TRANSGENIC PLANTS

Introduction
These are plants whose DNA is modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in this species. Examples include resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent.

Introduction
During the last couple of decades, considerable progress has been made to understand the function of genes, isolation of novel genes and promoters as well as the utilization of these genes for the development of transgenic crops with improved and new characters. In fact, in 2002, more than 5.5million farmers worldwide cultivated about 58.7million hectares the crops that were genetically manipulated for herbicide tolerance, insect resistance, delayed fruit ripening and improved oil quality.

Stress Tolerance
Crop plants are very productive under ideal culture conditions, but ideal growing conditions rarely occur. They face so many strategies like soil nutrient depletion, water scarcity, increase in salt level, effect of so many microorganisms and other biological factors, etc.. Based on this stress tolerant transgenic plants are of two types: - Biotic (viral, bacterial, fungal, pathogens, nematodes, and insect pests resistance) - Abiotic (salinity, drought, extreme temperatures, nutrient deficiency resistance)

Stress tolerance- Biotic

1. Insect Resistance:

All crop plants are affected by a variety of insects, mites and nematodes, that significantly reduce their yield and quality. To minimize these losses, farmers use synthetic pesticides extensively which cause severe effects on human health & environment. The transgenic technology provides an alternative & innovative method pest control management which are eco friendly, effective, sustainable & beneficial in terms of yields. The 1st gene available for GE of crop plants for pest resistance were Cry gene (Bt gene) from Bacillus thuringiensis.

Insect resistant plants

Clone gene coding for BT toxin - pesticide
Protein toxin from Bacillus thuringiensis Kills larvae of

Lepidopterans (butterflies, moths, boll worms) Dipterans (2 winged flies (gnats, mosquitos)) Coleopterans (beetles) orthoptera (grass hoppers) Homoptera (aphids)

Agricultural importance - Kills corn borer, corn root worm and cotton bollworm larvae

Corn borer

Corn root worm

 By using insect control protein genes Insect resistant transgenic plants contain either a gene from bacterium B.thuringiensis or some other gene Insect resistance - First reported in Tobacco(Vaeck 1987) & Tomato (fischhoff 1987)

 Insect resistance transgenes of plant, bacterial or other origin can be introduced in to plants to increase level of insect resistance

Insect-resistant plants
Bt toxin Bt gene from B.thuringiensis Ipt(Isopentyl transferase) from Agrobacterium tumefaciens cholesterol oxidase from streptomyces fungus Pht gene from photorhabdus luminescens Cowpea trypsin inhibitor Combinations of the above (e.g., Bt toxin and proteinase inhibitor II)

RESISTANCE GENES FROM HIGHER LANTS

2 categories 1) Proteinase inhibitor II & a-amylase inhibitor 2) Lectins: - Snow drop lectin (Pea lectin, rice lectin) Resistance genes of animal origin Serine proteinase inhibitors from mammals & tobacco hornworm (Manduca sexta)

Bacillus thuringiensis
Discovered by Ishiwaki (1901) Gram ve soil bacterium Produces a parasporal crystalline proteinous toxin with insecticidal activity

 Proteins produced are referred to as ICP Insecticidal crystalline protein B.thuringiensis used since world war I in europe to control insect pests Classified based on serological tests 30 different types

CRY GENE
The Bt gene of B.thuringiensis - encode the toxins called Endotoxins, such as betaendotoxin & delta-endotoxin - which pose cidal effect on certain insect pests. Cry gene of B.thuringiensis produces a protein Protein forms crystalline inclusions in the bacterial spores during sporulation These crystal proteins responsible for insecticidal activities of the bacterial strains

 They are converted to active form upon infection by susceptible insect They kill the insect by disruption of ion transport across the brush border/membranes of susceptible insect

 Cry genes grouped in to 18 groups which either code for a 130 kDa or a 70 kDa protein Available as Bt gene powder

Different Bt Crys
Cry 1skills caterpillars (lepidoptera) Cry 2skills caterpillars (lepidoptera) Cry 3skills beetles (coleoptera)
Canola plant expresses a Bt cry1Ac gene

Bt COTTON CRY PROTEIN The insecticidal toxin of Bt cry proteins has been classified 4 main groups as: Crystal proteins - cry I, cry II, cry III & cry IV based on insecticidal activities cry V, cry VI groups were also added These proteins nematicidal in action They do not harm beneficial insects.

Bt Cry structure

III

I
II

BT ACTION

 Cry proteins are solubilized in alkaline environment of insect midgut Then proteolytically processed to yield a 60kDa toxic core fragment (except cry IVD) The toxin function is localised in N terminal half of the 130 kDa proteins C terminal half of these proteins highly conserved involved in crystal formation None of the truncated proteins crystallizes in the typical bipyramidal shape of most of the 130kDa proteins

 12c terminal aminoacid residues of toxic fragment are highly conserved among cry proteins Cry IA(c) are packaged in vivo in association with 20 kb non specific chromosomal DNA DNA protein complex forms virus like structure in which central DNA core is surrounded by N terminal half of the cry protein C terminal half of protein extends outward

Bt toxin

Insect midgut cells that have bound Bt toxin.

Same gut cells a few hours later note the damage and leakage.

Bt

Insect midgut cells that have bound Bt toxin.

Mutated receptors cannot bind Bt toxin.

Stewart, 2004. Genetically Modified Planet 2004

Receptors are not present cells cannot bind Bt

TOXIC ACTION OF CRY PROTEINS

When cry proteins are ingested by insects they are dissolved in alkaline juices present in the midgut lumen The gut proteases process them hydrolytically to release the core toxic fragments The toxic fragments believed to bind to specific high affinity receptors present in the brush border of midgut epithelial cells

 As a result brush border membranes develop pores nonspecific in nature Permitting influx in to the epithelial cells of ions & water causes their swelling & eventual lysis

Toxic Action of Cry Proteins:

Expression of cry Genes in Plants:

Approach 1
Isolation of Bt toxin gene( Bt 2 from ) B.thuringiensis strain Berlkiner 1715 It produces a 1155 aa Bt2 protein Inserting into plant genome, here its the cotton plant, to produce insect resistant plants. This was done using Ti-plasmid transfer method introduction of toxin gene in to Ti DNA plasmid of Agrobacterium tumefaciens The genetically modified A.tumefaciens was allowed to infect the desired plant Ti plasmid mediated transformation of several plants have been done Tobacco, corn, Tomato, Cotton brinjal, cauliflower, cabbage, canola

 Field experiments with Manducta sexta pest of tobacco 75-100% larvae of M.sexta Died The insects die when they chew the leaves of transgenic tobacco. Control plants (not transgenic) severly damaged Tobacco plants crossed with normal control plant resistance gene was inherited as per mendelian principle Segregates as a single dominant gene

A binary T-DNA plasmid for delivering the Bt gene to plants (not a cointegrate vector)
(NPT or kanr) (35S-Bt gene-tNOS)

(Spcr)

2. KURSTAKI HD-1
Several genes encoding lepidopteran type toxins have been isolated Fischhoff 1987 One gene from B.thuringneisis subsp Kurstaki HD-1 contains an open reading frame of 3468bp encoding a protein of 1156 aa Chimeric B.thuringneisis Kurstaki genes containing Camv 35S promoter & a sequence coding for an active truncated variant as well as full length Constructued & expressed in tomato plants

 Level of insecticidal protein was sufficient to kill larvae of Manduca sexta, Heliothis virescenes & heliothis zea Analysis of progeny plants showed B.thuringneisis Kurstaki gene segregated as a single dominant mendelian marker

Bt corn

Plant cells are totipotent

Bt Corn from Phillipines

Mechanism of toxin action: Binds to receptors in insect gut Ionophore- ion channel that allows ions to flow across plasma membrane Note: organic farmers spray crops with intact Bt bacterium

Strategies to avoid Bt resistant insects

Use of inducible promoters (that can be turned on only when there is an insect problem) Construction of hybrid Bt toxins Introduction of the Bt gene in combination with another insecticidal gene Spraying low levels of insecticide on Bt plants Use of spatial refuge strategies

Genetically engineered Bt-plants in the field

Product Corn Corn Corn Institution(s) Bayer Dow/Mycogen Dow/Mycogen DuPont/Pioneer Corn Corn Corn Corn Corn Corn (pop) Monsanto/DeKalb Monsanto Monsanto Syngenta Syngenta Syngenta Bt toxin to control insect pests (European corn borer) Bt toxin to control insect pests (European corn borer) Resist glyphosate herbicide to control weeds/Bt toxin to control insect pests (European corn borer) Bt toxin to control insect pests (European corn borer) Bt toxin to control insect pests (European corn borer) Bt toxin to control insect pests (European corn borer) Bacteria Bacteria Arabidopsis, bacteria, virus Bacteria Corn, bacteria, virus Corn, bacteria, virus Bt-Xtra-1997 YieldGard-1996 ?-1998 Bt11-1996 Knock Out-1995 Knock Out-1998 Engineered Trait(s) Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (European corn borer) Bt toxin to control insect pests (European corn borer) Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (Lepidopteran) Sources of New Genes Bacteria, virus Corn, bacteria, virus Corn, bacteria, virus Name StarLink-1998 (animals only) NatureGard-1995 Herculex I-2001

Corn (sweet)
Cotton

Syngenta
Monsanto/Bayer

Bt toxin to control insect pests (European corn borer)

Resist bromoxynil herbicide to control weeds/Bt toxin to control insect pests (cotton bollworms and tobacco budworm)

Bacteria
Bacteria

Bt11-1998
?-1998

Cotton Potato Potato Potato

Monsanto Monsanto Monsanto Monsanto

Bt toxin to control insect pests (cotton bollworms and tobacco budworm) Bt toxin to control insect pests (Colorado potato beetle) Bt toxin to control insect pests (Colorado potato beetle)/resist potato virus Y Bt toxin to control insect pests (Colorado potato beetle)/resist potato leafroll virus

Bacteria Bacteria Bacteria, virus Bacteria, virus

Bollgard-1995 NewLeaf-1995 NewLeaf Y-1999 NewLeaf Plus-1998

Mode of treatment
B. thuringensis formulations

Resistance developed in
Diamonback moth (Plutella xylostella) population in several countries Lepidoptera Helicoverpa armigera, spodoptera exigua, S. Littorallis, P. Xylosetella, Ephestia kuehniella, Cadra cautella, Indian meal moth(Plodia interpunctella), Christoneura fumiferana Coleoptera Chrysomella scripta, Leptinotarsa decemlineata Diptera Aedes aegypti, Culex quniquefasciatus, Drosophila melanogester, Musca domestica

Laboratory selection(Cry protein resistance)

Agriculture Transgenics On the Market

Insect resistant cotton Bt toxin kills the cotton boll worm transgene = Bt protein

Insect resistant corn Bt toxin kills the European corn borer transgene = Bt protein
Normal Transgenic

Cry gene designation

CryIA(a), CryIA(b), CryIA(c) Cry1B, Cry1C, Cry1D
CryII CryIII CryIV CryV

Toxic to these insect orders

Lepidoptera Lepidoptera
Lepidoptera, Diptera Coleoptera Diptera Lepidoptera, Coleoptera

RESISTANCE GENES FROM HIGHER PLANTS

Discovery of non bt toxin genes having insecticidal activity Non bt insecticidal proteins interfere with nutritional needs of the insect Retarding insect growth & development

PROTEINASE
Plants contain peptides acting as protease inactivating proteins(PIPs) Different proteinases serine, cysteine, aspartic, metallo proteinases They catalyze the release of aa from dietary protein thereby providing the nutrients crucial for normal growth & development of insects

PROTEINASE (PROTEASE) INHIBITORS

PROTEINASE (PROTEASE) INHIBITORS

Proteinase inhibitors proteins that inhibit the activity of proteinase enzymes Certain plants naturally produce Proteinase inhibitors to provide defence against herbivorous insects Possible since inhibitors when ingested by insects interfere with digestive enzymes of the insect This results in nutrient deprivation causing death of insects

 Proteinase inhibitors deprive the insect of nutrients by interfering with digestive enzymes of the insect 2 types

 Possible to control insects by introducing proteinase inhibitor genes in to crop plants that normally do not produce these proteins

1.cowpea trypsin inhibitor (CpTI) gene

cowpea trypsin inhibitor (CpTI) gene

Wild species of cowpea plants growing in africa were resistant to attack by a wide range of insects Insecticidal protein was a trypsin inhibitor capable of destroying insects belonging to orders of lepidopters & coleoptera It has no effect on mammalian trypsins non toxic to mammals

 CpTI found in cowpea (vigna unguiculata) active inhibitor Inhibitor gene produces antimetabolite substances which provide major protection against the major storage pest Bruchid beetel (callosobruchus maculatus) Also harmful to various lepidopteran insects (Heliothis virescens), spodopteran insects (Manduca sexta), Coleopteran insects (Callosobruchus, Anthonomus grandis), orthopteran insects (locusta migratoria)

 CpTI has been cloned & constructs containing CaMV 35S promoter & a full length cDNA clone 550bp long were used to transform leaf discs of tobacco Bioassay for insecticidal activity of transgenic tobacco plants was done with cotton bollworm (helicoverpa zea) Insect survival & plant damage decreased in transgenic plants compared with control CpTI gene also introduced in to potato & oil seed rape

Binary cloning vector carrying a cowpea trypsin inhibitor (CTI) gene

(pNOS-NPT-tNOS) (35S-CTI-tNOS)

(Kanr)

ADVANTAGES
Many insects not controlled by Bt can be effectively controlled Use of proteinase gene along with Bt gene help to overcome Bt resistance development in plants

LIMITATIONS
High levels of proteinase inhibitors are required to kill insects Necessary that expression of proteinase inhibitors should be low in plant parts consumed by humans High in parts of plants utilised by insects

2. ALPHA AMYLASE INHIBITOR

ALPHA AMYLASE INHIBITOR

Genes for three alpha amylase inhibitors expressed in tobacco Transferring the gene of alpha amylase inhibitor (alpha AI-Pv) isolated from Adzuki bean (phaseolus vulgaris) transferred & expressed in tobacco Works & provides resistance against coleoptra - Zabrotes subfasciatus & callosobruchus chinensis

 Inhibitor blocks larval feeding in the midgut The insect larvae secrete a gut enzyme called alpha amylase that digests starch By adding a protein that inhibits insect gut alpha amylase & blocking the activity of this enzyme by alpha amylase inhibitor weevil undergoes starvation & dies

LECTINS
LECTINS are plant glycoproteins They provide resistance to insects by acting as toxins Lectin gene (GNA) from snow drop (Galanthus nivalis) has been transferred & expressed in potato & tomato, oil seed rape Activity against aphids

 Also act against piercing & sucking insects LIMITATION Works well only when ingested in large quantities

Stress tolerance- Biotic

2. Virus Resistance: Several approaches have been used to engineer plants for virus resistance, which are as follows: (1) coat protein gene, (2) cDNA of satellite RNA, (3) defective viral genome, (4) antisense RNA approach, and (5) ribozyme mediated protection. Of these strategies, use of coat protein gene has been the most successful. Transgenic plants having virus coat protein gene linked to a strong promoter have been produced m many crop plants, tobacco, tomato, alfalfa, sugarbeet, potato, etc. The disease rasistance generated by employing pathogen genes is called pathogen derived resistance(PDR).

VIRUS RESISTANCE
Virus infections of crops result in retarded cell division (hypoplasia), excessive cell division (hyperplasia) & cell death (necrosis) Strategy to integrate or create new resistance factors in plant virus systems Approach to identify those viral genes or gene products when present at an improper time or in wrong amount will interfere with normal functions of the infection process & prevent disease development

 Possible to immunize plants against viral damages by expressing viral proteins in plant cells

COAT PROTEIN MEDIATED CROSS PROTECTION

Cross protection the ability of one virus to prevent or inhibit the effect of a second challenge virus If susceptible strain of a crop is inoculated with mild strain of a virus then the susceptible strain develops resistance against more virulent strains Transgenic plants having virus coat protein gene linked to promoter produced in many crops

 The first transgenic plant of this type was tobacco produced in 1986; it contained & expressed the coat protein gene of tobacco mosaic virus (TMV) strain U I. Exhibited high levels of resistance to TMV When these plants were inoculated with TMV U I, symptoms either failed to develop or were considerably delayed. Further, there was a much less accumulation of virus than in the control plants in both inoculated and systemically infected leaves.

 In addition, these plants showed delayed expression of disease symptoms when inoculated with the related tomato mosaic virus (ToMV) and with tobacco mild green mosaic virus (TMGMV) Inhibition of viral replication at initial point of infection Initial step in viruslife cycle is disrupted Most likely the resistance generated by CP is due to the blocking of the process of uncoating of virus particles, which is necessary for viral genome replication as well

 It appears to be a common feature that expression of a virus coat protein gene not only confers resistance to the concerned virus but also gives a measure of resistance to related viruses. The effectiveness of coat protein (CP) gene in conferring virus resistance can be affected by both the amount of coat protein produced in transgenic plants and by the concentration of virus inoculum. In most cases virus resistance is produced by the virus coat proteins & not by the mRNA transcript of the genes

 However, other effects seem to be involved in producing coat protein mediated virus resistance; one such mechanism appears to be the prevention or delay of systemic spread of the viruses. But at least in some cases, the resistance mechanism does not involve the coat protein itself since CP genes even in antisense orientation produce resistance to the virus.

Transgenic papaya plants show resistance (right) while nontransgenic plants (left) are susceptible to papaya ringspot virus under field conditions. Photo courtesy of Dennis Gonsalves, Cornell University.

 Transgenic plant providing coat protein mediated resistance to virus are rice, potato, wheat, tobacco, peanut, sugar beet, alfalfa, tomato. Viruses include Alfalfa mosaic virus(AIMV), cucumber mosaic virus (CMV), Potato virus X (PVY), Citrus tristeza virus (CTV) and R rice stripe virus (RSV)

MECHANISM OF ACTION
As transgenic plant expresses the gene for coat protein of a given virus the ability of the same virus to infect the plants again is drastically reduced Molecular mechanism not unknown

OTHER VIRAL GENES

Viral genes other than that for coat proteins used to generate virus resistance in plants Poty viruses & genes that were used for encoding these poty virus genome linked proteins (VPg), VPg proteinases (Nla), RNA dependent RNA polymerase(Nlb), cylindrical inclusion proteins (CI)

cDNA OF SATELLITE RNA

Some RNA viruses have small RNA molecule s called satellites They depend on viral genomes for their replication not necessary for viral functions In many cases satellite may increase or decrease the severity of disease produced by the virus carrying it

 cDNA copies of the satellites that reduce disease severity have been integrated in to host genomes Expression of the satellites shown to reduce disease symptoms as well as virus accumulation under both green house & firld conditions Tobacco plants expressing the satellites of cucumber mosaic virus showed reduced disease symptoms when infected with CMV or with the related tomato aspermy virus (TAV)

 Transgenic pepepr, tomato, tobacco plants expressing CMV satellite grown in field showed reduced disease symptoms & less virus accumulation than the control plants inoculated with CMV

DEFECTIVE VIRAL GENOMES

Defective or deleted genomes of some RNA & DNA viruses disrupt the replication of complete genomes of those viruses with which they are associated Eg African cassava mosaic virus (ACMV) genome consists of two ss DNA molecules designated as A & B DNAs

 In addition a 50% deleted B DNA is also found associated with ACMV particles Tobacco plants containing this deleted B DNA integrated in their genomes showed reduced systemic spread when they were infected with ACMV Disease severity - reduced

 ANTISENSE RNA APPROACH RIBOZYME MEIDATED PROTECTION

ANTISENSE RNA APPROACH

Stress tolerance- Biotic

3. Fungi & Bacteria Resistance:
In case of bacterial and fungal pathogens, resistance has been sought to be generated by expression of the following transgenes: 1) genes encoding insensitive target enzymes, (2) genes specifying toxin inactivation, (3) expression of antibacterial peptides, (4) expression of bacterial lysozymes, (5) genes specifying artificially programmed cell death (in items 1-5, transgenes are from non plant sources), (6) expression of heterologous phytoalexins, (7) genes encoding ribosome inactivating proteins, (8) expression of heterologous thionins, (9) ectopic (out of the natural place) expression of pathogenesis related proteins, and (10) ectopic expression of chitinases (items 6-10 use plant genes). In almost all the approaches, transgenic plants showed increased resistance to the concerned diseases.

 The strategy of artificially programmed cell death has been designed to mimick hypersensitive response. A programmed cell death is brought about by endogenous gene action, particularly in response to some specific stimulus, e.g., the elicitor specified by (avirulence) avr genes of the pathogen in the case of hypersensitive response. However, hypersensitive response depends on specific pairs of avr genes of pathogens and R (resistance) genes of the host. Therefore, each such pair specifies resistance to a single race of a pathogen and is not of general applicability. In contrast, the artificially programmed cell death is so designed as to cover all the races of a pathogen and possibly, more than one pathogen as well. There are two schemes for artificial cell death, viz., 1) two-component and (2) single-component systems.

 The single-component system is based on the expression of a toxic polypeptide in response to pathogen infection. The transgenes usable in this scheme may be those that encode toxins, ribonucleases, or other enzymes, whose products are toxic to plant cells. The barnase gene from Bacillus amyloliquefaciens was placed under the control of infection specific promoter prp1-1and was transferred into potato. Transgenic potatoes showed effective control of Phytophthora infestans. Promoter prp1-1j ensures the expression of barnase gene in such cells that are infected by a fungal pathogen. Synthesis of Barnase protein, an RNase, in such cells leads to their death; the pathogen would also die along with the dying host cells. Obviously, the strategy of artificially programme cell death will be effective against obligate parasites, but not against facultative parasites; in fact, facultative parasites may be pleased to use it to their own advantage.

TWO COMPONENT SYSTEM

2 component system : 2 precisely matched transgenes are expressed in the same cell 1 transgene avr gene avr 9 driven by a promoter inducible by a nonspecific elicitor produced during by pathogen invasion Other transgene R Gene corresponding to the avr gene used cf9 This transgene - driven by a constitutive promoter

 Expression of avr gene precisly regulated It must be expressed immediately following pathogen attack but only in the infected cells Expression of avr gene would produce elicitor that would be recognised by the coressponding R gene product This recognition initiate & culmiante hyper sensitive response

Stress tolerance-Abiotic
1. Herbicide tolerance: Development of transgenic plants resistant to certain biodegradable herbicides was the 1st major achievement from genetic engg in plants. Infact this activity generated the basic tools and techniques for gene transfer in plants, and several gene that confer herbicide resistant serve as useful selectable marker. Transgenic plants resistant to several herbicides,eg.,Glyphosate,glufosinate,sulfonylur ease, etc., have been successfully developed and are commercial cultivation in U.S.

Strategies for Herbicide Resistance: Herbicide resistance in plants can be generated by expressing in them transgenes that serve one of the following purposes. 1.Overproduction of EPSPS enzyme This strategy effectively involves titrating the herbicide out by overproduction of the target protein. For eg, if the herbicide is a specific inhibitor of one particular enzyme, production of sufficient excess enzyme will partially overcome the inhibition. Over expression can be achieved by the integration of multiple copies of the genee and/or the use of a strong promoter plus translational enhancer to drive expression of the gene.

2. Mutation of the target protein: The logic behind this approach is to find a modified target protein that substitutes functionally for the native protein and which is resistant to inhibition by herbicide, and to incorporate the resistant target protein gene into the plant genome. Several sources of resistant proteins can be exploited. 3. Detoxification of the herbicide, using a single gene from a foreign source: Detoxification is a means of converting the herbicide to a less toxic form and/or removing it from the system. This strategy can be contrasted with the previous two bcos it does not require a detailed knowledge of the site of action.

4. Enhanced plant detoxification: The aim here is to improve the natural plant defences toxic compounds. This requires detailed information about endogenous plant detoxification pathways and the mechanism by which compounds are recognized and targeted for detoxification by the plant.

HERBICIDE RESISTANT PLANTS

Glyphosate Resistance: Glyphosate is a broad spectrum herbicide that inhibits the enzyme 5-enolpyruvylshikimate-3phosphate synthase (EPSPS). Enzyme EPSPS is involved in aromatic amino acid biosynthesis in plants. Thus the killing action of glyphosate results primarily from starving the cells of aromatic amino acid, which disrupts their protein synthesis. This is reputedly effective against 76 of the worlds worst 78 weeds and is marketed as Roundup by American chemical company Monsanto.

 Glyphosate binds more tightly to the EPSPSshikimate-3-phosphate complex than does PEPits dissociation rate from the complex is 2300 times slower than PEP. EPSPS is inactivated once the glyphosate binds to the enzyme-substrate complex. EPSPS is a key enzyme in the biosynthetic pathway of the aromatic amino acids Phe, Tyr, Trp. Thus, the herbicidal activity of the glyphosate result from inhibition of the biosynthesis of aromatic amino acids and other products of the shikimate pathway.

Stress tolerance-Abiotic
2. Drought Resistance:
A number of such genes have been identified; isolated, cloned and expressed in plants, which are potential sources of resistance to abiotic stresses. These genes include Rab (responsive to abscisic acid) and SalT (induced in response to salt stress) genes of rice; genes for enzymes involved in proline biosynthesis in bacteria (proBA and proC in E. coli) and plants, spinach genes involved in betaine synthesis, etc. In plants, proline is preferentially produced from ornithine under normal conditions. However, under stress it is made directly from glutamate, the first two reactions of the pathway being catalyzed by a single enzyme 1-pyrroline 5-carboxylate synthetase (P5CS).

 The gene encoding P5CS has been isolated from soybean and moth bean, and cloned. The moth bean P5CS gene has been transferred and over expressed in tobacco. The transgenic plants produced 10- to 18fold more proline than the control plants. The leaves of transgenic plants retained a higher osmotic potential and showed a greater root biomass under water stress than did the control plants.

 These findings indicates that over expression of P5CS in plants enhances their tolerance to osmotic stress. The primary function of accumulation of proline and other solutes, e.g., glycine betaine appears to be the regulation of intracellular water activity; under water stress, they may induce the formation of strong H-bonded water around proteins, thereby preserving the native state of cell biopolymers. But it should be kept in mind that accumulation of proline is only one of the factors, which enable plants to sustain growth under water stress. Other factors also allow plants to overcome osmotic stress.

Stress tolerance-Abiotic 3. Salt Resistance:

Plant species vary in how well they tolerate saltaffected soils. Some plants will tolerate high levels of salinity while others can tolerate little or no salinity. The relative growth of plants in the presence of salinity is termed their salt tolerance. Salt tolerances are usually given in terms of the stage of plant growth over a range of electrical conductivity (EC) levels. Electrical conductivity is the ability of a solution to transmit an electrical current. To determine soil salinity EC, an electrical current is imposed in a glass cell using two electrodes in a soil extract solution taken from the soil being measured (soil salinity). The units

 Table 1 categorizes salinity into general ranges from non- saline to very strongly saline. These values are used for plant selection for saline soils. Salinity levels vary widely across a saline seep. Salinity also varies from spring to fall. Salinity usually appears on the soil surface just after spring thaw.
Table 1. Salinity rating and electrical conductivity value Weakl Nony Saline Sal ine
<2 ds/m* 2-4 ds/ m

Soil Depth

Moderat Strongly ely Salin Saline e

4-8 ds/m 8-16 ds/m

Very Strongly Saline

>16 ds/m

0-60 cm (0-2 ft)

60-120 cm(2-4 ft)

<4 ds/m

4-8 ds/ m

8-16 ds/m

16-24 ds/m

>24 ds/m

ds/m = decisiemens per metre.

 The dominant salts in prairie saline seeps are calcium (Ca), magnesium (Mg), sodium (Na) cations and sulfate (SO4) anions. If Na levels are high or not balanced with the Ca and Mg, soil tilth can also be effected. The positively charged Na cations attach to the negatively charged clay particles in the soil, causing the soil to be sticky when wet, and hard and impermeable when dry. Table 2 gives salinity tolerance ratings for a range of plant species and a range of salinity levels. New research underway may modify the rating of some plant types. As a general rule, plants that have low drought tolerance will have low salinity tolerance.

Table 2. Salt tolerance of various types of plants Salt Tolerance EC (ds/m) Very High 20 High 16 kochia sugar beets Field Crops Forages beardless wildrye fulks altai grass levonns alkaligrass alkali sucatan altai wildrye tall wheatgrass Russian wildrye slender wheat grass birdsfoot trefoil sweetclover alfalfa bromegrass garden beets asparagus spinach Siberian salt tree sea buckthorn silver buffaloberry hawthorn Russian olive American elm Siberian elm villosa lilac laurel leaf willow spreading juniper poplar ponderosa pine apple mountain ash common lilac Siberian crab apple Manitoba maple Viburnum Vegetables Trees, Shrubs

6-row barley safflower sunflower 2-row barley fall rye winter wheat spring wheat oats yellow mustard meadow fescue flax canola

Moderate

crested wheatgrass intermediate wheatgrass reed canary grass

tomatoes broccoli cabbage

corn

sweet corn potatoes

Delayed Fruit Ripeneing

Ripening is a normal phase in the maturation process of fruits and vegetables. Upon its onset, it only takes about a few days before the fruit or vegetable is considered inedible. This unavoidable process brings significant losses to both farmers and consumers alike. Scientists have been working to delay fruit ripening so that farmers will have the flexibility in marketing their goods and ensure consumers of fresh-from-thegarden produce. A notable example of this kind is the Flavr Savr transgenic tomatoes, which are commercialized in U.S. about 6 years ago.

The Fruit Ripening Process Ethylene is a natural plant hormone associated with the growth, development, ripening and aging of many plants. This phytohormone is said to promote ripening in a variety of fruits including bananas, pineapples, tomatoes, mangoes, melons, and papayas. When the concentration of ethylene reaches 0.11.0 ppm (parts per million), the ripening process in climacteric fruits is considered irreversible. In tomatoes, it takes about 45-55 days for the fruit to reach full maturity. After which, it starts to undergo the ripening process

Controlling the Ripening Process There are several ways by which scientists can control the ripening process by genetic modification. 1. Regulation of Ethylene Production The amount of ethylene produced can be controlled primarily by switching off or decreasing the production of ethylene in the fruit and there are several ways to do this. They include: Suppression of ACC synthase gene expression. ACC (1aminocyclopropane-1-carboxylic acid) synthase is the enzyme responsible for the conversion of Sadenosylmethionine (SAM) to ACC; the second to the last step in ethylene biosynthesis. Enzyme expression is hindered when an antisense (mirror-image) or truncated copy of the synthase gene is inserted into the plants genome.

 Insertion of the ACC deaminase gene. The gene coding for the enzyme is obtained from Pseudomonas chlororaphis, a common nonpathogenic soil bacterium. It converts ACC to a different compound thereby reducing the amount of ACC available for ethylene production. Insertion of the SAM hydrolase gene. This approach is similar to ACC deaminase wherein ethylene production is hindered when the amount of its precursor metabolite is reduced; in this case SAM is converted to homoserine. The gene coding for the enzyme is obtained from E. coli T3 bacteriophage. Suppression of ACC oxidase gene expression. ACC oxidase is the enzyme which catalyzes the oxidation of ACC to ethylene, the last step in the ethylene biosynthetic pathway. Through anti-sense technology, down regulation of the ACC oxidase gene results in the suppression of ethylene production, thereby delaying fruit ripening.

2. Control of Ethylene Perception Since ethylene signals the onset of fruit ripening, delayed ripening on some plants can be achieved by modifying their ethylene receptors. The gene ETR1 is one example, and it has been shown to encode an ethylene binding protein. Plants with modified ETR1 lack the ability to respond to ethylene.

3. Suppression of Polygalacturonase Activity

Polygalacturonase (PG) is the enzyme responsible for the breakdown of pectin, the substance that maintains the integrity of plant cell walls. Pectin breakdown occurs at the start of the ripening process resulting in the softening of the fruit. To produce a fruit with DR trait using this method, scientists insert an anti-sense or a truncated copy of the PG gene into the plants genome resulting in a dramatic reduction of the amount of PG enzyme produced thereby delaying pectin degradation.

Male Sterility
Male sterility can be produced by transferring certain genes from other species against endogenous genes,e.g., rolB and rolC genes from Agrobacterium rhizogenes, barnase gene from Bacillus amyloliquefaciens, etc.,. Gene barnase is the 1st transgene that was used to produce male sterility by Mariani & coworkers in 1990. Its an effective fertility restoration system in barstar.

Barnase-barstar system: In 1990, C. Mariani and others from Belgium, successfully used a gene construct having an anther specific promoter (from TA29 gene of tobacco) and bacterial coding sequence for a ribonuclease (barnase gene from Bacillus amyloliquefaciens) for production of transgenic plants in B. napus. The results were spectacular in the sense that the transferred gene prevented normal pollen development leading to male sterility. The product of barnase gene is cytotoxic, killing the tapetal cells, thus preventing pollen development. Utilizing this male sterility barnase gene construct (TA-29- RNase), it was possible to introduce male sterility in other crops also.

 These crops include tobacco, lettuce, cauliflower, cotton, tomato, corn, etc. The same group of workers (Mariani el al., 1992) used another gene construct later involving the same anther specific promoter i.e. T A 29 and the barstar gene from B. amyloliquefaciens, for production of transgenic plants in B. napus. The product of barstar gene is a ribonuclease inhibitor. It forms a complex with ribonuclease and neutralizes its cytotoxic properties. In Bacillus, the ribonuclease is active extracellularly and the bacterium itself is protected by a ribonuclease inhibitor protein coded by barstar gene.

 When transgenic male sterile plants (with barnase gene) were crossed with transgenic male fertile plants (with barstar gene), the F1 plants expressed both genes so that male fertility was restored due to suppression of cytotoxic ribonuclease activity in the anther by the formation of cell specific RNase/RNase inhibitor complexes. This system of transgenic plants should facilitate hybrid seed production in crop plants in general.

Methods of Gene Transfer

1. 2. 3. 4. 5. 6. 7. 8. 9. These include: Electroporation Particle Gun Microinjection Agrobacterium-mediated transfer, Co-cultivation method Leaf disc transformation method Virus-mediated transformation Pollen-mediated transformation Liposome-mediated transformation, etc..

Applications
1. They have proved to be extremely valuable tools in studies on plant molecular biology, regulation of gene action, identification of regulatory/promotary sequences, etc. 2. Specific genes have been transferred into plants to improve their agronomic and other features. 3. Genes for resistance to various biotic stresses have been engineered to generate transgenic plants resistant to insects, viruses, etc. 4. Several gene transfers have been aimed at improving the produce quality. 5. Transgenic plants are being used to produce novel biochemicals like hirudin, etc. which are not produced by normal plants. 6. Transgenic plants can be used vaccines for immunization against pathogens; this is fast emerging as an important objective.