Includes all the techniques to develope transgenic plants resistant to pests and herbicides.

1. TISSUE CULTURE IN PLANT PROTECTION
Submitted By :- Jayant Yadav, C.C.S.H.A.University, Hisar, Haryana

2. Plant Tissue Culture : Terminology
Totipotency : Ability of a cell to divide into any type of cell.
Explant : Mass of tissue or cells
Solid medium – Callus culture. Tissue can be immature embryo, apical meristem,
root tip
Liquid medium – suspension culture. Tissue should be protoplast (cells with no cell
wall), micro or macrospores.
Nutrients and hormones are used for growth and development. Eg : 2,4
dichlorophenoxyacetic acid (analogous to auxin)
Callus : Undifferentiated cell which form a crystalline white layer on solid medium.

3.  It is the aseptic method of growing cells and organs
such as meristems, leaves, roots etc either in solid or
liquid medium under controlled conditions.
 Small pieces of viable tissues called ex-plant are
isolated from parent plants.
 These pieces are grown in a defined nutritional
medium and maintained in controlled environment
for prolonged period under aseptic conditions.

4.  Steps Involved In Plant Tissue Culture Technique
1. Selection of plant
2. Isolation of Explant
3. Sterilization of Explant
4. Inoculation of Explant
5. Incubation
6. Initiation of Callus
7. Sub Culturing
8. Regeneration
9. Hardening

5. Fig:- Steps involved in Plant Tissue Culture

6. The process of regenerating a plant from a single cell may cause three types of
Alterations :-
1. Temporary Physiological change
2. Epigenetic change
3. True genetic changes
An Entire Plant Can Be Regenerated from a Single Cell. Small samples of tissue, or even
single plant cells may be cultured in vitro. Under appropriate conditions, these may
regenerate into complete plants.

7. Callus or Liquid Culture of Plant Cells Can Regenerate Entire Plants. In callus culture a
mass of undifferentiated cells grows on a solid surface. In liquid culture, separated single
cells are grown. Both types of cultures can develop shoots and roots with appropriate
manipulation of plant hormone levels.
FIGURE 14.3

8. Applications Of Plant Tissue Culture In Crop Improvement
1. Helps in mass multiplication of plants which are difficult to
propagate through conventional methods.
2. Helps in rapid multiplication of ornamentals , fruits and
aromatics which can not be propagated through conventional
methods.
3. Virus free plants can be produced through plant tissue culture.
4. Production of secondary metabolites. Eg:- Nicotine from
Nicotiana rustica .
5. Inter specific and inter generic hybrids can be produced through
embryo rescue technique which is not possible through
conventional methods.
6. Development of transgenic plants through genetic engineering.

9. Tissue Culture is also used when
plants are Genetically Engineered
Plants that receive a new gene are
called Transgenic plants or GMOs
A genetically engineered plant has a
new gene.

10.  Transfer of gene from an organism into a plant cell and its integration
into the genetic material of the later usually employing recombinant-
DNA technique is known as Genetic Engineering of plants.
 The plant obtained through genetic engineering contain a gene (or)
genes usually from an unrelated organism.
 Such genes are called as ‘ Transgenes’ and plants containing transgenes
are known as ‘ Transgenic plants’ .
 The development of transgenic plants is the result of an integrated
application of rDNA technology , gene transfer methods and tissue
culture techniques.
 First transgenic plant was produced in 1983 when a tobacco line
expressing Kanamycin resistant was produced.
 Flavr Savr tomato was first transgenic variety to reach market . Fruit of
this variety remain fresh for a prolonged period.

11. TRANSGENIC PLANTS
NUTRITIONAL
QUALITY
BIOTIC STRESS
TOLERANCE
ABIOTIC STRESS
TOLERANCE
PHARMACEUTICALS
& EDIBLE VACCINE
HYBRID DEVELOPMENT
FOR HIGHER YIELD
ENHANCED
SHELF LIFE
INDUSTRIAL
PRODUCTS

12. Why gene transfer?
• Crop improvement
• Disease resistance
• Stress tolerance
• Improved performance
• Value-added traits
Basic studies
• Gene expression
• Reverse genetics - understanding functioning of unknown genes
• Biochemistry and metabolism
Gene transfer strategies: Systems and vectors
• Agro bacterium
• Direct DNA uptake
• Virus-based vectors

13.  Indirect Methods
• Agro-bacterium mediated gene transfer
• Viral vectors
Direct Methods
• Electroporation
• Particle bombardment
• Lypofection
• Micro injection
• Pollen transformation
• Chemical methods

15. Characteristics of an ideal vector:
 Should be of small size ( low molecular weight).
 Confer a selectable phenotype on the host cells so that
transformed cells can be selected.
 Contain single sites for a large number of restriction enzymes to
enable the efficient production of recombinant vectors.

16.  A. tumefaciens is a gram-negative soil bacterium which naturally transforms
plant cells, resulting in crown gall (cancer) tumors on plants like grapes, walnuts,
apples and roses.
 Tumor formation is the result of the transfer, integration and expression of
genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA
(transferred DNA) .
 The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid
found in A.tumefaciens . Crown gall formations in plants depends on the
presence of Ti plasmid.

18. Essential Elements for Carrying a Transgene on Ti Plasmids
The T-DNA segment contains both a transgene and a selective marker or reporter gene. These
have separate promoters and termination signals. The marker or reporter gene must be expressed
all the time, whereas the transgene is often expressed only in certain tissues or under certain
circumstances and usually has a promoter that can be induced by appropriate signals.
Ti plasmid
structure & function

19. 19
Transfer of Modified Ti Plasmid into a Plant
Agrobacterium carrying a Ti plasmid is added to plant tissue growing in culture. The T-DNA
carries an antibiotic resistance gene (neomycin in this figure) to allow selection of successfully
transformed plant cells. Both callus cultures (A) and liquid cultures (B) may be used in this
procedure.
FIGURE 14.6

21.  Several insect resistant transgenic varieties have been
tested and released in crops like tomato, potato, cotton
and maize.
Three approaches have been adopted to develop insect
resistance transgenic plants by transferring insect
control protein genes :-
1) Introduction of resistant genes from higher plants.
2) Introduction of resistant genes from animals.
3) Introduction of resistant genes from micro organisms.

22.  Proteinase inhibitors
o Deprive the insects by interfering with the digestive
enzymes of the insects. Eg:- Cowpea trypsin inhibitor
(CpTi) gene found in cowpea ( V.unguiculata ) produces
antimetabolite substances that provide protection
against the major storage pest Bruchid beetle
(Callosobruchus maculatus).
 Amylase inhibitors
o The α-amylase inhibitor gene (α-A1-Pv) isolated from P.
vulgaris has been expressed in tobacco. This α-amylase
inhibitor protein blocks the larval feeding in the mid gut.
The larva generally secretes a gut enzyme called α-
amylase that digests the starch. By adding a protein that
inhibits insect gut α-amylase the insect can be starved
and it dies.

23. Lectins
These are plant glycoproteins used as insect toxins. Lectin
from snowdrop (Galanthus nivalis) is known as GNA because
it has shown the activity against aphids.
2. Introduction of resistance genes from animals
Work in this area has, so far, involved primarily serine-
proteinase-inhibitor genes from mammals and the tobacco
hornworm (Manduca sexta). Bovine pancreatic trypsin
inhibitor (BPTI), α1-antitrypsin (α1AT) and spleen inhibitor (SI)
have been identified like possible mammals genes which can be
introduce into plants to do them resistant to insects.

24.  The cry gene of Bacillus thuringenesis (Bt) produces a
protein which forms crystalline inclusions in the bacterial
spores. These crystal proteins are responsible for the
insecticidal activities of the bacterial strains.
 Different Cry proteins produced by Bacillus :
 Cry I : kills Butterflies and moths
 Cry II : kills Butterflies and flies
 Cry III : kills beetles
 Cry IV : kills only flies.

25. Domain
I
•7 α-helix
•Helps in membrane
insertion
Domain
II
• β-prism of 3
antiparallel β- sheets
•Helps in receptor
recognition
Domain
III
• β-sandwich of
antiparallel β -sheeets
Crystal structure of Cry
protein

27. Insect Larvae Are Killed by Bt Toxin
Bacterial spores of Bacillus are found on food eaten by the caterpillar. The crystalline
protein is released by digestion of the spore and its breakdown produces a toxin that
kills the insect larvae.
Bacillus ------ Cry proteins ------ Insects eat ------ Cry realases delta endotoxins
(Bt toxin) ------ Toxin binds to intestinal lining ------ holes generated ------ digestive
system disturbed ------ Death of the insects.

28.  Over expression of the target protein: Involves the
titrating out of herbicide by overproduction of the
target protein.
 Mutation of the target protein: The logic behind this is
to find a modified target protein that substitutes
functionally for the native protein.
 Detoxification of the herbicide using a single gene from
a foreign source: Means converting the herbicide to a
less toxic form and removing it from the system.

29. Roundup Ready™ Soybeans
A problem in agriculture is the reduced growth of crops imposed by
the presence of unwanted weeds. Herbicides such as RoundupTM and
Liberty LinkTM are able to kill a wide range of weeds and have the
advantage of breaking down easily. Development of herbicide
resistant crops allows the elimination of surrounding weeds without
harm to the crops.

30.  is a broad spectrum herbicide that is effective against 76 of the
world’s worst 78 weeds.
 Marketed as “ round up” by the American chemical company
Monsanto.
 Is a simple glycine derivative , acts as a competitive inhibitor of the
enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS).
 EPSPS is a key enzyme in the biosynthetic pathways of the aromatic
amino acids phenylalanine, tyrosine and tryptophan.

31.  Isolation of petunia cDNA from Glyphosate resistant tissue
cultures.
 Stepwise selection of petunia cells capable of growing in
presence of the increased amounts of Glyphosate led to the
isolation of cultures in which the levels of EPSPS enzyme was
much higher than normal.
 The resistance was due to higher amounts of the enzyme
produced.

32.  Mutated EPSPS genes have been isolated from a number of
Glyphosate resistant bacteria.
 A mutated aroA gene from Salmonella typhimurium was inserted
between the promoter and the terminator sequences of the ocs
gene of the Agro bacterium tumifaceins Ti plasmid.
 Only a moderate increase in the herbicide tolerance was obtained.

33.  In soil micro organisms, Glyphosate can be degraded by cleavage
of the C-N bond, catalyzed by an oxido reductase, to form amino
methyl phosphonic acid (AMPA) and glyoxylate.
 Gene encoding the enzyme Glyphosate oxidase (GOX) has been
isolated from a soil organism, Ochrobactrum anthropi strain LBAA.
 Transgenic crops such as oilseed rape transformed with this gene
show very good Glyphosate resistance in the field.

34.  Pathogen Derived Resistance (PDR).
a. Interactions involving viral proteins.
b. Involving viral RNA.
 RNA Effects:
a. Satellite sequences.
b. Antisense and Ribozymes.
c. Gene silencing /Co repression.

35.  is the first and the main antiviral transgenic approach used; originally
known as parasite-derived resistance.
 Pathogen sequences are deliberately engineered into the host plants
genome.
 Cross-protection forms the basis of PDR i.e., the presence of the
pathogen sequence may directly interfere with the replication of the
pathogen or may induce some host defense mechanism.

36.  Most successful transgenic approach; involves the expression of the
coat protein (CP) coding sequence.
 CP mediated resistance was first reported with a TMV-tobacco
model system in 1986.
 Some degree of resistance has been found in many cases.
 Variations in the levels of expression are due to transcriptional gene
silencing, transgene position effects and the relationship between
coding sequence and target virus.

37. Satellite sequences:
 Plant viral satellites RNAs are small RNA molecules that are
unable to multiply in host cells without the presence of a
specific helper virus.
 Satellite RNA is not used for viral replication but affects disease
symptoms.
 It was noted that cucumber mosaic cucumovirus (CMV)
symptoms were reduced when the virus was carrying a satellite.
 Transgenic Tobacco and tomato plants expressing CMV
satellite RNA were tested in field in China (1990-1992).

38.  Although some reduction was seen but it was not strong enough
to protect the plants.
 To overcome this a strategy was developed in which satellite RNA
PDR was developed in combination with CMV CPMR.
 The resistance obtained was stronger than that of either CPMR
or satellite –PDR alone.

39.  For Fungal pathogens the genes that code for chitinase and
glucanase enzymes have been isolated.
 These enzymes degrade the cell walls of many fungi without
affecting mammals.
 Genes for the enzymes have been isolated from a number of
sources like plants (rice , barley); bacteria (Serratia marcescens) and
fungi ( Trichoderma harzianum).
 Glucanases (PR proteins) have been used against fungal infection.

40.  When β-1,3 – glucanase (from barley) is expressed in transgenic
tobacco plants under the control of 35S promoter, increased
resistance was seen towards soil borne fungal pathogen Rhizoctonia
solani.
 Ribosome inhibiting proteins (RIP’s) are also used in the defense
strategy. These enzymes remove an adenine residue from a specific
site in the large rRNA of eukaryote and prokaryote ribosome's,
thereby inhibiting protein synthesis.
 Few antimicrobial proteins are used as well .

41.  It may lead to monoculture and threaten crop genetic
diversity with a possible genetic erosion over a period of
time.
 Potential transfer of genes from herbicide-resistant crops
to wild or weedy relatives thus creating "superweeds".
 Insect pests may develop resistance to crops with Bt
(Bacillus thuringenesis) toxin.
 Genetic recombination to generate new virulent strains of
virus, especially in transgenic plants engineered for viral
resistance with viral genes.
 Vector-mediated horizontal gene transfer and
recombination to create new pathogenic bacteria.