TISSUE CULTURE IN PLANT PROTECTION

Submitted By :- Jayant Yadav, C.C.S.H.A.University, Hisar, Haryana

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.

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.

Plant Tissue Culture

Steps Involved In Plant Tissue Culture Technique1. 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

Fig:- Steps involved in Plant Tissue Culture

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.

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

Applications Of Plant Tissue Culture In Crop Improvement1. 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.

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.

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.

Transgenic Plants

TRANSGENIC PLANTS

NUTRITIONALQUALITY BIOTIC STRESS

TOLERANCE

ABIOTIC STRESSTOLERANCE

PHARMACEUTICALS & EDIBLE VACCINE

HYBRID DEVELOPMENTFOR HIGHER YIELD

ENHANCED SHELF LIFE

INDUSTRIAL PRODUCTS

Gene transfer in plantsWhy 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

Indirect Methods• Agro-bacterium mediated gene transfer• Viral vectors

Direct Methods

• Electroporation• Particle bombardment• Lypofection• Micro injection• Pollen transformation• Chemical methods

Gene Transfer Methods

Vector based gene plant transformation:

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.

Plant transformation with the Ti plasmid of Agrobacterium tumefaciens

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.

Ti plasmid:

Essential Elements for Carrying a Transgene on Ti PlasmidsThe 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

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Transfer of Modified Ti Plasmid into a PlantAgrobacterium 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

Engineering For Insect Resistance 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.

Proteinase inhibitorso 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 inhibitorso 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.

1. Introduction of resistance genes from higher plants

LectinsThese 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.

3. Introduction of resistance genes from micro organismsThe 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.

Structure of Cry proteinDomain 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

Bt mode of action

Insect Larvae Are Killed by Bt ToxinBacterial 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.

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.

Engineering For Herbicide Resistance

Roundup Ready™ SoybeansA 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.

Glyphosate Resistance: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.

1. Overexpression of a plant EPSPS gene:

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.

2. Mutant EPSPS genes:

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.

3. Detoxification by heterologous genesIn 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.

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.

Engineering for Virus resistance

Pathogen Derived Resistance:

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.

Interactions involving viral proteins:

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.

RNA Effects:

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).

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.

Engineering for Bacterial and Fungal Resistance

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.

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 .

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.

Limitations of Transgenic Plants

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