RIDDHI KARNIK

APPLICATIONS OF GENETIC ENGINEERING TECHNIQUES IN AGRICULTURE

Was under trial and error for almost 9900 years.The first genetically modified plant was produced in

1982, using an antibiotic-resistant tobacco plant.The first genetically modified crop approved for

sale in the U.S., in 1994, was the FlavrSavr tomato, which had a longer shelf life, as it took longer to soften after ripening.

As of mid-1996, a total of 35 approvals had been granted to commercially grow 8 transgenic crops and one flower crop of carnations, with 8 different traits in 6 countries plus the EU. In 2000, with the production of golden rice, scientists genetically modified food to increase its nutrient value for the first time.

HISTORY

• To improve the agricultural, horticultural or ornamental value of a crop plant

• To serve as a bioreactor for the production of economically important proteins or metabolites

• To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes

WHY GENETICALLY ENGINEER PLANTS?

Applications of Plant Genetic Engineering

A.Crop Improvement B.Genetically Engineered Traits: The

Big Six 1.Herbicide Resistance 2.Insect Resistance 3.Virus Resistance 4.Altered Oil Content 5.Delayed Fruit Ripening 6.Pollen Control

C.Biotech Revolution: Cold and Drought Tolerance and Weather-Gaurd Genes

D.Genetically Engineered Foods 1.Soybeans 2.Corn 3.Cotton 4.Other Crops

An Overview of the Crop Genetic Engineering cycle

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Leaf Disc Method for A. t. Mediated Transformation

Leaf Disk Preparation Co-cultivation with Agrobacterium Selection for Transformation

Regeneration of Shoots

Genetic engineering techniques applied to plants

METHOD SALIENT FEATURES1.VECTOR MEDIATED GENE TRANSFERa. Agrobacterium

mediated gene transfer

b. Plant viral vectors

Very efficient but limited to a selected group of plants

Ineffective, hence not widely used2.DIRECT OR VECTORLESS DNA TRANSFERa. Electroporation

b. Microprojectile

c. Liposome fusion

d. Silicon carbide fibres

Mostly confined to protoplasts that can be regenerated to viable plants

Limited use only one cell can be microinjected at a time

Confined to protoplasts that can be regenerated into viable whole plants

Requires regenerable cell suspensions

3 CHEMICAL METHODSa. Polyethylene glycol mediated

b.Diethylaminoethyl(DEAE)dextran- mediated

Confined to protoplasts. Regeneration of fertile plants is frequently problematical

Very less results

Herbicides are generally non-selective (killing both weeds and crop plants) and must be applied before the crop plants germinate

Four potential ways to engineer herbicide resistant plants

1. Inhibit uptake of the herbicide2. Overproduce the herbicide-sensitive target

protein3. Reduce the ability of the herbicide-sensitive

target to bind to the herbicide4. Give plants the ability to inactivate the

herbicide

HERBICIDES AND HERBICIDE-RESISTANT PLANTS

HERBICIDE-RESISTANT PLANTS: REDUCING THE ABILITY OF THE HERBICIDE-

SENSITIVE TARGET TO BIND TO THE HERBICIDE Herbicide: Glyphosate (better known as

Roundup) Resistance to Roundup (an inhibitor of the

enzyme EPSP involved in aromatic amino acid biosynthesis) was obtained by finding a mutant version of EPSP from E. coli that does not bind Roundup and expressing it in plants (soybean, tobacco, petunia, tomato, potato, and cotton)

5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a chloroplast enzyme in the shikimate pathway and plays a key role in the synthesis of aromatic amino acids such as tyrosine and phenylalanine

Genetic engineering here is more challenging; however, some strategies are possible:

Individually or in combination express pathogenesis-related (PR) proteins, which include b1,3-glucanases, chitinases, thaumatin-like proteins, and protease inhibitors

Overexpression of the NPR1 gene which encodes the “master” regulatory protein for turning on the PR protein genes

Overproducing salicylic acid in plants by the addition of two bacterial genes; SA activates the NPR1 gene and thus results in production of PR proteins

FUNGUS- AND BACTERIUM-RESISTANT PLANTS

Modification of plant nutritional content: increasing the vitamin A content of plants

• 124 million children worldwide are deficient in vitamin A, which leads to death and blindness

• Mammals make vitamin A from b-carotene, a common carotenoid pigment normally found in plant photosynthetic membranes

• Here, the idea was to engineer the b-carotene pathway into rice

• The transgenic rice is yellow or golden in color and is called “golden rice”

*Expression of enzymes of β-carotene pathway in rice endosperm

*Amelioration of Vitamin A deficiency

Edible Vaccines – Ongoing Research Areas

Hepatitis BDental caries - Anti-tooth decay AbAutoimmune diabetesCholeraRabiesHIVRhinovirusFoot and MouthEnteritis virusMalariaInfluenzaCancer

EDIBLE VACCINES FROM PLANTS

Two strategies for production

1) Expression of foreign antigens in plant via stable transformation

2) Delivery of vaccine epitopes via plant virus (Mason and Arntzen, 1995)

Strategy for the production of candidate vaccineantigens in plant tissues

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RABIES VIRUS G PROTEIN IN TOMATO

• Gene linked to CaMV35S promoter• Introduced to tomato plants by Agrobacterium- mediated transformation• Expression of recombinant glycoprotein in leaves and fruits• Protein localized in Golgi bodies, vesicles and plasma lemma

Norwalk virus (cold virus) capsid protein in potato and tobacco

• Causative agent for acute epidemic gastroenteritis

• NVCP was fused to CaMV35S promoter• Transformation by Agrobacterium• Expression level: varies with plant (

DEVELOPMENT OF STRESS- AND SENESCENCE-TOLERANT PLANTS: GENETIC ENGINEERING OF SALT-

RESISTANT PLANTS Overexpression of

the gene encoding a Na+/H+ antiport protein which transports Na+ into the plant cell vacuole

This has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)

Frost Resistance• Ice-minus bacteria

• Ice nucleation on plant surfaces caused by bacteria that aid in protein-water coalescence forming ice crystals @ 0oC (320F)

• Ice-minus Pseudomonas syringae• Modified by removing genes responsible

for crystal formation• Sprayed onto plants

• Displaces wild type strains• Protected to 23oF

• Dew freezes beyond this point• Extends growth season• First deliberate release experiment –

Steven Lindow – 1987- sprayed potatoes

Development of stress- and senescence-tolerant plants: genetic engineering of flavorful tomatoes

Fruit ripening is a natural aging or senescence process that involves two independent pathways, flavor development and fruit softening.

Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow for safe shipping of the fruit.

Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and contribute to fruit softening.

In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping of the fruit, polygalacturonase gene expression was inhibited by introduction of an antisense polygalacturonase gene and created the first commercial genetically engineered plant called the FLAVR SAVR tomato. Flavor development pathway

Fruit softening pathway

Green Red

Hard Softpolygalacturonaseantisense polygalacturonase

Crop Organization Gene Brinjal IARI, New Delhi cry1Ab, cry1Ac MAHYCO, Mumbai Caul iflower MAHYCO, Mumbai cry1Ac

Sungrow Seeds Ltd. , New Delhi Cabbage Sungrow Seeds Ltd. , New Delhi cry1Ac Chickpea ICRISAT, Hyderabad cry1Ac, cry1Ab Groundnut ICRISAT, Hyderabad IPCVcp, IPCV replicase, Maize Monsanto, Mumbia CP4 EPSPS Mustard IARI, New Delhi CodA, Osmotin,

NRCWS, Jabalpur bar, barnase, barstarTERI, New Delhi Ssu-maize, Psy, Ssu-tpCrtIUDSC, New Delhi bar, barnase, barstar

Okra MAHYCO, Mumbai cry1Ac Pigeonpea ICRISAT, Hyderabad cry1Ab + SBTI

MAHYCO, Mumbai cry1Ac Potato CPRI, Simla cry1Ab

NCPGR, New Delhi Ama-1 Rice Directorate of Rice Research, Bacterial bl ight res, Xa-21,

HyderabadOsmania University, Hyderabad cry1Ab, gna gene,IARI, New Delhi gnaMAHYCO, Mumbai Bt, chit inase, cry1Ac and AaMKU, Madurai cry1AcMSSRF, Chennai chit inase, B-1,3-glucanaseTNAU, Coimbatore chit inase

Sorghum MAHYCO, Mumbai cry1Ac

Transgenic crop under development and field trials in India

• improved nutritional quality• increased crop yield• insect resistance• disease resistance• herbicide resistance• salt tolerance• biopharmaceuticals• saving valuable topsoil• ability to grow plants in harsh environments

ADVANTAGES OF GM CROPS

• Damage to human health• allergies• horizontal transfer and antibiotic resistance• eating foreign DNA• changed nutrient levels

• Damage to the natural environment• crop-to-weed gene flow• leakage of GM proteins into soil• reductions in pesticide spraying: are they real?

• Disruption of current practices of farming and food production in developed countries• crop-to-crop gene flow

• Disruption of traditional practices and economies in less developed countries.

• Lack of research on consequences of transgenic crops.

DISADVANTAGES OF GM CROPS

 Foods produced using biotechnology has not been  established as safe and are not adequately regulated.

 Crops produced using biotechnology will negatively impact the environment.

The long term effects of foods developed using biotechnology are unknown.

MYTHS RELATED TO GENETIC MODIFICATION

Genetically-modified foods have the potential to solve many of the world's hunger and malnutrition problems, and to help protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides. Yet there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy and food labeling. Many people feel that genetic engineering is the inevitable wave of the future and that we cannot afford to ignore a technology that has such enormous potential benefits. However, we must proceed with caution to avoid causing unintended harm to human health and the environment as a result of our enthusiasm for this powerful technology.

CONCLUSION

Principles of genetic manipulations. PRIMROSE 5 th EDITION INTERNETMOLECULAR BIOTECHNOLOGY by GLICKhttp://en.wikipedia.org/wiki/Genetically_modified_crop

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REFERENCES