and tingling in the toes and fingers,
gradually spreading to include the hands and feet. Pain may be present, as well as muscle
weakness and sensitivity to touch. Acute peripheral neuropathy occurs within a few weeks of
being exposed. The VA cites peripheral neuropathy as another symptom of exposure to Agent
Orange.
Effects on Children

Children and infants are at a higher risk for illnesses from herbicides than adults. According to
the EPA, because children are still developing, their immune systems are less able to protect
them from damage from herbicides. Children are also more likely to play in areas that expose
them to chemicals, such as rolling on the floor or lawn. Mild exposure can result in complaints
of dizziness and nausea, but herbicides may also cause neurological and developmental
damage to children.
Pets

Pets can be poisoned by herbicides by coming into contact with the chemicals when they are
outside, but herbicides kept in the home may also be a problem if they are stored where pets
can get to them. Pets can ingest herbicides by chewing on plants or toys that have been
contaminated, or when they lick themselves after coming into contact with the chemical.
Animals that bring herbicides into the house may spread the chemicals around the home and
leave residue on furniture and carpets.

56. Herbicide Resistance

Weeds are unwanted & useless plants that grow along with the crop plants. Weeds compete
with the crops for light & nutrients, besides harboring various pathogens. So it is estimated
that the worlds crop yield is reduced by 10 – 15 % due to the presence of weeds. To tackle the
problem of weeds , modern agriculture has developed a wide range of weed killers (herbicides).
Herbicides are broad spectrum as they can kill wide range of weeds.
An ideal herbicide is to posses the following characters:

 Capable of killing weeds with out affecting crop plants

 Not toxic to animals & microorganisms
 Rapidly trans located with in the target plant
 Rapidly degraded in the soil
  Commercially available herbicides is that they can not discriminate weeds from the
crop plants .
 For this reason , crops are also affected by herbicides hence the need to develop
herbicide resistance plants

Herbicide resistance

Herbicide resistance is the inherited ability of an individual plant to survive a herbicide

application that would kill a normal population of the same species. Herbicide resistance does
not equate to poor performance of a herbicide. Resistant weeds can often survive application
of herbicide at rates that are much greater than the recommended rate. Herbicide resistance is
the inherited ability of a plant to survive and reproduce following exposure to a dose of
herbicide normally lethal to the wild type. In contrast, tolerance can be defined as the inherent
ability of a plant to survive a herbicide treatment at a normal use rate. In a plant, resistance
may be naturally occurring or induced by such techniques as genetic engineering. Resistance
may occur in plants by random and infrequent mutations; no evidence has been presented to
demonstrate herbicide-induced mutation. Through selection, where the herbicide is the
selection pressure, susceptible plants are killed while herbicide resistant plants survive to
reproduce without competition from susceptible plants. Thus, the appearance of herbicide
resistance in a field is an example of rapid weed evolution."
Factors Leading to the Development of Herbicide Resistance

Because weeds contain a tremendous amount of genetic variation that allows them to survive
under a variety of environmental conditions the development of a resistant species is brought
about through selection pressure imposed by the continuous use of an herbicide. Factors that
can lead to or accelerate the development of herbicide resistance include weed characteristics,
Herbicide characteristics and cultural practices.
Weed characteristics

 Annual growth habit.

 High seed production.
 Relatively rapid turnover of the seed bank due to high percentage of seed germination
each year (i.e., little seed dormancy).
 Several reproductive generations per growing season.
 Extreme susceptibility to a particular herbicide.
 High frequency of resistant gene(s), (e.g. Lolium rigidum).

Herbicide characteristics

 Herbicide characteristics which lead to rapid development of herbicide resistance in

weed biotypes include:
  A single site of action
 Broad spectrum control.
 Long residual activity in the soil.
Cultural practices

Cultural practices can also increase the selective pressure for the development of herbicide
resistant biotypes. In general, complete reliance on herbicides for weed control can greatly
enhance the occurrence of herbicide resistant weeds.
Other factors include:
 Shift away from multi crop rotations towards mono cropping.
 Little or no cultivation or tillage for weed control or no elimination of weeds that escape
herbicide control.
 Continuous or repeated use of a single herbicide or several herbicides that have the same
mode of action.
 High herbicide use rate relative to the amount needed for weed control.
 Orchard and vineyard systems.
 Roadsides.

57. Mechanisms of Herbicide Resistance

For a herbicide to reach its active site in the plant, it must be taken into the plant and moved in
lethal concentrations to the site where it has activity. Once it reaches the target site, it must be
able to bind to the active site and stop that particular pathway. For a plant to be resistant, there
must be a change that will allow it to avoid one or more of these steps. Theoretically, there
could be a change in any one of these necessary steps beginning with uptake of the herbicide
into the plant.
Potential mechanisms that could be responsible for resistance

Target-site mutation – there is a change in the binding site that prevents the herbicide from
binding or interacting.
Metabolism – the herbicide is modified into a nontoxic molecule before it reaches the target
site.
Sequestration – the herbicide is physically removed from the target site.
Reduced uptake – the herbicide is not taken up in lethal quantities.
Reduced translocation – the herbicide is not transported to the site in the plant where it has
activity.
Altered target site
• An herbicide has a specific site (target site of action) where it acts to disrupt a particular
plant process or function (mode of action).
• If this target site is somewhat altered, the herbicide no longer binds to the site of action
and is unable to exert its phytotoxic effect.
• This is the most common mechanism of herbicide resistance.
Enhanced metabolism:

Metabolism within the plant is one mechanism a plant uses to detoxify a foreign compound
such as an herbicide. A weed with the ability to quickly degrade an herbicide can potentially
inactivate it before it can reach its site of action within the plant.

Compartmentalization or sequestration

Some plants are capable of restricting the movement of foreign compounds (herbicides) within
their cells or tissues to prevent the compounds from causing harmful effects.
In this case, an herbicide may be inactivated either through binding (such as to a plant sugar
molecule) or removed from metabolically active regions of the cell to inactive regions, the cell
wall, for example, where it exerts no effect.
Over-expression of the target protein:

If the target protein, on which the herbicide acts, can be produced in large quantities by the
plant, then the effect of the herbicide becomes insignificant.
 58. Mechanisms of Herbicide Resistance 2
Glyphosate

It is a broad spectrum herbicide , effective against 76 of worlds worst 78 weeds. Less toxic to
animals , is rapidly degraded & short life span. The American company (Monsanto) market it
as round up .
Mechanism of Glyphosphate action

Capable of killing the plants in low concentration. Rapidly transported to growing tissues. It
is competitive inhibitor of EPSPS (a key enzyme shikimic acid path way
Shikimic acid pathway results in the formation of amino acids, phenols, metabolites.
Glyphosate binds with EPSPS & blocks metabolism. Thus biosynthesis of aa & other
products is inhibited. So cell division & plant growth is blocked. Shikimic acid pathway
doesn't occur in animals.
So it is not toxic to animals

Advantages of using herbicides

 Broad spectrum of weeds controlled

 Reduced crop injury
 Reduced herbicide carryover
 New mode of action for resistance management
 Crop management flexibility and simplicity
 Use of herbicides that are more environmentally friendly
Disadvantages of herbicides

 Mammalian toxicity
 Eco toxicity
  Weeds become super weeds
 Reduced crop yield
 Creates soil and air pollution
 Herbicides also damage the Crop plants along with weed
Herbicide resistant Crops/Plants

 A number of biological manipulations involved in genetic engineering are in use to

develop herbicide resistance plant
 Over expression of EPSPS gene
 Use of mutant EPSPS gene
 Detoxification of herbicide by a foreign gene

59. Herbicide Resistance Crops/Plants

How to produce herbicide resistant crops?

A number of biological manipulations involved in genetic engineering are in use to develop

herbicide resistance plant by Over expression of EPSPS gene, Use of mutant EPSPS gene,
Detoxification of herbicide by a foreign gene.
Glyphosphate resistance in crop/plants

1.Over expression of EPSPS gene

An over expression gene of EPSPS was detected in petunia. Gene from petunia was isolated
& introduced in to other plants. The transgenic plants can tolerate glyphosphate 2 -4 times
higher than that required to kill wild type weed plants
Transfer of petunia gene into bacterium and then into plant
2.Use of mutant EPSPS

EPSPS mutant gene resistant to glyphosphate was found in s. typhimurium it was found that
single base substitution ( C to T ) change in amino acid from proline to serine. This enzyme
cannot bind to glyphosphate using agrobacterium as vector mutant EPSPS was introduced in
to tobacco plants but this is failed. It was later known that shikimic acid Pathway occurs in
chloroplast, mutant EPSPS was produced in cytoplasm. This gene is not capable of
transported to chloroplast. Later years mutant EPSPS gene was tagged with chloroplast
specific transit EPSPS enzyme that freely enter chloroplast & confer resistance against
herbicide
3.Detoxification of glyphosphate

The soil microorganisms possess enzymes glyphosphate oxidase that converts to glyphosate
to glyoxylate .
That gene was isolated from ochrobactrum anthropy & was introduced in to crop plants e.g:
oil seed rape
glyphosate glyphosate oxidaseglyoxylate + amp

Use of combine strategy

High resistance is acquired when the above 3 strategies combine together by this approach
mutant, detoxification, over expression genes were employed in the same organism thus
provides resistance.
 60. Level of Herbicide Resistance
The level of herbicide resistance in weeds varies by weed biology and resistance mechanism.
In some cases, resistance occurs when the species survives application of a labeled rate, while
in other cases, the species can survive up to 1000 times the labeled rate. (1X equals the labeled
rate.) This is important in terms of being able to identify herbicide resistance in the field.
Herbicide Resistance Characteristics

• Low‐Level Resistance
• High‐Level Resistance
Low‐Level Resistance

• A continuum of plant responses from slightly injured to nearly dead

• The majority of plants display an intermediate response
• Susceptible plants will be present in the population, especially when herbicide resistance
is determined early
High‐Level Resistance

• Plants are slightly injured to uninjured

• Few plants have an intermediate response
• Susceptible plants can be present in the population
Low‐Level Resistance

• A continuum of plant responses from slightly injured to nearly dead

• The majority of plants display an intermediate response
• Susceptible plants will be present in the population, especially when herbicide resistance
is determined early
High‐Level Resistance

Plants are slightly injured to uninjured

Few plants have an intermediate response
Susceptible plants can be present in the population
Herbicide Resistance Types

 Single Herbicide Resistance

 Cross Herbicide Resistance
 Multiple Herbicide Resistance
 Cross Resistance

Single Herbicide Resistance

Resistant to only one herbicide

Cross Herbicide Resistance

Resistant to two or more herbicide families with same mechanism of action

Single resistance mechanism
Multiple Herbicide Resistance

Resistant to two or more herbicides with different mechanisms of action. May be the result of
two or more different resistance mechanisms
Cross Resistance: same mechanism of action

Multiple Resistance

Multiple resistance can occur following repeated applications of a single herbicide and
selection for herbicide‐resistant biotypes followed by repeated applications of another
herbicide and selection for herbicide‐resistant biotypes.