Madhusudhan RPAL 0061

Conventional Methods

Non Conventional Methods

The overall process of genetic transformation involves introduction, integration and expression of foreign gene(s) in the recipient host plant.

Result of integrated application of DNA technology, gene transfer methods and tissue culture techniques.

MAS

Genetic Engineering

TransgenicsTransgenics

Plant improvementPlant improvement

The capacity to introduce and express diverse foreign genes in

plants was first described in tobacco by Agrobacterium mediated

(Horsch et al 1984; De Block et al 1984) and vectroless approach

(Paszhkowki et al 1984)

Genetic Transformation has led to the possibility of

transforming crops for enhanced resistance to insects and

pathogens and it is rapidly moving towards commercialization.

STRESS

Biotic StressBiotic Stress

Abiotic Stress

InsectsInsects

WeedsWeeds

HerbivoresHerbivores

PathogensPathogens FungiBacteriaVirusesNematodes

Insect resistance

Viral resistance

Fungal and bacterial disease resistance

Insect resistance

Viral resistance

Fungal and bacterial disease resistance

Resistance to biotic stressesResistance to biotic stresses

Helicoverpa armigeraHelicoverpa armigera

L arboanlisL arboanlis P xylostellaP xylostella

Crop Insect pest Family

Rice Yellow stem borer Scirpophaga incertulasBrown plant hopper Nilaparvata lugens

LepidopteraHemiptera

Mustard Mustard aphid Lipaphys erysimi Hemiptera

Chickpea Gram pod borer Helicoverpa armigera Lepidoptera

Pigeonpea Gram pod borer H. armigera Lepidoptera

Cotton Cotton boll worm H. armigera Lepidoptera

Sugarcane Top borer S. novella Lepidoptera

Groundnut Leaf miner Stomopterix nertaria Lepidoptera

Potato Tuber moth Phthorimaea operculella Lepidoptera

Tomato Fruit borer H. armigera Lepidoptera

Brinjal Shoot and fruit borer Leucinodes orbonalis Lepidoptera

Cauliflower and

Cabbage

Diamondback moth Plutella xylostella Lepidoptera

Important pests of major crops of India Important pests of major crops of India

Insect ResistanceInsect Resistance

Genes conferring insect resistance to plants have been obtained from

Micro –organismsBt gene from Bacillus thuringenesis Ipt (isopentyl transferase) gene from A. tumefaciens Cholesterol oxidane gene from Streptomyces fungus.Pht gene from Photorhabdus luminescens

Higher plants Proteinase inhibitors

Cowpea trypsin inhibitor geneα amylase inhibitors

Lectins

AnimalsSirine proteinase inhibitors from mammals

BT toxin gene - Bacillus thuringenesis is G+ve, aerobic, sporulating bacteria which produces crystalline proteins during sporulation

These crystalline proteins are highly insecticidal.

Protoxins are acted upon by midgut proteases which cleave them into 2 halves, the N-terminal half (65-68 kDa) is the toxin protein.

This toxin fragment has 3 domains,1st domain is involved in pore formation2nd domain determines the receptor binding3rd domain protects the toxic from proteases

Bt strains contain a great diversity of δ endotoxins encoding genes.

The cloning and sequencing of the first insecticidal genes was published in 1981.

Today more than 100 crystal protein gene sequences have been published.

Both full length and truncated forms of Bt δ endotoxins have been introduced in plants, conferring demonstrable resistance to tobacco pests (M. sexta), tomato pests (Heliothis virescens) cotton pests (H. armigera) etc.

The first plants produced were capable of synthesizing the entire protoxin, but expression of the gene was weak and the resulting small quantity of δ endotoxin gave little or no resistance.

Further development ultimately led to optimization of the cry gene expression in plants.

What is Bt Brinjal?Bt Brinjal is a transgenic brinjal created by inserting a genecry1Ac from the soil bacterium Bacillus thuringiensis into Brinjal.This is said to give the Brinjal plant resistance againstlepidopteran insects like the Brinjal Fruit and Shoot BorerLeucinodes orbonalis and Fruit Borer Helicoverpa armigera.

What is the controversy about?Bt Brinjal has generated much debate in India. The promoterssay that Bt Brinjal will be beneficial to small farmers because itis insect resistant, increases yields, is more cost-effective andwill have minimal environmental impact. On the other hand, concerns about Bt Brinjal relate to its possible adverse impact on human health and bio-safety, livelihoods and biodiversity.

Cholestrol oxidase (CO) present in the Streptomyces culture filtrate showed acute toxicity to boll weevil larvae. This gene has been engineered into tobacco.

Cholestrol oxidase (CO) present in the Streptomyces culture filtrate showed acute toxicity to boll weevil larvae. This gene has been engineered into tobacco.

Isopently transferase (ipt) gene from A. tumefaciens codes for a key enzyme in the cytokinin biosynthetic pathway.

Expression of ipt in tobacco and tomato by a wound inducible promoter has resulted in a decrease in leaf consumption by the tobacco hornworm (M. sexta) and reduced survival of the peach potato aphid (Myzus persicae)

Isopently transferase (ipt) gene from A. tumefaciens codes for a key enzyme in the cytokinin biosynthetic pathway.

Expression of ipt in tobacco and tomato by a wound inducible promoter has resulted in a decrease in leaf consumption by the tobacco hornworm (M. sexta) and reduced survival of the peach potato aphid (Myzus persicae)

Resistance genes from higher plants Resistance genes from higher plants

Number of non Bt insecticidal proteins interfere with the nutritional needs of the insect.

Proteinase inhibitorsPlants contain peptides acting as protease inhibitors (PIPs)

Different proteinases are serine, cysteine, aspartic and metallo proteinases.

They catalyze the release of amino acids from dietary protein, thereby providing the nutrients crucial for normal growth and development of insects.

The proteinase inhibitors deprive the insect of nutrients by interfering with digestive enzymes of the insect.

Cowpea trypsin inhibitor gene (CpTi)

CpTi, found in Cowpea (Vigna ungiculata) is the most active inhibitor identified to date.

This inhibitor gene produces antimetabolite substances that provide protection against major storage pest Bruchid beetle (Callosobruchus maculatus)

Besides, this gene is also harmful to various lepidopteran, Coleopteran and Orthopteran insects.

Bioassay for insecticidal activity of transgenic tobacco plants was done with cotton bollworm (Helicoverpa zea). Insect survival & plant damage were clearly decreased in transgenic plants compared with control.

Cowpea trypsin inhibitor gene (CpTi)

CpTi, found in Cowpea (Vigna ungiculata) is the most active inhibitor identified to date.

This inhibitor gene produces antimetabolite substances that provide protection against major storage pest Bruchid beetle (Callosobruchus maculatus)

Besides, this gene is also harmful to various lepidopteran, Coleopteran and Orthopteran insects.

Bioassay for insecticidal activity of transgenic tobacco plants was done with cotton bollworm (Helicoverpa zea). Insect survival & plant damage were clearly decreased in transgenic plants compared with control.

α amylase inhibitor gene for α amylase inhibitor has been isolated from adzuki bean (Phaseolus vulgaris)This amylase inhibitor protein blocks the larval feeding in the midgut.The larvae secrete a gut enzyme called α amylase that digests the starch. This protein inhibits insect gut α amylase, the insect starves and dies. It works against Zabrotes subfasciatus and Callosobruchus chinensis.

α amylase inhibitor gene for α amylase inhibitor has been isolated from adzuki bean (Phaseolus vulgaris)This amylase inhibitor protein blocks the larval feeding in the midgut.The larvae secrete a gut enzyme called α amylase that digests the starch. This protein inhibits insect gut α amylase, the insect starves and dies. It works against Zabrotes subfasciatus and Callosobruchus chinensis.

Lectins are plant glycol proteinsRecent interest has mainly concentrated on the lectin from sundrop (Galanthus nivalis) also known as GNA, because it has shown activity against aphids.Laboratory tests with modified potato showed that GNA increase the mortality but considerably reduced fecundity.

Lectins are plant glycol proteinsRecent interest has mainly concentrated on the lectin from sundrop (Galanthus nivalis) also known as GNA, because it has shown activity against aphids.Laboratory tests with modified potato showed that GNA increase the mortality but considerably reduced fecundity.

Resistance genes from animalsResistance genes from animals

Serine proteinase inhibitors from mammals and the tobacco hornworm (Manduca sexta)

Bovine pancreatic trypsin inhibitor (BPTI), α antitrypsin, and spleen inhibitor (SI) have been identified as promising insect resistance proteins.

Manduca sexta derived proteinase inhibitors viz. anti -chymotrypsin and anti elastase expressed in cotton and chitinase in tobacco were found to reduce reproduction of Bemesia tabaci and Heliothis virescens respectively

Important Viral diseases of crops in IndiaImportant Viral diseases of crops in India

VIRUS RESISTANCEVIRUS RESISTANCE

The development of molecular strategies for the control of viral diseases has been especially successful because of the relatively small genomic size of plant viruses.

There are a number of different strategies for using molecular technology to integrate or create new resistance factors in plant virus systems.

The approach is to identify those viral genes or gene products, which when present at an improper time or in the wrong amount, will interfere with the normal functions of the infection process and prevent disease development.

The development of molecular strategies for the control of viral diseases has been especially successful because of the relatively small genomic size of plant viruses.

There are a number of different strategies for using molecular technology to integrate or create new resistance factors in plant virus systems.

The approach is to identify those viral genes or gene products, which when present at an improper time or in the wrong amount, will interfere with the normal functions of the infection process and prevent disease development.

Coat protein mediated cross protection Coat protein mediated cross protection

Based on concept of cross protection

Powell – Abel et al (1986) – 1st demonstrated that transgenic tobacco expressing TMV coat protein showed resistance similar to that occurring in viral mediated cross protection.

Cp mediated resistance is correlated with the inhibition of virus replication at the initial point of infection.

Kouassi et al. (2006) showed resistance rice against rice yellow mottle virus by expressing its coat protein gene.

Non-structural protein mediated resistanceNon-structural protein mediated resistance

Viruses encode non-structural proteins that are necessary for replication.

Several of these non-structural replicase proteins have been found to provide a high degree of resistance to virus infection when expressed in transgenic plants.

Pinto et al. (1999) developed rice YMV resistant transgenic plants by expressing replicase gene.

Ribozymes are small RNA molecules capable of highly

specific catalytic cleavage of RNA; therefore, they have

enormous potential to inhibit gene expression.

Ribozymes mainly have been used in attempts to inhibit

RNA viruses that have an exclusively cytoplasmic replication

cycle

Sano et al. (1997) developed transgenic potatoes resistant

against potato spindle tuber viroid through this approach.

Ribozyme mediated resistanceRibozyme mediated resistance

Anti- viral proteinsAnti- viral proteins

A class of polypeptides called anti-viral or ribosome inactivating proteins (RIPs) have been identified in a number of plants; of which best known source is poke weed (Phytolacca americana) .

The ribosome inhibiting function is due to their ability to modify ribosomal RNA and there by to interfere with polypeptide translation.

Lodge et al. (1993) generated transgenic tobacco plants which were resistant to PVX, PVY and CMV.

Wang et al. (1998) found that transgenic tobacco plants expressing pokeweed anti-viral protein was resistant to TMV.

Mammalian oligoadenylate synthase geneMammalian oligoadenylate synthase gene

In mammals, virus infections are fought via induction of the interferon system. Interferons induce additional proteins that defend the animal directly against viruses .

One of these proteins is oligoadenylate synthase. This enzyme is activated by dsRNA, the replication intermediate of RNA viruses.

After activation, the enzyme polymerizes ATP to oligoadenylate which in turn activates a latent endoribonuclease (Rnase L), an enzyme that degrades RNA.

Ogawa et al. (1996) generated transgenic tobacco expressing the mammalian oligoadenylate synthase for resistance against Cucumber Mosaic Virus.

Contribution of fungal diseases towards yield loss in some major crops of India

Contribution of fungal diseases towards yield loss in some major crops of India

Disease ResistanceDisease Resistance

A large number of plant defense response genes encoding anti-microbial proteins have been cloned.

Most of these are transcriptionally activated in response to infection or exposure to microbial elicitor macromolecules.

The products of defense response genes may includeHydrolytic enzymes like chitinases and glucanasesPR ProteinsRibosome inactivating proteins (RIPs)Antifungal proteinsPhytoalexins

Pathogenesis-related proteinPathogenesis-related protein

These are low molecular weight proteins, which accumulate to significant levels in infected plant tissues.

Van Loon and Van Kammen showed that a set of proteins is induced in tobacco plants after tobacco mosaic virus infection.

Later, PR proteins were shown to be induced not only by pathogens but also by wounding, fungal cell wall elicitors, ethylene, UV light, heavy metals, etc.

PR proteins have been grouped into five families based on primary structure, serological relatedness and enzymatic and biological activities

The family of PR-1 proteins consists of low molecular weight (15–17kDa) proteins. Their biological function is not known, nevertheless, constitutive expression of PR1A gene in tobacco enhances resistance of the plant to Peronospora tabacina.

PR2 and PR3 type proteins are the fungal cell wall hydrolysing enzymes, glucanase and chitinase respectively13,14. These proteins can inhibit the fungal growth in vitro by causing lysis of hyphal tips.

PR 4 show in vitro antifungal activity particularly in combination with other antifungal proteins.

PR5 proteins (thaumatin-like or AP24 or osmotin), in all probability, cause lysis of the pathogen by permeabilizing the fungal cell wall.

Plant ribosome-inactivating proteins (RIPs) Plant ribosome-inactivating proteins (RIPs)

Plant ribosome inactivating Proteins (RIPs) have N-glycosidase activity and they remove an adenine residue from 28S rRNA.

As a consequence, the 60S ribosomal subunit is not able to bind to elongation factor. resulting in inhibition of protein elongation.

Plant RIPs inactivate foreign ribosomes of distantly related species and of other eukaryotes including fungi.

Ex: Tobacco plants constitutively expressing a RIP encoding DNA sequence of barley showed better resistance to R. solani

Plant ribosome inactivating Proteins (RIPs) have N-glycosidase activity and they remove an adenine residue from 28S rRNA.

As a consequence, the 60S ribosomal subunit is not able to bind to elongation factor. resulting in inhibition of protein elongation.

Plant RIPs inactivate foreign ribosomes of distantly related species and of other eukaryotes including fungi.

Ex: Tobacco plants constitutively expressing a RIP encoding DNA sequence of barley showed better resistance to R. solani

PhytoalexinsPhytoalexins

Phytoalexins are antimicrobial low molecular weight secondary metabolites produced in plants following pathogen attack and are believed to have a role in plant defense.

The expression of stilbene synthase (or resveratrol synthase) gene resulted in the production of resveratrol, a stilbene-type phytoalexin. Such transgenics showed enhanced resistance to B. cinerea.

Host genotypePath

og

en

genoty

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Host-pathogen interactions: individual-level

Attempts to isolate disease resistance (R) and avirulance (AVR) genes have gained momentum in the past few years because of the map based cloning and gene tagging strategies

HM 1gene from Maize, which confers resistance to Cochilobolus carbonum, has been cloned by transposon tagging. (Johal and Briggs, 1992)

Resistant (R) genes like Arabidopsis Rps2 and RPM1; Pto, Cf9, Cf2,Cf4 from tomato; tobacco N gene; flax L6 and rice Xa21 have been cloned.

A number of avirulence (AVR) genes have also been cloned viz. avr9 and Avr4 of Cladosposium fulvum, N1p1 of Rhynchosporium secalis etc

Martin et al. (1993) developed tomato plants with Pto resistance gene that confers resistance against P. syringae pv tomato

Song et al. (1995) generated rice transgenic plants resistant to Xanthomonas expressing avrXa21.

Some examples of transgenic plants expressing genes encoding PI, α amylase inhibitor, & lectins

Some examples of transgenic plants expressing genes encoding PI, α amylase inhibitor, & lectins

REFERENCES

1. An introduction to Plant Biotechnology by HS Chawla

2. Biotechnolgy- A new horizon by BD Singh3. Biochemistry and Molecular Biology of plants by

Buchannan4. Microbial Biotechnology-Fundamentals of Applied

Microbiology-Second edition by Alexander N. Glazer & Hiroshi Nikaido