GPB 316 Plant Biotechnology (2+1) - Online Study Material

B.Sc. Hons. (Agri.) / Dr. T.
Sabesan / GPB 316 Plant Biotechnology /

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Online Study Material Collection
THEORY MATERIAL
GPB 316 PLANT BIOTECHNOLOGY
Prepared by
Dr. T. SABESAN, Ph.D.
DEPARTMENT OF GENETICS & PLANT BREEDING

FACULTY OF AGRICULTURE
ANNAMALAI UNIVERSITY
B.Sc. Hons. (Agri.) / Dr. T. Sabesan / GPB 316 Plant Biotechnology /
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1. INTRODUCTION
Biotechnology broadly refers to development of techniques for the application of

biological principles to make products by biological agents. For example, the production of
wine by fermentation. The term biotechnology was coined by Karl Ereky, a Hungarian
engineer in 1919. The term plant biotechnology refers to application of engineering techniques
to modify plants for the benefit of humans.
In a narrow definition, biotechnology refers to genetic manipulation of organisms for

specific purposes. Biotechnology has two branches namely, tissue culture and genetic
engineering. Tissue culture refers to the in vitro cultivation of cells, tissues, organs,
embryos, seeds and protoplasts on nutrient media under aseptic conditions in a
laboratory. The term genetic engineering refers to manipulation of organisms at the
molecular level directly involving the DNA.
Genetic engineering can modify bacterial cells to synthesize completely new substances,
e.g. hormones, vaccines, monoclonal antibodies, etc., or introduce novel traits into plants or
animals.
HISTORY OF PLANT TISSUE CULTURE
Gottlieb Haberlandt is regarded as the Father of plant tissue culture as he predicted

the totipotency of plant cells and attempted in vitro culture of plant mesophyll cells as early
as 1902. Totipotency is the ability of a plant cell to multiply, differentiate and grow into a
complete plant. The first embryo culture was attempted by Hanning in 1904. In 1925,
Laibach recovered hybrid progeny from an interspecific cross in Linum by using zygotic
embryos of seeds as explant.
The first plant growth hormone indoleacetic acid (IAA) was discovered in 1930s by F.
Kogl et al. In 1934, Professor Philip White successfully cultured tomato roots on the medium,
later called as White’s medium. In 1939, Gautheret successfully cultured carrot tissues and
the possibility of cultivating plant tissues for an unlimited period was independently
endorsed by Gautheret, White and Nobecourt in 1939. In 1955 Miller and Skoog published
their discovery of the hormone kinetin, a cytokinin. The first plant from a mature plant cell
was regenerated by Braun in 1959.
The focus of the scientists later shifted to preparation of single cell cultures. Muir
(1953-54) demonstrated that, callus tissues in liquid medium when subjected to shaking,
broke into single cells. In 1960, Bergmann developed the method for cloning of these single
cells by filtering suspension cultures. This technique called Plating technique is widely used
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for cloning isolated single protoplasts.
In 1962, Toshio Murashige and Skoog published the composition a plant tissue
culture medium known as MS medium, which became the most widely used medium for
tissue culture now.
For the first time in the world, haploid plants from anthers of Datura were first
produced by the Indians, Guha, S and Maheshwari, S.C. (Nature, 204:497 (1964) & Nature,
212:97-98 (1966)). This discovery received significant attention since, plants recovered from
doubling of haploids are homozygous and express all recessive genes thus making them
ideal for pure breeding lines.
The classical work of Steward (1966) on induction of somatic embryos from free cells
in carrot suspension cultures finally demonstrated totipotency of somatic cells, thereby
validating the ideas of Haberlandt. Morel utilized this application for rapid propagation of
orchids and Dahlias. He was also the first scientist to develop virus free orchid and Dahlia
plants by cultivation of the shoot meristem of infected plants.
Protoplast (a cell without cell wall) was produced by Cocking in 1960 by using cell
wall degrading enzymes. The first somatic hybrid plants by fusing the protoplasts of N.
glauca x N. langsdorfli was produced by Carlson et al. in 1972.
DEVELOPMENT OF BIOTECHNOLOGY IN INDIA
The promote biotechnology in India the Department of Biotechnology (DBT) was

started in 1986. It was initially started as National Biotechnology Board (NBTB) i n 1 982
under Department of Science and Technology. Later, the International Center of Genetic
Engineering and Biotechnology (ICGEB) was established by the United Nations to help
the developing countries like India. ICGEB has two centers, one in New Delhi and the
other in Trieste (Italy).
SCOPE AND IMPORTANCE OF BIOTECHNOLOGY
Biotechnology is controlled use of biological agents for beneficial use. It involves the
integrated application of biochemistry, molecular biology and microbiology to develop
technological application for enhancement of the capabilities of biological agents. Thus,
biotechnology has emerged as a science with immense potential for human welfare which will
be evident from following examples:
Biotechnology in Medicine: Production of monoclonal antibody, DNA and RNA probes for
diagnosis of various diseases; synthesis of valuable drugs like insulin and interferon from
bacteria for treatment of human diseases; DNA fingerprinting for identification of parents and
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criminals; Development of recombinant vaccines like human hepatitis B etc. by genetically

engineered microbes are some of the notable achievements.
Industrial Biotechnology: It involves large scale production of alcohol and antibiotics by

microorganisms. A variety of pharmaceutical chemicals, with better quality and quantity, like
lactic acid, glycerine etc. are being produced by genetic engineering. Protein engineering i.e.,
remodeling of existing proteins and enzymes for a specific function or for increasing efficiency
of their function is gaining momentum.
Biotechnology and Environment: Environmental problems like pollution control, depletion

of non-renewable energy resources, conservation of biodiversity etc are being dealt with using
biotechnology. For e.g. bacteria are being utilized for detoxification of industrial effluents, in
removing oil spills, for treatment of sewage and for biogas production. Bio-pesticides give
environmentally safer alternative to chemical pesticides for control of insect pests and
diseases.
Biotechnology and Agriculture: In agriculture, plant cell, tissue and organ culture is used
for rapid and economic clonal multiplication of fruit and forest trees, for production of virus
free genetic stocks and planting material as well as in the creation of novel genetic variations
through somaclonal variation.
Genetic engineering techniques are utilized to produce transgenic plants with desirable
genes like disease resistance, herbicide resistance, increased shelf life of fruits etc. Also,
molecular breeding has hastened the process of crop improvement for e.g. molecular markers
like RFLP, SSRs provide powerful tools for indirect selection of both qualitative and
quantitative traits and also for studying genotypic diversity.
Landmark discoveries in the field of molecular biology

YEAR CONTRIBUTION
1970 Smith and Nathans discovered first restriction enzyme from Haemophilus
influenza (HindIII)
1972 First recombinant DNA molecule produced by Berg et al. combining SV40
virus and λ virus.
1972 Discovery of reverse transcriptase by Termin
1974 Biotransformation in plant tissue culture by Reinhard
1974 Discovery of Ti plasmid as tumour inducing principle by Zaenen et al., and
Larebeke et al.
1977 Successful integration ofTi plasmid DNA from Agrobacterium tumefaciens in
plants by Chilton et al.
1977 DNA sequencing methods based on degradation of DNA by Maxam and
Gilbert.
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1978 Crossing potato and tomato to produce pomato by somatic hybridization by

Melchers et al.
1980 Commercial production of human insulin in bacterial cells through genetic
engineering by Eli Lilly and Co.
1980 Restriction Fragment Length Polymorphism (RFLP) technique developed.
1981 Coining of the term somaclonal variation by Larkin and Scowcroft.
1983 Polymerase Chain Reaction (PCR), a chemical DNA amplification process
conceived by Kary Mullis.
1984 Transformation of tobacco with Agrobacterium and development of
transgenic plant by De Block et al., and Horsch et al.
1984 Development of genetic fingerprinting technique for identification of
individuals by analyzing DNA polymorphism by Jeffreys.
1987 Insolation of Bt gene from Bacillus thuringiensis bacterium by Barton et al.
1990 Launch of Human Genome Project formally.
1990 Development of Random Amplified Polymorphic DNA (RAPD) technique by
Williams et al., and Welsh & McClelland.
1991 Development of DNA microarray system by Fodor.
1995 Development of DNA fingerprinting by Amplified Fragment Length
Polymorphism (AFLP) technique by Vos et al.
1997 Completion of DNA sequencing of E.coli genome by Blattner et al.
2001 Successful completion of Human Genome Project by Craig Venter et al. of
Celera genomics
Questions
1. Define plant biotechnology.

2. Who coined the term biotechnology?
3. Haploid plants were first produced in vitro by _________________.
4. __________________ is considered as the father of plant tissue culture.
5. Polymerase chain reaction was conceived by ______________________.
6. Write an essay about the scope and applications of biotechnology?
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2. NUTRITIONAL REQUIREMENTS OF TISSUE

CULTURE
The plants growing in the field requires a medium (eg. soil) containing nutrients.
The isolated plant tissues called explants are grown on an artificial nutrient medium. The
nutrient medium is composed of a physical support system, macro nutrients,
micronutrients, carbon source, organic supplements and growth regulators. The nutrient
media has to supply all the essential mineral required for in vitro growth and
morphogenesis of the plant tissue.
I. SUPPORT SYSTEM
In vitro culture occurs either in liquid medium or on solid medium. In liquid medium
(suspension culture), the tissues or cells are cultured in water containing nutrients. The
liquid medium has to be frequently agitated for aeration. The solid media are prepared by
using gelling agents. Agar (0.5% - 1.0%) is the most widely used gelling agent as it is
resistant to enzymes and does not react with media components. Agarose, a pure form of
gel, gellan gums are also used.
Alternative methods of support include perforated cellophane, filter paper bridges,

filter paper wicks, polyurethane foam, and polyester fleece.
II. MACRONUTRIENTS
The nutrient elements n a m e l y , nitrogen (N), phosphorus (P), potassium (K),

calcium (Ca), magnesium (Mg), and sulfur (S) required in concentration m o r e t h a n 0.5
ml/lit. are referred to as macro nutrients. Most media contain N and K at 20-30 mM.
while P, Mg, S, and Ca range from 1-3 mM.
III. MICRONUTRIENTS
The nutrient elements n a m e l y , iron (Fe), manganese (Mn), zinc (Zn), boron (B),
copper (Cu), and molybdenum (Mo) which are required in concentration less than 0.5
ml/lit. are considered as micro nutrients. Iron is the most critical of all the micronutrients.
Iron and zinc are commonly used in chelated form.
IV. CARBON SOURCE

Sucrose (2-3%) is the most preferred carbon source. Glucose and fructose can also
be used. Fructose is less effective. Other carbohydrates include Myo-inositol, maltose,
lactose, galactose, rafinose and starch.
V. ORGANIC SUPPLEMENTS
a. Vitamins : The most commonly used vitamins are thiamin (B1), nicotinic acid,
pyridoxine (B6), and myo-inositol. Thiamin is basically required by all cells for growth.
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b. Aminoacids: It is important for stimulating cell growth in protoplast cultures and for
establishing cell cultures. Glycine is most commonly used amino acid. Glutamine aspargine,
arginine, cystine are the other common sources.
c. Other organic supplements: It include organic extracts like protein (casein)

hydrolysate, coconut milk, yeast & malt extract, ground banana, orange juice, tomato juice,
activated charcoal. The addition of activated charcoal helps in removal of toxic compounds.
d. Antibiotics: To prevent the infection of microbes, antibiotics such as Streptomycin or

Kanamycin are added to the medium.
V. GROWTH REGULATORS
Four broad classes of growth regulators are important in plant tissue culture. These
include auxins, cytokinins, gibberillin and ABA.
a. Auxins: The only naturally occurring auxin found in plant tissues is IAA. Auxins
induce cell division, cell elongation, elongation of stem, internodes, tropism, apical
dominance, abscission and rooting. The commonly used auxins are:
IAA (Indole 3-Acetic Acid),
IBA (Indole 3-Butyric Acid)
2,4-D (Dichloro Phenoxy Acetic Acid)
NAA (Naphthylene Acetic Acid)
NOA (Naphthoxy Acetic Acid)
The 2,4-D is used for callus induction whereas, the other auxins are used for
root induction.
b. Cytokinins
Cytokinin stimulate cell division, to induce shoot formation and axillary shoot
proliferation, and to inhibit root formation. They have been shown to activate RNA
synthesis and to stimulate protein and the enzymatic activity. The commonly used
cytokinins are
BAP (6-Benzyl Amino Purine)
BA (Benzy adenine)
2ip (Isopentyl adenine)
Kinetin and Zeatin
EFFECT OF GROWTH REGULATORS:

 More cytokinin / low auxin ratio regenerated to shoot part.
 Low cytokinin / more auxin regenerated to only root part.
 Medium cytokinin / medium auxin regenerated to both shoot and root ,
 Medium cytokinin. low auxin only regenerated to callus.

The concentrations of auxins and cytokinins are equally as important as their ratio.
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c. Gibberillins and Abscisic acid
GA3 is most commonly used gibberillin. I t e nhances callus growth and

simulates the elongation of dwarf or stunted plantlets. ABA in culture medium
either stimulates or inhibits culture growth depending upon the species and to
inhibit latter stages of embryo development.
pH
pH range of 5.0 to 6.0 is suitable for in vitro culture. Autoclaving f nutrient media
reduces pH by 0.3 to 0.5 units. pH above 6.0 leads to a hard medium while a pH less
than 5.0 prevents gelling of agar. Hence, it must be adjusted by adding 0.1N NaOH
or HCl.
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NUTRIENTS AND THEIR PHYSIOLOGICAL ROLE
Element Functions
Nitrogen (N) Component of proteins, nucleic acids some co-enzymes
Phosphrous (P) Component of nucleic acids, energy transfer, component of

intermediate in respiration and photosynthesis
Potassium (K) Regulates osmatic potential, principle inorganic cation.
Calcium (Ca) Cell wall synthesis, membrane function cell signaling.
Magnesium (Mg) Enzyme co-factor, component of chlorophyll.
Sulphur (S) Component of some amino acids (Methionine, cysteine) some

co-factors.
Chlorine (Cl) Required for photosynthesis
Iron (Fe) Electron transfer as a component of cytochromes
Managanese (Mn) Enzyme co-factor
Cobalt (Co) Component of some vitamins
Copper (Cu) Enzyme co-factor electron transfer reaction
Zinc (Zn) Enzyme co-factor chlorophyll biosynthesis
Molybdenum (Mo) Enzyme co-factor component of nitrate reductase.
PREPARATION OF NUTRIENT MEDIUM
The nutrients required for optimal growth of plant organ tissue and
protoplast in vitro generally vary from species to species. No single media can be
suggested as for all types of in vitro culture. In order to formulate a suitable medium for
a new system a well known basal medium such as MS medium (Murashigel and Skoog),
B5 (Gamborg et al), White media etc.
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The composition of MS (Murashige & Skoog) media is given below

MACRO NUTRIENTS CONCENTRATION
Na 4NO3 - 1.65 g
KNO 3 - 1.90 g
CaCl2 2 H2O - 0.44 g
MgSO 4 7H2O - 0.37 g
KH2PO4 - 0.17 g
Micro Nutrients -
FeSO4 7 H2O - 27.80 mg
Na2EDTA 2 H2O - 33.60 mg
Kl - 0.83 mg
K 3BO4 - 6.20 mg
MnSO 4 4H2O - 22.30 mg
ZnSO4 7 H2O - 8.60 mg
Na2MoO4 2 H2O - 0.25 mg
CuSO4 5 H2O - 0.025 mg
CoCl2 6 H2O - 0.025 mg
Organic supplements -
Myo-inositol - 100.00 mg
Nicotinic acid - 0.05 mg
Pyridoxine HCl - 0.05 mg
Thiamine HCl - 0.05 mg
Glycine - 0.20 mg
Sucrose - 20.00 mg
Growth regulators - As needed
Gelling agent - Only for solid medium
Agar
Agar - (0.5-1%) 6-8 g/lit
pH - 5.8
Introduction to Plant Biotechnology
T. Sabesan, Dept. of Genetics & Plant Breeding 11
By making minor quantitative and qualitative changes a new media can develop to
accommodate the specific requirements of the desired plant material
Methods of media preparation
The most suitable method for preparing media now is to use commercially
available dry powdered media. The powder is dissolved in distilled water generally 10%
less than final volume of medium and after adding sugar, agar and other desired
supplements. The final volume is made up with distilled H 2O. The pH is adjusted and
media is autoclaved,
Another method of preparing media is to prepare concentrated stock solutions. The

use of stock solutions reduces the number of repetitive operations involved in media
preparation and, the chance of human or experimental error. Also, direct weighing of
media components (e.g., micronutrients and hormones) required only in milligram or
microgram quantities in the final formulation cannot be performed. For these components,
preparation of concentrated stock solutions by dissolving required quantities of chemicals
in distilled water and subsequent dilution into the final media is standard procedure. All
the stock solutions are stored in proper containers at low temperature in refrigerators at
2°- 4°C.
Stock solutions of macronutrients can be prepared at 10 times the concentration of

the final medium. A separate stock solution for calcium salts may be required to prevent
precipitation.
Micronutrient stock solutions are generally made up at 100 times their final strength
and can be stored in a refrigerator for up to 1 year. Vitamins are prepared as 100X or 1000X
stock solutions and stored in a freezer at -20°C for 2-3 months. Auxin stock solutions are
generally prepared at 100-1000 times the final desired concentrations. The auxins NAA and
2,4-D are considered to be stable and can be stored at 4°C for several months;
Stock solution of Iron is stored in amber coloured bottles. Substances which

are unstable in frozen state must be freshly added to the final mixture of stock solution at
the time of medium preparation, Contaminated (or) precipitated stock solution should not
be used.
3.TECHNIQUES IN PLANT TISSUE CULTURE
Depending upon the plant parts used as explants, plant tissue culture techniques are
classified following types viz. meristem culture, embryo culture, anther culture, ovule
culture etc. The whole process can be summarized into the following stages :
STAGE 0: SELECTION OF EXPLANT

The plant part used to initiate tissue culture is called as explant. The success of the
explant depends on its location on the plant, age, or developmental phase. Explants that
contain shoot primordia (e.g., meristems, node buds, shoot apices) are preferred. Also,
explants from younger (juvenile) plants are more successful.
STAGE I: INITIATION OF ASEPTIC CULTURE
In this stage the explant is surface sterilized to remove the microbial contaminants
and transferred into nutrient medium. The most commonly used disinfectants are sodium
hypochlorite, calcium hypochlorite, ethanol and mercuric chloride (HgCl 2). The cultures are
incubated in growth chamber under light or dark conditions.
STAGE II: PROLIFERATION OF AXILLARY SHOOTS
Axillary shoot proliferation is induced by adding cytokinin to the shoot culture
medium. Cytokinin to auxin ratio of about 50:1 produces shoots with minimum callus
formation. New shoots may be subcultured at an interval of about four weeks.
STAGE III: ROOTING
Addition of auxin to the medium induces root formation. Roots must be induced on
the shoot to produce plantlets for transfer into the soil. The rooting stage may occur
simultaneously in the same culture media used for multiplication of the explants. However,
in some cases it is necessary to change media to induce rooting and the development of
strong root growth.
STAGE IV: HARDENING
At this stage, the in vitro plants are transferred to an appropriate substrate (sand,
peat, compost etc.) and gradually acclimatized to field condition by exposure to low
humidity and high light intensity in green house. This process is called as hardening.
APPLICATIONS OF PLANT TISSUE CULTURE

1. Tissue culture is useful in producing large
numbers of identical individuals from a mother
plant.
2. Used to rescue hybrid embryos in wide
hybridization.
3. To conserve rare or endangered plant species.
4. Useful in creation of transgenic plants.
5. Useful in propagation of orchids by culture of
immature embryos
6. Plant Breeder may use tissue culture to screen
cells rather than plants for advantageous
characters like herbicide resistance/tolerance.
7. Useful in production of haploid plants by
anther or pollen culture.
8. Large-scale growth of plant cells in liquid
culture in bioreactors for production of valuable compounds, like plant-derived
secondary metabolites and recombinant proteins used as biopharmaceuticals.
9. To cross distantly related species by protoplast fusion.
10. Biosynthesis of secondary products and biotransformation.
11. Useful in rapid evaluation of molecular basis for physiological, biochemical, and
reproductive mechanisms in plants, Eg. in vitro selection for stress tolerant plants, and
in vitro flowering studies
12. For chromosome doubling and induction of polyploidy, Eg. Doubled
haploids, tetraploids etc. by application of colchicine or oryzalin.
13. Meristem tip culture can be used to produce virus free plants even from virus infected
plants or stock as in potatoes and fruit crops.
14. Tissue culture is useful in production of identical, sterile hybrid species.
4. MAINTENANCE OF ASEPSIS
Asepsis literarily means absence of contamination. A knowledge of the source
and types of contamination is essential for detection and prevention.
SOURCES OF CONTAMINATION
Laboratoryware and media: The containers used for media preparation, if not properly
sterilized, can cause contamination. Also, the medium, which contains all nutrients for
growth may also harbor microorganisms.
Explant: It is a major source of contamination. Plants may harbor microorganisms either

on the surface, or between cells or even within the cells. When the organism is present
within the plant tissue it is called endogenous or systemic contamination. They may
remain latent during several subcultures. Eg. Corynebacterium and Xanthomonas.
Environment of the transfer area: Even if all the precautions are taken by sterilizing the
media, laboratorywares etc., if the environment is not clean, the material could be
contaminated by atmospheric dust particles, fungal spores and bacteria.
Worker: Dirty hands and clothing of the worker may also become a source of
contamination. Talking or sneezing during work spreads microbes leading to
contamination.
TYPES OF CONTAMINATION
Bacteria : They are usually killed at high temperature. But few genera like Clostridium
produces heat-resistant endospores. Common genera which act as contaminant includes
Agrobacterium, Bacillus, Beijerinckia, Pseudomonas, Staphylococcus, Acinetobacter etc.
Identification of bacteria involves morphological, biochemical and physiological
characterization.
Fungi: Most fungi reproduce by means of spores, which may be sexual or asexual.
Common fungal contaminants in plant tissue culture are Neurospora, Pensillium, Fusarium
and Cladosporium, Identification of fungi is much easier than bacteria as it is mainly based
on morphology of colonies, mycelia and fruiting bodies.
Yeast: Common genera associated with plant tissue culture are Candida and Rhodotorula.
Actinomycetes are also prokaryotic in nature which resemble fungi in morphology and
spore production is also a source of contamination.
Viruses: Unlike bacterial or fungal contaminants, the presence of viruses in tissue culture
plants cannot be visually seen. Infected plants may carry virus particles, but look healthy.
If the mother plant is infected, the virus will be passed on to the tissue culture plantlets
through explants which may exhibit disease at a later stage during hardening.
Insects like mites and thrips also pose a problem. Though they themselves are not much
serious, the microbial contamination carried by them is highly dangerous. Insect invasion
is most serious when they reproduce within cultures.
DETECTION OF CONTAMINATION
Contamination occurs at some point in any tissue culture process. The
contamination may be slow growing if the media are not ideal for the organism. Testing of
contamination should be carried out whenever necessary. After a plant culture or medium
has been contaminated, the container should be autoclaved before the content is discarded.
Bacterial contamination can be recognized by a turbidity in the liquid media and sometimes
by unusual odour. Yeast growth often appears as a heavy milky turbidity in liquid media
and have a distinctive odour. Fungi forms mycelia which appears as ‘balls’ in liquid media.
EFFECT OF CONTAMINANTS ON TISSUE CULTURE PLANTS

The contaminants affect tissue culture plants in following ways:
 As microorganisms are fast growing, they often overrun the explants.
 Carbohydrate fermentation by the contaminant leads to accumulation of toxic
metabolites like acetic acid and ethanol.
 They bring down the pH of the medium to less than 3.0.
 Low pH of the medium leads to non-availability of certain nutrients.
5. MICROPROPAGATION
Propagation of plants vegetatively by cutting, budding, grafting etc. involves only

mitotic cell division. The progeny obtained by vegetative propagation of a single plant is
called as a clone.
Tissue culture also enables rapid clonal propagation of plants. In vitro clonal
propagation of plants by tissue culture is called as micropropagation. The process involves
selection of plant tissues (explant) from a healthy and vigorous mother plant. Any part of
the plant such as leaf, apical meristem, bud and root can be used as explant. The main
objective of micropropagation is to produce progeny plants which are identical to the parent
plants in genotype. This is achieved by the following three pathways:
PATHWAYS OF REGENERATION
(i) Proliferation from preexisting meristems (Axillary bud proliferation)
(ii) Organogenesis and
(iii) Somatic embryogenesis
(i). PROLIFERATION OF PRE-EXISTING MERISTEM/AXILLARY BUD PROLIFERATION

This method makes use of already existing meristem to initiate in vitro culture (eg.
shoot-tip / nodal explant). The merit of using axillary bud proliferation from a node or bud
is that, the shoot has already differentiated and only its elongation and root differentiation
are required. The size of the shoot tip ranges between 1 and 10 mm in length. Cytokinin in
the media stimulates pre-existing meristem in the explant (apical meristem in shoot tips
and axillary buds in nodal explants) to develop into shoots. Each leaf on such shoot has an
axillary bud which are sub-cultured after 4-6 weeks onto a fresh medium. In most plant
species, each explant produces 5-6 shoots in 4-5 weeks which would result in 510 to 612
plants in one year from a single explant, assuming 100% survival.
In some species, (Eg. Blueberry) when the axillary buds do not produce new shoots
then the shoot bud developed from the explant is excised and cut into small pieces to obtain
nodal explants which are then subcultured to initiate a new cycle of micropropagation. This
is termed as single node culture.
Shoot tips are easy to excise from the plant. They are genetically stable and have high
survival and growth rates. They contain preformed incipient shoot and are phenotypically
homogeneous. Axillary and terminal buds also have the advantages of shoot tips but they
are more difficult to disinfect.
(ii). ORGANOGENESIS
Organogenesis refers to the formation of plant organs such as roots and shoots
directly on the explant which lacks a preformed meristem or de novo origin from callus and
cell suspension culture induced from the explant. Plant production through organogenesis
can be achieved by two modes:
i. Emergence of adventitious organs directly from the explant (Direct organogenesis /

Adventitious regeneration) and
ii. Emergence of adventitious organs through callus formation with de novo origin
(Indirect organogenesis).
Organogenesis is of two types namely,

a). Direct organogenesis: Direct organogenesis or Adventitious regeneration refers to
the development of organs such as roots, buds, shoots, flowers etc. or embryo like
structures on an explant directly, bypassing the callus stage. The shoots or roots are
induced on tissues that normally do not produce these organs. This pathway is less
common.
Fig. Direct organogenesis / Adventitious regeneration

b). Indirect organogenesis: In this pathway, explant gives rise to initiation of callus,
which is an unorganized mass of undifferentiated cells, from which shoots and roots are
formed. Hence, it is called as indirect organogenesis. The callus consists of an aggregation of
meristem-like cells that are developmentally plastic. This process involves formation of
callus from a matured explant (by dedifferentiation) and formation of various organs from
the callus or adventitious meristems (by redifferentiation).
Fig. Indirect Organogenesis via callus formation

It is made possible by altering the concentration of plant growth hormones in the

nutrient medium. Skoog and Muller demonstrated that high ratio of cytokinin to auxin
stimulated the formation of shoots in tobacco callus while high auxin to cytokinin ratio
induced root regeneration. Thus the organ formation depends upon the ratios rather than
the absolute concentration of auxin and cytokinin.
The negative side of this method is that it introduces mutations in vitro (somaclonal
variations). The callus phase also makes it more technically challenging than shoot tip
micropropagation.
(iii). SOMATIC EMBRYOGENESIS

Normally a zygote is formed after an egg has been fertilized by a sperm. The
zygote then develops into an embryo (zygotic embryo). The process by which the
embryos are regenerated from somatic cells, tissues or organs is called as somatic
embryogenesis. Such embryos are also called as non-zygotic embryos. Somatic
embryogenesis is the opposite of zygotic or sexual embryogenesis. Somatic embryos are
formed from a single cell and it requires a single hormonal signal to induce a bipolar
structure capable of forming a complete plant. The bipolar structure of the somatic embryo
contains both shoot and root meristems. The embryos develops by forming structural steps
of the globular, heart, torpedo, cotyledonary and mature stages. While in organogenesis, it
requires two different hormonal
signals to induce first a shoot
organ and then a root organ. No
endosperm or seed coat is
formed around a somatic
embryo.
Somatic embryos could be
induced either directly from the
explant tissue bypassing the
callus formation stage or via the
formation of callus from the
explant.
It is of two types:
a). Direct somatic
embryogenesis: It can be
initiated directly from the
explants through ″pre-
embryogenic determined cells.″ Fig. Somatic embryogenesis showing globular, heart and torpedo
Such cells are found in stages of embryo development
embryonic tissues of scutellum,
hypocotyls and nucellus.
b). Indirect somatic embryogenesis: It is done through the establishment of callus from
which embryos are developed. Here the embryo arises from ″induced embryogenic
determined cells.”
Somatic embryogenesis encompasses various stages from callus initiation,

development of somatic embryos, maturation, plantlet formation and transfer to soil.
Somatic embryogenesis has been reported in cactus, grapes, rose etc.
Advantages of Somatic embryogenesis

It is useful in clonal propagation of genetically uniform plant material; elimination of
viruses; provision of source tissue for genetic transformation; generation of whole plants
from single cells called protoplasts; development of synthetic seed technology. The process
can also be used to develop the plants that are resistant to various kinds of stresses
It is also used to introduce the genes by genetic transformation Eg. A successful
protocol has been developed for regeneration of cotton cultivars with resistance to Fusarium
and Verticillium wilt.
PROBLEMS ASSOCIATED WITH MICROPROPAGATION

1. Microbial contamination
Bacterial and fungal contamination in culture do not allow propagules to grow and
contaminated cultures have to be usually discarded. It can be overcome by growing the
donor plant in growth chamber, by effective sterilization of explants, by performing
inoculation in the laminar air flow cabinets and by using sterilized surgical
instruments. Fumigation of inoculation with dilute formaldehyde solution also helps.
2. Callusing
Callus formation is highly undersirable as it often effects the normal development of
shoots and roots and may lead to generation of variability among the regenerated plants.
Addition 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

Micropropagated 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, and controlling the
cultural environment favouring slow multiplication rates
4. Browning of medium
In many woody species (eg. sugarcane) phenolic substances
leach from the cut surfaces of explant into the medium. These
phenolics turn brown on oxidation and lead to browning or blackening
of medium and explants, leading to necrosis and eventually death of
the cultures.
It can be overcome by frequent subculture (every 3-7 days);
growth of culture in liquid medium for 3-10 days; use of antioxidants like citric acid
(150 mg / lit) to check the oxidation of polyphenols; use of adsorbents like activated
charcoal (0.5-2g/lit) (or) PVP (poly vinyl pyrrolidon) and incubation of cultures in dark since
light enhances polyphenol oxidation as well as polyphenol bio-synthesis.
5. Vitrification
Some shoots developed in vitro appear brittle, glassy and water soaked. This
condition is called as vitrification or hyper-hydration. The plants appear abnormal because
of abnormal leaf morphology leading to poor photosynthetic efficiency, malfunctioning of
stomata and reduced epicuticular waxes.
Vitrification can be reduced by reducing the relative humidity in culture vessels;
reducing the cytokinins level or NH4 level or salt concentration in the medium, and by
addition of flurorizin, fluroroglucinol or CaCl2 in the medium.
6. Transplantation shock
High mortality rate of tissue culture derived plants to soil continues to be a major
bottleneck in micropropagation of many plant species. Conservation of moisture by creating
high humidity, partial defoliation, application of antitranspirants ahs given good results.
SOLUTION TO SOME COMMON PROBLEMS IN TISSUE CULTURE
 In preparation of media, formation of insoluble precipitates due to calcium, phosphate

and magnesium compounds creates a problem. This can be avoided by dissolving each
compound completely before adding the next compound. Also it can be avoided, if each
compound is dissolved separately and added as a solution rather than as the salt.
 Colour of the final medium is a very useful quality indicator. Media with agar are usually
of golden colour. Liquid media are very pale yellow colour due to the presence of iron
compound. Any abnormal change in colouration indicates that the failure in autoclaving
or irregularity in media composition. In such cases, discarding of the media and
preparation of a new media is a way.
 If a batch of agar or other gelled medium fails to solidify, the medium may not be
properly mixed or the pH is too acidic in nature.
 If the surface sterilization procedures do not yield clean cultures, then the procedure
needs to be more stringent. Increased prewashing time, use o strong detergent,
increased time in ethanol (rarely recommended), concentration of bleach solution and
time in bleach solution.
 In contrast, if the surface sterilization procedures yield brown or black explants with no
growth, then the procedure needs to be less stringent. This can be done by reducing the
strength of the detergent or decrease the amount of handling during pre-washing,
eliminate exposure to ethanol and /or decreasing the time in bleach or concentration of
bleach solution.
 Stage I becomes a difficult step in micropropagation when endophytic microorganisms

are present in the plant species. Repeating the surface sterilization procedure, or use of
an antibiotic treatment followed by the bleach will help in establishment of few sterile
cultures.
 In stage II, the important problem is the lack of shoot multiplication after cutting the
tissues into small pieces for subculture. This can be due to lack of a true shoot meristem
in the subcultured piece. This can be overcome by the use of larger pieces.
 Recovery of plants from the culture vessel into the soil is the most difficult step. The
plantlets were grown under in vitro conditions with 100% relative humidity with no need
for photosynthesis or to regulate respiration process. The plantlets transferred to the soil
must perform its own photosynthesis and regulate stomatal functions. The hardening
process must be gradual. Large plantlets survive better as they are better developed. For
herbaceous plants, watering should be regulated as little water leads to permanent wilt
and too much water leads to rotting.
ADVANTAGES OF MICROPROPAGATION
 Production of large number of genetically uniform plants.
 A small explant is enough to produce millions of true to type plants
 Rapid multiplication of rare and elite genotypes in a small area.
 This technique is possible alternative in plants species which do not respond
to conventional bulk propagation technique
 In plants with long seed dormancy, micro propagation is faster than seed
propagation.
 Useful to obtain virus free stocks
 In dioecious species plants where one sex is more desirable than the other sex. eg:-
Male asparagus and Female papaya etc. In such cases plants of desired sex can be
selectively multiplied by micropropagation.
 This technique is carried out through out the year independent of seasons.
CLASSIFICATION OF TISSUE CULTURE TECHNIQUES
Plant tissue culture refers to all types of aseptic plant culture which can be classified as
follows:
i). Seed culture: Culture of seeds in vitro to regenerate seedlings/plants.
ii). Embryo culture: Culture of isolated mature or immature embryos
iii). Callus culture: Culture of a differentiated explants, reversing it from adult to juvenile
stage, by dedifferentiation to produce callus is called as callus culture.
v). Cell culture: Culture of isolated cells or very small cell aggregates which are dispersed
in liquid medium.
vi). Protoplast culture: Culture of the plant protoplasts i.e., cells devoid of their cell walls.
vii) Organ culture: Culture of isolated plant organs in vitro is termed as organ culture. It
can be given different names depending upon the organ used as explants. For example, if we
use organs such as such as meristem, shoot tip, shoot bud, root, anther, pollen ovule, ovary
etc., accordingly they are called as meristem culture or shoot tip culture or shoot bud
culture or root tip culture or anther culture etc.
Fig. Types of culture

1).
MERISTEM CULTURE & SHOOT TIP CULTURE
Meristem culture refers to regeneration of whole plant from tissues of an actively

dividing plant part such as stem tip, root tip or axillary bud. The apical meristem refers to
dome like extreme shoot tip of 0.25 to 0.30 mm in length and 0.1 mm in diameter. To grow
virus free plants meristem tips of 0.2-0.3 mm is used. For shoot tip culture large explants
measuring up to 2 cm in length is used. This technique is widely used in vegetatively
propagated plants such as sugarcane, potato, banana and several timber species.
Morel and Martin (1952) isolated 100µm long meristem from virus -infected
plants, and cultured them to obtain virus-free shoots in dahlia. Plants free from viruses,
viroids, mycoplasma and even fungi and bacteria in a range of crops can be accomplished
by this technique. Virus free clones of potato, sugarcane have been produced from valuable
virus infected stocks through meristem culture.
Procedure for production of virus free plants by Meristem culture
1. Dissect out the shoot apical meristem (100-500 µm in length) with one or two leaf
primordia.
2. The larger the meristem explant, the greater the chances of its survival and shoot
development. But the risk of infection by the virus also increases with explant size.
Therefore, a compromise has to be reached between these two opposing forces in
deciding the explant size.
3. Viruses are eliminated by thermotherapy of whole plants, in which plants are exposed
to temperatures between 35-400C for a few minutes to several weeks depending on the
host-virus combination.
4. In general, it is preferable to excise larger shoot-tips from heat-treated plants. Also,
cultured meristems may also be given thermotherapy.
5. A prolonged exposure to a low temperature (50C), followed by shoot-tip culture, has
also proved quite successful in virus elimination. This technique is called cryotherapy.
6. Some chemicals, e.g., virazole (ribavirin), cyclohexamide, actinomycin D, etc., which
interfere with virus multiplication, may be added into the culture medium for making
the shoot-tips free from Viruses; this is known as chemotherapy.
Applications of meristem culture in crop improvement

1. It is used for micro propagation of banana, strawberries, citrus etc.
2. Virus free plants can be obtained through this technique as meristematic cells remain
free from virus even in the virus infected plants.
3. Useful in germplasm exchange of asexually propagated plant species as the plantlets
obtained by meristem culture are free from pathogens.
4. Meristems are suitable for cryopreservation by storing the germplasm at – 196° С in the
liquid nitrogen for long period of time.
5. Virus-free plants serve as excellent experimental materials for evaluating the detrimental
effects of infections by various viruses.
6. The virus free bulbs grew more rapidly, plants were more vigorous, and they produced a
greater number of larger flowers that had richer colour than the virus infected stock.
7. The virus-free plants are deliberately infected by known viruses to study the effects of
the infection on performance of the host.
8. Meristem culture can also help eliminate other pathogens like mycoplasma, bacteria and
fungi. Bacteria and fungi present in explants show up when they are cultured in vitro
since tissue culture media provide excellent nutrition for the microbes.
9. Meristem culture has been used to eliminate systemic bacteria from Diffenbachia and
Pelargonium, and Fusareum roseus from carnations.
Fig. Meristem tip

EMBRYO CULTURE
Embryo culture is a type of plant tissue culture in which the plantlets develops
directly from the embryo or indirectly through the formation of callus with subsequent
formation of shoots and roots. The technique of embryo culture has been widely used to
produce hybrids which were not able to develop through conventional method due to
embryo abortion. The first attempt to grow embryos was made by Hannig(1904).
Embryos of appropriate stage are removed from the seed and are transferred to the
culture medium. This technique is used when there is disharmony between embryo and
endosperm.
Types of Embryo culture: Based on the stage of the embryo it can be classified as
a. Mature Embryo culture: It is culture of mature embryo derived from ripe seeds. It
requires simple medium. This is done when embryos do not survive in vivo or become
dormant for longer periods of time or to eliminate the inhibition of seed germination. Some
species (Eg: Iris, orchids) produce sterile seeds due to incomplete embryo development.
Such embryos can be cultured and viable seedlings can be produced.
b. Immature embryo culture / Embryo rescue technique - Culture of immature

embryos to rescue the embryos of wide crosses is used to avoid embryo abortion and
produce viable plants. It requires complex media which includes special aminoacids,
hormones, endosperm extract like coconut milk etc.
The most important aspects of embryo culture are excision of embryo and cultural
requirements.
Excision of embryo: The mature embryos can be isolated by splitting open the seed. Seeds
with hard seed coat are dissected after soaking them in water overnight.
To excise immature embryos from single ovule, the ovule is split longitudinally to isolate
the half containing the embryo. By carefully keeping the part, the ovular tissue the entire
embryo along with attached suspension should be removed. Presence of suspensor is
critical for the survival of young embryos.
For excising older embryo, a small incision is made in the ovule on the side lacking the
embryo and than a slight pressure is applied with a blunt needle to release the intact
embryo.
Cultural requirements: The most important aspects of embryo culture is the selection of a
suitable medium that would support the development of embryos excised at different
stages of growth. The zygotic embryos develop through the following stages:
Proembryo- Globular stage - Heart stage - Torpedo stage - Cotyledonary stage.
A fully developed embryo undergoes a period of maturation during which the embryo
becomes hardy. Upto a certain stage, for example, upto globular stage in capsella the
embryo is heterotropic, as it derives some part of its nutrition from endosperm. Beyond this
stage the embryo becomes autotropic and is able to synthesis its biochemical needs from
simple nutrients like salt and sugar. In general, the older the embryo, the simpler is its
nutritional needs.
Applications of embryo culture

1. Useful in making wide hybridization (interspecific and intergeneric crosses) successfully.
2. Embryo culture is applied to get viable hybrids from interspecific hybridization in
Trifolium, Lycopersicon.
3. It is also used to grow progenies from intergeneric hybrization between Hordeum &
Secale, Triticum & Secale and Triticum & Aegilops etc.
4. Useful in obtaining haploids e.g. barley and wheat.
5. The technique has been developed to break long term seed dormancy and obtain viable
seedlings in Prunus and Taxus species.
6. Embryo culture is also applied to test the viability of seeds, production of rare species
and haploid plants.
7. Conservation of endangered species can also be done by embryo culture technique.
8. In orchids, where the seeds serve to store food and are unable to propagate embryo
culture is useful.
9. It is applied in forestry for propagation of elite plants in which the selection and
improvement of natural population is difficult.
EMBRYO RESCUE
Fig. Embryo culture (A). Proembryo dissected 3-5 days after pollination (B). Proembryo
culture in agar medium (C). Plantlet development from embryo (D). Plantlet transplanted
into soil
In wide hybridization which involve crossing between two different species or

genus, the embryo abortion may occur and hybrid seeds cannot be recovered. The
breeder may dissect the flower to remove the immature embryo and nurture it into a
full plant by using tissue culture technology. This technique is called embryo rescue.
The fertilized ovary is excised within several days of fertilization to avoid an
abortion The development of the embryo goes through several stages with certain
distinct features. The globular stage is undifferentiated, while the heart stage is
differentiated and capable of independent growth. The torpedo stage and
cotyledonary stage of embryo development follow these early stages.
Prior to differentiation, the developing embryo is heterotrophic and dependent
on the endosperm for nutrients. Excising the embryo prematurely gives it less a
chance of surviving the embryo rescue process. Just like all tissue culture work,
embryo rescue is conducted aseptically and cultured on the medium appropriate
for the species.
ANTHER / POLLEN CULTURE
Regeneration of whole plant from anther (or) pollen in the culture medium is called
anther culture. Haploid plants can be developed by anther culture. The optimum stage
differs from species to species.
Production of haploid
plants through anther
culture is known as
“androgenesis”
Haploids can be identified

by cytological studies at callus
stage (or) at plant level by bio
chemical studies or marker
genes linked with haploidy. In
general, the haploids are
much weaker highly sterile
and difficult to maintain
when compare to the normal
plants of concerned species.
Therefore, chromosome
number of all haploids are
doubled usually by treating
with colchicine to produce
doubled haploids which have
the normal somatic
chromosome complement (2n)
of the species and are fully
fertile.
The double haploid
plants are completely
homozygous and fully vigorous
and can be used for the
evaluation of performance and
selection for desirable traits. Fig. Anther culture
FACTORS AFFECTING ANDROGENESIS
i. Genotype of donor plant: Genotypic differences among the donor plants greatly affect
the ability of pollen grains to form haploid plants. For example, In tobacco, N. langsdorffii
only few pollen embryos could be induced than in other species. Also, in rice, japonica types
respond better than indica types.
ii. Physiological status of donor plants: Physiology of the donor plant is affected by
a) Age: The buds from the first flush of flowers show better respond than those born
subsequently.
b) Environment: The response in culture is predominantly influenced by different external

conditions like light intensity, photo period temperature, nutritional status and
concentration of CO2. Androgenic response was greater when the anthers were taken from
plants are grown in short day (8hrs) with high light intensity as compared to those grown in
long days (16hrs) with low light intensity.
c). Stage of pollen development: The optimum stage of pollen varies with the species.
Usually the anthers containing pollen at early to mid uninucleate stage is used. Generally
the bud size is used as an index of the pollen stage.
d). Size of anthers: Spikelets and texture of spikelets are correlated with the optimum
development stage of the pollen which influences the culture.
e). Anther wall factors: The anther wall, whole anthers (or) extract of anthers were found to
play an important role in androgenic response by acting as a conditioning factor.
f). Culture medium: Sucrose is essential for androgenesis, the usual level of sucrose is 2-
4%. However higher concentration of 6-12% favours androgenesis in cereals. The media
requirements vary with the genotype, age of anther and the conditions under which the
donor plants are grown. Basal medium of MS, Nitsch and Nitsch, white, N6 for solonaceous
crops, B5 and its modifications for Brassica and B5, N6, for potato are commonly used.
After pre-treatment, the anthers are dissected under sterile conditions. In plants with
minute flowers Eg:- Brassica and Trifolium it may be necessary to use a stereo microscope
for dissecting the anthers. In case of cereals whole panicles may be inoculated in the
medium. Anthers should be placed horizontally and not in upright position usually about
50-60 anthers should be placed in 10ml of liquid medium. Anthers can also be plated on
solid agar media at the rate normally 10-20 anthers in a 6 cm petridish.
Applications of anther culture

1. To obtain haploids plants
2. Homozygous diploids are obtained simply by doubling chromosomes or obtained
spontaneously from anther culture.
3. Anther culture is applied in Mapping population
4. It is also applied to shorten the breeding cycle.
Achievements in anther culture

In Japan, a commercial tobacco variety N. tabaccum F-211 has been produced by
anther culture which is resistant to bacterial wilt and has mild smoking quality. In china,
81 varieties and strains of rice have been developed through anther culture. Eg. Hua yu-1,
Xin-Xiu, Xhonghua -8 and xhonghua -9 with high yield and blast resistance have been
produced.
DOUBLED HAPLOID (DH) PRODUCTION
A doubled haploid (DH) is a genotype formed by chromosome doubling of

haploid cells. The 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. If the original
plant was diploid, the haploid cells are monoploid, and the term doubled monoploid may
be used for the doubled haploids. Haploid organisms derived from tetraploids are
called dihaploids (and the doubled dihaploids are tetraploids).
Doubled haploids can be produced in vivo or in vitro. Haploid embryos are

produced in vivo by parthenogenesis, pseudogamy, or chromosome elimination after wide
hybridization. The haploid embryo is rescued, cultured, and chromosome-doubling of it
produces doubled haploids. The in vitro methods include gynogenesis (ovary and flower
culture) and androgenesis (anther and microspore culture). Androgenesis is preferred
method. Another method of producing the haploids is wide crossing.
In barley, haploids can be

produced by wide crossing with the
related
species Hordeum bulbosum;
fertilization is affected, but
during the early stages of seed
development the H.
bulbosum chromosomes are
eliminated leaving a haploid
embryo. In tobacco, wide
crossing of N. tabacum with N.
africana results in haploids
which are later doubled using
colchicine.
Advantages of DH Production of Doubled Haploids

 Artificial production of doubled haploids is important in plant breeding.
 Conventional inbreeding procedures take six generations to achieve approximately
complete homozygosity, whereas doubled haploidy achieves it in just one
generation. Dihaploid plants derived from tetraploid crop plants may be important for
breeding programs which involve diploid wild relatives of the crops.
CALLUS CULTURE
Callus refers to unorganized mass of undifferentiated cells. In some cases, it is

necessary to go through a callus phase prior to regeneration via somatic embryogenesis or
organogenesis. Callus is formed as a result of wounding and hormones (auxin, high
auxin/low cytokinin). Genotype, composition of nutrient medium, and physical growth
factors influence the formation of callus. The size and shape of the explants is also
important. Callus cultures need to be sub-cultured every 3-5 weeks in view of cell growth,
nutrient depletion and medium drying. Therefore, calluses are easy to maintain and are the
most widely used.
Callus differs in compactness

or looseness, i.e. cells may be
tightly joined and the tissue
mass is one solid piece or cells
are loosely joined and
individual cells readily
separate (friable). This can be
due to the genotype or the
medium composition. A friable
callus is often used to initiate
a liquid cell suspension
culture. Friable callus is a
source of protoplasts.
Application of callus culture
Callus is ideal material for in vitro selection of useful somaclonal variants (genetic or
epigenetic).
Friable callus is a source for protoplast
In vitro mutation or somaclonal variation is possible due to the formation of callus
under in vitro condition.
A friable callus is often used to initiate a liquid cell suspension culture for production
of metabolites.
OVULE CULTURE
Regeneration of whole plant from the ovule in the nutrient medium is called ovule culture.
This technique is however used to a limited extent.
Applications:
1. To obtain interspecific and intergeneric hybrids Eg. Cotton. The hybrid embryo
between Tetraploid and diploid was rescued and obtained plants.
Gossypium barbadense x G. arborium
Gossypium hirsutum x G. herbaceum
2. Orchid seed germinate only in association with fungus, but the cultured fertilized
ovules germinate even in the absence of fungus
3. Test tube pollination and fertilization is possible through ovule culture
4. It helps in the development of several embryos in Citrus and other crops
5. Culture of unfertilized ovules helps in the formation of haploid callus.
SUSPENSION CULTURES
A cell suspension culture refers to culturing of cell aggregates which are dispersed
and growing in a moving liquid media. It is normally initiated by transferring pieces of
undifferentiated and friable callus to a liquid medium, which is continuously agitated by a
suitable device. The cells are suspended in the liquid culture by constant agitation by
keeping in a gyratory shaker at 100-250 rpm which facilitates aeration and dissociation of
cell clumps into smaller pieces.
A good suspension culture is one which

consists of a high percentage of single cells along
with small cluster of cells. Orbital shakers are
widely used for the initiation and serial propagation
of plant cell suspension culture. They should have a
variable speed control (30-150 rpm) and the stroke
range should be of 4-8 cm orbital motion.
Suspension cultures grow much faster than

callus cultures and it should be sub-cultured about
every week. The suspension cultures are broadly
grouped as follows:
(a) Batch cultures,

(b) Continuous cultures, and
(c) Immobilized cell cultures.
(a). Batch Culture

Batch culture is a type of cell suspension culture that is grown in a fixed volume of
nutrient culture medium. Here, the cell suspension increases in biomass by cell division
and cell growth until a factor in the culture medium becomes limiting and then the growth
ceases.
The cells in batch culture exhibits a typical sigmoidal curve with the following five
phases of a growth cycle (Fig.).
i. Lag phase, where the cells prepare to divide.
ii. Exponential or log phase, where the rate of cell division is the highest.
iii. Linear phase, where cell division slows down but the rate of cell 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.
The lag phase duration depends mainly on inoculum size and growth phase of the
culture from which the inoculum is taken. The log phase lasts about 3-4 cell generations (a
cell generation is the time taken for doubling of cell number), and the duration of a cell
generation may vary from 22-48 hr, depending mainly on the plant species. The stationary
phase is forced on the culture by depletion of the nutrients and possibly due to an
accumulation of cellular wastes. If the culture is kept in stationary phase for a prolonged
period, the cells may die.
Batch cultures are maintained by sub-culturing at weekly intervals. The exact time
and dilution required must be determined for each cell line. Dilutions of 1:4 after one week
or 1:10 after two weeks are commonly used. It is recommended that a small sample should
be withdrawn to determine the cell density before subculturing.
Batch cultures are unsuitable for studies on cell growth and metabolism as there is a
constant change in cell density and nutritional status of the medium. But batch cultures
are much more convenient than continuous cultures and, hence are routinely used.
(b). Continuous Culture
In a continuous culture, the cell population is maintained in a steady state by

regularly replacing a portion of the used or spent medium by fresh medium. Continuous
cultures are of two types : (1) Closed type or (2) Open type.
In a closed continuous culture, the cells which are separated while the used medium
is taken out for replacement are added back to the culture so that cell biomass keeps on
increasing. In open continuous cultures, both cells and the used medium are taken out and
replaced by equal volume of fresh medium. The replacement volume is so adjusted that the
cultures remain at submaximal growth indefinitely.
The open cultures are of either turbidostat or chemostat types. In a turbidostat, cells
are allowed to grow upto a preselected turbidity (usually, measured as OD) when a
predetermined volume of the culture is replaced by fresh normal culture medium. But in a
chemostat, a chosen nutrient is kept in a concentration so that it is depleted very rapidly to
become growth limiting, while other nutrients are still in concentrations higher than
required. In such a situation, any addition of the growth-limiting nutrient is reflected in cell
growth. Chemostats are ideal for the determination of effects of individual nutrients on cell
growth and metabolism.
(c). Immobilized Cell Culture
Plant cells and cell groups may be encapsulated in a suitable material, e.g., agarose
and calcium alginate gels, or entrapped in membranes or stainless steel screens. The gel
beads containing cells may be packed in a suitable column or, alternatively, cells may be
packed in a column of a membrane or wire cloth.
Liquid medium is continuously run through the column to provide nutrients and
aeration to cells. Immobilization of cells changes their cellular physiology in comparison to
suspension culture cells. The advantages of immobilized cell reactors are: i). No risk of cell
washout ii) protection of cells from liquid shear by protective covering iii) low contamination
iv) better control on cell aggregate size v) regular removal of cellular wastes.
SUBCULTURE
After a period of time, it becomes necessary to transfer organs and tissues to fresh
media chiefly due to nutrient depletion and medium drying. This is particularly true of
tissue and cell cultures where a portion of tissue is used to inoculate new culture tubes or
flasks; this is known as sub-culturing. In general, callus cultures are sub-cultured every 4-
6 weeks, while suspension cultures need to be sub-cultured every 3-14 days. Plant cell and
tissue cultures may be maintained indefinitely
by serial sub-culturing.
In case of suspension cultures, sub-culturing

should be done about or somewhat prior to
the time of their maximum growth. The
inoculums volume should be 20-25% of the
fresh medium volume; in any case, the initial
cell density of the fresh culture (just after
inoculation) should be around 5 x 104 cells
m1-1 or higher otherwise the cells may fail to
divide.
Estimation of growth
Cell number is the most informative measure of cell growth. This measurement is
applicable to only suspension cultures, and even their cell aggregates must be treated, e.g.,
with pectinase, to dissociate them into single cells before counting the cell number in a
haemocytometer. Therefore, cell number is estimated only where information obtained
justifies the efforts.
In contrast, packed cell volume of suspension cultures is easily determined by

pipetting a known volume into a 15 ml graduated centrifuge tube, spinning at 2000 × g for 5
min and reading the volume of cell pellet, which is expressed as ml pellet/ml of culture.
Culture fresh and dry weights are the most commonly used measures of growth of
both suspension and callus cultures.
In case of callus cultures, the cell mass is placed on a preweighed dry filter paper or
nylon filter and weighed to determine fresh weight. Cells from suspension cultures are
filtered onto a filter paper or nylon filter, washed with distilled water, excess water removed
under vacuum and weighed along with the filter; the filter is preweighed in wet condition.
For dry weight determination, the cells and the filter are dried in an oven at 60°C for
12 hr and weighed; the filter is pre-weighed in dry condition. Cell fresh and dry weights may
either be expressed as per ml (suspension culture) or per culture.
OVARY CULTURE
Culture of unfertilized ovaries to obtain haploid plants from egg cells or other
haploid cell of the embryo sac is called ovary culture and this process of haploid production
is termed as gynogenesis. The first report of gynogenesis was by San Noem Lu 1976 in
case of barley subsequently haploid plants were obtained from ovary / ovule cultures of
rice, wheat, maize, sunflower, sugar beet, tobacco etc.
Application : ovary culture is often used for in vitro pollination and fertilization and for
embryo rescue when embryo culture and ovule culture fail (or) not feasible due to their
very small size.
ENDOSPERM CULTURE
Culture of endosperm to regenerate whole plants under in vitro condition is termed as

endosperm culture. Endosperm is formed in most cases by the fusion of two polar nuclei with
one of the male gamete. It is the main source of reserve food for the developing embryo.
Endosperm is generally short lived-structure and is consumed during the development of
embryo (exalbuminous seed). In plants like Castor, it exists as a massive tissue even in the
meture seed (albuminous seed). Seedless triploid plants can be produced by endosperm
culture. The first attempt on endosperm culture was made by La Duel (1949). He reported
growing tissue of young immature maize endosperm.
Applications of Endosperm culture
1) Endosperm culture technique is applied to economically important cultivars for

raising superior triploid plants
2) Triploid plants are seed sterile which can be exploited for crop improvement. Eg:-
Apple, Banana, Mulberry, Sugarbeet, Peach, Watermelon etc, which
are commercially important for their edible parts.
3) In some cases triploids are superior in quality than diploid. Eg:- Triploids of
populus have better quality pulpwood.
4) To exploit in the biosynthesis of some natural products. Eg:- Cultured endosperm
of coffee synthesizes caffeine. The level of this alkaloid in callus is synthesized by
three times after two weeks and by 6 times after 4-5 weeks
5) Various trisomics developed from triploids may also be useful in gene
mapping for cytogenetic studies
6) Endosperm can be used as a nurse tissue for raising hybrid embryos. Eg:- using
Hardeum endosperm as a nurse tissue the young embryos of hybrid between
Hardeum x Triticum Hardeum x Cicale Hardeum x Agropyron
These can be induced to germinate and form normal hybrid plants
Limitations
1. Triploid production through endosperm culture technique has been successful only in
a limited number of species. In majority of species mature endosperm proliferation
resulted in a callus tissue of unlimited growth. But the induction of organogenesis in
endosperm culture has always being a challenging problem.
2. In cereals (or) crops where grains (or) seeds are used, triploids are undesirable.
SOMACLONAL VARIATION
Variations that arise through the cell and tissue culture process under in vitro
condition are termed as somaclonal variations. The plants derived from such cell and
tissue cultures are termed as ‘somaclones', and the plants displaying variation as
‘somaclonal variants'. Variants obtained using callus cultures are referred as “Calliclones”
(Skirvin, 1978) while variants obtained using protoplast cultures are known as
“Protoclones” (Shepard et al. 1980).
Evans et al. (1984) suggested the term ‘gametoclonal variation' for those variations
arising in cell cultures of gametic origin like, in pollen and microspores cultures, to
distinguish them from somatic cell derived regenerants.
The basic cause of these variations may be attributed to changes in karyotype
(chromosome number and structure), chromosome rearrangements, somatic crossing over,
sister chromatid exchange, DNA amplification and deletion, transposable elements and DNA
methylation.
Somaclonal variation can be characterized based on morphological, biochemical
(isozymes) and DNA markers such as, Random Amplified Polymorphic DNA (RAPDs),
Restriction Fragment Length Polymorphism (RFLPs) and Inter-Simple Sequence Repeats
(ISSR).
Steps in creation of somaclonal variation
i. Growth of callus or cell suspension cultures for several cycles.
ii. Regeneration of a large number of plants from such long term cultures.
iii. Screening for desirable traits in the regenerated plants and their progenies. For
example, in vitro selection to select agronomically desirable somaclones for tolerance to
various biotic and abiotic stresses, herbicides, high salt concentration and extremes of
temperature.
iv. Testing of selected variants in subsequent generations for desirable traits.
v. Multiplication of stable variants to develop new breeding lines.
Applications of Somaclonal Variations

i. Variability generated at the genetic level proves to be a source for crop improvement
ii. Distinctive mutations may give rise to elite characters in the regenerants which cannot
be achieved by conventional methods of breeding.
iii. Disease resistant genotypes of various plants can be attained. Resistance was first
reported in sugarcane for eye spot disease (Heliminthosporium sacchari) and Fiji virus
disease by regenerating plants from callus of susceptible clones.
iv. Plants with characteristic resistance to abiotic stress (cold, draught, acidic or alkaline
soil) can be obtained as somaclones.
v. Somatic genome exchange may give rise to regenerants where a part of alien genome can
be introgressed thereby leading to germplasm widening.
Limitations of Somaclonal variations

i. Poor plant regeneration from long-term cultures of various cell lines.
ii. Regeneration being limited to specific genotypes which may not be of much interest to
breeders.
iii. Some somaclones have undesirable features, such as aneuploidy, sterility etc.
iv. Unpredictable variations that are often generated are of no use.
v. Variations attained may not always be stably integrated.
PROTOPLAST CULTURE
A plant cell without its cell wall is known as a protoplast. It is called as a naked plant
cell because the cell wall has been removed either by a mechanical or an enzymatic method.
Protoplast can be isolated from almost all plant parts viz. root, leave fruits, tuber,
endosperm, pollen etc. Protoplast culture refers to the aseptic isolation and in vitro culture
of protoplast to obtain viable
plants.
Methods of protoplast isolation:
1. Mechanical method
In this method large &
highly vacuolated cells (eg. onion
bulbs, scales, radish root & beet
root tissue) are plasmolysed in an
osmotic solution, causing the
protoplast to shrink away from
the cell wall.
2. Enzymatic method
In enzymatic method, the
isolated cells are macerated with
macro enzyme (Pectinase) in 13%
mannitol. Pectinase mainly
degrades the middle lamella while
cellulose are required to digest the
cell wall. The cells are purified by
filtration through nylon mesh.
Then the cells are incubated in
2% cellulose for about 90 min.
After the digestion of cell
wall the isolated protoplast is
subject to osmotic stress. If an
osmotic stabilizing agent is not Fig. Steps in Protoplast culture
included in the medium the
isolated protoplast would take in water by the process of osmosis & would eventually burst
as there is no cell wall to constrain the cell.
PROTOPLAST CULTURE
Fully developed leaves are initially processed with effective surface sterilant (dipping into
70% ethanol for 1 min. then in 2% sodium hypochloride for 20-30 min.) and the lower
epidermis is removed with a pair of fine forceps then it is cut into small pieces and
incubated in a plasmolytic solution for a certain period. After a specific hour of enzymatic
treatment with enzyme mixture containing cellulose, pectinase, protease and lipase, the
digested mixture containing sub-cellular debris, undigested cell, broken protoplast &
healthy protoplast has to purified by filtration and washing.
Isolated protoplasts are usually cultured in either liquid or semi-solid agar media
plates. Protoplasts are sometimes allowed to regenerate the cell wall in liquid media before
they are transferred to any agar media. Protoplast in culture generally starts to regenerate a
cell wall with a few hours after isolation & may take several days to complete the process
under suitable condition. After 3-4 weeks, small cell colonies will be visible. Colonies will
reach approximately 1 mm in diameter within 5-6 weeks. Once small colonies have formed,
these are transferred to an osmotic free medium to develop callus. The callus then
undergoes organogenic or embryogenic differentiation leading to the formation of plants.
PROTOPLAST FUSION
It refers to the fusion of protoplasts of two different genomes followed by the selection
of desired somatic hybrid cells and regeneration of hybrid plants. To achieve induced fusion,
a suitable chemical agent (fusogen) like, NaNO3, high Ca2+, polyethylene glycol (PEG), or
electric stimulus is needed.
i. Fusion by means of NaNO3: Kuster in 1909 showed that hypotonic solution of
NaNO3 induces fusion of isolated protoplast forming heterokaryon (hybrid).
ii. High pH and Ca++ treatment: It was demonstrated by. In this technique the
isolated protoplasts from two plant species are incubated in 0.4 M mannitol solution
containing high Ca++(50 mM CaCl2.2H2O) with highly alkaline pH of 10.5 at 37°C for about
30 min. Aggregation of protoplasts takes place at once and fusion occurs within 10 min.
iii. Polyethylene glycol treatment: Polyethylene glycol (PEG) is the most popularly
known fusogen due to ability of forming high frequency, binucleate heterokaryons with low
cytotoxicity. The freshly isolated protoplasts from two selected parents are mixed in
appropriate proportions and treated with 15-45% PEG (1500-6000MW) solution for 15-30
min followed by gradual washing of the protoplasts to remove PEG. Protoplast fusion occurs
during washing. The washing medium may be alkaline (pH 9-10) and contain a high Ca++
ion concentration (50 mM). This combined approach of PEG and Ca ++ is much more efficient
than the either of the treatment alone.
Importance of protoplast culture

1. Two or more protoplasts can be induced to fuse & then fusion product carefully
nurtured to produce a hybrid plant. In some cases, hybrids that can not be produced
by conventional plant genetics because of sexual or physiological incompatibility can
be produced by protoplast fusion.
2. After removal of cell wall the isolated protoplast is capable of ingesting foreign
material into the cytoplasm by a process similar to endocytosis as described for certain
animal cells & protozoans.
3. The cultured protoplast rapidly regenerates a new cell wall & this developmental
process offers a novel system for the study of wall biosynthesis & deposition.
4. Population of protoplasts can be studied as a single cellular system that is their

manipulation is similar to that of microorganisms.
CYBRIDS OR CYTOPLASMIC HYBRIDS
Somatic hybrids containing nuclear genome of one parent but cytoplasm from both
the parents, are termed as cybrids. Production of somatic hybrids involves the following
steps namely,
(i) Islolation of protoplasts from two different speices
(ii) Fusion of protoplasts from two different species
(iii) Isolation of fused protoplasts, and
(iv) Regeneration of fertile hybrid plants from the fused protoplasts.
Applications of somatic hybridization

1. Novel interspecific and intergeneric
crosses which are difficult to produce by
conventional methods can be easily
obtained.
2. Important characters, such as resistance

to diseases, ability to undergo abiotic stress
and other quality characters, can be
obtained in hybrid plant by the fusion of
protoplasts of plant bearing particular
character to the other plant which may be
susceptible to diseases.
3. Protoplasts of sexually sterile haploid,

triploid, aneuploid plants can be fused to
obtain fertile diploids and polyploids.
4. Most of the agronomically important

traits, such as cytoplasmic male sterility,
antibiotic resistance and herbicide resistance, are cytoplasmically encoded, hence can be
easily transferred to other plant.
5. Plants in juvenile stage can also be hybridized by means of somatic hybridization.
6. Somatic hybridization can be used as a method for the production of autotetraploids.

Limitations of somatic hybridization

1. Application of protoplast methodology requires efficient plant regeneration system from
isolated protoplasts. Protoplasts from two species can be fused, however, production of
somatic hybrids is not easy.
2. The end product of somatic hybridization are often unbalanced (sterile, misformed and
unstable)
3. Somatic hybridization of two diploids leads to formation of amphidiploids which is
unfavorable.
4. It is not sure for a character to completely express after somatic hybridization.
SYNTHETIC SEEDS / ARTIFICIAL SEEDS
Somatic embryo (embryoids), shoot buds or any other plant material obtained as a
result of in vitro culture are covered (encapsulated) with a chemical membrane. Such
encapsulated materials behave as seeds. These are called artificial seeds or synthetic seeds.
The artificial covering acts as an artificial seed coat. Such seeds are bead like and can
“germinate” and plantlets are also formed.
Several substances are used as artificial seed coats. Some of them are agar, agarose,
carrageenin, polyacrylamide, introcellulose, ethyl cellulose and sodium alginate. Sodium
alginate is most commonly used.
Advantages of Artificial Seeds

1. The size of the artificial seeds is smaller when compared to the natural seeds of the plant.
2. Storage and transportation of such seeds is easier.
3. Viability of seeds is 100%.
4. Artificial seeds can be made to germinate uniformly on a suitable substratum.
5. Such seeds do not show dormant.
6. The plant grower can grow the desired plant any time and this is not season dependent.
7. Large scale production of seeds from any kind of plant part is possible.
Disadvantages
1. The artificial seeds cannot be stored for longer time and it is temperature
dependent.
2. The initial cost for the production of artificial seed is more than that for the natural
seeds.
3. Production and germination of artificial seeds require aseptic conditions. Any
deviation will affect the quality of the seeds and their subsequent development.
IN VITRO GERMPLASM CONSERVATION

Storage of plant species to make them available for future breeding programmes is
called as germplasm storage. Usually, seeds are used for storage. But it has the following
limitations:
1. The seeds may lose viability with the passage of time
2. The seeds may be damaged by seed borne pathogens
3. This method cannot be effectively used for vegetatively propagated crops.
Storage of cells or tissues in liquid nitrogen at -196°C in a frozen state for very long
period of time is called as cryopreservation or in vitro germplasm conservation. When there
is a need, whole plants can be regenerated from the tissues in cryopreservation.
In general the tissues are stored in liquid nitrogen at – 196°C. Certain substances are
added to the culture media before freezing. These protect the tissues against ice damage.
Such substances are called cryoprotectants, e.g., Glycerol, Proline, Mannitol, Sucrose,
Glucose, Polyethylene glycol. Cryopreserved tissues can be recultured to produce whole
plants or they can be sub-cultured. The viability of cryopreserved tissues depends upon the
plant species.
NATIONAL CERTIFICATION SYSTEM FOR TISSUE CULTURED PLANTS
(NCS-TCP)
The Tissue Culture Certification Agency (DBT) is responsible for implementing the
National Certification System for Tissue Culture raised Plants (NCS-TCP) in the Country. A
NCS-TCP Management Cell (NMC) has been setup for assisting DBT in Accreditation of Test
Laboratories for testing of Virus and Genetic Fidelity/ Uniformity and also Recognition of
Tissue Culture Production Facilities, based on the established guidelines and criteria.
Referral Laboratories have been identified for carrying out confirmatory tests, if required,
and also for developing standard protocols, validating protocol and diagnostic reagents,
maintenance of referral material, training of technical personal working at accredited test
laboratories (ATLs), providing diagnostic reagents to ATLs etc. The Certification Agency is
overall responsible for developing standard tests, production protocols/guidelines and
manuals.
National Certification System for Tissue Culture raised Plants (NCS-TCP) in India
is comprised of the following agencies:
• Tissue Culture Certification Agency (TCCA)
• NCS-TCP Management Cell (NMC)
• Accreditation Panel (AP)
• Referral Laboratory (RL)
• Biotechnology (NRCPB),
• Accredited Test Laboratories (ATLs)
• Recognized Tissue Culture Production Facility
• Appellate Authority (AA)
NCS-TCP Management Cell (NMC):

NMC has been established for assisting DBT in implementation of NCS-TCP in
country. It is also responsible for Accreditation of Test laboratories for virus diagnosis and
genetic fidelity/ uniformity testing of tissue culture raised plants and Recognition of Tissue
Culture Production Facilities.
Accreditation Panel (AP):

The panel of experts undertake assessment of test laboratories for virus diagnosis and
genetic fidelity/ uniformity testing for Accreditation and periodical auditing. The panel
comprises experts specialized in plant tissue culture/plant biotechnology/plant
virology/plant bacteriology/ molecular biology / phytosanitary.
Referral Laboratory (RL):

The DBT has designated Referral laboratories for virus diagnosis/genetic fidelity testing
of tissue cultures plants.
• Referral Center for Virus Diagnosis – Indian Agriculture Research Institute (IARI),
New Delhi
• Referral Centers for Genetic Fidelity/ Uniformity – National Research Center on
Plant Biotechnology (NRCPB), New Delhi
The Referral Laboratory is responsible for carrying out confirmatory tests in the
event of dispute or nonconformity of test results, developing standard protocols, validating
protocol and diagnostic reagents, maintenance of referral material, training of technical
personal working at accredited test laboratories (ATLs), providing diagnostic reagents to
ATLs.
Accredited Test Laboratories (ATLs):

Test laboratories are accredited entities, responsible for testing the Tissue Culture
material for Virus diagnosis and Genetic fidelity/ uniformity, for the purpose of
certification. The Test laboratory prepares a Test Report. Based on the Test Report, each
Accredited Test Laboratory (ATLs) is authorized to issue the Certificate of Quality for the
Tissue Culture Plant (CQ-TCP) along with certification label on behalf of the Tissue Culture
Certification Agency. ATLs are responsible for maintaining/ procuring all diagnostic kits,
primer, probes etc required for routine testing. Each ATL would perform both tests-for
virus diagnosis and true-to-type.
Recognized Tissue Culture Production Facility:

Commercial Tissue Culture Production Facility with minimum production capacity
of 0.5 million plants per annum may get recognition for a period of 2 years after assessed
by the Accreditation Panel. All the activities of tissue culture production facility including
hardening facility needs to be operational at the time of assessment by the AP. Recognized
Tissue Culture Production Facility should adopt Standard Operating Procedure (SOP) and
maintain all relevant records.
Appellate Authority (AA):

An Appellate Authority under the Chairpersonship of Secretary, DBT established to
review the decision taken with regard to Accreditation of Test laboratories, Recognition of
Tissue Culture production facilities and also for Certification of tissue culture material. The
Nodal Officer designated by the Tissue Culture Certification Agency will act as Member
Secretary. The members represented in the appellate panel will include:
 Chairman - Secretary/Additional Secretary, DBT or his nominee

 Not less than two Co-opted non-officio experts in the area of Virus Indexing and
 Genetic fidelity/ uniformity Testing and or expertise in the field concerned
Representative from Ministry of Agriculture, Govt. of India
 Nodal Officer designated by the Certification Agency ofNCS-TCP would act as Member
Secretary.
National Certification System for Tissue Culture Plants in India

THE STRUCTURE OF DNA

The structure of DNA can be studied in three levels as primary, secondary and tertiary
structure. The primary structure refers to the structure of nucleotide and how they are joined
together. The secondary structure refers to the double helical structure proposed by Watson
and Crick. The tertiary arrangement refers to the complex packing of the double stranded DNA
in chromosomes.
I. PRIMARY STRUCTURE OF DNA
Nucleic acids (DNA and RNA) are polymers made up of repeating units of
nucleotides. A nucleotide consists of three basic components: 1. Pentose sugar, 2.
Nitrogenous base, and 3. Phosphate group.
1. PENTOSE SUGAR
The sugar is a cyclic five-carbon

structure. It contains a hydroxyl group
attached to 2’-carbon atom and called as
ribose in RNA. In DNA, the sugar lacks
oxygen atom and hence called deoxyribose.
This minor chemical difference is recognized
by all the cellular enzymes that interact with
DNA or RNA, thus yielding specific functions Fig. The chemical structure of sugars. RNA has
for each nucleic acid. Further, the
ribose and DNA has deoxy ribose sugar.
additional oxygen atom in the RNA
nucleotide makes it more reactive and less
chemically stable than DNA.
The carbon atoms are numbered as 1' (called one prime), 2', 3', 4' and 5' in order to
differentiate them from the carbon atoms in the DNA and RNA bases. The 5' and 3' carbons
of the pentoses forms the phosphodiester linkage, while the l' carbon is always occupied by
an organic base.
2. NITROGENOUS BASES
There are two kinds of bases: purines and pyrimidines.

Purines are double ringed structure with a six-sided ring
attached to a five-sided ring whereas, a pyrimidine consists of a
six-sided ring only. There are two purines, adenine (A) and
guanine (G), and three pyrimidines, cytosine (C), thymine (T),
and uracil (U). Thymine occurs only in DNA, while uracil occurs
only in RNA. The letters A, C, T, G, are usually referred to as the
alphabets of life.
Fig. . Structure of purine and pyrimidines bases. The carbon atoms are assigned
unprimed numbers
When a base is linked to a sugar, the product is called a nucleoside. A nucleoside

linked to a phosphate is called as a nucleotide (Nucleotide = Nucleoside + Phosphate) (Fig.
). In a nucleotide, the nitrogenous base always forms a covalent bond with the 1’-carbon
atom of the sugar.
3. PHOSPHATE GROUP
The third component of a nucleotide is the phosphate group, which consists of a

phosphorus atom bonded to four oxygen atoms. Phosphate groups are found in every
nucleotide and frequently carry a negative charge, which makes DNA acidic. The phosphate
is always bonded to the 5-carbon atom of the sugar in a nucleotide.
The DNA nucleotides are properly known as deoxyribonucleotides or

deoxyribonucleoside 5-monophosphates. Because there are four types of bases,there are
four different kinds of DNA nucleotides. The equivalent RNA nucleotides are termed
ribonucleotides or ribonucleoside 5-monophosphates.
POLYNUCLEOTIDES
Two nucleotides are linked by a phosphodiester group i.e. the 5’-phosphate group of
one nucleotide joins to the 3’-carbon atom of the next nucleotide. These bonds, called
phosphodiester linkages, are relatively strong covalent bonds. Shorter chains (consisting
of less than 20 nucleotides) are called oligonucleotides while longer chains are called
polynucleotides.
An important characteristic of the polynucleotide strand is its direction or polarity. At

one end of the strand a phosphate group is attached only to the 5’-carbon atom of the sugar
in
the
Fig. The four types of DNA nucleotides
nucleotide. This end of the strand is therefore referred to as the 5’ end. The other end of the
strand, referred to as the 3’ end, has an OH group attached to the 3-carbon atom of the
sugar.
RNA nucleotides also are connected by phosphodiester linkages to form similar

polynucleotide strands.
II. SECONDARY STRUCTURE OF DNA
The secondary structure refers to the double helical

structure of DNA proposed by Watson and Crick at Cambridge.
The X-ray diffraction analysis of Rosalind Franklin and

Maurice Wilkins revealed that the DNA was a double helix with
a width of 2 nm. The purine and pyrimidines bases were
stacked 0.34 nm apart in a ladder. Since the width of the helix
is 2 nm it can accommodate only two strands. Also, Erwin
Chargaff found that A=T and G=C and that the number of
purine bases (A + G) is equal to the number of pyrimidine bases
(C + T).
Using the Chargaff’s data and X ray diffraction studies

Watson and Crick, built the molecular model of DNA using
metal wires. Watson and Crick along with Maurice Wilkins,
were awarded a Nobel Prize in 1962.
Fig. Watson & Crick’s
The key features about the double helical structure of double helical structure
of DNA
DNA molecule are as follows:
1. The DNA double helix (DNAdh) consists of two polynucleotide chains coiled around a
central axis in a spiral fashion.
2. The polynucleotide chains are antiparallel; one chain runs in the 5’ to 3’ orientation and
the other in 3’ to 5’ direction. This anti-parallel orientation of the two strands is essential
for the formation of hydrogen bonds between the pairs of DNA bases.
3. The two bases in each base pair lie in the same plane which is perpendicular to the axis
of the helix. Neighbouring bases lie 3.4 A apart. There are 10 base pairs per helical turn
i.e. the helix repeats itself at an interval of 34 A.
4. The helix has two kinds of alternating external grooves: a deep groove (called the major
groove) and a shallow groove (called the minor groove).
5. The nitrogenous bases on one strand pair with

those on the other strand in complementary
fashion (A always pairs with T, while G pairs with
C). The most common natural form of DNA is a
right-handed ouble helix of diameter 2.0nm, called
the B-DNA. DNA can also assume other forms like
Z DNA, A DNA.
In addition to these features described above,

certain implications deserve emphasis:
1. Complementary base pairing means that

the replicate of each strand is given the base sequence
of its complementary strand when DNA replicates.
2. Because the strands are antiparallel, when

two nucleotides are paired, the sugar portions of these
molecules lie in opposite directions (one upward and
the other downward along the chain) (Fig. ).
3. Because the strands are antiparallel, the

convention for writing the sequence of bases in a
strand is to start from the 5’ P terminus at the left
(e.g., GAC refers to a trinucleotide 5’ -P-GAC-3’ -OH).
Fig. DNA consisting of two
4. The conventional way of expressing the base polynucleotide chains running
composition of an organism is by the percentage of [G] antiparallel to each other &
+ [C]. This value is approximately 50% for most Composed of complementary
strands. RNA has single nucleotide
eukaryotes with only minor variations among species. chain.
In simpler organisms, there are significant variations
(e.g., 27% for Clostridium, 50% for Escherichia coli, and 76% for Sarcina, all of these
organisms being bacteria).
5 The chains of the double helix are held together by hydrogen bonds between base
pairs in opposite strands. The bond between A and T is a double bond, while the bond
between G and C is a triple hydrogen bond.
The DNA molecule satisfies the requirement of genetic material in the following ways:
1. It can replicate itself accurately during cell growth and division.
2. Its structure is sufficiently stable so that heritable charges i.e., mutations
can occur only very rarely.
3. It has a potential to carry all kinds of necessary biological information.
4. It transmits all the biological information to the daughter cells.
Thus the essential functions of DNA are the storage and transmission of genetic
information and the expression of this information in the form of synthesis of cellular
proteins.
Denaturation: The hydrogen bonds between the DNA strands break on heating
the DNA to high temperature (nearly 100 oC). The process of separation of DNA
strands is known as denaturation.
Renaturation: Reunion of the separated or denatured DNA strands on cooling is

called renaturation or annealing. The optimum temperature for renaturation is 20 –
25 oC.
DNA REPLICATION
The process by which a DNA molecule makes identical copies of itself is called as
DNA replication.
THREE MODELS OF DNA REPLICATION
1. Dispersive: In dispersive mode of replication, the old DNA molecule would break into
several pieces, each fragment would replicate and the old and new segments would
recombine randomly to yield the progeny DNA molecule. Each progeny molecule would have
both old and new segments along its length.
2. Conservative: According to conservative scheme, after

replication the two newly synthesized strands would form a
double helix, while the two old parental strands would form
another double helix.
3. Semi conservative: In the semi conservative model of DNA

replication, one of the two parental DNA strand serves as a
template for the synthesis of new, complementary daughter
strand. Each progeny DNA molecule would consist of one old
and one newly synthesized strand. As one of the parental strand
is conserved in the new daughter DNA molecule it is called as
semi conservative model and is the universally accepted model.
EVIDENCE FOR SEMICONSERVATIVE REPLICATION
The experimental evidence for the semiconservative

method of replication was provided by Meselson and Stahl
(1958). They cultured E.Coli in a medium containing radioactive
15N. and labeled its DNA with 15N. This radioactively labelled
E.coli was transferred to a medium containing normal nitrogen,

14N, and allowed to divide. Fig. Semiconservative
The DNA of newly formed daughter cells contained one model of replication
DNA strand labelled with 15N and the other strand labelled with
14N. This showed that each progeny DNA molecule would consist of one old and one newly
synthesized strand.
REQUIREMENTS OF REPLICATION
Although the process of replication includes many components, they can be

combined into three major groups:
1. A template consisting of single-stranded DNA,
2. Substrates for new nucleotide strand, and
3. Enzymes and other proteins that read the template and assemble the substrates
into a DNA molecule.
Table - Major components required for DNA replication in bacteria
Sl. COMPONENTS FUNCTION

No
1. Initiator protein Initiates replication by binding to origin and unwinds
DNA strands
2. DNA helicase Unwinds DNA at the replication fork
3. Single-stranded Attach to single stranded DNA and prevent reannealing
binding proteins (re-joining)
4. DNA gyrase Moves before replication fork, cutting and resealing
breaks in the DNA to release the torque developed in it
due to unwinding of DNA
5. DNA primase Synthesizes short RNA primers
6. DNA polymerase III Elongates a new nucleotide strand
7. DNA polymerase I Replaces RNA primers with DNA
8. DNA ligase Joins Okazaki fragments by sealing sugar-PO4 backbone
MAJOR STEPS IN DNA REPLICATION
1). Replication begins when an initiator protein binds to origin of replication and unwinds a
short stretch of DNA, to which DNA helicase attaches.
2). DNA helicase then unwinds the DNA at the replication fork and single-strand-binding
proteins bind to single nucleotide strands to prevent them from reannealing.
3). DNA gyrase (a topoisomerase) removes the strain ahead of the replication fork that is
generated by unwinding.
4). Primase synthesizes short primers of RNA nucleotides, providing a 3’-OH group to which
DNA polymerase can add DNA nucleotides.
5). DNA polymerase adds new nucleotides to the 3’ end of a growing polynucleotide strand.
6). The leading strand is continuously synthesized.
7). The other strand, which is synthesized discontinuously in short pieces is called as
lagging strand or Okazaki
fragments.
8). The Okazaki fragments are joined
together by DNA ligase. In bacteria,
Okazaki fragments are ~1,000 to
2,000 nucleotides long. In
eukaryotic cells, they are 150 to 200
nucleotides long.
9). DNA ligase seals the nicks that
remain in the sugar-phosphate
backbones when the RNA primers are
replaced by DNA nucleotides.
The high accuracy of DNA replication is maintained by (1) Base selection by the
polymerase, (2) Proofreading by exonuclease from 3’ to 5’ activity and (3) Repair systems
which replaces mismatches in replication.
EUKARYOTIC DNA REPLICATION
Although not as well understood, eukaryotic replication resembles bacterial

replication in many respects. The most important differences are that eukaryotes have: (1)
Multiple origins of replication in their chromosomes; (2) Different types of DNA polymerases,
with different functions; and (3) Assembly of nucleosome immediately after DNA replication.
Precise replication at multiple origins is ensured by a licensing factor that must attach to an
origin before replication can begin. The licensing factor is removed after replication is
initiated and renewed after cell division. The ends of linear eukaryotic DNA molecules are
replicated by the enzyme telomerase.
THE GENETIC CODE

A sequence of three nucleotides in mRNA, codes for an amino acid. This triplet is
referred to as codon. For example, the mRNA sequence AUG codes for the aminoacid
methionine. The set of all the codons that specify the 20 amino acids is termed as the
genetic code.
The set of bases in tRNA which base pair with a codon of mRNA is known as anti-
codon. The sequence of bases in an anti-codon is exactly the opposite of that present in the
codon.
There are 20 amino acids involved in protein synthesis and there are only four bases
(A,T,G,C) in the DNA coding for all the amino acids. Thus the 4 bases when arranged in the
form of triplet code (43) can generate 64 codons. Of these, three codons, UAA, UAG and UGA
do not code for any amino acid and serve as stop codons (nonsense codons or termination
codons). One codon, AUG serves as initiation or start codon as it starts the synthesis of
polypeptide chain. This codon also codes for amino acid methionine. In eukaryotes, the
starting amino acid is methionine, while in prokaryotes it is N-formyl methionine.
It is proved that a sequence of

three nucleotides in the mRNA codes for an
amino acid (a triplet code or codon),
and the code is non-overlapping and
commaless (Fig.). A commaless code
means that all the bases in a
polynucleotide are parts of codons and
that no base serves as a punctuation
mark.
The genetic code is said to be

degenerate because nearly all amino
acids are specified by at least two
codons. Some (serine, arginine, leucine)
are encoded by six different codons.
Further, for a set of codons encoding the
same amino acid, the first two letters in
the figure are the same, with only the
third being different (called the wobble Fig. The genetic code
hypothesis).
CENTRAL DOGMA OF MOLECULAR BIOLOGY

The flow of genetic information from DNA to RNA by the process of transcription and
from RNA to proteins by the process of translation was formalized by Francis Crick in a
concept called the central dogma of molecular biology.
It was now found that

transfer of information from RNA to
DNA, (via reverse transcription) and the
transfer of information from RNA to
RNA (by RNA replication) also
occurs.
This flow of information can also

be reversed. Thus, once a protein is
known, the nucleotide sequence in the
prescribing DNA strand can be Fig. The central dogma of molecular biology
determined and synthesized. The
product is called a complementary DNA or cDNA.
TRANSCRIPTION
(mRNA SYNTHESIS)
The process of copying the genetic information of the DNA to mRNA (messenger RNA) is
called as transcription. The DNA strand which is transcribed is called as the template strand
or antisense strand and the other strand is called as sense strand. The mRNA thus
produced is complementary to the template stand and identical to the sense strand.
Major Steps in Transcription

(i) The process of transcription is
carried out by enzymes called RNA
polymerases which unwinds a
portion of DNA near a special DNA
sequence called promoter region.
(ii) Transcription always starts
with the start codon AUG. As it
codes for the amino acid
methionine, all proteins begin with
methionine.
Fig. Transcription
(iii) The end of the polypeptide
sequence is indicated by the stop
codons UAA, UAG, or UGA.
(iv) The transcription always
proceeds from 5’ to 3’ direction.
(v) The newly synthesized
mRNA remaining inside the
nucleus is called hn-RNA
(heterogeneous nuclear RNA).
(vi) This hnRNA is processed
addition of methyl guanosine cap
at the 5’ end and by addition of
several adenines (poly –A tail) at
3’ end. This capping and addition of poly A-tail protects the mRNA from degradation by
enzymes.
(vii) This molecule undergoes severe alterations to remove non-coding parts called introns,
leaving only the coding parts or exons to produce the mRNA. This mRNA is about 25% of the
original length.
(viii) The mRNA is then transported from the nucleus to the ribosomes in the cytoplasm
for protein synthesis.
TRANSLATION
(PROTEIN SYNTHESIS)
The process by which the nucleotides sequence present in the mRNA is translated into
aminoacid sequence is called as translation.
Ribosomes are the sites of protein

synthesis. Translation requires mRNA,
rRNA, ribosomes, 20 kinds of aminoacids
and their specific tRNAs and many
translation factors. The process of
translation (protein synthesis) consists of
five major steps viz.,
(1) Activation of aminoacids

(2)Transfer of activated aminoacids to
tRNA
(3) Chain initiation
(4) Chain elongation and
(5) Chain termination.
1. Activation of aminoacid
The mRNA moving out of the

nucleus binds to ribosome in the
cytoplasm. Ribosomes are the site for
protein synthesis. The amino acids in the
cytoplasm, are activated in the presence
of ATP. Then the enzyme amino acyl
synthetase links with amino acid ~
AMP to form amino acyl adenylate
enzyme complex. This complex is called
activated amino acid.
Amino acid + ATP Fig. Steps in Protein synthesis
AA~AMP + PP
AA~AMP + Amino acyl synthetase AA~AMP - Enzyme

2. Transfer of activated aminoacid to tRNA
When an activated aminoacid collides with a specific tRNA, it binds with the A
site (aminoacid attachment site) of tRNA, forming aminoacyl tRNA. The aminoacyl
tRNA then moves towards the ribosome.
3. Chain initiation
Translation begins when an aminoacylated tRNA base pairs with the start codon
(AUG) present in the mRNA located in the smaller subunit of ribosome. The initiation codon
and the small sub unit forms the initiation complex to which the larger subunit joins.
4. Chain elongation
The ribosome has two distinct sites namely, A-site (acceptor or aminoacyl attachment
site) and P-site (peptidyl site).
Each new aminoacyl-tRNA enters the ribosome and attaches to A-site. The mRNA codon of
A-site determines which charged tRNA with aminoacid will attach next. As soon as the next
tRNA attaches at the A-site, a peptide bond is formed between the aminoacid (-COOH) on
the A-site and the polypeptide (-NH2) on the P-site. The peptide bond formation is
catalysed by the enzyme peptidyl transferase.
After the formation of peptide bond, the tRNA from P-site is released to the cytosol
and the polypeptide chain is transferred to tRNA on A-site. Then, the tRNA on A-site is
shifted to P-site, making A-site available for new tRNA. Then the ribosome complex moves
one codon towards the 3’ end on the mRNA, releasing the first tRNA from initiation point to
pick another methionine. The free initiation point can now form a new initiation complex.
During protein synthesis a number of ribosomes are attached to a single mRNA molecule,
each forming a different polypeptide chain. The complex thus formed is known as
polyribosome. The process is repeated until the whole mRNA is translated, and adjacent
amino acids are linked by peptide bonds.
5. Chain termination
The translation will proceed until a releasing factor binds to the stop codon (UAA,
UAG, UGA) and terminates translation, as they does not code for any aminoacid. The
interval between the start and stop codons is called the open reading frame (ORF).
The ribosome releases the polypeptide and mRNA and subsequently dissociates into
two subunits. Further processing of polypeptide chain into proteins and enzymes is done in
the cytoplasm itself depending on the bonding properties of the aminoacids in it. Most
of the mRNA molecules are unstable and degraded after the release of polypeptide chain,
but some mRNAs such as those coding for hemoglobin may be stable.
Differences between transcription in prokaryotes and eukaryotes
Transcription and translation in prokaryote and eukaryotes
Prokaryotes have only exons and no introns, so the translation can begin even
before transcription of the mRNA is complete. But, this is not possible in eukaryotes as the
mRNA must be processed to remove the introns and the mRNA must leave the nucleus.
INTRON refers to the non-coding region of the eukaryotic genes that are transcribed into
mRNA. They are removed by splicing of RNA.
EXON refers to the region of DNA that codes for a protein. In eukaryotes, the exons are
separated by many introns.
REGULATION OF GENE EXPRESSION

IN PROKARYOTES
Regulation of gene expression refers to the turning on and turning off of the genes at
appropriate time and to the desired level. It is important for proper growth and development.
The gene expression may be regulated during transcription, mRNA processing, translation,
and involve posttranslational modifications also.
An Operon is a group of structural genes whose transcription is regulated by the

action of a regulator gene ‘r’, a promoter gene ‘p’ and an operator gene ‘o’.
REGULATION OF OPERON
There are two basic

categories of gene regulation:
negative and positive.
1. In negative regulation, an
inhibitor which is bound to the
DNA must be removed for transcription to occur. In this case the transcription is normally
on and must be turned off. It is called as repressible operon.
2. In positive regulation, an activator has to bind to the DNA for transcription to occur.
In this case, the transcription is normally off and must be turned on. It is called as
inducible operon.
THE LAC OPERON OF E. COLI
The Operon concept was proposed by Jacob and Monod in 1961. The lac operon E.
coli bacterium consists of three structural genes of namely,
a). lacZ which codes for beta galactosidase,
b). lacY which codes for the enyme permease that allows the diffusion of lactose into
bacterial cell and
c). lacA which codes for the enzyme transacetylase.
Mechanism of lac operon when lactose is absent
When E. coli is cultured in a medium devoid of lactose, the enzymes for lactose
catabolism are not required and hence the lac-operon has to be switched off or repressed.
Under such conditions, the regulator gene produces a repressor protein which binds
to the operator site. Therefore, RNA polymerase cannot bind to the promoter and
transcription is stopped.
Mechanism of lac operon when lactose is present
When lactose is present in the medium, some of it is converted into allolactose which
binds to the repressor, making it inactive.
Therefore, RNA polymerase binds to the promoter and hence, the transcription of
lacZ, lacY and lacA takes place and lac enzymes are produced for the catabolism of lactose.
1. In the absence of lactose, the regulator protein (a repressor) binds to the operator and inhibits
transcription. 2. When lactose is present, some of it is converted into allolactose which binds to the
regulatory protein (repressor), making it inactive. Hence, structural genes are transcribed and translated.
REGULATION OF GENE EXPRESSION

IN EUKARYOTES
There are three major methods by which eukaryotic cells are known to regulate
translation by:
a) Altering the life of the mRNA,
b) Controlling the initiation of translation, and
C) Changing the overall rate of translation.
A typical eucaryotic mature mRNA consists of four major regions: A) a 5' noncoding
region (leader), B) a coding region, C) a 3' noncoding region (trailer), and D) a poly-A tail.
Each of the four segments may affect the half-life of mRNA molecules.
Eukaryotic genes contain introns (noncoding regions) interspersed among the coding
regions (exons). Part of the process that converts primary transcripts to complete mRNA
molecules involves removal of the introns and splicing the exons together. Variations in the
excision and splicing jobs can lead to different mRNAs and, following translation, to
different protein products.
Eukaryotic gene regulation is less well understood than bacterial regulation, partly
owing to the larger genomes in eukaryotes, their greater sequence complexity, and the
difficulty of isolating and manipulating mutations that can be used in the study of gene
regulation. Nevertheless, great advances in our understanding of the regulation of
eukaryotic genes have been made in recent years, and eukaryotic regulation continues to be
one of the cutting-edge areas of research in genetics.
Comparison between Eukaryotic and Prokaryotic gene regulation
Many features of gene regulation are common to both bacterial and eukaryotic cells. For
example, in both types of cells, DNA-binding proteins influence the ability of RNA
polymerase to initiate transcription. However, there are also some differences.
First, eukaryotic genes are not organized into operons and are rarely transcribed
together into a single mRNA molecule; instead, each structural gene typically has its own
promoter and is transcribed separately.
Second, chromatin structure affects gene expression in eukaryotic cells; DNA must
unwind from the histone proteins before transcription can take place.
Third, although both repressors and activators function in eukaryotic and bacterial gene
regulation, activators seem to be more common in eukaryotic cells.
Finally, the regulation of gene expression in eukaryotic cells is characterized by a
greater diversity of mechanisms that act at different points in the transfer of information
from DNA to protein.
POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is an enzymatic, in vitro method for rapid
amplification or multiplication of specific DNA. It was developed in 1984 by Karry Mullis
(Nobel prize, 1993). It is now a basic tool for molecular biology works. In this process, DNA
is heated to separate the two strands, short primers attach to the target DNA, and DNA
polymerase synthesizes new DNA strands from the primers. Each cycle of PCR doubles the
amount of DNA.
Requirements for PCR

1. A target DNA (100-35,000bp)
2. Primers complementary to flanking region of target DNA
3. dNTPs (Deoxyribonucleotides dATP, dCTP, dGTP, dTTP)
4. Taq polymerase from Thermus acquaticus which is stable at 950C
PCR consists of three steps:

(i) Denaturation: The reaction
mixture is heated to 950C for
a short time period (about
15-30 sec) to denature the
target DNA into single
strands that can act as
templates for DNA synthesis.
(ii)Primer annealing /
renaturation: The mixture
is rapidly cooled to 55 to
650C which allows the two
primers to bind to the
sequences on each of the two POLYMERASE CHAIN REACTION
strands flanking the target
DNA. This process is called annealing. One primer binds to each strand. The two
parental strands do not reanneal with each other because the primers are in large excess
over parental DNA.
(iii) Elongation: The temperature of the mixture is raised to 720 c (usually) and kept at this
temperature for a pre-set period of time to allow DNA polymerase to elongate each primer
by adding the dNTPs added in the mixture. Thus at the end of this incubation, both
single-stranded template strands have been made partially double stranded. The new
strand of each double-stranded DNA extends for a variable distance downstream.
The three steps of the PCR

cycle are repeated. Thus in the
second cycle, the four strands
denature, bind primers and are
extended. No other reactants
need to be added. The three
steps are repeated again and
again. By third cycle, the DNA
sequences between the two
primer sites alone are extended.
After 20 cycles, the original DNA has
been amplified a million-fold and
this rises to a billion-fold (1000)
million after 30 cycles.
Automated thermocyclers are now
routinely used to get a billion-fold
amplification of the target DNA
sequence (30 cycles) in less than one
hour.
As the three steps—
denaturation, primer annealing
and primer extension—are
carried out repetitively just by
changing temperature of the
reaction mixture PCR is also
called as a thermocycler.
Applications of PCR
PCR already has very
widespread applications, and new
uses are being devised on a
regular basis. Some of the
applications of PCR are as
follows:
i. PCR can amplify a single
DNA molecule from a complex
mixture, largely avoiding the
need to use DNA cloning to
prepare that molecule. Also, it is
now possible to amplify a POLYMERASE CHAIN REACTION
specific single RNA molecule
from a complex mixture.
ii. PCR is used for DNA sequencing.
iii. By using suitable primers, it is possible to use PCR to create point mutations,
deletions and insertions of target DNA which greatly facilities the analysis of gene
expression and function.
iv. Using appropriate primers, very small amounts of specified bacteria and viruses can
be detected in tissues, making PCR invaluable for medical diagnosis.
v. PCR is now invaluable for characterizing medically important DNA samples. For
example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs.
vi. Because of its extreme sensitivity, PCR is now fundamentally important to forensic
medicine. It is even possible to use PCR to amplify the DNA from a single human hair or a
microscopic drop of blood left at the scene of a crime to allow detailed characterization.
DNA LIBRARY
One method of finding a gene is to create and screen a DNA library. A genomic library
is created by cutting genomic DNA into overlapping fragments and cloning each fragment in
a separate bacterial cell. A cDNA library is created from mRNA that is
converted into cDNA and cloned in bacteria.
GENOMIC LIBRARY :
It refers to a large collection of

bacterial cells, each containing a random
piece of human genomic DNA. For
constructing a gene library, the entire DNA of
cell is cleaved into small pieces by using
different REs. All these cut fragments are
then introduced into appropriate vectors.
This forms a large collection of different
recombinant clones, which are then
introduced into the host bacterial cells to form the gene library. All the genes of an
organism are represented in the gene library. In order to produce a complete gene library
for E. coli, about 1500 fragments are required, whereas about 10 lakhs fragments are
required for human gene library. Genomic libraries contain all the DNA of an organism,
and cDNA libraries contain only expressed DNA.
cDNA Library:
In a genomic library, all the
genomic material is represented
but only about 3% of the cloned
DNA codes for proteins. If a
collection of only expressed DNAs
were to be made, then RNA and
not DNA would be the starting
point. cDNA library is a collection
of all the expressed DNA of a
particular cell type or tissue. For
example, a cDNA from pancreatic
_-cell contains clones with cDNA
for proinsulin. On the other and, a
cDNA library from bone marrow
cell contains many clones with
cDNA for and chains of
haemoglobin. Thus, for a cDNA
library the tissue of origin is important. For a genomic library, the tissue of origin is
unimportant because the genomic material is same in all cell types of an organism. Building
of cDNA library: The mRNA is extracted from a specifi c tissue. It is used as a template for
synthesis of complementary DNA strand; the enzyme catalyzing this synthesis is reverse
transcriptase, which yields a single stranded cDNA. Double-stranded cDNA is then obtained
from it by adding the DNA polymerase. The latter is incorporated in plasmid, λ phage or
cosmid and introduced into host bacterial cell.
SCREENING OF GENOMIC LIBRARY:

It is possible to screen the genomic library and obtain a gene of interest from it.
By the use of molecular markers. Then the identified DNA fragments can be inserted into
cloning vectors. Cloning vectors must have an origin of replication, one or more unique
restriction sites, and selectable markers. Plasmids are commonly used as cloning vectors.
SCREENING DNA LIBRARY TO IDENTIFY CLONED GENE BY COLONY HYBRIDIZATION
Libraries, once created, can be stored as permanent reference resources (which is

why they’re called libraries). Libraries are often used in the effort to isolate specific genes.
There are two ways to find the specific clone within the library that contains a DNA
fragment that includes a gene of interest. One is to look for a specific sequence of base pairs
(if known). The other is to look for the specific protein encoded by the
gene. In either case, the process is called screening.
There are many techniques, depending on the vector used and the gene being sought.
Bacterial colonies or phage plaques are transferred from an agar-filled Petri
dish to a solid disk, usually made of nitrocellulose, by the simple method of laying the disk
on the agar, then lifting it up again. This doesn’t remove the colonies from the agar, but it
does transfer enough material to the nitrocellulose for analysis.
Next, the DNA on the nitrocellulose disk is unwound by immersing the disk
in a chemical solution. Then the disk is placed in a solution that contains a single
strand of DNA or RNA tagged with a radioactive atom complementary to one of
the strands in the clone. This tagged single strand is called a probe. Wherever the probe
joins up with one of the recombinant strands, it leaves a radioactive spot on the disk which
can be registered on photographic film, and directly correlated to a location on the original
agar disk.
If the DNA sequence of the gene being sought is unknown, but a protein the
gene codes for is known, another screening method is to create clones using vectors cells
begin producing the protein the gene encodes, and researchers can then search the library
for the protein in question.
Once a desired clone is located, it can be picked off the agar plate with a needle
and allowed to multiply freely. The recombinant DNA can be chemically purified from the
cells for use in the laboratory, and the clone that produces it can be stored and regrown as
needed. This provides genetic engineers with an endless supply of a particular gene for
insertion into other organisms.
Fig. Use of antibiotic resistance in creation of genomic library for

easy selection of transformed cells
Fig. Colony hybridization for screening of clones to identify gene of interest

NUCLEIC ACID BLOTTING TECHNIQUES
Blotting techniques are very widely used analytical tools for the specific identification
of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the
process of immobilization of sample nucleic acids on solid support (nitrocellulose or nylon
membranes). The blotted nucleic acids are then used as targets in the hybridization
experiments for their specific detection. An outline of the nucleic acid blotting technique is
depicted in fig.
Types of blotting techniques

The most commonly used blotting techniques are listed below
(i) Southern blotting (for DNA)
(ii) Northern blotting (for RNA)
(iii) Dot blotting (DNA/RNA)
(iv) Western blotting (for proteins)
SOUTHERN BLOTTING
Southern blotting techniques is the first nucleic acid blotting procedure developed in
1975 by Southern. The genomic DNA isolated from cells/tissues is digested with one or
more restriction enzymes. This mixture is loaded into a well in an agarose or
polyacrylamide get and then subjected to electrophoresis. DNA, being negatively charged
migrates towards the anode (positively charged electrode); smaller DNA fragments move
faster.
The separated DNA molecules are denatured by exposure to a mild alkali and
transferred to nitrocellulose or nylon paper. This results in an exact replica of the pattern of
DNA fragments on the gel. The DNA can be annealed to the paper on exposure to heat
(800C). The nitrocellulose or nylon paper is then exposed to labeled cDNA probes. These
probes hybridize with complementary DNA molecules on the paper.
The paper after thorough washing is exposed to x-ray film to develop autoradiograph.
This reveals specific bands corresponding to the DNA fragments recognized by cDNA probe.
This procedure is called as Southern blotting.
Applications of Southern blotting

Southern blotting technique is extremely specific and sensitive, although it is a
simple technique. Some of the applications are listed.
(i) It is an invaluable method in gene analysis.
(ii) Important for the confirmation of DNA cloning results.
(iii) Forensically applied to detect minute quantities of DNA (to identify parenthood, thieves,
rapists etc.).
(iv) Highly useful for the determination of restriction fragment length polymorphism (RFLP)
associated with pathological conditions.
SOUTHERN BLOTTING
NORTHERN BLOTTING
Northern blotting is the technique for the specific identification of RNA molecules.
The procedure adopted is almost similar to that described for Southern blotting. RNA
molecules are subjected to electrophoresis, followed by blot transfer, hybridization and
autoradiography.
RNA molecules do not easily bind to nitrocellulose paper or nylon membranes. Blot-
transfer of RNA molecules is carried out by using a chemically reactive paper prepared by
diazobenzyloxymethyl (DBM) paper. The RNA can covalently bind to DBM paper.
Northern blotting is theoretically, a good technique for determining the number of
genes (through mRNA) present on a given DNA. But this is not really practicable since each
gene may give rise to two or more RNA transcripts. Another drawback is the presence of
exons and introns.
DOT-BLOTTING
Dot-blotting is a modification of Southern and Northern blotting techniques described
above. In this approach, the nucleic acids (DNA or RNA) are directly spotted onto the filters,
and not subjected to electrophoresis. The hybridization procedure is the same as in original
blotting techniques. Dot-blotting technique is particularly useful in obtaining quantitative
data for the evaluation of gene expression.
WESTERN BLOTTING
Western blotting can be used for detection of one or more antigens in a
mixture. The sample is electrophoresed on an SDS- polyacrylamide gel (SDS-PAGE)
that separates the proteins on the basis of size, resulting in a series of protein bands
down the gel. It is very useful to understand the nucleic acid functions, particularly
during the course of gene manipulations. The technique of western blotting involves the
transfer of electrophoresed protein bands from polyacrylamide gel to nylon or nitrocellulose
membrane.
These proteins can be detected by specific protein-ligand interactions. The western
blot is then reacted with labeled antibody, unreacted antibody is washed away and those
protein bands that have bound the antibody become visible and hence are identified. The
method of visualization depends on how the antibody was labeled. If it is labelled by
radioactive probe (e.g. 125I), then autoradiography is carried out to detect the radioactive
protein bands. Alternatively, the antibody may be detected by incubating the sheet with a
second antibody that recognizes the first antibody (e.g. if the first specific antibody was
raised in rabbits, the second antibody could be a goat anti-rabbit antibody). The second
antibody could be radiolabeled and its binding detected by autoradiography or it could be
conjugated to an enzyme that generates a colored product as in ELISA.
Western blotting can also be used to analyze specific antigens after two-dimensional
gel electrophoresis which resolves proteins as spots, separated on the basis of both charge
and size.
WESTERN BLOTTING
Autoradiography
Autoradiography is the
process of localization and
recording of a radiolabel
within a solid specimen,
with the production of an
image in a photographic
emulsion. These
emulsions are composed of
silver halide crystals
suspended in gelatin.
When a β-
particle or a γ-ray from a
radiolabel passes through
the emulsions, silver ions
are converted to metallic
silver atoms. This results
in the development of a
visible image which can be
easily detected.
COMPARISON OF BLOTTING TECHNIQUES

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)
The human genome contains hundreds of variations in base sequences that do not
affect the phenotype. The property of the molecules to exist in more than one form is known
as polymorphism. Difference in mutation and DNA polymorphism: If more
than 1% of the population has a particular alteration in the sequence, it is polymorphism. If
only a few individuals have it, then it is mutation. Polymorphism is normal variation, and
generally having no deleterious effect. Mutation is abnormal,
and sometimes will have defective function, e.g., phenylketonuria. A polymorphic gene is
one, in which the variant alleles are common in more than 1% of the total population. The
existence of two or more types of restriction fragment patterns is called restriction fragment
length polymorphism (RFLP). This can be used as a genetic marker. DNA is treated with
restriction enzymes, which cleave DNA into fragments of defined lengths. Then
electrophoresis is done in agarose gels, when the fragments are separated. Finally, the DNA
from the agarose gel is transferred on to nitrocellulose
paper (Southern blotting) and hybridized with labelled probe sequences. Genotypic changes
can be recognised by the altered restriction fragments.
A RFLP represents a stretch of DNA that serves as a marker for mapping a specified
gene. RFLPs are located randomly throughout a person’s chromosomes and have no
apparent function.
A DNA molecule can be cut into different fragments by a group of enzymes called
restriction endonucleases (see table). These fragments are called polymorphisms (literally
means many forms).
An outline of RFLP is depicted in fig. The DNA molecule 1 has three restriction sites
(R1, R2, R3), and when cleaved by restriction endonucleases forms 4 fragments. Let us now
consider DNA 2 with an inherited mutation (or a genetic change) that has altered some base
pairs. As a result, the site (R2) for the recognition by restriction endonuclease is lost. This
DNA molecule 2 when cut by restriction endonuclease forms only 3 fragments (instead of 4
in DNA 1).
As is evident from the above description, a stretch of DNA exists in fragments of
various lengths (polymorphisms), derived by the action of restriction enzymes, hence the
name restriction fragment length polymorphisms.
RFLPs in the diagnosis of diseases
If the RFLP lies within or even close to the locus of a gene that causes a particular
disease, it is possible to trace the defective gene by the analysis of RFLP in DNA. The
person’s cellular DNA is isolated and treated with restriction enzymes. The DNA fragments
so obtained are separated by electrophoresis. The RFLP patterns of the disease suspected
individuals can be compared with that of normal people (preferably with the relatives in the
same family). By this approach, it is possible to determine whether the individual has the
marker RFLP and the disease gene. With 95% certainity, RFLPs can detect single gene-
based diseases.
Methods of RFLP scoring

Two methods are in common use for the detection of RFLPs.
(i) Southern hybridization:
The DNA is digested with appropriate restriction enzyme, and separated by agarose gel
electrophoresis. The so obtained DNA fragments are transferred to a nylon membrane.
A DNA probe that spans the suspected restriction site is now added, and the hybridized
bands are detected by autoradiograph. If the restriction site is absent, then only a single
restriction fragment is detected. If the site is present, then two fragments are detected
(fig).
(ii) Polymerase chain reaction:
RFLPs can also be scored by PCR. For this purpose, PCR primers that can anneal on
either side of the
suspected
restriction site are
used. After
amplification by
PCR, the DNA
molecules are
treated with
restriction
enzyme and then
analysed by
agarose gel
electrophoresis. If
the restriction site
is absent only one
band is seen,
while two bands are
found if the site is
found (fig).
Applications of
RFLPs:
The approach by
RFLP is very
powerful and has
helped many Restriction Fragment Length Polymorphism
genes to be
mapped on the chromosomes. E.g. sickle-cell anemia (chromosome 11), cystic fibrosis
(chromosome 7), Huntington’s disease (chromosome 4), retinoblastoma (chromosome
13), Alzheimer’s disease (chromosome 21).
MICROSATELLITES/ SIMPLE SEQUENCE REPEATS

Microsatellites are short repeat units (10-30 copies) usually composed of
dinucleotide or tetranucleotide units. These simple tandem repeats (STRs) are more popular
than minisatellites (VNTRs) as DNA markers for two reasons.
1. Microsatellites are evenly distributed throughout the genome.
2. PCR can be effectively and conveniently used to identify the length of
polymorphism.
Two variants (alleles) of DNA molecules with 5 and 10 repeating units of a dimer
nucleotides(GA) are depicted in fig. By use of PCR, the region surrounding the
microsatellites is amplified, separated by agarose gel electrophoresis are identified.
SINGLE NUCLEOTIDE POLMORPHISMS (SNPs):
SNPs represent the positions in the genome where some individuals have one
nucleotide (e.g. G) while others have a different nucleotide (e.g. C). There are large numbers
of SNPs in genomes. It is estimated that the human genome contains at least 3 million
SNPs. Some of these SNPs may give rise to RFLPs.
SNPs are highly useful as DNA markers since there is no need for gel
electrophoresis and this saves a lot of time and labour. The detection of SNPs is based on
the oligonucleotide hybridization analysis (fig).
An oligonucleotide is a short single-stranded DNA molecule, synthesized in the
laboratory with a length not usually exceeding 50 nucleotides. Under appropriate
conditions, this nucleotide sequence will hybridize with a target DNA strand if both have
completely base paired structure. Even a single mismatch in base pair will not allow the
hybridization to occur.
DNA chip technology is most commonly used to screen SNPs hybridization with
oligonucleotide.
CURRENT TECHNOLOGY OF DNA FINGERPRINTING
In the forensic analysis of DNA, the original techniques based on RFLPs and
VNTRs are now largely replaced by microsatellites (short tandem repeats). The basic
principle involves the amplification of microsatellites by polymerase chain reaction followed
by their detection.
It is now possible to generate a DNA profile by automated DNA detection system
(comparable to the DNA sequencing equipment).
DNA FINGERPRINTING / DNA PROFILING /DNA TYPING
The use of DNA sequences to identify a person is called as DNA fingerprinting. It is a

powerful tool for criminal investigations and other forensic applications. The technique was
discovered in England by Alec Jeffreys. The basic principles of DNA fingerprinting are briefly
described.
A sample of DNA from blood, semen, hair, or other body tissue is collected from the
crime scene. If the sample is very small, PCR can be used to amplify it so that enough DNA
is available for testing. Additional DNA samples are collected from one or more suspects.
Each DNA sample is cut with one or more restriction enzymes, and the resulting DNA
fragments are separated by gel electrophoresis. The fragments in the gel are denatured and
transferred to nitrocellulose paper by Southern blotting. One or more radioactive probes is
then hybridized to the nitrocellulose and detected by autoradiography. The pattern of
bands produced by DNA from the sample collected at the crime scene is then compared with
the patterns produced by DNA from the suspects. A match between the sample from the
crime scene and one from the suspect can provide evidence that the suspect was present at
the scene of the crime.
The probes most commonly used in DNA fingerprinting are complementary to short
sequences repeated in tandem that are widely found in the human genome. People vary
greatly in the number of copies of these repeats; thus, these polymorphisms are termed
variable number of tandem repeats (VNTRs).
The structure of each person’s genome is unique. The only exception being
monozygotic identical twins. The unique nature of genome structure provides a good
opportunity for the specific identification of an individual. Thus DNA fingerprinting is an
analysis of the base sequence of DNA in an individual.
Applications of DNA fingerprinting:

The amount of DNA required for DNA fingerprint is remarkably small. The minute
quantities of DNA from blood strains, body fluids, hair fiber or skin fragments are enough.
Polymerase chain reaction is used to amplify this DNA for use in fingerprinting. DNA
profiling has wide range of applications-most of them related to medical forensics. Some
important ones are listed below.
(i) Identification of criminals, rapists, thieves etc.
(ii) Settlement of paternity disputes.
(iii) Use in immigration test cases and disputes.
(iv) Identification of crop varieties in agriculture.
DNA FINGERPRINTING
MARKER ASSISTED SELECTION (MAS)
Selection of genotypes carrying desirable gene using linked-markers is called as

marker assisted selection. In conventional breeding, breeders cross two different parents
and select desirable genotypes in the F2 generation by visual evaluation. The visual marker
traits (eg. Black coloured stigma, purple stripes in leaves)that help in selection are called
morphological markers. When biochemical characters (eg. Proline content) are considered for
selection that character is called as biochemical marker. Both morphological and
biochemical markers differ according to the environment. This defect can be overcome with
the use of molecular markers.
Genetic markers are simply landmarks on chromosomes that serve as reference
points to the location of other genes of interest when a genetic map is constructed. The
markers that are currently widely used include RFLP, AFLP (amplified fragment length
polymorphism), SNPs (single nucleotide polymorphisms), and microsatellites or simple
sequence repeats (SSRs).
CLASSIFICATION OF MOLECULAR MARKERS:
1 Single-locus, multiallelic,
codominant markers.
Examples are RFLPs and
microsatellites (SSRs). Microsatellites
are capable of detecting higher levels
of polymorphisms than RFLPs.
2 Multilocus, single-allelic,
dominant markers.
Examples are AFLPs and RAPD
(random amplified polymorphic DNA).
POSITIVE AND NEGATIVE SELECTABLE MARKERS

 Positive selectable markers are selectable markers that confer selective advantage to
the host organism. An example would be antibiotic resistance, which allows the host
organism to survive antibiotic selection.
 Negative selectable markers are selectable markers that eliminate or inhibit growth
of the host organism upon selection.[6]An example would be thymidine kinase, which
makes the host sensitive to ganciclovir selection.
A distinction can be made between selectable markers (which eliminate certain
genotypes from the population) and screenable markers (which cause certain genotypes to
be readily identifiable, at which point the experimenter must "score" or evaluate the
population and act to retain the preferred genotypes). Most MAS uses screenable markers
rather than selectable markers.
PROPERTIES OF AN IDEAL MARKER:

 Easy recognition of all possible phenotypes (homo- and heterozygotes) from all
different alleles
 Demonstrates measurable differences in expression between trait types or gene of
interest alleles, early in the development of the organism
 Testing for the marker does not have variable success depending on the allele at the
marker locus or the allele at the target locus (the gene of interest that determines the
trait of interest).
 Low or null interaction among the markers allowing the use of many at the same time
in a segregating population
 Abundant in number
 Polymorphic
MARKER ASSISTED SELECTION

Broadly, marker assisted selection (MAS) can be divided into two categories, that of
marker-assisted backcrossing or introgression and that of marker-assisted recurrent
selection or population improvement. In the former, the goal is to incorporate one or few
major genes or QTL into elite breeding lines (or in some situations, a breeding population).
The second case involves using markers to improve the overall genetic value of a population
with respect to some trait or suite of traits. Of the two, marker-assisted backcrossing,
particularly of a single gene, is the easiest to put into practice; strategies to use markers in
recurrent selection are still being developed and the best strategy for a given situation is not
clear at the current time.
FOREGROUND SELECTION VS. BACKGROUND SELECTION

Foreground selection refers to using markers that are tightly linked to the gene of
interest in order to select for the target allele or gene. Background selection refers to using
markers that are not tightly linked to the gene of interest in order to select against other
DNA from the donor parent (i.e., to select for recurrent parent alleles at other loci than the
target).
STEPS IN MARKER ASSISTED SELECTION
Generally the first step is to map the gene or quantitative trait locus (QTL) of interest
first by using different techniques and then use this information for marker assisted
selection. The markers to be used should be close to gene of interest (<5 recombination unit
or cM). Use of two markers reduce the chances of an error due to homologous
recombination. For example, if two flanking markers are used at same time with an interval
between them of approximately 20cM, there is higher probability (99%) for recovery of the
target gene.
The basic procedure for conducting MAS with DNA markers is as follows:
a. Extract DNA from tissue of each individual or family in a population.
b. Screen DNA samples via PCR for the molecular marker (SSR, SNP, SCAR, etc.) linked
to the trait of interest.
c. Separate and score PCR products, using an appropriate separation and detection
technique.
d. Identify individuals exhibiting the desired marker allele.
e. Combine the marker results with other selection criteria (e.g., phenotypic data or
other marker results), select the best fraction of the population, and advance those
individuals in the breeding program.
MARKER ASSISTED BACKCROSSING OF QUANTITATIVE TRAITS

Marker assisted backcrossing uses DNA markers, which can be scored as a dominant
or codominant trait prior to flowering, to facilitate the backcrossing program, saving time if
progeny testing would need to be conducted and saving resources if phenotyping is difficult.
Markers can be used to select for the gene being introgressed into the recurrent parent and
to select against undesirable donor DNA. Markers enable the pyramiding of resistance
genes; that is, they enable the incorporation of alleles at multiple loci each of which confers
resistance to the same race of pathogen. This is difficult to do traditionally because one
locus masks the presence of the others.
The general method is as follows:
Season 1: Cross two lines or genotypes to produce an F1.
Season 2: Generate ≥100 BC1 individuals, select against clearly undesirable types but
otherwise backcross all to the recurrent parent.
Season 3-4: Generate ≥200 BC2 individuals (say 2-3 plants per BC1), select against
clearly undesirable types but otherwise self pollinate for one or two generations to develop
BC2F2 and BC2F3 families for phenotypic evaluation. Develop a genetic map of the
BC2F1 individuals; use the marker and phenotypic data to localize QTL controlling the
trait(s) of interest.
Season 5: Identify BC2F2 lines (families) that are segregating for these QTL and
continue backcrossing until a desired level of recurrent parent is reached followed by self-
fertilization to recover homozyous plants for the introgressed QTL region. Ideally, the
breeder could develop nearly isogenic lines for each QTL – called “QTL-NIL” – using markers
to narrow the introgressed region and to recover the recurrent parent region.
The goal of this process is to develop a set of lines each carrying a single desirable
QTL allele introgressed from the donor. With this set of lines, the effect of each QTL on the
phenotype can be assessed. Later, after evaluating individual QTL, crosses among NILs
carrying different QTL can be made to pyramid multiple QTL for the given trait into a single
line.
Advantages of molecular markers:
i. The desirable segregants can be scored at the seedling stage itself
ii. It is possible to screen for difficult and expensive characters such as tolerance to
drought, flood, salinity, mineral deficiency, toxicity, pest resistance, disease resistance etc.
iii. Selection can be practiced for several traits.
iv. Heterozygotes and homozygotes are easily identified without resorting for progeny
testing.
v. It speeds up breeding cycle by saving enormously on time.
vi. Space required for screening is very low.
Disadvantages of MAS
Currently, one of the most important barriers for MAS is the prohibitive
cost. Although there are only a small number of reports analyzing the economics of MAS
versus conventional breeding in the literature, the cost-effectiveness of using MAS compared
to conventional plant breeding varies considerably between studies.
Two additional factors need to be considered for cost-analysis:
(1) the equipment and consumables required to establish and maintain a marker lab
is considerable; and
(2) there is a large initial cost in the development of markers which is seldom
reported.
For marker assisted backcrossing, the initial cost of using markers would be more
expensive compared to conventional breeding in the short term however time savings could
lead to an accelerated variety release which could translate into greater profits in the
medium to long term.
Another important factor obstructing the successful application of markers for line
development is the low reliability of markers to determine phenotype. This is often
attributable to the thoroughness of the primary QTL mapping study. Even QTLs that are
detected with high LOD scores and explain a large proportion of the phenotype may be
affected by sampling bias (especially in small populations), and therefore may not be useful
for MAS. Furthermore, the effect of a QTL may depend on the genetic background. This
emphasizes the importance of testing the QTL effects and the reliability of markers (i.e.
QTL/marker validation) before MAS is undertaken.
Finally the level of integration between molecular geneticists and plant breeders (and
scientists from other disciplines) may not be adequate to ensure that markers are effectively
applied for line development.
RECOMBINANT DNA TECHNOLOGY
Genetic recombination which refers to the exchange of genetic information between

two individual organisms occurs in nature. When a gene (DNA fragment) of one species is
transferred to another organism artificially it is called recombinant DNA technology/genetic
engineering. Boyer and Cohen (1973) were the first to successfully recombine two plasmids
(pSC 101 and pSC 102) and clone the recombinant plasmid in E. coli. This marked the
beginning of modern rDNA technology. DNA manipulation is made possible by three key
factors namely, the availability of restriction enzymes that cut DNA at specific sequences,
the discovery of cloning vectors which carry the transgene into a host and the annealing
(rejoining) properties of nucleic acids,.
BASIC PRINCIPLES OF DNA CLONING USING rDNA TECHNOLOGY

The basic principles of involved in DNA cloning are as follows:
1. Generation of DNA fragments by restriction enzymes and selection of the desired
fragment of gene.
2. Insertion of the selected DNA fragment into a cloning vector to create a recombinant
DNA.
3. Introduction of the recombinant vectors into the host cells (eg. bacteria).
4. Multiplication and selection of clones containing the transgene.
5. Expression of the gene to produce the desired product.
OBJECTIVES OF RECOMBINANT DNA TECHNOLOGY

1. To obtain a large number of copies of specific DNA fragments
2. To recover large quantities of the protein production by the concerned gene
3. To integrate gene into the chromosome of target organism where it expresses itself.
Many enzymes which are involved in rDNA technology and their functions are given below.
Enzymes Name of the enzyme
1. Cleaving (cutting) DNA Nucleases
a) Endonucleases b) Exonucleases
S1 Nucleases
DNA ases
2. Joining the DNA fragments DNA ligases
3. Synthesis of DNA DNA polymerase I
Terminal transferase
Reverse transcriptase
Enzymes that modify ends of DNA Alkaline phosphatase and Kinase
molecule
Enzyme that degrade RNA RNA ases.
The various aspects involved in the recombinant DNA technology listed below are are
described in the following chapters:
 Restriction endonucleases (DNA cutting enzymes)

 Cloning vectors (carriers)
 DNA amplification and cloning in the host
 Methods of gene transfer
 Molecular diagnostic techniques (Hybridization and blotting techniques).
DNA Cloning by using rDNA technology

I. RESTRICTION ENDONUCLEASES / DNA CUTTING ENZYMES
Restriction enzymes are bacterial enzymes which cleaves (cuts) the DNA at specific
sites. Werner Arber discovered that certain enzymes protect the E. coli bacterium by cutting
and destroying the invading viral DNA. The enzymes that restrict the viral replication are
called as restriction enzymes eg. Eco RI. More than 800 enzymes are commercially
available now.
Nomenclature of Restriction enzymes

Restriction enzymes have a three letter name with the first letter indicating the genus
name, followed by the first two letters of the species name followed by the strain of the
organism with a Roman numeral indicating the order of discovery. For example, Eco RI is
derived from Escherichia coli, strain Ry 13 and the first endonuclease (I) to be discovered.
There are three categories of restriction endonuclease:

1. Type I enzymes : They have different subunits for recognition, modification, and
restriction or cleavage. The cleavage site is located more than 1,000 base pairs away from
the recognition site. As a consequence, cleavage does not occur at a specific sequence even
though certain regions are preferentially cleaved. It is not possible to define the recognition
sites by characterizing the broken ends of the DNA.
2. Type II enzymes: It occur in about one in three bacterial strains. The enzymes are
highly specific in action, as they are involved in only one act of restriction. The recognition
sites or sequences are usually short (4 to 6 bp) and often palindromic. They cleave at or
close to the target site and require no ATP for restriction. Some enzymes produce blunt ends
while others produce staggered cuts (or sticky ends). The type II enzymes are the
workhorses of recombinant DNA technology.
3. Type III enzymes: They have two subunits, one for recognition and methylation and the
other for restriction. Like type I, type III restriction sites consist of assymetrical sequences
that may be 5 to 7 bp long. Cleavage occurs some 24 to 26 bp downstream from the
recognition site.
Whereas all three types of enzymes previously described are proteins with a catalytic
effect, there is a unique class of non-protein enzymes called ribozymes. These are RNA
enzymes with the capacity for cleaving specific phosphodiester bonds. Recognition
sequences
Restriction enzymes recognize specific palindromic sequences in the double stranded
DNA which are 4-6 nucleotides long, and then cut both the strands of DNA at specific
locations. These sequences are called as recognition sequences.
Cleavage patterns
Most REs cleave recognition sequence at one or two base pairs away from the center
on both strands of DNA. This results in a double-stranded DNA with short, single-stranded
ends called sticky ends/cohesive ends. Examples Eco RI and Taq I. Other REs cleave the
recognition sequence without any overhanging nucleotides resulting in DNA with blunt ends
/ flush ends eg. Hae III or Hpa I. The DNA fragments with sticky ends can join easily with
other DNA fragments. The blunt ends can be joined with the help of adaptors. Adaptors are
short, chemically synthesized DNA double strands which can be used to link the ends of two
DNA molecules that have different sequences at their ends.
Restriction maps
Any double stranded DNA can be cut by a variety of restriction enzymes. After
separating the restriction fragments by gel electrophoresis and measuring their size, the site
where each restriction enzyme cleaved can be found out. A map showing the position of cut
sites for a variety of restriction enzyme is called the restriction map for that DNA fragment.
Restriction maps allows comparison between DNA molecules without the need to determine
the nucleotide sequence.
DNA LIGASES / DNA JOINING ENZYMES

The cut DNA fragments are joined together by DNA ligases. DNA ligase joins the DNA
fragments by forming phosphodiester bond between the 5’ phosphate group and 3’ hydroxyl
group.
GENE ISOLATION
Gene isolation is one of the major activities of biotechnology. Before a gene can be ge-
netically engineered, it must first be identified, isolated, and characterized (e.g., number
and position of introns, the promoter and its elements). Isolating a gene enables re-
searchers to determine its nucleotide sequence. From the DNA sequence several things can
be deduced, including the amino acid sequence and the protein structure and function of
the gene’s product. In order to transfer a gene from one individual to another, it must first
be identified and isolated. Isolation of a gene permits it to be amplified to obtain large
quantities for studies.
A number of strategies may be used to isolate or clone a gene.
1. Activation tagging
This strategy requires the availability of a well-characterized transposon system, something
that is lacking in many species, except species like corn. The gene to be isolated is first
inactivated by transposon insertion, resulting in the formation of a mutant. The DNA
sequence of the transposon is used to identify the clones that contain the gene of interest.
2.cDNA screening
A cDNA library is first created. A probe is then designed and used to screen the library to
hybridize to the sequence of interest.
3.Map-based gene cloning
Map-based cloning or positional cloning is an rDNA-based method for identify- ing a gene
without first knowing its product. The first step in this method is to produce a high-
resolution genetic map (average distance of less than 5 centi Morgans). This is followed by
the production of a physical map (a map of the location of identifiable landmarks on DNA
regardless of their inheritance). The principal procedures include physical mapping by
contig construction using BACs, YACs, STS-content mapping, DNA fingerprinting, and
pulse-field gel electrophoresis. Once a physical map is in place, the target gene may be
identified by chromosome walking, using RFLP or other molecular markers. This entails
starting with a closely linked RFLP probe and isolating genomic clones that it corresponds
with, and then walking from these clones to the target genes. Alternatively, molecular
markers that are tightly linked with the gene of interest are first identified. The DNA
markers are used to screen a genomic library to isolate clones that contain the target gene
(called chromosome landing). Genetic complementation through transformation is also
part of this process of gene identification.
4.Transformation-associated recombination
This method of gene isolation capitalizes on the natural ability of yeast cells to find and
combine similar DNAs, regardless of their origin. Yeast cells are transformed with pieces of
DNA along with a small fragment of the target DNA. As the yeast cells reproduce, only DNA
that complements the small piece of DNA introduced into the cell are maintained (cloned).
II. VECTORS
CARRIERS OF TRANSGENE
A vector is a DNA molecule which carries the foreign genetic material into another
cell. A vector carrying a foreign gene is a chimera and is called as the recombinant DNA.
A vector must contain:
(a) An ori site (Origin of replication)
(b) Multiple cloning sites which are the active sites of the RE enzymes and they allow
insertions of DNA into the vector to be targeted.
(c) Selectable markers: such as an antibiotic resistance [e.g. tetracycline] is often
carried by the vector to allow the selection of positively transformed cells.
Other desirable features that can be present in a suitable cloning vector may be
(i) vir genes for plant transformation,
(ii) intergrase sites for chromosomal insertion,
(iii) lacZ fragment for complementation and blue –white selection,
(iv) reporter genes flanking the MCS to facilitate the production of
recombinant proteins.
Properties of a good vector

 It should be able to replicate independent of the replication of host chromosome
 It should be easy to isolate, purify and introduce into the host cells.
 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.
 A vector should contain unique target sites for as many restriction enzymes as possible
into which the DNA insert can be integrated.
 When expression of the DNA insert is desired, the vector should contain suitable
regulatory elements like promoter, operator, ribosome binding sites.
There are four major types of vectors namely, plasmids, bacteriophages,

cosmids, and artificial chromosomes (BAC, YAC etc.), of which the first two are
most commonly used ones.
a). Plasmid vectors

A plasmid is a circular, double-stranded, self-replicating extrachromosomal
molecules present in some bacteria. In nature, plasmids confer antibiotic
resistance to the host bacteria. They replicate independent of the bacterial DNA
and can accept about 6-10 kb. Eg. pUC18. pUC refers to plasmid from University
of California.
b). Bacteriophages
The virus that infect and replicate inside the bacteria are called
bacteriophages or phages. About one-third of the phage genome is nonessential
and can be replaced with foreign DNA. These viral vectors can carry about 23 kb
of DNA or RNA, and contain viral promoters for translating the target gene in the
host cell. They often produce an identification mark to indicate successful
transfection. The commonly used phages are phage λ, phage M13.
c). Cosmids
A cosmid is a “hybrid” between a plasmid and a phage. It consists of the cos
sequence of phage lambda (required for packing the phage DNA into the phage
protein coat), the plasmid sequence for replication, and an antibiotic resistance
gene to identify the host cell carrying the cosmids. They can handle about 40 kb
of cloned DNA and can be maintained as either plasmids or bacteriophage λ
vectors because they have an E. coli origin of replication. They also have cohesive
ends (cos) sites found in phage. An example of a cosmid is the pJB8-5.
Cosmids are vectors possessing the characteristics of both plasmids and
bacteriophages. It can be constructed by adding a fragment of phage λ DNA
including cos site, to plasmids. They are capable of carrying about 42 kb of
inserts. They replicate intensely and their success rate is high.
d). Bacterial Artificial Chromosomes (BACs): Bacterial artificial chromosomes

are plasmids designed for the cloning of very long segments (100 to 300 kb) of
DNA. They include selectable markers such as resistance to the antibiotic
chloramphenicol, as well as a stable origin of replication (ori) that maintains the
plasmid at one or two copies per cell. The large circular DNAs are then introduced
into host bacteria by electroporation.
e). Yeast artificial chromosomes (YAC)

These are useful for cloning large fragments (100-300kb). They may be used
to even clone whole chromosomes. But they are notorious for generating chimeras
(recombinant molecules in which non-contiguous donor fragments are joined
together), which are laborious to separate from the desired recombinants.
CATEGORIES OF VECTORS BY FUNCTIONS
1. Cloning vectors
The vectors used for multiplication of DNA fragments in a suitable host are called
cloning vectors. A vector is used because it provides an Ori (origin of replication site). For
increased efficiency the original restriction sites of most cloning vectors are replaced
by a synthetic multiple cloning site (contains many restriction sites). Other
additional features that may be engineered into vectors include vir genes (for
plant transformation), integrase sites (for chromosomal insertion), and lacZa
fragment (for a complementation).
The choice of cloning vector to use for a particular project depends on the insert
size, copy number, cloning sites, selectable marker, and incompatibility. Two of the
most common vectors used in genetic engineering are the Plasmids and the Phage DNA.
2. Transcription vectors
Transcription is a necessary component of all vectors. Stable expression of an
insert depends on stable transcription which depends on the promoters in the
vector. Transcription vectors are designed to only be transcribed (replicated or
amplified) but not translated (expressed). They are relatively simple in their
construction. Plasmid transcription vectors cannot be used as transcription vector.
3. Expression vectors
When a vector is designed for the expression of i.e. production of the protein specified
by the DNA insert, it is known as expression vector. Sometimes the goal in gene cloning is
not just to replicate the gene, but also to produce the protein that it encodes. In addition to
the usual origin of replication, restriction sites, and selectable markers, contains sequences
required for transcription and translation in bacterial cells. These additional sequences may
include:
1. A bacterial promoter, such as the lac promoter. The promoter precedes a restriction site
where foreign DNA is to be inserted, allowing transcription of the foreign sequence to be
regulated by adding substances that induce the promoter.
2. A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome
binding site.
3. Prokaryotic transcription initiation and termination sequences.
4. Sequences that control transcription initiation, such as regulator genes and operators.
The bacterial promoter and ribosome-binding site are usually placed upstream of the
restriction site, which allows the foreign DNA to be inserted just downstream of the
initiation codon. When the plasmid is placed in a bacterial cell, RNA polymerase binds to
the promoter and transcribes the foreign DNA. Bacterial ribosomes attach to the ribosome-
binding site on the RNA and translate the sequence into a foreign protein. An expression
vector contains a promoter, a ribosome-binding site, and other sequences that allow a
cloned gene to be transcribed and translated in bacteria.
One of the first commercial products produced by recombinant DNA technology was
the protein insulin. The gene for human insulin was isolated and inserted into bacteria,
which were then multiplied and used to synthesize human insulin.
4. Shuttle vectors
Shuttle vectors are plasmids capable of propagating and transferring/shuttling

genes between two different organisms. Hence, they contain two origins of replication,
one specific for each host species, as well as those genes necessary for their replication
and not provided by the host cells. These vectors are created by recombinant DNA
technique. Some of them can be grown in two different prokaryotic species, while
others can propagate in prokaryotic species (E.coli) and eukaryotic one (yeast,
plants and animals) Since these vectors can be grown in one host and then moved into
another without any extra manipulation they are called shuttle vectors.
Shuttle vectors have been designed to specifically satisfy the need i.e. for the
initial cloning of DNA inserts in E.coli and sub-sequent functional tests in the species
to which the DNA inserts belong. Most of the eukaryotic vectors are shuttle vectors.
III. METHODS OF GENE TRANSFER
Introduction of a foreign gene into the host cell can be done indirectly or directly.
1. DIRECT GENE TRANSFER METHODS

The most commonly used methods includes transformation, conjugation,
electroporation and liposome mediated gene transfer.
i. Transformation:
Transformation is the method of introducing foreign DNA into bacterial cells. The
uptake of plasmid DNA by E.coli is carried out in 0 to 50C ice-cold CaCl2 and a
subsequent heat shock (37-450C for 90sec). the transformational frequency which is the
fraction of transformed cells is good (one cell per 1000 cells). Eg. Agrobacterium
mediated gene transfer.
ii. Conjugation:
Conjugation is a natural microbial recombination process. During conjugation,
two live bacteria (a donor and a recepient) come together, join by cytoplasmic bridges
and transfer single stranded DNA from the donor to the recipient cell. The new DNA
strand may integrate with the host chromosome or remain free. The natural
phenomenon is exploited for transferring plasmid-insert DNA to new recipient cells.
iii. Electroporation:
Electric shocks can also induce cellular uptake of exogenous DNA from the
suspended solution by means of pores formed by electric pulses. It is a simple and
rapid technique for introducing genes into the cells from various organisms.
iv. Liposome-mediated gene transfer:

Liposomes are positively charged, circular lipid molecules, with an aqueous
interior that can carry nucleic acids. Lipofection refers to the technique of
encapsulating the DNA fragments in liposomes. These liposomes can adhere to cell
membranes and fuse with them to transfer DNA fragments into the cells and then into
the nucleus.
2. DIRECT GENE TRANSFER METHODS

The direct gene transfer methods include protoplast transformation, tissue/cell
electroporation, silicon carbide fiber vortexing, and microprojectile bombardment.
i. Protoplast transformation was the first method used to demonstrate that direct
gene delivery to plants was feasible. But, this method poses some technical challenges
and hence is not widely used.
ii. Tissue/cell electroporation: Callus cultures or primary explants such as immature

embryos or inflorescence may be used as target material. The transformation process
occurs in an electropora- tor. Transformation efficiency levels by electroporation are
sufficiently high.
iii. Silicon carbide fiber vortexing: Silicon carbide fibers are mixed with a suspension
culture as explant along with plasmid DNA, and vortexed. The mixture is then cultured
on a medium with selectable markers. To use this technique, there must be a
regeneration system in place for regenerating plants from single cells.
iv. Microprojectile bombardment

/ Gene gun
In this method the target DNA is
coated in a carrier particle and
transferred by shooting by the gene
gun into the tissue. In this method
1 to 5µ diameter tungsten or gold
particles are coated with the DNA of
interest. Compressed helium gas at
the rate of 430m/s is used to propel
the DNA coated particles through a
barrel into the target cell. About 50
µg of tungsten is required for each
DNA transfer event. The carrier
particles are placed on a support
film and mounted in the particle A gene gun.
acceleration device. The support film is accelerated by gas pressure and then stopped
by a protective mesh. The carrier particles pass through the mesh, hitting the target
tissue mounted in a petridish below the biolistic device. The survival of target cell is
high when a low penetration number of projectiles (1 to 5 per cell) occurs.
Many factors such as chamber vacuum level, particle size range, and shot
distance affect the success of a gene gun. Other factors include the amount of DNA per
particle, explant type and physiological conditions, and gas type and pressure.
Biolistics has been used to transform both dicots (e.g., soybean, peanuts, and tobacco)
and monocots (e.g., corn, wheat, and rice). Organelle transformations have also been
reported with this technique.
v. Micro-injection:
Microinjection refers to the process of injecting DNA directly into the cell, or even
into the cell nucleus via an inserted cannula under a
microscope. The target cell is held in place by two
micromanipulators, one holding a pipette and one
holding a microcapillary needle (0.5 to 5 µm
diameter). The introduced DNA then integrates into
the plant genome during its own DNA repair process.
The main advantage is that the use of marker gene
for identification of successful transformation is not
needed. Micro injection
BLUE WHITE SCREENING
A more sophisticated procedure for screening for the presence of recombinant

screening plasmids, which can be carried out on a single transformation plate, is called
blue–white screening. This method also involves the insertional inactivation of a gene
and, as the name implies, uses the production of a blue compound as an indicator.
The blue–white screen is a screening technique that allows for the rapid and
convenient detection of recombinant bacteria in vector-based molecular
cloning experiments. DNA of interest is ligated into a vector. The vector is
then inserted into a competent host cell viable for transformation, which are then
grown in the presence of X-gal. Cells transformed with vectors containing recombinant
DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e.
only the vector) grow into blue colonies. This method of screening is usually performed
using a suitable bacterial strain, but other organisms such as yeast may also be used.
An LB agar plate showing the result of a blue white screen

Source: www.wikipedia.com
The gene in this case is lacZ, which encodes the enzyme 13-galactosidase, and is
under the control of the lac promoter (see Topic L1). If the host E. coli strain is
expressing the lac repressor, then expression of a lacZ gene on the vector may be
induced using isopropyl-13-D-thiogalactopyranoside (IPTG) (Topic L1), and the
expressed enzyme can utilize the synthetic substrate 5-bromo-4-chloro-3-indolyl-13-D-
galactopyranoside (X-gal) to yield a blue product. Insertional inactivation of lacZ in the
production of a recombinant plasmid would prevent the development of the blue
color. In this method the transformed cells are spread on to a plate containing
ampicillin (to select for transformants in the usual way), IPTG and X-gal, to yield a
mixture of blue and white colonies. The white colonies have no expressed β-
galactosidase and are hence likely to contain the inserted target fragment. The blue
colonies probably contain religated vector.
In practice, the vectors used in this method have a shortened derivative of

lacZ, lacZ', which produces the N-terminal a-peptide of β-galactosidase. These vectors
must be transformed into a special host strain which contains a mutant gene
expressing only the C-terminal portion of β-galactosidase which can then complement
the a-peptide to produce active enzyme. This reduces the size of the plasmid-borne
gene, but does not alter the basis of the method.
IV. DNA SEQUENCING
Sequencing a genome requires breaking it up into small overlapping fragments whose

DNA sequences can be determined in a sequencing reaction. The sequences can be ordered
into the final genome sequence by a map-based approach (large fragments are ordered with
the use of genetic and physical maps) or by whole-genome shotgun sequencing (overlap
between the sequences of small fragments is compared by computers). DNA sequencing
enables researchers to determine the base sequence of DNA found in genes and other
chromosomal regions. It is one of the most important tools for exploring genetics at the
molecular level.
During the 1970s, two methods for DNA sequencing were devised. One method,
developed by Allan Maxam and Walter Gilbert, involves the base-specific chemical cleavage
of DNA. Another method, developed by Frederick Sanger and colleagues, is known as
dideoxy sequencing. Because it has become the more popular method of DNA sequencing,
we consider the dideoxy method here.
The dideoxy procedure of DNA sequencing is based on DNA replication. DNA

polymerase connects adjacent deoxyribonucleotides by forming a covalent bond between
the 5 phosphate on one nucleotide and the 3ʹ –OH group on the previous nucleotide.
Chemists synthesize deoxyribonucleotides that are missing the –OH group at the 3ʹ
position. These synthetic nucleotides are called dideoxyribonucleotides (ddNTPs). The prefix
dideoxy- indicates that two (di) oxygens (oxy) are removed (de) from this sugar compared
with ribose.
Sanger reasoned that if a dideoxyribonucleotide is added to a growing DNA strand, the
strand cannot grow because the dideoxyribonucleotide is missing the 3ʹ –OH group. The
incorporation of a dideoxyribonucleotide into a growing strand is therefore referred to as
chain termination.
To detect the incorporation of dideoxynucleotides during DNA replication, the newly

made DNA strands are labeled with a different colored fluorescent molecule: ddA is green,
ddT is red, ddG is yellow, and ddC is blue.
Prior to automated DNA sequencing, the segment of DNA to be sequenced must usually
be obtained in large amounts. This is accomplished by using gene cloning. For example the
segment of DNA to be sequenced, called the target DNA, was cloned into a vector at a
defined location. The target DNA was inserted next to a site in the vector where a primer will
bind, which is called the primer-annealing site. The aim of the experiment is to determine
the base sequence of the target DNA.
First, a sample containing many copies of the single-stranded DNA is mixed with many
primers that will bind to the primer-annealing site. The primer binds to the DNA because
the primer and primer-annealing site are complementary to each other. All four types of
deoxyribonucleotides and DNA polymerase are then added to the annealed DNA fragments.
The tube also has a low concentration of each dideoxyribonucleotide (ddG, ddA, ddT, or
ddC), which are fluorescently labeled. The tube is then incubated to allow DNA polymerase
to make strands complementary to the target DNA sequence.
DNA synthesis continues until a dideoxynucleotide is incorporated into a growing

strand. For example, chain termination can occasionally occur at the sixth or thirteenth
position of the newly synthesized DNA strand if a ddT becomes incorporated at either of
these sites. Note that the complementary A base is found at the sixth and thirteenth
position in the target DNA. Therefore, we expect to make DNA strands that terminate at the
sixth or thirteenth positions and have a ddT at their ends. Because these DNA strands
contain a ddT, they are fluorescently labeled in red. Alternatively, ddA causes chain
termination at the second, seventh, eighth, or eleventh positions because a complementary
T base is found at the corresponding positions in the target strand. Strands that are
terminated with ddA are fluorescently labeled in green.
After the samples have been incubated for several minutes, mixtures of DNA strands
of different lengths are made, depending on the number of nucleotides attached to the
primer. These DNA strands can be separated according to their lengths by running them on
a slab gel or more commonly by running them through a gel-filled capillary tube. The
shorter strands move to the bottom of the gel more quickly than the longer strands.
Because we know the color of each dideoxyribonucleotide, we also know which base is at
the very end of each DNA strand separated on the gel. Therefore, we can deduce the DNA
sequence that is complementary to the target DNA by “reading” which base is at the end of
every DNA strand and matching this sequence with the length of the strand. Reading the
base sequence, from bottom to top, is much like climbing a ladder of bands. For this reason,
the sequence obtained by this method is referred to as a sequencing ladder.
Theoretically, it is possible to read this sequence directly from the gel. From a practical
perspective, however, it is faster and more efficient to automate the procedure using a laser
and fluorescence detector. As the gel is running, each band passes the laser and the laser
beam excites the fluorescent dye. The fluorescence detector records the amount of
fluorescence emission from the excited dye. The detector reads the level of fluorescence at
four wavelengths, corresponding to the four different colored dyes. An example of the
printout from the fluorescence detector is shown in Figure 18.19b. As seen here, the peaks
of fluorescence correspond to the DNA sequence that is complementary to the target DNA.
Note that ddG is usually labeled with a yellow
TRANSGENIC PLANTS
Plants with desirable characters created through gene transfer methods are called
transgenic plants. Though a number of methods have been developed to introduce the
cloned genes into plant cells. Ti plasmid of Agrobacterium tumefaciens has been widely
used as an effective vector for obtaining transgenic plants. Several transgenic plants have
been produced to meet specific needs of agriculture and human life. Some of these are
given below.
a. Transgenic crop plants having resistance to pathogens and pests:
1. Transgenic papaya is resistant to papaya ring spot virus
2. Bt. Cotton is resistant to insects.
3. Transgenic tomato plants are resistant to the bacterial pathogen Pseudomonas.
4. Transgenic potato plants are resistant to the fungus Phythophthora
b. Transgenic plants suitable for food processing technology:
Transgenic tomato ‘Flavr Savr’ is bruise resistant i.e., suitable for storage and
transport due to delayed ripening and offers longer shelf-life.
c. Transgenic plants with improved nutritional value:
Transgenic golden rice obtained from ‘Taipei’ is rich in vitamin A and prevents
blindness.
d. Transgenic plants useful for hybrid seed production:
Male sterile plants of Brassica napus are produced. This will eliminate the problem
of manual emasculation and reduce the cost of hybrid seed production.
e. Transgenic plants tolerant to abiotic stresses caused by chemicals, cold,
drought, sale, heat etc.
1. Basmati variety of rice was made resistant against biotic and abiotic stresses.
2. Round-up ready soybean is herbicide tolerant.
Besides these, genetically modified crops have evolved as alternative resources to industries,
in the form of starches, fuels and pharmaceuticals.
Transgenic plants have been shown to express the genes of insulin, interferon, human
growth hormones, antibiotics, antibodies etc. these biochemical produced by plants are as
food as or sometimes better than those produced in bacteria.
Utilization of plants as biofactories or bioreactors for obtaining commercially useful
products, specialized medicines, chemicals and antibodies on a large scale is described as
molecular farming. In the near future this field is expected to revolutionise both the farming
as well as biochemical industry.
DOLLY- THE TRANSGENIC CLONE
Dolly, the first ever mammal clone was developed by Wilmut and Campbell in 1997. It is a
sheep(female lamb) with a mother and no father.
The technique primarily involves nuclear transfer and the phenomenon of
totipotency. The character of a cell to develop into different cells, tissues, organs, and
finally an organism is referred to as totipotency or pluripotency. Totipotency is the basic
character of embryonic cells. As the embryo develops, the cells specialize to final give the
whole organism. As such, the cells of an adult lack totipotency. Totipotency was induced
into the adult cells for developing Dolly.
The cloning of sheep for producing Dolly, illustrated in fig. is briefly described
here. The mammary gland cells from a donor ewe were isolated. They were subjected to
total nutrient deprivation (starvation) for five days. By this process, the mammary cells
abandon their normal growth cycle, enter a dormant stage and regain totipotency character.
An ovum (egg cell) was taken from another ewe, and its nucleus was removed to form an
enucleated ovum. The dormant mammary gland cell and the enucleated ovum were fused
by pulse electricity. The mammary cell outer membrane was broken, allowing the ovum to
envelop the nucleus. The fused cell, as it had gained totipotency, can multiply and develop
into an embryo. This embryo was then implanted into another ewe which served as a
surrogate/foster mother. Five months later, Dolly was born.
As reported by Wilmut and Campbell, they fused 277 ovum cells, achieved 13
pregnancies, and of these only one pregnancy resulted in live birth of the offspring-Dolly.
CLONING OF PET ANIMALS

Some of the companies involved in transgenic experiments have started cloning
pet animals like cats and dogs. Little Nicky was the first pet cat that was cloned at a cost of
S50,00 by an American company (in Dec. 2004). More cloned cats and dogs will be made
available to interested parties (who can afford) in due course.
Some people who own pet animals are interested to continue the same pets
which is possible through cloning. There is some opposition to this approach as the cloned
animals are less healthy, and have shorter life span, besides the high cost factor.
GMOs IN AGRICULTURE
Genetically modified (GM) foods were first approved for human consumption in
the United States in 1995, and by 1999 almost 50 percent of the corn, cotton,
and soybeans planted in the United States were GM. By the end of 2010, GM crops covered
more than 9.8 million square kilometres (3.8 million square miles) of land in 29 countries
worldwide—one-tenth of the world’s farmland.
GMOs in Agriculture
Bt crops
Engineered crops can dramatically increase per area crop yields and, in some cases,
reduce the use of chemical insecticides. For example, the application of wide-spectrum
insecticides declined in many areas growing plants, such as potatoes, cotton, and corn, that
were endowed with a gene from the bacterium Bacillus thuringiensis, which produces a
natural insecticide called Bt toxin. Field studies conducted in India in which Bt cotton was
compared with non-Bt cotton demonstrated a 30–80 percent increase in yield from the GM
crop. This increase was attributed to marked improvement in the GM plants’ ability to
overcome bollworm infestation, which was otherwise common. Studies of Bt cotton
production in Arizona, U.S., demonstrated only small gains in yield—about 5 percent—with
an estimated cost reduction of $25–65 (USD) per acre due to
decreased pesticide applications. In China, a seven-year study of farms planting Bt cotton
demonstrated initial success of the GM crop, with farmers who had planted Bt cotton
reducing their pesticide use by 70 percent and increasing their earnings by 36 percent.
However, after four years, the benefits of Bt cotton eroded as populations of insect pests
other than bollworm increased, and farmers once again were forced to spray broad-
spectrum pesticides. While the problem was not Bt-resistant bollworms, as had been feared
initially, it nonetheless became clear that much more research was needed for communities
to realize sustainable and environmentally responsible benefits from planting GM crops.
Other GM plants were engineered for resistance to a specific chemical herbicide, rather than
resistance to a natural predator or pest. Herbicide-resistant crops (HRC) have been available
since the mid-1980s; these crops enable effective chemical control of weeds, since only the
HRC plants can survive in fields treated with the corresponding herbicide. However, because
these crops encourage increased application of chemicals to the soil, rather than decreased
application, they remain controversial with regard to their environmental impact.
By 2002 more than 60 percent of processed foods consumed in the United States
contained at least some GM ingredients. Despite the concerns of some consumer groups,
especially in Europe, numerous scientific panels, including the U.S. Food and Drug
Administration, have concluded that consumption of GM foods is safe, even in cases
involving GM foods with genetic material from very distantly related organisms. Indeed,
foods containing GM ingredients do not require special labeling in the United States,
although some groups have continued to lobby to change this ruling. By 2006, although the
majority of GM crops were still grown in the Americas, GM plants tailored for production
and consumption in other parts of the world were in field tests. For example, sweet
potatoes intended for Africa were modified for resistance to sweet potato feathery
mottle virus (SPFMV) by inserting into the sweet potato genome a gene encoding a viral
coat protein from the strain of virus that causes SPFMV. The premise for this modification
was based on earlier studies in other plants such as tobacco in which introduction of viral
coat proteins rendered plants resistant to the virus.
Golden Rice (Ingo Potrykus)
The “golden” rice intended for Asia was genetically modified to produce almost 20
times the beta-carotene of previous varieties. Golden rice was created by modifying the rice
genome to include a gene from the daffodil Narcissus pseudonarcissus that produces
an enzyme known as phyotene synthase and a gene from the bacterium Erwinia
uredovora that produces an enzyme called phyotene desaturase. The introduction of these
genes enabled beta-carotene, which is converted to vitamin A in the human liver, to
accumulate in the rice endosperm—the edible part of the rice plant—thereby increasing the
amount of beta-carotene available for vitamin A synthesis in the body.
Golden rice (right) compared to white rice (left) (Source:
www.wikipedia.com)
Bio-fortification
Another form of modified rice was generated to help combat iron deficiency, which
impacts close to 30 percent of the world population. This GM crop was engineered by
introducing into the rice genome a ferritin gene from the common bean, Phaseolus vulgaris,
that produces a protein capable of binding iron, as well as a gene from the
fungus Aspergillus fumigatus that produces an enzyme capable of digesting compounds that
increase iron bioavailability via digestion of phytate (an inhibitor of iron absorption). The
iron-fortified GM rice was engineered to overexpress an existing rice gene that produces a
cysteine-rich metallothioneinlike (metal-binding) protein that enhances iron absorption.
A variety of other crops modified to endure the weather extremes common in other parts of
the globe are also in production.
GMOs in medicine and research / Bio-farming / Biopharmaceuticals

GMOs have emerged as one of the mainstays of biomedical research since the 1980s. For
example, GM animal models of human genetic diseases enabled researchers to test novel
therapies and to explore the roles of candidate risk factors and modifiers of disease
outcome. GM microbes, plants, and animals also revolutionized the production of complex
pharmaceuticals by enabling the generation of safer and cheaper vaccines and therapeutics.
Pharmaceutical products range from recombinant hepatitis B vaccine produced by GM
baker’s yeast to injectable insulin (for DIABETICS ) produced in GM Escherichia
coli bacteria and to factor VIII (for hemophiliacs) and tissue plasminogen activator (tPA,
for heart attack or strokepatients), both of which are produced in GM mammalian cells
grown in laboratory culture. Furthermore, GM plants that produce “edible vaccines” are
under development. Such plants, which are engineered to express antigens derived from
microbes or parasites that infect the digestive tract, might someday offer a safe, cheap, and
painless way to provide vaccines worldwide, without concern for the availability of
refrigeration or sterile needles. Novel DNA vaccines may be useful in the struggle to prevent
diseases that have proved resistant to traditional vaccination approaches, including
HIV/AIDS, tuberculosis, and cancer.
GM Mosquitoes
Genetic modification of insects has become an important area of research, especially
in the struggle to prevent parasitic diseases. For example, GM mosquitoes have been
developed that express a small protein called SM1, which blocks entry of
the malaria parasite, Plasmodium, into the mosquito’s gut. This results in the disruption of
the parasite’s life cycle and renders the mosquito malaria-resistant. Introduction of these
GM mosquitoes into the wild may someday help eradicate transmission of the malaria
parasite without widespread use of harmful chemicals such as DDT or disruption of the
normal food chain.
Gene therapy
Finally, genetic modification of humans, or so-called gene therapy, is becoming a
treatment option for diseases ranging from rare metabolic disorders to cancer.
Coupling stem cell technology with recombinant DNA methods may someday allow stem
cells derived from a patient to be modified in the laboratory to introduce a desired gene. For
example, a normal beta-globin gene may be introduced into the DNA of bone marrow-
derived hematopoietic stem cells from a patient with sickle cell anemia, and introduction of
these GM cells into the patient could cure the disease without the need for a matched
donor.
Role of GMOs in environmental management

Another application of GMOs is in the management of environmental issues. For
example, somebacteria can produce biodegradable plastics, and the transfer of this ability
to microbes that can be easily grown in the laboratory may enable the wide-scale “greening”
of the plastics industry.Zeneca, a British company, developed a microbially produced
biodegradable plastic called Biopol. This plastic is made using a GM bacterium, Ralstonia
eutropha, to convert glucose and a variety of organic acids into a flexible polymer. GMOs
endowed with the bacterially encoded ability to metabolize oil and heavy metals may provide
efficient bioremediation strategies.
Genetic modification technologies may help save endangered species such as the giant
panda, whose genome is being sequenced in an international effort led by the Beijing
Genomics Institute at Shenzhen. Genetic studies of the panda genome may provide insight
into why pandas have such low rates of reproductive success in captivity. A likely set of
genes to consider for future genetic modification, should the goals of panda conservation
warrant it, is the major histocompatibility complex (MHC). The MHC genes play an
important role in regulating immune function and also influence behaviours and
physiological patterns associated with reproduction.
Sociopolitical relevance of GMOs

While GMOs offer many potential benefits to society, the potential risks associated
with them have fueled controversy, especially in the food industry. Many skeptics warn
about the dangers that GM crops may pose to human HEALTH . For example, genetic
manipulation may potentially alter the allergenic properties of crops. However, the more-
established risk involves the potential spread of engineered crop genes to native flora and
the possible evolution of insecticide-resistant “superbugs.” In 1998 the European

Union (EU) addressed such concerns by implementing strict GMO labeling laws and a
moratorium on the growth and import of GM crops. In addition, the stance of the EU on GM
crops has led to TRADE disputes with the United States, which, by comparison, has
accepted GM foods very openly. Other countries, such as Canada, China, Argentina, and
Australia, also have open policies on GM foods, but some African states have rejected
international food aid containing GM crops.
The use of GMOs in medicine and research has produced a debate that is more
philosophical in nature. For example, while genetic researchers believe they are working to
cure disease and ameliorate suffering, many people worry that current gene
therapy approaches may one day be applied to produce “designer” children or to lengthen
the natural human life span. Similar to many other technologies, gene therapy and the
production and application of GMOs can be used to address and resolve complicated
scientific, medical, and environmental issues, but they must be used wisely.
BIO-SAFETY AND ETHICAL ISSUES

Despite several advantages, genetic modifications of organisms can heave
unpredictable results when they are introduced into the natural ecosystem. Some of the
apprehensions towards bio-safety issues of genetically engineered crops are:
The major concern about GM crops and GM foods are
1. There is fear of transfer of allergens from genetically modified food to humans and
animals.
2. Due to molecular farming, there is a risk of changing the fundamental nature of
vegetables.
3. The GM crops are manipulated artificially and are not naturally evolved which may
affect their buffering capacity under adverse conditions.
4. GM crops poses threat on biodiversity due to monoculture and impact environment.
5. There is a risk of gene pollution, which may result in the development of super-weeds.
6. Plants generally adapt the fluctuations occurring in nature and evolve gradually. GM
plants may bring about changes in natural evolutionary pattern.
7. Use of herbicide-tolerant transgenic crops can lead to transfer of herbicide tolerance
genes to sexually compatible wild relatives or weeds, and can create “super weeds”.
8. It would actually increase the dependence on a few herbicides rather than reducing
herbicide usage.
9. Gene flow is the primary risk in releasing transgenic plants. Once released, the GM
crops cannot be recalled back due to gene flow.
10. There is fear of transferring allergins or toxins too humans and animals as
side effects.
There is a risk of changing the fundamental nature of vegetables.
1. They may pose a harmful effect on biodiversity and have an adverse impact on
environment.
2. There is a risk of gene pollution due to transfer of the new genes into related wild
species through natural out-crossing. This may result in the development of super-weeds
which may be fast-growing than the crops and may be resistant to weedicides.
3. They may bring about changes in natural evolutionary pattern.
Going beyond the morality of such issues, their biological significance is also important.
The manipulation of living organisms by the human race cannot go on any further without
regulation. Some ethical standards are required to evaluate the morality of all human
activities that might help or harm living organisms. Therefore, the Indian Government has
set up organisations such as GEAC (Genetic Engineering Approval Committee), which will
make decisions regarding the validity of GM research and the safety of introducing GM-
organisms for public services.
The modification/usage of living organisms for public services (as food and medicine
sources, for example) has also created problems with patents granted for the same.
There is growing public anger that certain companies are being granted patents for
products and technologies that make use of genetic materials, plants and other biological
resources that have long een identified, developed and used by farmers and indigenous
people of a specific region/country.
For example, rice is an important food grain, the presence of which goes back to
thousands of years in Asia’s agricultural history. There are an estimated 200,000 varieties
of rice in India alone. The diversity f rice in India is one of the richest in the world. Basmati
rice distinct for its unique aroma and flavor and 27 documented varieties of Basmati are
grown in India. There is reference to Basmati in ancient texts, folkore and poetry, as it has
been grown for centuries. In 1997, an American company got patent rights on Basmati rice
through the US patent and Trademark Office. This allowed the company to sell a ‘new’
variety of Basmati, in the US and abroad. This ‘new’ variety of Basmati rice had actually
been derived from Indian farmers varieties. Indian Basmati was crossed with semi-dwarf
varieties and claimed as an invention or a novelty. The patent extends to functional
equivalents, implying that other people selling Basmati rice could be restricted by the
patent. Several attempts have also been made to patent users, products and processes
based on Indian traditional herbal medicines, such as turmeric and neem. If we are not
vigilant and do immediately counter these patent applications, other countries/individuals
may encash on our rich legacy and we may not be able to do anything about it.
BIOSAFETY GUIDELINES AND REGULATION
Cultivation of genetically modified crops by the farmers is increasing rapidly

throughout the world. Inspite of impressive progress in this field, there is much uneasiness
among the public towards biosafety issues of genetically engineered crops.
Due to the alarming concerns of these GMCs in Indian agriculture, the Govt. of India
has evolved recombinant DNA safety guidelines for the manufacture, use, import, export
and storage of hazardous microorganisms / genetically engineered organisms cells etc.
These guidelines are being implemented through the Environmental Protection Act 1986
(EPA).
1. Institutional Biosafety Committees (IBCS) monitors research activity at institutional
level.
2. Review Committee on Genetic Manipulation (RCGM) will monitor research activities in
the laboratories and
3. Genetic Engineering Approval Committee (GEAC) of the Ministry of Environment and
Forest has the power to permit large scale use of GMO’s at commercial level and also
monitors field trials of transgenic materials including agricultural crops, industrial
products, health care products etc.
BIOPIRACY
Biopiracy is the term coined to refer to the use of bio-resources by multinational

companies and other organizations without proper authorization from the countries and
people concerned or without compensatory payment.
Most of the industrialized nations are rich financially but poor in biodiversity and
traditional knowledge. In contrast, the developing and underdeveloped world is rich in
biodiversity and traditional knowledge related to bio-resources. Traditional knowledge
related to bi-resources can be exploited to develop modern applications and can also be
used to save time, effort and expenditure during their commercialization.
There has been growing realization of the injustice, inadequate compensation and
benefit sharing between developed and developing countries. Therefore, some nations are
developing laws to prevent such unauthorized exploitation of their bio-resources and
traditional knowledge.
The Indian Parliament has recently cleared the second amendment of the Indian
Patents Bill, that takes such issues into consideration, including patent terms emergency
provisions and research and development initiatives.
BIOREACTOR
Obtaining the Foreign Gene Product:
When a piece of alien DNA is inserted into a cloning vector and transferred into a
host cell, the alien DNA gets multiplied. The ultimate aim is to produce a desirable protein.
If any protein encoding gene is expressed in a heterologous host, it is called as a
recombinant protein.
Any new biotechnological manufacturing process must first be tried on a laboratory
scale. It is useful to make a pilot plan first and then to scale it up for industrial production.
A bioreactor may refer to any device or system that supports a biologically active
environment. In one case, a bioreactor is a vessel in which is carried out a chemical process
which involves organisms or biochemically active substances derived from such organisms.
This chemical process carried out may be aerobic or anaerobic in nature. The bioreactors
are frequently cylindrical in shape. They may vary in size. The body of a bioreactor is
usually made up of stainless steel.
The bioreactor is also used to grow cell or tissues. The process of growing cells or
tissues is employed in plant cell/tissue culture. An another field of the application of the
bioreactor is the field of tissue engineering.
The classification of the bioreactors is based on their mechanism of working. They
may be (a) batch, (b) fed batch or (c) continuous.
One of the biggest problems is to maintain the optimum conditions within the
bioreactors and to maintain proper asepsis throughout the process. Any derivation from the
optimum conditions and any contamination of the bioreactor would lead to a sub-optimum
yield or no yield at all. Thus bioreactors have been considerably improved upon for ensuring
these conditions.
Fouling of the bioreactor can decrease the efficiency of the bioreactors considerably.
They may be especially detrimental to the heat exchangers that play a very important role in
the bioreactors. During the designing, due care is taken to ensure that the surface of the
bioreactors is smooth. Periodic cleaning of the bioreactors is mandatory.
A heat exchanger is an important component of bioreactors and it maintains the
constant temperature required to carry out the process in the bioreactor. A large amount of
heat can be produced in the fermentation carried out in a bioreactor. Therefore, to maintain
a constant temperature, a bioreactor commonly employs a source of refrigeration. The
refrigeration can be provided by an external mechanism such as an external jacket or by
internal mechanisms such as internal cooling coils.
If a bioreactor is carrying out an aerobic biological process, adequate and uniform
delivery of the oxygen is a major problem encountered. The delivery of oxygen is made
difficult by the fact that oxygen is poorly soluble in water. The medium may be agitated to
ensure uniform distribution of oxygen. Baffles are used for proper mixing of the medium.
But this method is not very effective for oxygen transfer. A sparger (a perforated tube) is
more commonly used to ensure adequate oxygen supply.
As the process of agitation may be detrimental to the microorganisms, care is taken

in employing bacteria or other simple organisms that can withstand the forces of agitation.
It is prudent to use organisms that have minimal nutritional requirements and those with a
higher growth rates.
Downstream processing
It is referred to the stage of purifying and recovering the product from the bioreactor.
FERMENTER OR BIOREACTOR
Fermenter is a vessel in which the microorganisms are grown. It is a vessel designed
to carryout fermentation process i.e. biological reactions under the controlled conditions,
Hence it is called as bioreactor. Several criteria should be taken into account for designing
a fermentor. They are
1. Long term operation in aseptic condition
2. Adequate aeration and agitation
3. pH controlling system
4. Sampling facility
5. Temperature controlling system
6. Minimum labour in operation, harvesting, cleaning and maintenance
7. Suitable for a variety of process
8. Fermentor is provided with limited amount of medium containing all the materials at
optimum environmental condition
FERMENTATION PROCESS
The word fermentation is derived from a latin verb “Fervete” means to boil. The
definition of fermentation is the breakdown of larger molecules (metabolism). For example
carbohydrates are breakdown into simple ones by micro organisms for their enzymes. In a
microbiological way, fermentation is defined as “any process for the production of useful
products through mass production of microorganisms. In a biochemical sense, the
fermentation means the several oxidation-reduction reactions in which organic compounds
are used as source of carbon and energy, act as acceptors or donars of hydrogen ions.
ANIMAL BIOREACTORS
Transgenesis is wonderfully utilized for production of proteins for
pharmaceutical and medical use. In fact, any protein synthesized in the human body can
be made in the transgenic animals, provided that the genes are correctly programmed. The
advantage with transgenic animals is to produce scarce human proteins in huge quantities.
Thus, the animals serving as factories for production of biologically important products are
referred to as animal bioreactors or sometimes pharm animals. Some transgenic animals
that serve as bioreactors are listed
 Transgenic cow for the production of lactoferrin and interferons.
 Transgenic goat to synthesize tissue plasminogen activator, and
antithrombin III
 Transgenic mouse for the production of immunoglobulins, and

urokinase.
 Transgenic pig to produce hemoglobin.
ANTIBODIES
Antibodies are proteins or Immunoglobulins (Igs) stimulated by specific antigen made
up of two heavy and two light chains creates two functionally important sites, one recognize
and bind the antigen (Fab)and the Fc portion at the other end of molecule which mediate
the function of antibody. Normally β-cells secrete antibodies in correspondence to antigene.
Individual antibody posses atleast two sites i.e. antigen binding site. The result of a
antibody antigen interaction depends on the nature of antigen and antibody and may
include
1. Enhanced phagocytosis
2. Complement activation
3. Neutralization of toxin
4. Inactivation of proteins
5. Inhibition on binding of toxins or bacteria to surface
STRUCTURE OF ANTIBODIES
Human antibodies have two binding sites therefore, they are bivalent which is also
called as monomer because it is the simplest antibody. Two monomers are interconnected
by joining (J) chain. Similarly in a pentamer Ig molecules five monomers are held in
position by a J-chain.
There are several types of antibody molecule, which differ in structure and function.
There are five classes
a. Ig G – gamma
b. Ig M – Mu
c. Ig A – Alpha
d. Ig D – Delta
e. Ig E – Episilon
ANTIGEN
The antigens (Ag) or Immunogen is a large organic molecule capable of stimulating
the production of specific antibody with which it may chemically combine. The ability of
antigens to induce antibody formation is known as antigenicity.
Nature and properties of Antigens:
1. Chemically different types of antigens are found
a) Proteins
b) Nucleo proteins (Nucleic acid + protein)
c) Lipo proteins (lipid + protein)
d) Glycoproteins (carbohydrate + protein)
e) Large polysaccharides
2. Many antigens posses different types of determinants (site in which antibody binds)
on their surface is the immune system may produce several antibodies against a single
antigen.
3. Most of antigens have molecular weight of 10000 daltons or more.
POLYCLONAL AND MONOCLONAL ANTIBODIES

POLYCLONAL ANTIBODIES
A preparation of antibody molecules that arises from several different clones of
cells is called a polyclonal antibody. It is a mixture of antibody molecules that bind
to different parts of the antigen and with different binding affinities.
If an antigen is injected into an animal, a number of antibody-producing cells
will bind that antigen, albeit with varying degrees of affinity, and so the antibody
which appears in the bloodstream will have arisen from several clones of cells, that
is it will be a polyclonal antibody. Different antibody molecules in a preparation of
polyclonal antibody will bind to different parts of the macromolecular antigen and
will do so with different binding affinities. The binding region recognized by any one
antibody molecule is called an epitope. Most antibodies recognize particular surface
structures in a protein rather than specific amino acid sequences (i.e. the epitopes
are defined by the conformation of the protein antigen). A preparation of polyclonal
antibodies will bind to many episodes on the protein antigen.
MONOCLONAL ANTIBODIES
Antibody produced by a single clone of cells is a monoclonal antibody; all the
antibody molecules are identical and bind to the same antigenic site with identical
binding affinities. Monoclonal antibodies can be generated in large amounts by
creating a cell fusion (called a hybridoma) between an antibody producing cell and a
myeloma cell.
If a single clone of antibody-producing cells could be isolated, then all of the

antibody produced from that clone would be identical; all antibody molecules in
such a monoclonal antibody preparation would bind of the same antigen epitope.
The problem is that if an individual anti-body producing cell is isolated and
grown in culture, its descendants have a limited lifespan that severely limits their
use for the routine preparation of monoclonal antibodies. In 1975, Milstein and
Kohler discovered how monoclonal antibodies of almost any desired antigen
specificity can be produced indefinitely and in large quantities. Their method was to
fuse a B lymphocyte producing antibody of the desired specificity with a cell derived
from a cancerous lymphocyte tumor, called a myeloma cell, which is immortal. The
cell fusion is called a hybridoma, which is both immortal and secretes the same
specific antibody originally encoded by the B lymphocyte.
Monoclonal antibodies produced using this technology are now common tools
in research because f their very high specificity for example, they can be used to
locate particular molecules within cells or particular amino acid sequences within
proteins. If they are first bound to an insoluble matrix,, they are also extremely
useful for binding to and hence purifying the particular molecule from crude cell
extracts or fractions. They are also increasingly of use in medicine, both for
diagnosis and as therapeutic tools, for example to inactivate bacterial toxins and to
treat certain forms of cancer.
HYBRIDOMA TECHNOLOGY
Conventional methods adopted in the laboratory for the production of antisera

against antigens lead to the formation of heterogeneous antibodies. Among these
antibodies a few may have the desired properties but are found with many other
antibodies which undoubtedly are not required. A simple, convenient and desirable
method for the large scale production of specific antibodies remained a dream for
immunologists for a long period. In 1975, George Kohler and Cesar Milstein (Nobel
Prize 1984) made this dream a reality. They created hybrid cells that will make
unlimited quantities of antibodies with defined specificities, which are termed as
monoclonal antibodies (McAb). This discovery, often referred to as hybridoma
technology, has revolutionized methods for antibody production.
Principle
This is based on the fusion between myeloma cells (malignant plasma cells)
and spleen cells from a suitably immunized animal. Spleen cells die in a short
period under ordinary tissue culture conditions while myeloma cells are adopted to
grow permanently in culture. Mutants of myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyltransferase (azaquinine resistant) or
thymidine kinase (bromodeoxyuridine resistant). These mutant cannot grow in a
medium containing aminopterin, supplemented with hypoxanthine and thymidine
(HAT medium). Hybrids between the mutant myeloma cells and spleen cells can be
selected and cultured in HAT medium.
From the growing hybrids, individual clones can be chosen that secrete the
desired antibodies (monoclonal origin). The selected clones like ordinary myeloma
cells can be maintained indefinitely. In short, the hybridoma technology for the
production of monoclonal antibodies involves the following steps.
1. Immunization of appropriate animals with antigen (need not be pure) under
study.
2. Fusion of suitable drug resistant myeloma cells with plasma cells, obtained
from the spleen of the immunized animal.
3. Selection and cloning of the hybrid cells that grow in culture and produce
antibody molecules of desired class and specificity against the antigen of
interest.
Hybridoma technology can make available highly specific antibodies in abundant
amounts. The clones once developed are far cheaper than the traditionally
employed animals(horses, rabbits) for producing antibodies. The clones developed
from the hybrids will also ensure constancy of the quality of the product and will
also avoid the batch to batch variation inherent in the conventional methods.
APPLICATIONS OF MONOCLONAL ANTIBODIES

The antibodies produced by hybridoma technology have been widely used for
a variety of purposes. These include the early detection of pregnancy, detection and
treatment of cancer, diagnosis of leprosy and treatment of autoimmune disease.
ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA)

Specific antibodies can also be used to quantify the amount of the
corresponding antigen in a biological sample. Several types of
immunological assays exist. An increasingly popular version is enzyme-
linked immunosorbent assay (ELISA) see fig. which can readily detect
and quantify less than a nanogram of a specific antigenic protein. In
ELISA, the specific antibody is coupled to a solid support. A convenient
format for ELISA is to use a plastic tray that has molded wells in it (a
microtiter tray) where the antibody has been coupled to the plastic
forming the wells. Samples to be assayed are added to the wells. If
antigen is present that is recognized by the antibody, it becomes bound
(fig). the wells are then washed to remove unbound protein and
incubated with a second antibody that recognizes the protein but a
different epitope than the first antibody (fig). The second antibody is
attached to an enzyme that can catalyze the conversation of a colorless
or nonfluoresecent substrate into a colored or fluorescent product. The
intensity of the color or fluorescene produced for each sample is then
measured to determine the amount of antigen present in each sample.
Several machines are now commercially available that scan the wells of
microtiter plates following ELISA and quantify the amount of antigen
bound in each well.
Enzyme-linked immunosorbant assay (ELISA) is a non-isotopic immunoassay.
An enzyme is used as a label in ELISA in place of radioactive isotope employed in
RIA. ELISA is an sensitive as or even more sensitive than RIA. In addition, there is
no risk of radiation hazards (as is the case with RIA) in ELISA.
Principle
ELISA is based on the immunochemical principles of antigen-antibody
reaction. The stages of ELISA, depicted in fig. are summarized.
1. The antibody against the protein to be determined is fixed on an inert solid
such as polystyrene.
2. The biological sample containing the protein to be estimated is applied on
the antibody coated surface.
3. The protein antibody complex is then reacted with a second protein specific
antibody to which an enzyme is covalently linked. These enzymes must be
easily assayable and produce preferably coloured products. Peroxidase,
amylase and alkaline phosphatase are commonly used.
4. After washing the unbound antibody linked enzyme, the enzyme bound to
the second antibody complex is assayed.
5. The enzyme activity is determined by this action on a substrate to form a
product (usually coloured). This is related to the concentration of the
protein being estimated.
The principle for the use of the enzyme peroxidase in ELISA is illustrated next.
Applications
ELISA is widely used for the determination of small quantities of proteins
(hormones, antigens, antibodies) and other biological substances. The most
commonly used pregnancy test for the detection of human chorionic gonadotropin
(hCG) in urine is based on ELISA. By this test, pregnancy can be detected within
few days after conception. ELISA is also been used for the diagnosis of AIDS.
GENE TRANSFROMATION
AGROBACERIUM MEDIATED GENE TRANSFER
The “Ti” plasmid, or tumor-inducing plasmid is the most commonly used plant-
cloning vector used for cloning genes in plants. This plasmid is found in cells of the
bacterium known as Agro-bacterium tumefaciens. This bacterium normally lives in soil.
Agrobacterium causes crown gall tumors in plants at the site of infection. The Ti plasmid is
responsible for the development of the disease.
The Ti plasmid consists of a segment called as the T-DNA or the transforming DNA.
When Agrobacterium infects a plant, it does not enter the cells of the plant. Instead, it
lodges itself in the intercellular spaces. The T-DNA separates from the Ti plasmid and gets
incorporated into the host genome.
This property makes the Ti plasmid a useful plant-cloning vector and it is this
property that is exploited to shuttle a foreign gene into the plant cells. The tumor causing
genes of the Ti plasmid are excised so that the bacterium can no longer cause the disease.
The desirable foreign gene that needs to be incorporated into the plant cells is inserted in
the same place from where the disease causing genes were removed from the plasmid. The
Agrobacterium is made to infect the plant by artificially creating an injury to the plant. The
modified Ti plasmid, carrying the desirable exogenous genes and the intact T-DNA enters
the protoplast of the plant cell.
The foreign DNA with the
desirable genes and the T-DNA gets
incorporated into the host genome.
New plants can be generated through
these engineered protoplasts. Each
cell of the such generated plants will
carry the desirable exogenous gene.
A major drawback of
Agrobacterium was that in nature it
infects only the dicots. Most of our
important crops are monocots. Thus,
it used to be difficult to modify our
important crop plants.
Using the processes of
microinjection, electroporation,
particle bombardment and biolistics
we can now insert the desirable
exogenous genes into plant cell types that are not susceptible to A. tumefaciens transfection.
Fig. Agrobacterium mediated gene transformation

BIOPLASTICS
Bio-based plastic : This is a very broad term that basically means a substance was derived
from plant-based material, whether wholly or in part. Starch and cellulose are two of the
most common renewable feedstocks used to create bioplastics; these typically come from
corn and sugarcane. Bio-based plastics are distinguished from much more common
petroleum-based polymers (visit our Plastics page to learn more about conventional types of
plastics). Although many would assume that anything “bio-based” is biodegradable, this is
not the case.
Biodegradable plastic: Whether a plastic is biomass- or petroleum-based is a different

question than whether it will biodegrade (a process by which microbes break down material
if conditions are suitable). Technically, all materials are biodegradable, but for practical
purposes, only those that degrade within a relatively short period of time (weeks to months,
usually) are considered biodegradable. As mentioned in the previous bullet, not all bio-
based plastics are biodegradable; bioplastics that don’t degrade within a few months or
years are sometimes called “durable.” Conversely, there are petroleum-based plastics that
will degrade faster under optimal conditions than will their organic biomass counterparts.
Compostable plastic: According to the American Society for Testing and Materials,
compostable plastics are those which are "capable of undergoing biological decomposition in
a compost site as part of an available program, such that the plastic is not visually
distinguishable and breaks down to carbon dioxide, water, inorganic compounds, and
biomass, at a rate consistent with known compostable materials (e.g. cellulose), and leaves
no toxic residue." The requirement for no toxic residue is one of the distinguishing
characteristics between compostable and biodegradable. Also of note, some plastics can be
composted in home gardens, whereas others require commercial composting (where
temperatures get much higher and the composting process happens faster).
The first known bioplastic, polyhydroxybutyrate (PHB), was discovered in 1926 by a

French researcher, Maurice Lemoigne, from his work with the bacterium Bacillus
megaterium. The significance of Lemoigne’s discovery was overlooked for many decades, in
large part because, at the time, petroleum was inexpensive and abundant. The petroleum
crisis of the mid-1970s brought renewed interest in finding alternatives to petroleum-based

products. The rise ofmolecular genetics and recombinant DNA technology after that time
further spurred research, so that by the beginning of the 21st century the structures,
methods of production, and applications for numerous types of bioplastics had become
established.
Bioplastics that were either in use or under study included PHB and
polyhydroxyalkanoate (PHA), both of which are synthesized within specialized microbes, as
well as polylactic acid (PLA), which is polymerized from lactic acid monomers produced by
microbial fermentation of plant-derived sugars and starches. Degradation of the chemical
links between the monomers in these plastics is brought about by microorganisms or by
water, making bioplastics highly desirable materials for fabrication into biodegradable
bottles and packaging film. In addition, because the degradation products are natural
metabolites, the polymers are of interest in medical applications, such as controlled-release
drug packaging and absorbable surgical sutures.
Bioplastics currently make up an insignificant portion of total world production of

plastics. Commercial manufacturing processes are plagued by low yields and are expensive.
However, improvements in metabolic and genetic engineering have produced strains of
microbes and plants that may significantly improve yields and production capabilities while
reducing overall costs. These factors, when added to increasing oil prices and growing
environmental awareness, may expand the market for bioplastics in the future.
Polylactic acid (PLA): One of the most common bioplastics
Polylactic Acid is biodegradable and has characteristics similar to polypropylene

(PP), polyethylene (PE), or polystyrene (PS). It can be produced from already existing
manufacturing equipment (those designed and originally used for petrochemical industry
plastics). This makes it relatively cost efficient to produce. Accordingly, PLA has the second
largest production volume of any bioplastic (the most common typically cited as
thermoplastic starch, which is commonly used in food storage bags and food utensils).
There are a vast array of applications for polylactic acid. Some of the most common
uses include plastic films, bottles, and biodegradable medical devices (e.g. screws, pins,
rods, and plates that are expected to biodegrade within 6-12 months). For more on medical
device prototypes (both biodegradable and permanent), read here. PLA constricts under heat
and is thereby suitable for use as a shrink wrap material. Additionally, the ease with which
polylactic acid melts allows for some interesting applications in 3D printing. On the other
hand, its low glass transition temperature makes many types of PLA (for example, plastic
cups) unsuitable to hold hot liquid.
HERBICIDE TOLERANT CROPS
The most significant and widespread applications of recombinant DNA technology

in practical plant breeding to date are the development of Bt cultivars and herbicide
tolerance in plants. The soil-borne bacterium, Bacillus thuringiensis, is the source of
the gene used in the development Bt products. The gene encodes the inactive form of a
protein, Bt toxin, this is toxic to various herbivorous insects when ingested and
converted to it toxic form (delta endotoxin) in the gut of the insect. Over 100 different
variation of the Bt toxin have been identified, as well as a variety of associated target
insect specificity. The toxins classified as Cry1a group target Lepidoptera or butterfly
group, while the toxins in Cry3 group target beetles. Scientists have cloned the bacterial
genes, which are then transferred into plants to provide resistance to target pest,
thereby eliminating the need for pesticides. The major crops that have received such
treatment include corn, cotton, and potato.
Researchers are pursuing additional naturally occurring insecticidal compounds

as alternatives to the Bt technology. These included chitinase, lectins, alpha-amylase
inhibitors, cystatin, and proteinase inhibitors.
Why engineer herbicide resistant crops?

A successful herbicide should destroy weeds only, leaving the economic plant
unharmed. Broad-spectrum herbicides (non-selective) are attractive but their use in
crop production can be problematic, especially in the production of broadleaf crops
such as soybean and cotton. There is a general lack of herbicides that will discriminate
between dicot weeds and crop plants.
Pre-plant applications may be practical to implement. However, once the crop is
established and too tall for the safe use of machinery, chemical pest management
becomes impractical. Grass crops (e.g., wheat, corn) may tolerate broadleaf herbicides
better that the reverse situation. Consequently, when cereal crops and broadleaf crops
are grown in rotation or adjacent fields, the broadleaf plants are prone to damage from
residual herbicides in the soil, or drift from herbicides applied to grasses. When a crop
field is infested by weed species that are closely related to the crop (e.g., red rice in rice
crop or nightshade in potato crop), herbicides lack sensitivity enough to distinguish
between the plants.
To address these problems, one of two approaches may be pursued: (i) the
development of new selective post emergent herbicides or (ii) genetic development of
herbicide resistance in crops to existing broad-spectrum herbicides. The latter strategy
would be advantageous to the agrochemical industry (increased market) and
farmers (safer alternative of pesticides that are already in use). New herbicides are
expensive to develop and take time.
Modes of action and herbicide resistance mechanisms
Most herbicides are designed to kill target plants by interrupting a metabolic stage in
photosynthesis. Because all higher plants photosynthesize, most herbicides will
kill both weeds and desirable plants. Plants resist phytotoxic compounds via one of
several mechanisms:
• The plant or cell does not take up toxic molecules because of external barriers
such as cuticles.
• Toxic molecules are taken but sequestered in a sub- cellular compartment away
from the target (e.g., protein) compounds the toxin was designed to attack.
• The plant or cell detoxifies the toxic compound by enzymatic processes into
harmless compounds.
• The plant or cell equipped with resistance genes against the toxin may produce
a modified target compound that is insensitive to the herbicide.
• The plant or cell overproduces the target compound for the phytotoxin in large
amounts such that it would take a high concentration of the herbicide to
overcome it.
Environmental impact
One of the complaints launched against the deploy- ment of GM crops was that
using plants as “pesticides” could have ecological consequence by being harmful to
non-target organisms. This concern has not been significantly substantiated, at least
in the case of Bt products. The tendency is to increase the dose rate of pesticides as
pests develop resistance to them. However, this is counterproductive in that such an
action only serves to intensify the selection pressure for resistant pests.
Pest resistance
Repeated and widespread use eventually results in resistance of pests to pesticides.
The high rates of pesticide use create a situation for intense selective pressure that
increases resistance to pesticides. Also, whereas older pesticides were designed to
attack multiple sites in their target organism, modern pesticides are designed to be
more specific in action (often one biochemical pathway), thereby increasing the
chance for development of resistance.
Resistance of insects to the defense mechanisms of plant is well-documented.

Resistance to a number of commonly used insecticides has been reported in
hundreds of insects and mites. There has been the concern that, sooner or later,
resistance of insects to Bt cultivars would surface. Laboratory resistance to Bt has
been demonstrated for some major pests, such as the tobacco budworm, Colorado
potato beetle, and the diamondback moth. Some reports have indicated field
resistance for the diamondback moth to Bt. Also, resistant populations of the
bollworm (Helicoverpa zea) have been reported in some fields in Mississippi and
Arkansas.
Researchers are investigating how to extend the Bt durability in transgenic

cultivars. Current approaches include the engineering of cultivars with very high
levels of insecticidal crystal proteins. This way, only insects that have a high-level
resistance gene can survive after feeding on these new cultivars. Another
approach is to search for new insecticidal genes for developing new transgenic
plants that can express multiple insecticidal genes that target different sites in the
insect. Insects that can overcome this strategy are those with multiple resistance
genes.
Resistance to herbicides is also growing, the confirmed cases approaching 300.
The concern for growers and researchers is that some pests are resistance to multiple
pesticides, while some are resistant to all the pesticides that are legally approved for
their control!
BASIC CONCEPTS
Aseptic culture refers to in vitro culture of tissues free from micro-organisms like
bacterium, fungi etc.
Auxins refers to a group of growth hormones which causes cell elongation, apical
dominance, root initiation Eg : NAA, IAA, 2,4 –D etc.
Batch culture refers to growth of cell suspension culture in fixed volume of liquid
medium.
Browning: Discolouration of the in vitro culture due to a pathogen or nutritional problem
or phenolic oxidation. Leads to death of the culture.
Callus refers to a unorganized mass of undifferentiated cells derived from explants.
cDNA cloning: A method of cloning the coding sequence of a gene, starting with its mRNA
transcript. Normally used to clone a DNA copy of a eukaryotic mRNA.
cDNA library : A collection of cDNA clones
cDNA: Complementary DNA, a fragment of DNA produced from mRNA by reverse
transcription.
Cell culture refers to growing of cells in vitro.
Central dogma: The concept that genetic information generally can flow only from DNA to
RNA to protein. It is now known that information can flow back from RNA to DNA as
in retroviruses.
Clonal propagation refers to asexual propagation of plants.
Clone refers to a group of cells, tissues or plants which are genetically identical to the
mother cell or plant.
Continuous culture : When suspension culture is continuously supplied with nutrients
by continuous flow of fresh medium it is called as continuous culture. The volume
of t h e culture medium is normally constant.
Cosmids refers to plasmid vectors into which phage lambda cos sites were inserted. As a
result the plasmid DNA can be packed in the phage coat. It can carry around 40kb of
DNA fragment. Its replication is same as plasmid.
Culture refers to growing of cells or tissues o f plant in nutrient medium under aseptic
conditions. Depending upon the explant source it can be named as anther culture,
pollen culture, embryo culture, protoplast culture, callus culture etc.
Cybrid refers to cytoplasmic hybrid. A somatic hybrid with nucleus from one parent and
cytoplasm from both the parents.
Cybrid: It refers to a cytoplasmic hybrid produced by fusion of t w o protoplasts.
Cytokinins refers to a class of growth hormones which causes cell division, shoot
differentiation, breaking of apical dominance etc. Kinetin, Zeatin etc.
de novo means from the beginning, arising anew.
Dedifferentiation : Reversion of differentiated cells to non-differentiated condition.
Differentation is a process in which unspecialized cells develop into organs with specific
function like roots, shoots etc.
DNA amplification : Multiplication of a piece of DNA into millions of copies by

polymerase chain reaction (PCR).
DNA fingerprinting: A technique in which an individual’s DNA is analysed
DNA ligase : The enzymes which joins DNA fragments
Embryo culture refers to the culture of embryos in vitro on nutrient medium.
Embryogenesis : The process by which an embryo develops from a fertilized egg cell or
asexually from a group of cells.
Embryoids refers to embryo like structures formed in culture. They are also called as
somatic embryos.
Excise: Cut out (with knife or scalpel) and prepare a tissue or organ for culture.
Explant refers to a piece of tissue or callus used to initiate tissue culture.
Genetic Engineering refers to changing the genetic architecture of an organism directly

at the DNA level by molecular techniques.
Hardening refers to process of gradual acclimatization of tissue culture plants in green

house.
In vitro refers to any process which is carried out in sterile condition in the laboratory.
In vitro means “in glass”.
In vivo refers to any process occur in a whole organism under field condition. In vivo
means “in living”.
Meristem refers a group of actively dividing cells from which other tissue such as
root, shoot, leaf, flower etc. are derived.
Meristemoid r e f e r s t o a group of cells in a callus giving rise to adventitious
shoots or roots.
Micropropagation refers to clonal propagation of plants under in vitro conditions.
Nutrient medium : A solid or liquid medium containing nutrients such as inorganic
salts, a carbon source, vitamins and growth regulators used for culturing plants or
microorganisms is called as nutrient medium.
Organ culture refers to culture of plant organs i n v i t r o l e a d i n g t o l i k e root or
shoot tips.
Organogenesis refers to formation of organs directly from a non -meristematic
explant.
Parasexual hybridization refers to hybridization by non-sexual methods like protoplast
fusion.
Passage time: The time interval between two successive sub-cultures is called as passage
time.
Plant tissue culture refers to the in vitro cultivation of cells, tissues, organs, embryos,
seeds and protoplasts on nutrient media under aseptic conditions in a laboratory.
Plasmids are extrachromosomal, autonomous circular DNA found in certain bacteria,
capable of autonomous replication. They can transfer genes between bacteria and
act an important tool in genetic engineering.
Protoplast refers to a cell without cell wall. The cell wall may be removed mechanically or
by using cell wall digesting enzymes.

Recognition sites refers to specific nucleotide sequence composed of 4-8 nucleotides to
which restriction endonuclease binds.
Recombinant DNA or rDNA refers to the technique of cutting and recombining DNA from
different organisms.
Restriction enzyme is an endonuclease which has the ability to cut (cleave) DNA at the
point where a certain base sequence occurs.
Sub culture refers to establishment of a new culture by transfer of some of the cells
from a previous culture to a fresh medium aseptically.
Suspension culture refers to culturing of cells in agitated liquid medium.
Totipotency is the ability of a s i n g l e c e l l to develop into a whole organism.
Transformation: The process of transfer of foreign DNA to an individual using vectors.
Transgenic refers to organisms with foreign DNA inserted in its genome. Also called as
Genetically Modified Organism (GMO). It may be a transgenic plant or animal of
microbe.
Vector refers to a small self replicating DNA molecule in which DNA of interest is inserted.
Vectors carry the inserted DNA into the host cell for multiplication. Eg plasmid, phage
etc.

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B.Sc. Hons. (Agri.) / Dr. T.

Sabesan / GPB 316 Plant Biotechnology /

GPB 316 PLANT BIOTECHNOLOGY

Dr. T. SABESAN, Ph.D.

DEPARTMENT OF GENETICS & PLANT BREEDING

Biotechnology broadly refers to development of techniques for the application of

In a narrow definition, biotechnology refers to genetic manipulation of organisms for

HISTORY OF PLANT TISSUE CULTURE

Gottlieb Haberlandt is regarded as the Father of plant tissue culture as he predicted

for cloning isolated single protoplasts.

DEVELOPMENT OF BIOTECHNOLOGY IN INDIA

The promote biotechnology in India the Department of Biotechnology (DBT) was

SCOPE AND IMPORTANCE OF BIOTECHNOLOGY

criminals; Development of recombinant vaccines like human hepatitis B etc. by genetically

Industrial Biotechnology: It involves large scale production of alcohol and antibiotics by

Biotechnology and Environment: Environmental problems like pollution control, depletion

Landmark discoveries in the field of molecular biology

1978 Crossing potato and tomato to produce pomato by somatic hybridization by

1. Define plant biotechnology.

2. NUTRITIONAL REQUIREMENTS OF TISSUE

Alternative methods of support include perforated cellophane, filter paper bridges,

The nutrient elements n a m e l y , nitrogen (N), phosphorus (P), potassium (K),

IV. CARBON SOURCE

c. Other organic supplements: It include organic extracts like protein (casein)

d. Antibiotics: To prevent the infection of microbes, antibiotics such as Streptomycin or

EFFECT OF GROWTH REGULATORS:

 Low cytokinin / more auxin regenerated to only root part.

 Medium cytokinin / medium auxin regenerated to both shoot and root ,

 Medium cytokinin. low auxin only regenerated to callus.

c. Gibberillins and Abscisic acid

GA3 is most commonly used gibberillin. I t e nhances callus growth and

NUTRIENTS AND THEIR PHYSIOLOGICAL ROLE

Nitrogen (N) Component of proteins, nucleic acids some co-enzymes

Phosphrous (P) Component of nucleic acids, energy transfer, component of

Potassium (K) Regulates osmatic potential, principle inorganic cation.

Calcium (Ca) Cell wall synthesis, membrane function cell signaling.

Magnesium (Mg) Enzyme co-factor, component of chlorophyll.

Sulphur (S) Component of some amino acids (Methionine, cysteine) some

Chlorine (Cl) Required for photosynthesis

Iron (Fe) Electron transfer as a component of cytochromes

Managanese (Mn) Enzyme co-factor

Cobalt (Co) Component of some vitamins

Copper (Cu) Enzyme co-factor electron transfer reaction

Zinc (Zn) Enzyme co-factor chlorophyll biosynthesis

Molybdenum (Mo) Enzyme co-factor component of nitrate reductase.

PREPARATION OF NUTRIENT MEDIUM

The composition of MS (Murashige & Skoog) media is given below

Methods of media preparation

Another method of preparing media is to prepare concentrated stock solutions. The

Stock solutions of macronutrients can be prepared at 10 times the concentration of

Stock solution of Iron is stored in amber coloured bottles. Substances which

3.TECHNIQUES IN PLANT TISSUE CULTURE

STAGE 0: SELECTION OF EXPLANT

APPLICATIONS OF PLANT TISSUE CULTURE

Explant: It is a major source of contamination. Plants may harbor microorganisms either

EFFECT OF CONTAMINANTS ON TISSUE CULTURE PLANTS

Propagation of plants vegetatively by cutting, budding, grafting etc. involves only

(i). PROLIFERATION OF PRE-EXISTING MERISTEM/AXILLARY BUD PROLIFERATION

i. Emergence of adventitious organs directly from the explant (Direct organogenesis /