NOTES

H/ BIOT-361

FUNDAMENTALS OF
BIOTECHNOLOGY
Complied by Prof.A.S.Raskar & Prof.A.R.Phadtare Depatment of
Agril.Botany Shreemant shiwajiraje College of Horticulture,Phaltan
 H/BIOT-361

DEPARTMENT OF GENETICS AND PLANT BREEDING

1. Course No. : H/BIOT-361

2. Course Title : Fundamentals of Plant Biotechnology
3. Credit Hours : 2 (1+1)

1. Biotechnology

The term Biotechnology was coined by Karl Ereky a Hungarian engineer in 1919.
This term is derived from a fusion of Biology and Technology.
Biotechnology is not a pure science but an integrated affect of these two areas, the
root of which lies in biological sciences.

Definition-
Biotechnology is the application of scientific and engineering principles to
the processing of materials by biological agents to provide goods and services.
OR
The controlled use of biological agents such as microorganisms or cellular
components beneficial use of mankind.

Scope-
Medical Biotechnology –
a) Disease prevention - Recombinant vaccines are developed against Hepatitis B and
influenza virus.
b) Disease Diagnosis – Production of monoclonal antibodies for diagnosis of various
diseases.
c) Detection of genetic diseases- Different markers are used for detection of genetic
disorders.
d) Gene therapy- It is used to cure genetic diseases like Sickle cell anemia.
e) Forensic studies- To assists in resolution of crimes, identity of victims, Paternity
disputes by DNA fingerprinting.

Industrial Biotechnology-
a) Metabolite Production-1) Antibiotics : Penicillin, Streptomycin.
2) Enzymes : Protease and Amylase.
3) Organic Acids : Lactic acid, Tartaric acid, Citric acid.
4) Amino Acids : Lysine
b) Fuel- Ethanol from Bagas.
c) Microbial mining- Mineral extraction.
d) Immobilization of Enzymes.
e) Food industry- Dairy, fruit juice and brewing Industry.
F) Desulphurization of coal.

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Animal Biotechnology-
a) In vitro fertilization and embryo transfer in production of test tube babies.
b) Production of transgenic animals for increase milk production and disease resistance.

Environmental Biotechnology- Biotechnological approaches are use to manage

environmental problems, sewage treatment, biogas production, detoxification of industrial
waste and degradation of petroleum.

Plant Biotechnology –
a) Plant tissue culture technology-1) Rapid multiplication of homozygous lines.
2) To recover haploid plants.
3)Germplasm conservation (Cryopreservation at -1960c)
4) Isolation of stable somaclonal variants.

b) Genetic engineering- 1) Herbicide resistance e.g.Glyphosate in soyabean

2) Insect resistance e.g. Bt cotton to ball worm
3) Production of biodegradable plastic by transgenic plants.
4) Seed quality improvement e.g.Golden rice rich in Vit „A‟
5) Induction of male sterility.

Importance of Biotechnology for improvement in horticultural Crops-

1) Mass scale production of banana tissue culture plants.
2) Development of export grade quality of cut flowers like Gerbera, Gladiolus, Roses,
Chrysanthemum and Geranium.
3) Development of virus free plants of Dahlia.
4) Development of virus resistance plants in Citrus, Orange and melons.
5) Modified flowers colour in carnation.
6) Increase vase life of Carnation etc.
7) Rapid clonal multiplication through Meristem culture e.g. fruits and forest trees.
8) Micro propagations in Orchids.

2. Plant tissue culture

Definition -
Plant tissue culture is the aseptic method of growing cells and organ such as leaves,
roots, meristems, etc either in solid or liquid medium under controlled condition.
In this technique small pieces of viable tissues called ex-plant are isolated from parent
plants and grown in a defined nutritional medium and maintained in controlled environment
for prolonged period under aseptic condition.

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The general technique of plant tissue culture involve four main stages
1) Initiation of culture
2) Multiplication (or) sub culture
3) Development and differentiation
4) Hardening

Initiation of culture –
The most important factor in tissue culture technique is the maintenance of
aseptic condition. For this purpose the culture medium generally, a GR-free medium is
used immediately after preparation the culture vessel has to be plugged and autoclaved at
1210c 15 psi (pounds per sq. inch) for an about 15-20 min. The plant material has to be
surface sterilized with a suitable sterilent. The transfer area should also maintained free of
micro organisms. Strict precautions are to be taken to prevent the entry of micro organisms.
The plug of a culture vessel is removed carefully to transfer plant material to the
nutrient medium during sub culturing. After inoculation the cultures are incubated in culture
room under controlled condition at 25 ± 20C temperature and 1000 lux light intensity
generated by florescent tube and at a constant photoperiod regulated by automatic timers.

Multiplication (or) sub culture –

After 2-3 weeks the explants show visible growth by forming either callus (or)
differentiated organs like shoots, roots (or) complete plantlets, depending upon the
composition of the medium. Periodically sub-culturing of callus (or) organs (or) plantlets to
the fresh medium is done to multiply the callus (or) organs (or) to obtain large number of
plantlets from the callus.

Development and Diffentiation / organogenesis-

The concentration of phytoharmones in the medium are altered to induce
differentiation in callus. A high cytokinins to auxin ratio induces shoot formation
(caulogenesis) (basal medium + low cytokinins / GA3 medium is used before they can be
rooted. Higher concentration (>2 mg/l BAP) of cytokinins induce adventitious shoot buds and
retard shoot growth. Very high auxins to cytokinin ratio induces root formation
(Rhizogenesis). The development of organ structures like shoot, roots etc. from the cultured
cells (or) tissues is known as organogenesis. Alternatively media composition can also be
altered to induce the development of somatic embryos and the process is known as somatic
embryogenesis. Further, an entire plantlet can be induced to grow on culture media by
manipulating the phytoharmone balance correctly and the process is called Regeneration. The
regeneration may be either direct or callus mediated. The in vitro induced shoots must be
transferred to the culture media that supports root induction.

Hardening-
The in vitro cultured rooted plants are first subjected to acclimatization before
transferring to the field. The gradual acclimatization of in vitro grown plant to in vivo
conditions is called hardening. The plantlet is taken out from the rooting medium and is
washed thoroughly to remove entire agar from the surface of plantlet as agar may attract

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microbes to grow and destroy the plantlets. The plantlet is now kept in a low minimal salt
medium for 24-48hrs and transferred to a pot that contains autoclaved sterilized mixture of
clay soil, coarse sand and leaf moulds in 1 : 1 : 1 ratio proportion. The pot containing plantlet
is covered generally with the transparent polythene cover having holes for aeration to
maintain the humidity. The plantlets are maintained for about 15-30 days in this condition.
The plantlets are then transferred to the soil and are ready for transfer either to the green
house or main field.

Steps involved in plant tissue culture –

Selection of Mother plant

Isolation of Explant

Sterilization of Explant

Inoculation of Explant

Incubation

Initiation of callus

Sub culturing

Regeneration

Hardening

Transfer of plantlets to Green house or open field

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Applications of plant tissue culture in crop improvement –

1) Micro propagation helps in mass multiplication of plants which are difficult to propagate
through conventional methods.

2) Some perennial crop plants like ornamental and fruit crops can not be propagated through
seeds. The vegetative propagation like grafting, budding are tedious and time consuming. In
such crops micro propagation helps in rapid multiplication.

3) Rapid multiplication of rare and elite genotypes such as Aromatic and Medicinal plants.
Isolation of in vitro mutants for a large number of desirable character Eg:- Isolation of
biochemical mutants and mutants resistant to biotic (pest and disease) abiotic (salt and
drought, cold, herbicide etc) stresses through the use of somaclonal variation

4) Screening of large number of cells in small space.

5) Cross pollinated crops like cardamom, Eucalyptus, coconut, oil palm do not give true to
type plants, when multiplied through seed. Development of genetically uniform plants in
cross pollinated crops is possible through tissue culture

6) In case of certain horticultural crops orchids etc seed will not germinate under natural
conditions, such seed can be made to germinate in vitro by providing suitable environment.

7) Induction of flowering in some trees that do not flower or delay in flowering. Eg:- Bamboo
flowers only once in its life time of 50 years

8) Virus free plants can be produced through meristem culture

9) Large amount of Germplasm can be stored within a small space and lesser cost for
prolonged periods under in vitro condition at low temperature. The preservation of cells
tissues, organs in liquid Nitrogen at – 196 0 c is called cryopreservation

10) Production of secondary metabolites. Eg:- Caffine from Coffea arabica, Nicotine from
Nicotiana rustica.

11) Plant tissue culture can also be used for studying the biochemical pathways and gene
regulation.

12) Anther and pollen culture can be used for production of halploids and by doubling the
chromosome number of haploids using cholchicine homogygous diploids can be produced.
They are called dihaploids.

13) In case of certain fruit crops and vegetative propagated plants where seed is not of much
economic important, triploids can be produced through endosperm culture.

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14) Inter specific and inter generic hybrids can be produced through embryo rescue technique
which is not possible through conventional method. In such crosses in vitro fertilization helps
to overcome pre-fertilization barrier while the embryo rescue technique helps to over come
post fertilization barrier.

15) Somatic hybrids and cybrids can be produced through protoplast fusion (or) somatic
hybridization.

16) Ovary culture is helpful to know the physiology of fruit development.

17) Development of transgenic plants.

Advantages of tissue culture -

1) Rapid multiplication within a limited space.

2) It is not time bound and not season bound.

3) Free from pests and diseases.

Limitations (or) Disadvantages –

1) Laborious and costly method.

2) Special risk is required.

Factors influence the plant tissue culture -

1) Genotype or variety of material.

2) Explant selection and its type.

3) Medium : a) Nutrients
b) Growth regulators
c) Other additives

4) Culture environment : a) Temperature

b) Relative humidity
c) Light

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3. Media

Definition -
It is a substrate used for plant growth such as soil, sand, Agar-agar etc

Introduction –
Culture medium is a general term used for the liquid (or) solidified formulations upon
which plant cells, tissues (or) organs develop in the plant tissue culture. Thus normally the
explants are grown in two different types of media
1) Solid Medium
2) Liquid Medium

1) Solid Medium-
A solidifying or a gelling agent is commonly used for preparing semisolid (or) solid
tissue culture medium. The plant material is placed on the surface of the medium. The tissue
remains intact and the cell multiplication is comparatively slow.

Advantages
1) Solid medium is most widely used in plant tissue culture because of its simplicity and easy
handling nature.
2) Acquires sufficient aeration without a special device since the plant material is placed on
the surface of the medium.

Disadvantages
1) Only a part of the explant is incontact with the surface of the medium. Hence there may be
inequality in growth response of tissues and there may be a nutrient gradient between callus
and medium.
2) There will be a gradiation in the gaseous exchange
3) Solid medium represent a static system. Hence there will be polarity of the tissues due to
gravity and there will be variation in the availability of light to the tissues
4) Considerable damage to the tissues may occur during sub culturing
5) Some physiological experiments which requires the immersion of tissues in the culture
medium can not be conducted by using the solid medium.

2) Liquid medium:-
All the disadvantage of solid medium can be overcome by use of liquid medium. It
does not contain a gelling or solidifying agent. So the plant material is immersed in the
medium either partially or completely. Liquid medium is used for suspension cultures and for
a wide range of research purposes.

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Advantages
1) The tissue is more easily supplied with nutrients.
2) The culture of plant tissue in an agitated liquid medium facilitates
a) Gaseous exchange.
b) Removes any polarity of the tissue due to gravity.
c) Eliminates nutrient gradient within the medium and at the surface of the cells.
3) Toxic waste products can be easily removed.
4) Growth and Multiplication of cells tissues occur at a faster rate.
5) There will be less damage to the tissues while sub-culturing.
6) Isolation of secondary metabolites is easy.
7) Liquid media are suitable for studies on the effect of any selective agent on individuals
cells.
8) Therefore screening can be done at the cellular level for resistance to biotic and abiotic
stresses.
9) Liquid medium can be easily changed without re-culturing and are preferred for some
plant species whose explants exude phenols from their cut surfaces.

Disadvantages
1) The explant gets submerged in liquid medium hence it requires some special devices for
proper aeration. Usually filter paper bridge may be used to keep the explant raised above the
level of the medium.
2) The cultures may be regularly aerated either by bubbling sterile air / gentle agitation on a
gyratory shaker.
3) Needs to be sub-cultured frequently.
4) Recovery is difficult.

Commonly used tissue culture media-

1) MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog, 1972) media are used for
regeneration of both monocots and dicots.
2) B5 (Gamborg et al., 1969) developed for culture of soybean cell suspensions but also has
been effectively used for variety of plant regeneration. B5 and its various derivatives have
been valuable for cell and protoplast cultures.
3) SH: Schenk and Hildebrandt (1972) introduced this for culture of monocots and dicots.
Widely used for legumes.
4) WPM: Lloyd and McCown (1980-1981). This is post MS media. WPM is increasingly
used for propagation of Ornamental shrubs and trees in commercial labs.
5) N6 was developed by Chu for cereal anther culture and also used for anther culture of
other species.

Composition of Nutrient media :

The composition of medium for the tissue culture is the most important key factor in
the successful culture of plant cells. The medium should be accurately defined of inorganic
and organic chemical additives so as to provide i) the nutrients for the survival of the plant

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cells, tissues and organs under culture and ii) the optimal physical condition of pH, osmotic
pressure, etc.
Nutrients
A standard basal medium consists of a balanced mixture of macronutrients and
micronutrients (usually salts of chlorides, nitrates, sulphates, phosphates and iodides of Ca,
Mg, K, Na, Fe, Zn and B, a carbon source, vitamins, phytohormones and organic additives.
Among the above mentioned nutrients some are essential and some are optional. The
essential components include inorganic nutrients and organic nutrients like carbohydrates
besides phytohormones and vitamins, organic additives like natural extract and liquid
endosperm are optional.
 Inorganic salts :
Inorganic nutrients of a plant cell culture are those required by the normal plants. The
optimum concentration of each nutrient for achieving maximum growth rates varies
considerably.
 Macro elements - The major elements are N, P, K, S, Mg and Ca
 Micro elements - Co,Fe, B, Zn, Mo, Cu, I are microelements.
 Organic nutrients :
 Carbohydrates –
Carbohydrates are used as carbon sources. The standard carbon source is sucrose at a
concentration of 2-5 per cent. Monosaccharides like glucose or fructose can also be used as
carbon sources but are generally less suitable. Sucrose is the best source, since sucrose is
dehydrolysed into usable sugars during autoclaving.
 Vitamins -
Vitamins are supplemented with medium to achieve the best growth of the tissues.
Among the vitamins only thiamine HCL (B1) seems to be universally required. Other
vitamins are pyridoxine HCL (B6), nicotinic acid (B3) and calcium pantothanate (B5).
Specific requirement of each one varies with the plant species subject to culture.
 Amino acids - L-asparagine, L-glutamine, L-cystine etc
 Others – Coconut milk, Corn milk, Yeast extract, Malt extract, Potato extract, Tomato
juice and phenolic compounds.
 Phytohormones -
These are organic compounds, other than nutrients, which influence growth,
differentiation and multiplication. They required in very minute quantity in the media. There
are many commercially available synthetic substances that mimics the PGR specific to
certain species.
1) Auxin -
In nature, the hormones of this group are involved with elongation of stem,
internodes, tropism, apical dominance, abscission, rooting etc. In tissue culture auxins have
been used for cell division and root differentiation. The commonly used auxins in tissue
culture are
1. Indole-3-acetic acid (IAA)
2. Indole-3-butyric acid (IBA)
3. Naphthalene acetic acid (NAA)
4. Dichlorophenoxyacetic acid (2, 4-D)

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2) Cytokinins -
These hormones are essential for cell division, modification of apical dominance,
shoot differentiation etc. In tissue culture media, cytokinins are incorporated mainly for cell
division, differentiation of adventitious shoots from callus and organ & shoot proliferation.
Commonly used cytokinins are
1. Benzylamino purine (BAP)
2. Isopentenyl adenine (2-ip)
3. Furfurylamino purine (kinetin)
4. Zeatin
3) Gibberellins -
Naturally occurring plant hormones involved in internodes elongation, enhancement
of flower, fruit and leaf size, germination and vernalization in plants. Among the 20 known
gibberellins, GA3 is used widely.
4) Ethylene -
A gaseous plant hormone involved in fruit maturation, abscission, and senescence. All
kinds of plant tissue cultures produce ethylene and the rate of production increases under
stress conditions.
5) Abscisic acid -
A plant hormone involved in abscission, enforcing dormancy and regulating early
stages of embryo development. It is required for normal growth and development of somatic
embryos and promotes morphogenesis.

6) Brassinosteroids -
It promotes shoot elongation at low concentrations and strongly inhibits root growth
and development. It also promotes ethylene biosynthesis and epinasty.
7) Jasmonates -
Jasmonates are represented by jasmonic acid and it is a methyl ester. Jasmonic acid is
considered to be a new class of plant growth substance. It inhibits many processes such as
embryogenesis, seed germination, pollen germination, flower bud formation, chlorophyll
formation. It is involved in differentiation, adventitious root formation, breaking of seed
dormancy and pollen germination.
8) Polyamines -
There is some controversy as to whether these compounds should be classified with
hormones. They appear to be essential in growth and cell division.
9) Salicylic acid -
It is thought to be a new class of plant growth substances. It promotes flowering,
inhibits ethylene biosynthesis and reverses the effects of ABA.
 Gelling agents & Solidifying agents-
Generally tissue culture media are solidified with any of the gelling agents. Agar is
widely used for solidification of the medium. The optimum concentration of agar used ranges
from 0.8-1.0 per cent (W/V). If the concentration of the agar is increased, the medium
becomes hard and does not allow the diffusion of nutrient into the tissues. Gelatin, silica gel,
acryl amide gel and starch copolymers are also used as substitutes for agar.
 Antibiotics – Steptomycine, Riboflavin etc

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 Antioxidants – Activated charcoal, Thio-urea,Citric acid, Ascorbic acid etc.

 Hydrogen ion concentration (pH)
The pH of the medium is usually adjusted between 5.0 and 6.0 prior to the addition of
agar and autoclaving. The extremes of pH should be avoided as this will block the availability
of some of nutrients to the inoculum. A pH of 5.8 is found to be optimum for plant tissue

The following sequential steps are followed for preparation of media

1) Appropriate quantity of Agar and sucrose is dissolved in distilled water.
2) Required quantity of stock solution, heat stable growth harmones (or) other substances are
added by continuous stirring
3) Additional quantity of distilled water is added to make final volume of the medium.
4) While stirring the pH of the medium is adjusted by using 0.1 NaoH (or) HCL
5) If a gelling agent is used heat t he solution until it is clear.
6) medium is dispensed into the culture tubes, flasks, (or) any other containers.
7) The culture vessels are either plugged with non-absorbant cotton wool rapped in cheese
cloth or closed with plastic caps.
8) Culture vessels are sterilized in autoclave at 121oC 15Psi (1.06kg / cm2)for about 15- 20
min
9) Heat labile constituents are added to the autoclaved medium after cooling to 30-40oC
under a Laminar airflow cabinet.
10) Culture medium is allowed to cool at room temperature and used or stored at 4oC (1or2
days)

3. Micro propagation

Introduction –
Multiplication of genetically identical copies of a cultivar by asexual reproduction is
called clonal propagation. In nature, clonal propagation occurs by apomixis (seed
development without meiosis and fertilization) and/or vegetative propagation (regeneration of
new plants from vegetative parts). Tissue culture has become popular method for vegetative
propagation of plants. It is the fact that micropropagation is the only commercially viable
method of clonal propagation of most of the horticultural crops. E.g. Orchids.

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Definitions –
Clonal propagation through tissue culture is called as micropropagation.
OR
In vitro clonal propagation of plants is called as Micro propagation.

Explants used in Micro propagation –

Different kinds of explants were used in micropropagation. For example, in case of
orchids, shoot tip (Anacamptis pyramidalis, Aranthera, Calanthe, Dendrobium), axillary bud
(Aranda, Brassocattleya, Cattleya, Laelia), inflorescence segment (Aranda, Ascofinetia,
Neostylis, Vascostylis), lateral bud (Cattleya, Rhynocostylis gigantean), leaf base (Cattleya),
leaf tip (Cattleya, Epidendrum), shoot tip (Cymbidium, Dendrobium, Odontioda, Odontonia),
nodal segment (Dendrobium), flower stalk segment (Dendrobium, Phalaenopsis) and root
tips (Neottia, Vanilla) are being used in micropropagation.

Stages of Micro propagation –

The process of Micro propagation involves 05 distinct stages

Stage - 0
Selection of mother plants and aseptic maintenance of stock plants.
Stage - I
Selection of suitable explants and sterilization.
Stage - II
Proliferation of multiple shoots from the explants.
Stage - III
transfer of shoots to rooting medium.
Stage - IV
Establishment under Green house condition or ex vitro establishment.

1) Selection of mother plants and aseptic maintenance of stock plants –

This stage consists of identification of mother plant and their preparation in such a
way that they provide more responsive explants suitable for establishment of contamination
free cultures. Cultures are initiated from various kind of explants such as meristem shoot tips,
nodal buds, intermodal segments leaves, young inflorescence etc. but meristem, shoot tips
and nodal buds are most prefer for commercial micro propagation.

2) Selection of suitable explants and sterilization –

In this stage selection of appropriate explants and the chosen explants is surface
sterilizes and wash before use.The most common explants used shoot tips & auxillary
buds.Initiation and establishment of culture in suitable medium.

3) Proliferation of multiple shoots from the explants –

Effective explants from stage I are subcultured on to a fresh medium. The time and
concentration of auxins and cytokinins in multiplication medium is an important factor
effecting the extent of multiplication. In vitro multiplication of shoots can be achieved by the

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following main approaches

1. Multiplication through callus culture
2. Multiplication by adventitious shoots
3. Multiplication by apical and axillary shoots
4. Multiplication by somatic embryo genesis

4) Transfer shoots in rooting medium –

Shoots proliferated during stage II are transferred to a rooting medium. In general
rooting medium has low salt. All cytokinins inhibit rooting. Eg:- half (or) even 1/4th salts of
M.S medium and reduced sugar levels.

5) Establishment under green house condition or ex vitro establishment –

Transfer of plantlets to soil is the most critical step in micropropagation. The plantlets
are maintained under highly protected conditions in in vitro i.e. high humidity,
low irradiance, low CO2 levels and high sugar content. The ultimate success of micro
propagation on commercial scale depends on the capacity in the transfer of plants to the soil
at low cost and high survival rates. The heterotrophic mode of nutrition and poor
physiological mechanisms. lack of cuticle on leaves to control water loss, tender the micro
propagation plants vulnerable to the transplantation, plants are acclamatised in suitable
compost mixture (or) soil in pots under controlled conditions of light temperature and
humidity. Inside the Green house the plants increase their resistance to moisture stress and
disease. The plantlets have to become autotrophic in constrast to their heterotrophic state
induced in micro propagation culture.

Problems of Micro propagation –

1) Microbial contamination
Bacterial and fungal contamination in culture do not allow propagules to grow and
contaminated cultures have to be usually discarded. Such a problem can be overcome by
growing the donar plant in growth chamber, by effective sterilization of explants, by
performing inoculation in the laminar air flow cabinets and by using sterlised surgical
instrument. Fumigation of inoculation with dilute formaldehyde solution helps to minimize
this problem.
2) Callusing
Callus formation is highly undersirable as it often effects the normal development of
shoots and roots and may lead to variability among the regenerated plants additon of tri-iodo-
benzoic acid, flurogauicinol and flurorizin into the culture medium (or) reduction of
inorganic salt concentration helps in overcoming this problem
3) Tissue culture induced variation
The Micro propagation plants exhibit genetic (or) epigenetic variations which may be
a major problem in getting true to type plants. It can be controlled by careful selection of
initial explant, tha t is selecting meristems and controlling the cultural environment favouring
slow multiplication rates

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4) Browning of medium
In may species phenolic substances leach into the medium from the cut surfaces of
explant. These phenolics turn brown on oxidation and lead to browning (or) blackening of
medium and or explants.
5) Vitrification
Some shoots developed in vitro appear brittle glassy and water soaked. This is called
vitrification (or) hyper hydration. The plants appear abnormal because of abnormal leaf
morphology. Poor photosynthetic efficiency malfunctioning of stomata, reduced epicuticular
waxes. It can be reduced by reducing the relative humidity in culture vessels. Reducing the
cytokinins level (or) NH4 levels (or) salt concentration in the medium, addition of flurorizin,
fluroroglucinol (or) Cacl2 in medium etc
6) Vulnerability of micro propagation plants to transplantation shocks
High mortality rates upon transferring the tissue culture derived plants to soil
continuously to be a major bottle neck in micro propagation of many plants species.
Conservation of moisture by creating high humidity around the plant, partial defoliation,
application of antitransperants have met with good suc cess.

Advantages of Micro propagation –

1. To get genetically uniform plants in large number.
2. Only a small explant is enough to get millions of plants with extremely high multiplication
rate.
3. Rapid multiplication of rare and elite genotypes.
4.This technique is possible alternative in plants species which do not respond to
conventional bulk propagation technique.
5. In plants with long seed dormancy micro propagation is faster than seed propagation.
6. Useful to obtain virus free stocks.
7. In dioecious species plants of one sex is more desirable than those of other sex Eg:- Male
asparagus and Female papaya, In such cases plants of desired sex can be selectively
multiplied by this technique.
8. This technique is carried out throughout the year independent of seasons
9. Undesirable juvinile phase associated with seed raised plants does not appear in micro
propagation plants of some species.
10. Considerable reduction in period between selection and release of new variety.
11. Maintenance of parental lines (male sterile lines especially) for the production of F1
hybrid seed.
12. Facilitates speedy international exchange of plant materials
13. Meristems have been identified as an excellent material for germplasm preservation of
some species
14. In case of ornamentals tissue culture derived plants give better growth, more flowers and
less fall out.

Limitations of Micro propagation –

1. This technique has limited application because of high production cost.
2. At each stage the technique has to be standardized.

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3. Suitable techniques of micro propagation are not available for many crop species.
4. Somaclonal variation may arise during in vitro culture especially when a callus phase is
involved e.g.- banana.
5. Vitrification may be problem in some species.
6. Browning of medium is a problem in woody (Adult trees) perennials.
7. Requires highly advanced skills.
8. Requires a transitional period before the plants are capable of independent growth.
9. The plants obtained are photosynthetically not self sufficient.
10. The plantlets are susceptible to water losses in external environment and they have to be
hardened to the external atmosphere.
11. Acclimatization is difficult process to get high percentage of suruvial of plants.
12. Continuous propagation from same material for many generations may lead to many off
types in culture.
13. This is available for lab scale not for commercial scale.
14. Inspite of great care taken during culture there are chances of contamination by various
pathogens which could cause vary high losses in a short time.

Applications of Micro propagation –

1) Micro propagation of a hybrid has the greatest multiplication advantage since it can be
result in large number of elite plants from a very small tissue clump taken from the hybrid
plant.
2) Maintenance of inbred lines for producing F1 hybrids.
3) Maintenance of male sterile genotypes of wheat and onion are useful in hybridization.
4) Selective propagation of dioecious plants Eg:- female plants of papaya, male plants of
Asparagus.
5) Multiplication of particular heterozygous superior genotype with increased productivity
Eg:- oil palm.
6) Shoot cultures of some species are maintained as slow growth culture for Germplasm
conservation.
7) Rapid production of disease free material.
8) Tissue culture can be used to minimize the growing space in commercial nurseries for
maintenance of shoot plant.

4. Somaclonal variation

Introduction –
This term is introduced by Larkin and Scowcroft in 1981. However first report of
morphological variation in plants regenerated from cell culture was made by Heinz and Mee
in 1971 in sugarcane. Larkin and Scowcraft proposed the term Somaclonal variation to
describe all those variations which occurs in plants regenerated from any form of cell culture
(or) it refers to the heritable changes which accumulated in the callus from somatic explants
and expression in the progeny of invitro regenerates obtained from callus.

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Definitions –
The genetic variability present among the cultured cells, plants derived from such
cells or progeny of such plants is called somaclonal variation.
OR
The genetic variability regenerated during plant tissue culture is called as somaclonal
variation.
OR
The variation observed in plant tissue culture is called as somaclonal variation.

Types of somaclonal variation

Based on the tissue from which variation originate Somaclonal variation can be
divided into the following types
1) Gametoclonal variation : variation observed among the plants regenerated from gametic
cultures.
a) Androclonal variation:- observed among the plants regenerated from anther (or) pollen
culture.
b) Gynoclonal variation:- from ovule (or) ovary culture.
2) Protoclonal variation:- variation observed among the plants regenerated from protoplast
Cultures.
3) Calliclonal variation:- variation observed among the plants regenerated from callus
cultures.

Causes of Somaclonal Variation –

1) Physiological
2) Genetic
3) Biochemical

Factors affecting Somaclonal variation –

1) Source of explants
2) Genotype of plant.
3) Duration of cell culture.
4) Number of sub cultures.
5) Culture medium and plant Growth regulators.

Procedure for obtaining Somaclonal variation –

Selection of Mother plant

Isolation of Explant

Sterilization of Explant

Inoculation of Explant

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Incubation

Initiation of callus

Sub culturing

Regeneration

Hardening

Transfer of plantlets to Green house or open field

Screening for desirable traits

Selection for Somaclonal variant

Agronomical trail

Steps involved in somaclonal variation

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Advantages of Somaclonal variation –

1) Frequency of variation under in vitro condition are often considerably higher than the
incidence of spontaneous mutation or Chemically induced mutation.
2) Some times unique mutations have been generated through tissue culture which could not
be obtained through crossing because of their non – availability in Germplasm Eg:- Jointless
pedicel mutant in tomato.
3) Use of Somaclonal variation may reduce the time required for release of new variety by
two years as compared to mutation breeding
4) It can be used to isolate new genotype that retain all the favourable characters of the
existing cultivers while adding one additional trait that means it may not involve a drastic
change in genetic background.
5) It occurs for trait both nuclear (or) cytoplasmic origin.
6) Saves time by reducing lengthy procedures of hybridization and selection.
7) In wide crosses it provides a mechanism of gene integration.
8) Mature embryos of the wide cross can be callused and the desirable gene integrated
plants can be selected.
9) Highly efficient as it can screen very large, number of cells rapidly with small effort time,
cost labour, and space requirement.
10) Characters can be selected at the cell level which can not be allowed at plant level.
11) This is the only approach for isolation of biochemical mutants in plants.
12) It is a simple and cheap form of plant biotechnology as compared with somatic
hybridization and genetic transformation.
13) The in vitro regenerates Eg:- of tomato and potato showed increased rates of
recombination. This could be useful in generating novel genetic variation by breaking
undesirable close linkages and by shuffling the genes linked in the repulsion phase.

Limitations of somaclonal variation -

1) It is a serious limitation in exploitation of the full potential of in vitro technique for micro
propagation where the main aim is production of large number of uniform and true to type
plants.
2) Reduced (or) No regeneration capacity of resistant clones.
3) This technique is applicable to only those species whose cell cultures could regenerate
complete plantlets.
4) Somaclones show undesirable genetic changes such as reduced fertility, growth and even
overall performance.
5) The phenotype expressed in selected cells may not be expressed by plants regenerated
from them.
6) Certain genes may not be active at the single cell level specific tissue function may occur
only when the cell is an integrated part of the intact plant. Therefore the characters for which
the cells have been selected must be recheck at the whole plant level.
7) Usually a large portion of variant clones are unstable and loose their variant phenotypes
once the selection pressure is withdrawn (epigenetic variance).
8) Only a small and variable proportion of clones are stable and the plants regenerated from
them express variant phenotypes and transmit variant feature to their progeny.
9) Selection approach can be applied only to those traits which are expressed at cell level and
whose expression at the cell level is highly correlated with that at the plant level.

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10) Most of the variation may not be novel and may not be useful.
11) Many somaclonal variants arise as a result of pleotrophic effects and may not be true
variants.
12) Superior variants for most agronomic traits Eg:- yields and quality can be selected only
by screening the progeny of tissue culture derived plants.
13) There is generally a poor correlation between glass house and field performances of
somaclonal variants.

Applications of Somaclonal variation –

1. Somaclonal variation has been described for a variety of both qualitative and quantitative
Traits.
2. Isolation of regenerants resistant to diseases.
3. A tomato line resistant to bacterial wilt caused by pseudomonas solanacearum were
isolated by screening of plants regenerated from un selected calli.
4. A fiji disease resistant sugarcane line were isolated from the variety pindar is released as a
new variety called “ono”
5. Variation may arise for useful morphological characters. An improved scented Geranium
variety named velvet Rose has been developed from Rober‟s lemon rose.
6. Isolation of variants resistant to abiotic stresses.
7. Low temperature is another important environmental factor effecting survival and
performance of crop plants.
8. Development of varieties with improved seed quality. A variety Ratan of Lathyrus which
has low neurotoxin content has been develop through somoclonal variation.
9. Isolation of mutants for efficient nutrient utilization. Tomato cell lines which are able to
grow normally in phosphate deficient condition due to high secretion of enzyme. Acid
phosphatage have been isolated through in vitro.

Achievements in Somaclonal variation –

A list of somaclonal variants released as variety for commercial cultivation.

Sr.No Crop Somaclonal Parent variation Salient features

Variety
1 Blackberry Lincoln logan Rubus Thorn less variety
2 Geranium Velvet rose Robbers lemon Polyploid somaclone,sturdiness
vigor and attractiveness.
3 Ciltronella Bio-13 --- 37 % more oil content ,39%
more citronella content.
4 Mustard Pusa jai kisan Varuna Bolder seeds.
5 S. Potato Scarlet --- Darker and more stable skin
colour.
6 S.cane Ono Pindar Resistant Fiji disease

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5. Embryogenesis

Introduction -
An embryo is defined as a plant in its initial stage of development. Each embryo
possesses two distinct poles, one to form root and the other shoot, and is the product of fusion
of gametes. In some plant species, embryos are produced without the fusion of gametes and
termed as asexual embryogenesis or adventitious embyrony. Haccius (1978) defined Somatic
embryos as a non-sexual development process which produces a bipolar embryo from
somatic tissue. Steward and Reinert first reported the production of embryos from cell
suspensions of carrots.

Definition –
The process of embryo development is called embryogenesis.

Types of Embryogenesis –
1) Somatic embryogenesis – a) Direct embryogenesis
b) Indirect embryogenesis
2) Zygotic embryogenesis

Somatic embryogenesis –
Plant cell also induced to form embryos in plant tissue culture these embryo is
called somatic embryo.

Pathway of Somatic embryogenesis

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Differences between direct and indirect embryogenesis

Direct embryogenesis Indirect embryogenesis

Embryos form from the explants Embryos arise from the callus
directly. induced from the explants.
A promoting substance to induce Auxin is need to induce callus, and
the embryo formation is needed. cytokinin is needed to induce
differentiation.
The embryogenic nature of a cell is The embryogenic nature of a cell
Predetermined. is induced in the culture.
The origin of embryos mostly from The origin may be either from
individual cells; sometimes from a single cells or from a group of
group of cells. cells called proembryonal
complex.

Zygotic embryogenesis –
Embryogenesis may occurs naturally in the plant as a result sexual fertilization &
these embryo is called as zygotic embryo.

Plant regeneration via somatic embryogenesis –

1) Initiation of embryonic culture.
2) Proliferation of embryonic culture.
3) Pre-maturation of somatic embryo.
4) Maturation of somatic embryo.
5) Plant development on non-specific media.

Factors affecting somatic embryogenesis

1) Growth regulators -
In most species an auxin (generally, 2,4-D at 0.5-5 mg/1) is essential for somatic
embryogenesis.
2) Nitrogen source.
3) Genotype of explants.

Applications –
1) Somatic embryogenesis may replace micropropagation for the rapid propagation of
economically important plants.
2) Somatic embryos can meet specific breeding objectives by rapidly multiplying germplasm
that is initially present as embryonic material Eg:- maternal embryos, haploid embryos and
interspecific hybrid embryos that normally abort due to non availability of endosperm tissue
3) Raising somaclonal variations from tree species Embryos formed directly from
preembryonic determined embryonic cells appear to produce relatively uniform to clonal
material, where as the indirect pathway involving in callus proliferation and differentiation of
embryogenic cells generate a high frequency of somaclonal variants.
4) synthesis of artificially synthetic seeds.

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5) Source of regenerable protoplast system.

6) Embryogenic callus, suspension culture and somes have been employed as sources of
protoplast isolation for a range of species.
7) The nucellus usually degenerated during the development of seeds but in citrus species
embryogenic callus derived from nucellus remains totipotent for many years, can be used as
source material for regeneration of plants.
8) Non-chemical mutants may be obtained through adventive embryogenesis in tissue
cultures as somatic embryos develop from a single cell.
9) Disease free plants can be obtained by using the nucellar embryos. In citrus certain viruses
which infect the vegetative tissue are eliminated from nucellar cells during ovule
development. Somatic embryos developed from nucellar cells produce rejuvenated clones
which are therefore, also virus free
10) The possibility of chimeric embryos arising from transformed and non transformed
tissues of the callus can be by-passed through the process of repetitive somatic
embryogenesis
11) The repetitive embryogenesis is of potential use in the synthesis of metabolites such as
pharmaceuticals and oils

Limitations -
1) In many species somatic embryo maturation and convertion remain problematic and
resolution of this bottle neck is critical to the practical utilization of somatic embryogenesis.
2) Occurance of somaclonal variations in indirect somatic embryo genesis.
3) Somatic embryos are without seed coat.
4) Abnormalities exhibited by somatic embryos which include double and triple vascular
system, secondary embryogenesis and pluri-cotyledonary.
5) Large scale production is difficult.
6) SE quality is often poor.
7) Field conversion frequencies of SEs and artificial seeds are low (15-20%)
8) Synchronization of somatic embryogenesis is inadequate.

6. Artificial Seed / Synthetic Seed

Introduction -
Large scale production of somatic embryos and their encapsulation is referred to as
Artificial or synthetic seed production. It is an alternative to traditional micro propagation for
production and delivery of cloned plantlets. Artificial or synthetic seed is a bead of gel
containing somatic embryo or shoot bud and the nutrients, growth regulators. Pesticides,
antibiotics etc needed for the development of a complete plantlet from the enclosed somatic
embryos or shoot bud. Artificial seed is used for substitute for Natural seed.

Definition –
Artificial seed is living seed like structure which are made experimentally by
technique where somatic embryoid derived from plant tissue culture.

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Types of Artificial Seed – a) Hydrated seed

b) Desiccated seed

1.) Hydrated Artificial Seed –

Somatic embryos induced in appropriate nutrient medium is mixed with sodium
alginate (0.5-5%) . Then embryo loaded sodium alginate is dropped in to a solution of
calcium chloride (30-100 min ) drop by drop. Each droplets form calcium alginate capsule
around each embryo within 10-60 min .Excess calcium ions removed by rinsing of water.

2.) Desiccated Artificial Seed –

Desiccated synthetic seed are coated with a water soluble resin polyethylene oxide S
E suspension is mixed with equal volumes of 5% solutions of polyethylene oxide, with
subsequently dried from polyembryonic desiccated wafers. The survival of the encapsulated
embryos was further achieved by embryo hardening treatment with 12% Sucrose followed by
chilling at inoculums density.

Steps for making Artificial Seed –

1) Establishment of callus culture.
2) Induction of somatic embryogenesis in callus culture.
3) Maturation of somatic embryos.
4) Encapsulation of somatic embryos.
( After encapsulation the artificial seeds are tested by two steps )
1) Test for embryoid to plant conversion.
2) Green house and field Planting.

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Applications / Importance -
1) seed propagation of sterile plants.
2) High efficiency in multiplication.
3) Fixation of hybrid vigour, eleminate the need of inbred lines to produce F1 hybrids.
4) Elimination of the need of edible seeds or tubers for propagation
5) Multiplication of Genetically engineered individuals, which may be sterile and unstable
during sexual production.
6) Production of virus and disease free plants.
7) Protection of seedlings by encorporating useful chemicals in the encapsulation material.
8) provide the advantages of true seed (case of handling and transportation) for vegetative
propagation.

Limitations -
1) Large scale production of high quality somatic embryos is a costly affair
2) Poor germination of synthetic seeds due to lack of supply of nutrients, sufficient oxygen,
microbe invasion and mechanical damage of somatic embryos.
3) Occurrence of somaclonal variation.
4) Special skills are required to carry out the work.

Problems
1. Artificial seeds that are stable for several months requires the procedures for making the
embryos quiescent.
2. Artificial seeds need to be protected against desiccation.
3. Recovery of plants from Artificial seed is often very low due to incomplete embryo
formation or difficulties in creating an artificial endosperm.
4. The embryo must be protected against microorganisms

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7. PCR – Procedure and applications

The PCR technique for quickly cloning a particular piece of DNA in the test tube
rather than living cells like E. coli / in -vitro method for the amplification of DNA fragments.
PCR technique is developed by Kary mullis 1985. It is one of the most powerful molecular
biology technique used to multiply minute (or) trace amounts (μg microgram quantities) of
DNA copies of the desired DNA by multiple cycles of cooling and heating in a reaction
catalysed by a heat stable DNA polymerase enzyme. (The PCR is carried out in vitro). PCR is
based on the features of semiconservative DNA replication carried out by DNA polymerase
in prokaryotic and eukaryotic cells.
The PCR utilizes the following :
1) DNA preparation containing the desired segments to be amplified must have known
nucleotide sequence so that oligonucleotide primers can bind and synthesize the DNA.
2) Two nucleotide primers (about 20 bases long) specific i.e. complementary to the two
5‟ - 3‟ borders of desired segment
Procedure of PCR
Amplification of DNA is achieved by a repetitive series of cycles involving 3 steps.
1. Denaturation
The DNA double helix separated into two complimentary single strands by heating
reaction mixture to temparature between 90-980C that ensures DNA denaturation. The
duration of this step in the first cycle of PCR is usually, 2 min at 940C, but in subsequent
cycles it is of only 1min duration.
2. Annealing
1. The mixture is now cooled to a temparature of generally 40-60oC that permits
annealing of primer to the complimentary sequences in the DNA. The duration of annealing
step is usually 1 min during the first as well as the subsequent cycles of PCR.
2. The annealed primers are then extended (i.e. synthesis of DNA) with Taq DNA
polymerase.
3. Primer Extension (or) synthesis
It involves heating the mixture to 72oC at which a special polymerase synthesizes the
new DNA strand by using the original strand as the template starting by the primer utilizing
their 31OH free ends and continuing in the 31 direction. The completion of extension step
completes the first cycle 01- amplification and these three steps are repeated 25-40 times to
produce millions of exact copies of the target region of DNA. Thus at the end of each cycle
the number of copies of desired segment becomes twice the number present at the end of
previous cycle. Thus at the end of „n‟ cylces 2n copies of the segments are expected. In case
of automated PCR machines, called thermal cycles, the researcher specify only the number
and duration of cycle etc. after placing the complete reaction mixture for incubation, and the
machine performs the entire operations preciously. After PCR cycles the amplified DNA
segment is purified by gel electrophoresis and can be used for cloning, DNA
sequencing, etc.

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Application
DNA cloning for sequencing
DNA based phylogenetic studies.
Functional analysis of gene
Diagnosis of hereditary diseases
Identification of genetic finger prints used in forensics and paternity studies
The detection and Diagnosis of infectious diseases
It can be used to determine the sex of embryos

8. DNA Fingerprinting

Every year in court cases all over the world the ability to establish a person‟s identity
is essential for a just decision. Genetics has come to the rescue of the courts and now the
following new questions are routinely asked in the courts: (1) Is the drop of blood found at
the crime scene from suspect on trait? Who is the child‟s father? Until recently, there was no
foolproof test. In a criminal case, if there was no identifiable fingerprint left behind at the
crime scene, there was no case. Blood tests can determine who is not the parent, not who is.
A test has now been developed that provides hundred percent positive identification. The test
is called DNA fingerprinting. The test of DNA fingerprinting can show conclusively
whether the genetic material in a drop of blood matches that of the suspect, or it can be used
to solve paternity case. The technique of DNA fingerprinting relies on developments from
recombinant DNA technology and allows an examination of each individual‟s unique genetic
blueprint – DNA. The technique was discovered in England by Alec Jeffreys. It is based on
the fact that the DNA of each individual is interrupted by a series of identical DNA sequences
called repetitive DNA or tandem repeats. The pattern, length, and number of these repeats are
unique for each individual. Jeffreys developed a series of DNA probes, which are short pieces
of DNA that seek out any specific sequence they match, and base pair with that sequence.

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Such molecular probes are used to detect the unique repetitive DNA patterns characteristic of
each individual.

Definition – DNA fingerprinting is a technique by which an individual can be identified

at molecular level.

The procedure of DNA fingerprinting has the following steps:

1. DNA is purified from a small sample of blood, semen, or other DNA-bearing cells, and
digested into smaller fragments with restriction endonucleases.
2. The fragments are separated by agarose gel electrophoresis.
3. The separated fragments are transferred to a nylon membrane by the technique of Southern
blotting.
4. The DNA probes labeled with radioactive material are added to a solution containing the
nylon membrane.
5. Wherever the probes fit a band containing repetitive DNA sequences, they attach.
6. The X-ray film is pressed against the nylon filter and exposed at bands carrying the
radioactive probes attached to the fragments.
7. The patterns of bands obtained on the film is 100 per cent unique for each person, except
for identical twins who would have the same pattern.

Advantage of DNA Fingerprinting –

1) Every individual has unique DNA fingerprint which remain same throughout life even
after death.
2) DNA fingerprint can not be altered or erased by any means.
3) DNA fingerprint is primary and reliable method of identification.
4) Reliable information can be obtained even from degrade sample of DNA.
5) Very small sample required for testing.
6)
Drawback of DNA Fingerprinting –
1) DNA fingerprinting is not 100% reliable.
2) Misleading results observed due to contaminated blood sample.
3) Error from probing.
4) Error by technician.
5) Testing cost very high.
Applications of DNA fingerprinting –
1) Diagnosis and curing of genetic disorders.
2) Identification of parental and maternal status.
3) Confirming the legal nationality.
4) Identification of exchanged child.
5) Identification of Criminals.
6) Identification of Transgenics.

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9. Genetic transformation - Gene transfer methods

Indirect method of gene transfer
Agro-bacterium mediated gene transfer method

Methods of Gene transfer -

1) Direct Method – a) Physical method i) Electroporation

ii) Biolistic
iii) Macroinjection
iv) Microinjection
v) Lipofection
vi) Silicon Carbide mediated
vii) Ultrasound
viii) Laser indused
ix) DNA transfer via pollen
x) Partical Gun method.
b) Chemical method – PEG method

2) Indirect method – a) Agrobacterium mediated gene transfer.

b) Virus mediated gene transfer – i) Gemini virus
ii) RNA virus
The process of transfer, intigration and expression of transgene in the host cells is
known as genetic transformation. Various genetic transfer techniques are grouped into two
main categories.
1) Vector mediated and Indirect gene transfer.
2) Vector less and Direct gene transfer

Vector mediated and indirect gene transfer.

In this approach the transgene is combined with a vector which takes it to the target
cells for inteigration. The term plant gene vector applies to potential vectors both for transfer
of genetic information between plants and the transfer of genetic information from other
organisms (bactieria fungi and animals) to plants. The vector mediated transfer is strongly
linked to regeneration capabilities of the host plant. The plant gene vectors being exploited
for transfer of genes are plasmids of Agrobacterium viruses and transposable elements.
Agrobactreium mediated transformation
The Agrobacterium system was historically the first successful plant transformation
system, marking the break through in plant Genetic engineering in 1983. The Agrobacterium
is naturally occurring gram negative soil bacterium with two common species A Tumifacience
and A rhizogenes There are known as natural gene engineers for their ability to transform
plants. A tumifacience induces tubers called crown galls, where as A rhizogenes causes hairy
root diseases. Large plasmids in these bacteria are called tumer inducing (Ti plasmil) and root
inducing (Ri plasmid) respectively. The Ti plasmid has two major segments of interest in
transformation that is T DNA and virus region. The T DNA region of the Ti plasmid is the

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part which is transferred to plant cell and incorporated into nuclear genome of cells. The
transfer of T DNA is mediated by genes in the another region of Ti plasmid called virs genes
(virulence genes). Modified Ti plasmid are constructed that lack of undesirable Ti genes but
contain a foreign gene (resistant to a disease) and a closely linked selectable marker gene
(Eg:- for antibiotic resistance). With in the T DNA region any gene put in T DNA region of
plasmid cysts transferred to the plant genome. The T DNA is generally integrated in low copy
number per cell. Transfer of gene through to wounded plant organs A. tumifacience has
limited range of host. It can infest about 60% gymnosperms and Angiosperm. Hence
Agrobacterium mediated transformation is the method of choice in dicotyledonous plant
species, where plant regeneration system are well established, However, Monocotyledons
could not be successfully utilized for Agrobacterium mediated gene transfer.
Advantages
It is a natural means of gene transfer
Agrobacterium is capable of infecting infact plant cells and tissue and organs.
Agrobacterium is capable of transfer of large fragments of DNA very efficiently
Integration of T DNA is a relative precise process.
The stability of gene transferred in excellent.

Limitations
Host specificity
Soma clonal variation
Slow regeneration
Inability to transfer multiple genes

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Vector less and Direct gene transfer-

a) Physical methods –
1) Electroporation -
Induction of DNA into cell by exposing them for a very brief period to high voltage
electrical pulses to induce transiant pores in the plasma lemma is called Electroporation.
Generally protoplasts are used since they have expand plasma membrane. A suspension of
protoplast with a desired DNA is prepared. Then a high voltage current is applied through the
protoplast DNA suspension. The electric current leads to the formation of small temporary
holes in the membrane of the protoplasts through which the DNA can pass. After entry into
the cell, the Foreign DNA gets incorporated with the host genome, resulting the genetic
transformation The protoplasts are then cultured to regenerate in to whole plants. This
method can be used in those crop species in which regeneration from protoplast is possible
2) Particle bambordment / microprojectile / biolistic / gene gun / particle acceleration -
The process of partical acceleration (or) biolistics acceleration of DNA into cells with
sufficient force such that a part of it gets integrated in to DNA of target cells. The process of
transformation employes foreign DNA coated with minute 0.2-0.7 μm gold (or) are tungstun
particles to deliver into target plant cells. Two procedures have been used to accelerate the
minute particles · By using pressurized helium gas · By electro static energy released by a
droplet of water exposed to a high voltage This method is being widely used because of its
ability to deliver foreign DNA into regenerable cells, tissue (or) organs irrespective of
monocots (or) dicots Because of the physical nature of process there is no biological
limitation to the active DNA delivery that makes it, genotype independent. This method
allows the transport of genes into many cells of nearly any desired position in an
experimental system without too much manual labour. The method was first used by Klein et
al. in 1987, and Sanford et al 1987.

3) Lypofection -
Introduction of DNA into cells via lyposomes is known as lipofection, lioposomes are
small lipid artificial vesicles. The procedure of liposome encapsulation was developed to
protect the foreign DNA during the transfer process The DNA enclosed in the lipid vesicles

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when mixed with protoplast under appropriate condition penetrates into the protoplast where
lipase activity of the protoplast dissolves the lapid vesicles and DNA gets released for
integration into the host genome. This method has not been commonly used as it is difficult
to construct the lipid vesicles. The success depends upon the protoplast regeneration
4) Microinjection -
The DNA solution is injected directly inside the cell using capillary glass micropipetts
with the help of micromanipulators of a microinjection assembly. It is easier to use protoplast
than cells since cell wall interferes with the process of microinjection. The protoplast are
usually immobilized in agarose (or) on a glass slides coated with polylysine or by holding
them under suction by a micropipette. The process of microinjection is technically
demanding and time consuming a maximum of 40-50 protoplasts can be microinjected in one
hour.
5) Macroinjection -
The injection of plasmid DNA into the lumen of developing inflorescence using
hypodermic syrange is known as macro injection An aqueous solution of DNA was
introduced into the developing floral tillers 14 days prior to meiosis. Transformed seeds were
obtained from these injected tillers after cross pollination with other and injected tillers.
However the mechanism by which the DNA entered the zygotic tissue yet unknown.
6) Pollen transformation -
Involves the gene transfer by soaking the pollengrains in DNA solution prior to their
use for pollination. The method is highly attractive in view of its simplicity and general
applicability but so far there is no definite evidence for a transgene being transferred by
pollen soaked in DNA solution

8) DNA Delivary via growing pollen tubes -

The stigmas were cut after pollination exposing the pollen tubes, the DNA was
introduced onto the cut surface that presumably diffused through the germinating pollen tube
into the ovule. This method is simple easy and very promising provided consistent result and
stable transformations are achieved The mechanism of DNA transfer into zygote through this
method is not yet established.

9) Laser induced transformation -

It is method of introducing DNA into plant cells with a laser microbeam. Small pores
in the membrane are created by laser microbeam. The DNA from the surrounding solution
may than enter into the cell cytoplasm through the small pores Lasers have been used to
deliver DNA into plant cells But there is no information on transient expression or stable
integration.

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10. Transgenic plants – Applications in crop improvement – Limitations

Transfer of gene from an organism into a plant cell and its integration into the genetic
material of the later usually employing recombinant-DNA technique is known as Genetic
engineering of plants It is the most potent biological approach which constitute the transfer of
specifically constructed gene assembles through various transformation techniques. The
plants obtained through genetic engineering contain a gene (or) genes usually from an
unrelated organisms. Such genes are called transgenes and plants containing transgenes are
known as transgenic plants. The first transgenic plant was produced in 1983 when a tobacco
line expressing Kanamycin resistant was produced. Soon after transgenic crop varieties
resistant to herbicides, insects (or) viruses (or) expressing male sterility, delayed ripening,
and slow fruit softening were developed. Flavr savr tomato was first transgenic variety to
reach market. Fruits of this variety remain fresh for a prolonged period.

Role of transgenic plants in crop improvement:-

Transgenic plants are those which carry additional stability integrated and expressed
foreign genes, usually transferred from unrelated organisms. The combined use of
recombinant-DNA technology, gene transfer methods and tissue culture technique has lead to
efficient productions of transgenics in a variety of crop plants. Transgenic plants or
genetically modified plants (GMP) have both basic and applied uses which are briefly
summerised below…………
1) Transgenes will be important in increasing the efficiency of crop production systems. For
instance tragenic plants resistant to herbicides, insects, viruses and other biotic and abiotic
stress have already been produced.
2) Transgenic breeding is an effective means of inducing male sterility in crop plants . Eg:-
Barnase and barstar systems in Brassica napus
3) Transgenic plants which are stable for food processing have also been produced Eg:-
Bruise resistant and delay ripening in tomato
4) Several gene transfers have been aimed in improving the produce quality. Eg:- Protein and
lipid quality : improved quality may be achieved by either supression or over production of
endogenous genes.
5) Transgenic plants are aimed to produce novel biochemicals like interferon, Insulin,
Immunoglobin etc useful biopolymers like poly hydroxy butyrate which are not produced by
normal plants. These compounds are extracted from plants can be used as pharmaceutical (or)
industrial substrates
6) The utilization of transgenic plants as bioreacters (or) factories for the production of
special chemical and pharmaceuticals is known as Molecular forming.
7) Transgenic plants have been produced that express a gene encoding antigenic protein from
a pathogen. Use of transgenic plants as vaccines for immunization against pathogens fast
emerging as an important objective.

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8) Transgenic plants have proved to be extremely valuable tools in studies on plant molecular
biology. Regulation of gene action, identification of regulatory, promoters sequences etc,

Limitations:
1.Transgenic plants some times exhibit instable performance for character under
consideration.
2.The transformants had the undesirable side effects of trans genes
3.The position and integration of foreign gene in host genome effects the expression of
transgene in the transformant
4. Inability to transfer polygenic traits
5. Transgenic breeding is a very expensive method of crop improvement
6. Requires high technical skills.
7. Transgenic cells are recovered at a very low frequency in cell culture, more over regene
ration of transgeniccells into whole plants is also difficult and time consuming task.
8. Transgenic breeding acts against natural evolution.
9. There are chances of developing new weed species through transgenic breeding
10. Sometimes the foreign genes has adverse effects on genome of the recipient parent, in
such cases it may give rise useless gene combinants which may become a problem.

11. Totipotency – Growth and differentiation in cultures

Types of cultures – callus and suspension cultures

Explant - A plant organ (or) an exised part used to initiate Tissue culture
Growth:- An increase in size (vol/wt/length) due to cell division and subsequent enlargement
Re-differentiation - The ability of component cells of the callus to differentiate into a whole
plant
Callus may be defined as an unorganized mass of loosely arranged parenchymatous tissue
which develop from parent cells due to proliferation of cells.
Cellular Totipotency
The inherent capacity of a plant cell to give rise to a whole plant is known as cellular
totipotency.
Differentiation: The phenomenon of mature cells reverting to a meristematic state and
forming
undifferentiated callus tissue is termed as „De differentiation‟
Differentiation may be categorized into 2 groups – 1) Structural 2) physiological
1) Structural differentiation
It is further distinguish into external and Internal differentia tion.
a) External
Most common example is root and shoot differentiation another familiar example of is
vegetative and reproductive phases of life cycle Further differentiation in reproductive organs
results in male and female organs
b) Internal

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This inc ludes differentiation of various types of cells and tissues. Differentied cells mostly
occur into groups forming different type of tissues Eg:- Vascular tissues

c) Physiological
The variations in the structure between root and shoot are the expressions of fundamental
physiological differences

Callus growth pattern

Growth of callus is measured in terms of increasc in fresh weight, dry weight (or) cell nember
A generalized growth pattern takes the form of a sigmoid curve
Three district phases can be observed during growth of the callus
Lag phase: a period of little (or) no cell division (Biomass remain unchanged)
Cell division followed by linear phase (log phase) :- a period of cell division and expansion
rate of division.
Stationary phase (or) regeneration phase:- As the nutrient supply of medium depleats, a
gradual cessation of cell division occurs. This phase is associated with the initiation of
structural organization of the cell which increases the production of secondary metabolites.

Types of Cultures
1) Callus culture:-
callus culture may be derived from a wide variety of plant organs roots, shoots, leaves
(or) specific cell types. Eg:- Endosperm, pollen. Thus when any tissue (or) cell cultured on an
agar gel medium forms an unorganized growing and dividing mass of cells called callus
culture. In culture, this proliferation can be maintained more (or) less indefinitely by sub
culturing at every 4-6 weeks, in view of cell growth, nutrient depletion and medium drying.
Callus cultures are easy to maintain and most wide ly used in Biotechnology. Manipulation of
auxin to cytokinin ratio in medium can lead to development of shoots or somatic embryos
from which whole plants can be produced subsequently.
Callus culture can be used to initiate cell suspensions which are used in a variety of
ways in plant transformation studies.
Callus cultures broadly speaking fall into one of the two categories.
1) Compact 2) Friable callus
In compact callus the cells are densly aggregated. Where as in friable callus the cells
are only loosly associated with each other and callus becomes soft and break a part easily. It
provides inoculum to form cell suspension culture.
Suspension culture
When friable callus is placed into a liquid medium (usually the same composition as
the solid medium used for callus culture) and then agitated single cells and / or small clumps
of few to many cells are produced in the medium is called suspension culture Liquid cultures
may be constantly agitated generally by a gyratory shaker of 100-250 rpm to facilitate
aeration and dissociation of cell clumps into small pieces. Suspension cultures grow much
faster than callus cultures, need to be sub-cultured at every week, allow a more accurate
determination of the nutritional requirement of cells and even somatic embryos.
The suspension culture broadly grouped as 1) Batch culture 2) Continuous culture

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1) Batch culture
A batch culture is a cell suspension culture grown in a fixed volume of nutrient
culture medium. Cell suspension increases in biomass by cell division and cell growth until a
factor in the culture environment (nutrient or oxygen availability) becomes limiting and the
growth ceases. The cells in culture exhibit the following five phases of a growth cycle.
i. Lag phase, where cells prepare to divide
ii. Exponential phase, where the rate of cell division is highest.
iii. Linear phase, where cell division shows but the rate of cells expansion increases.
iv. Deceleration phase, where the rates of cell division and elongation decreases.
v. Stationary phase, where the number and size of cells remain constant.
When cells are subcultured into fresh medium there is a lag phase. It is the initial
period of a batch culture when no cell division is apparent. It may also be used with reference
to the synthesis of a specific metabolite or the rate of a physiological activity. Then follows a
period of cell division (exponential phase). It is a finite period of time early in a batch culture
during which the rate of increase of biomass per unit of biomass concentration (specific
growth rate) is constant and measurable. Biomass is usually referred to in terms of the
number of cells per ml of culture. After 3 to 4 cell generations the growth declines. Finally,
the cell population reaches a stationary phase during which cell dry weight declines. It is the
terminal phase of batch culture growth cycle where no net synthesis of biomass or increase in
cell number is apparent. In batch culture, the same medium and all the cells produced are
retained in the culture vessel (Eg. culture flask 100-250 ml). the cell number or biomass of a
batch culture exhibits a typical sigmoidal curve. Batch cultures are maintained by sub-
culturing and are used for initiation of cull suspensions.
2) Continuous culture:-
These cultures are maintained in a steady state for a long period by draining out the
used (or) spent medium and adding the fresh medium. such subculture systems are either
closed (or) open type.
1) Closed:-
The cells separated from used medium taken out for replacement and added back to
the suspension culture. So that the cell biomass keeps on increasing
2) Open:-
Both cells and the used medium are taken out from open continuously cultures and
replaced by equal volume of fresh medium. The replacement volume is so adjusted that
cultures remain at sub-maximal growth indefinitely.

12. Vectors for gene transfer – Properties of a good vector –Properties of

good host.

A vector is a DNA molecule that has the ability to replicate in an appropriate host
cell, and into which the DNA insert is integrated for cloning. Therefore a vector must have a
origin of DNA replication (Denoted as ori) that functions efficiently in the concerned host
cell. The vector is a vehicle (or) carrier which is used for cloning foreign DNA in bacteria.

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The cloning vehicles are called vectors. Any extra-chromosomal small genome, eg. Plasmid,
phage and virus may be used as vector.

Properties of a good vector

1) It should be able to replicate autonomously that is independent of the replication of host
chromosome.
2) It should be easy to isolate and purify.
3) It should be easily introduced into the host cells.
4) The vector should have suitable marker genes that allow easy detection or / and selection
of the transformed the host cell. Eg. Genes for ampicillin and Tetracycline resistance.
5) The cells transform with recombination DNA should be indentifiable (or) selectable from
those transformed by the unaltered vector.
6) A vector should contain unique target sites for as many restriction enzymes as possible
into which the DNA insert can be integrated.
7) When expression of the DNA insert is desired, the vector should contain suitable
regulatory elements like promoter, operator, ribosome binding sites.

Types of vectors for gene transfer –

1) Plasmid
2) Bacteriophages ( Natural vector)
3) Cosmids
4) Binary and shuttle vectors ( Constructed by man)
5) Phasmid / Phagemid vector
6) Yeast vector
7) Transposons
8) Artificial Chromosomes a) Yeast artificial chromosome (YAC)
b) Bacterial artificial chromosome (BAC)
9) Plant and animal viruses
10) Ti plasmid

Properties of a good host

1) It should be easy to transform
2) It should support the replication of recombinant DNA
3) It should be free from element that interfere with replication of recombinant DNA\
4) It should lack activity of restriction enzyme.
5) It should not have methylase since these enzyme methylate the replicated recombinant
DNA.

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13. Gel Electrophoresis and Different blotting techniques

Gel Electrophoresis
Separation of charged molecules (usually DNA RNA (or) proteins) in a gel
electrophoresis is the technique of under the influence of an electrical field. This technique
separates DNA fragments on the basis of their size and base composition. The nucleic acid
(or) DNA to be analysed is made into small segments through the action of restriction
enzymes. The fragmented nucleic acid is applied at one end of the glass (or) plastic plate on
which a thin layer of agarose (or) polyacrylamide is solidified. After adding a suitable buffer
solution of the plate a high voltage (60-100 volts) electric current is passed across the gel. On
according of their negative charge of the nucleic acid fragments move from cathode to anode
on gel with a speed according to the size of fragment such that shortest fragment lie at
farthest end of the gel. The segments with different placement on the gel are detected by
radioactive labeled probe hybridization and then exposing to x-ray film. The molecular size
of DNA fragments can be estimated by comparing the migration of bands with that of size of
the standards separated on the same gel.

Southern blotting
This technique was launched by E.M Southern the transfer of DNA fragment from an
electrophototic gel to the nitrocellulose filter or nilon membrane by capillary action is known
as Southern blotting.
It involves DNA-DNA hybridization and the basic steps in southern blotting.
1) Isolation of genomic DNA
2) Digestion of DNA with endonuclease and separation of fragments by agarose gel
electrophorosis
3) Denaturation of separated fragments into single strands form by alkali treatment
4) Transfer and blotting of these segments on to a Nitro cellulose filter membrane from
agarose gel by capilallary action
5) The Nitro cellulose membrane is now removed from the blotting stack and DNA is
permanently immobilized on the membrane by baking it at 80 0C.
6) The baked membrane is treated with a solution containing 0.2% each of Ficoll, polyvinyl
pyrolidone, and Bovine serum albumin, to prevent the non specific binding of the radio active
probe (pre treatment).
7) The pretreated membrane is placed in a solution of radio active single standard DNA or an
oligonucleotide called probe.
8) This probe hybridizes with complimentary DNA on the membrane resulting in DNA DNA
hybridization.
9) After the hybridization the membrane is washed to remove the unbound probes
10) The autoradiography or X-ray film reveals the positions of DNA segments in the gel that
are complimentary to the radio active probes

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Application of Southern blotting

1) It can be used to map the restriction site around a single copy gene sequence in any
genome.
2) It is used for DNA finger printing preparation of RFLP maps, detection and identification
of transferred genes in the transgenic individuals etc.
3) Use to locate particular gene to entire genome.
4) Used to determine copy number of gene in the genome.
5) Used in genome mapping ( to detect linkage)
6) Used to gene cloning and chromosome walking.
7) It can identify related other DNA sequences in the genome

Advantages of Southern blotting

1) This technique is very easy and highly sensitive ( High degree if freedom)
2) It can detect single gene from entire genome.
3) It can analyze DNA from specific DNA clones or PCR products.
4) The robes can be labelled with radioactive or non radioactive chemicals.
5) It can use both DNA as well as RNA probes.

Limitations of Southern blotting

It is a time consuming and expensive method.

Northern Blotting
In this technique the concept of southern hybridization has been used to explore the
sequence of m-RNA, which after separation into segments through electrophoresis is blotled
into the filter support known as northern blot.
Steps in Northern Blotting

Isolation and purification of RNA

Denaturation and separation by gel electrophoresis

Blotting/ Transfer on membrane

Hybridization with labeled probes

Washing and detection

Application of Northern blotting

1) It is use to detect gene expression in the particular tissue or cell
2) It detects individual mRNA in a populations of RNAs
3) It determine size and amount of particular mRNA molecule in the sample.
4) It provides rough idea about physical abundance of a specific mRNA.
5) It provide information about conditions that affects processing of mRNA.

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Advantages of Northern blotting

1) The procedure is simple and can be erased many times with proper equipment‟s and
solutions.
2) It is well suited both radioactive and non - radioactive probes.
3) Any tissue can be used for analysis.
4) It can compare results obtained from different tissue.
5) It has high degree of accuracy.

Limitations of Northern blotting

Multiple probes can not be used to detect more than one sequences in a sample.

Western Blotting
The transfer of proteins from on electrophoretic gel to a nitrocellulose and nilon
membrane by means of an electric force is known as western blot the proteins are
electrolyzed in polyacrylamide gel transferred on to a Nilon membrane (or) Nitro cellulose
membrane and proteins bands are detected by their specific interaction with antibodies lectins
and some other compounds.

Applications of Western Blotting

1) Used for medical diagnostic test for viral, bacterial and genetic diseases.
2) Used to determine expression of integrated gene in transgenic organisms.
3) It is use to determine in which tissue under contain physiological condition a gene is
expressed.
4) Used the study of Protein Protein interactions.

Advantages of Western Blotting

1) It is highly specific and it can test several samples at a time.
2) It can analyze changes in protein levels and detect intensity of protein expression.

Limitations of Western Blotting

1) It requires highly sensitive equipment.
2) Some blotting conditions can destroy the antibody binding site.
3) It can‟t detect protein if degraded quickly.

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