Madhusudhan R PAL 0061

Plant improvement
Conventional Methods Non Conventional Methods MAS Genetic Engineering The overall process of genetic transformation involves introduction, integration and expression of foreign gene(s) in the recipient host plant.

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

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.

Weeds Herbivores Biotic Stress Insects Pathogens

STRESS

Fungi Bacteria Viruses Nematodes

Abiotic Stress

Resistance to biotic stresses

Insect resistance Viral resistance Fungal and bacterial disease resistance

Helicoverpa armigera

L arboanlis

P xylostella

Important pests of major crops of India

Crop Rice Mustard Chickpea Pigeonpea Cotton Sugarcane Groundnut Potato Tomato Brinjal Cauliflower and Cabbage Insect pest Yellow stem borer Brown plant hopper Mustard aphid Gram pod borer Gram pod borer Cotton boll worm Top borer Leaf miner Tuber moth Fruit borer Shoot and fruit borer Diamondback moth Scirpophaga incertulas Nilaparvata lugens Lipaphys erysimi Helicoverpa armigera H. armigera H. armigera S. novella Stomopterix nertaria Phthorimaea operculella H. armigera Leucinodes orbonalis Plutella xylostella Family Lepidoptera Hemiptera Hemiptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera

Insect Resistance
Genes conferring insect resistance to plants have been obtained from
Micro organisms
Bt 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 gene amylase inhibitors Lectins

Animals
Sirine 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 formation 2nd domain determines the receptor binding 3rd domain protects the toxic from proteases

Bt strains contain a great diversity of endotoxins encoding genes. The cloning and sequencing of the first insecticidal genes was published in 1981. Today more than 100 crystal sequences have been published. protein gene

Both full length and truncated forms of Bt 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 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 gene cry1Ac from the soil bacterium Bacillus thuringiensis into Brinjal. This is said to give the Brinjal plant resistance against lepidopteran insects like the Brinjal Fruit and Shoot Borer Leucinodes orbonalis and Fruit Borer Helicoverpa armigera.
What is the controversy about? Bt Brinjal has generated much debate in India. The promoters say that Bt Brinjal will be beneficial to small farmers because it is insect resistant, increases yields, is more cost-effective and will 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.

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

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

Proteinase inhibitors
Plants 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.

 amylase inhibitor
gene for 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 amylase that digests the starch. This protein inhibits insect gut amylase, the insect starves and dies. It works against Zabrotes subfasciatus and Callosobruchus chinensis.

Lectins are plant glycol proteins

Recent 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 animals

Serine proteinase inhibitors from mammals and the tobacco hornworm (Manduca sexta) Bovine pancreatic trypsin inhibitor (BPTI), 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 India

VIRUS 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.

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 resistance

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

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.

Ribozyme mediated resistance

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.

Anti- 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 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

Disease 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 include Hydrolytic enzymes like chitinases and glucanases PR Proteins Ribosome inactivating proteins (RIPs) Antifungal proteins Phytoalexins

Pathogenesis-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 (1517kDa) 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) have Nglycosidase 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

Phytoalexins
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-pathogen interactions: individ

Host genotype

R
Pathogen genotype

r
Compatible (disease) Compatible (disease)

Avr

Incompatible (no disease) Compatible (disease)

avr

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, amylase inhibitor, & lectins

REFERENCES
1. An introduction to Plant Biotechnology by HS Chawla 2. Biotechnolgy- A new horizon by BD Singh 3. Biochemistry and Molecular Biology of plants by Buchannan 4. Microbial Biotechnology-Fundamentals of Applied Microbiology-Second edition by Alexander N. Glazer & Hiroshi Nikaido