Role of biotechnology in Veg. Improvement

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PROSPECTIVE ROLE OF BIOTECHNOLOGY IN VEGETABLE CROP IMPROVEMENT KOUNDINYA, A.V.V . 2010 A 88 M M.Sc (Vegetable Science)

Origin of Biotechnology :

Origin of Biotechnology Term Biotechnology was coined by Karl Ereky, a Hungarian engineer, in 1919 According to Rai and Rai, the origin of Biotechnology traced back to prehistoric times when farmers and gardeners started the use of grafting and hybridization among species with a view to bring an improvement in existing attributes of a species or variety.

ANY HOW???????:

ANY HOW??????? “Applications of microorganisms or biological systems to the production of goods and services that are beneficial to human welfare (Malik, 1989)”

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Already we have……

Locks of conventional breeding:

Locks of conventional breeding Long time trails Involving high cost Transfer of undesirable genes along with desirable gene and in the process of transfer we may loss some other useful genes from original genome. Distant Hybridization barriers Inbreeding depression in cross pollinated plants Ineffective phenotypic selection Difficult to produce viral resistant plants in case of clones. Not possible to transfer a gene from micro organisms Ineffective diagnosis of diseases

Pushing us……:

Pushing us…… Green revolution to Gene revolution

It unlocks!!!!!!!!:

It unlocks!!!!!!!! Eliminates long term trails 100% achievement of gene transfer, no transfer of undesirable genes, no loss of genes from original genome. Over coming Distant Hybridization barriers Development of inbreds without Inbreeding depression in cross pollinated plants Effective Marker Assisted selection Production of viral resistant plants in case of clones. No species/genus barriers Effective disease diagnosis Production of genetically true to type plants

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HOW TO????

GENETIC ENGINEERING:

GENETIC ENGINEERING PLANT TISSUE CULTURE

Principle of PTC:

Principle of PTC The capacity of a plant cell to regenerate into a whole plant. - Vochting (1878) Totipotency

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Establishment Explant collection Hardening Sterilization Sodium hypo chlorite Out door planting Acclimatization Green house MEDIA (Sugar-energy PGR, Agar) Regeneration of plantlets May be repeated Steps Involved in PTC

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PARENT PLANT CALLUS CELL SUSPENSION SOMATIC EMBRYOGENESIS EMBRYO TRANSPLANTATION AFTER HARDENING GENERAL SCHEME FOR MICRO PROPAGATION

Meristem or other explant culture:

Meristem or other explant culture Explant – somatic meristamatic tissue To get genetically true to type plants To propagate MS, SI, Inbred lines and Haploids that are not easily propagated by seed - Cole Vegetables Clonal propagation of F1 hybrids in vegetatively propagated plants – Potato To propagate superior manually produce hybrids for several generations with out any change in the genetic make up (without loss in heterosis). Propagation of virus free stocks – Onion, potato.

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Crop Purpose Ref. Onion Elimination of onion yellow dwarf virus Walkey et al . 1987 Shallot Elimination of onion yellow dwarf virus Walkey et al . 1987 Pea Elimination of pea seed borne mosaic virus Kartha and Gamborg 1978 Brinjal Elimination of Mosaic Virus Raj et al. 1991 Achievements of Meristem culture in various vegetables

Anther culture:

Anther culture Regeneration of large numbers of haploid plants saves time and cost. From which, by chromosome doubling via colchicine treatment or via further in vitro culture, diploid plants, homozygous at all loci, can be obtained. Avoids long generation inbreeding and selection after hybridization. Lines produced from anther culture of hybrids are obtained in less time and show greater variability than those obtained by self-pollination. 100% homozygous Inbred lines - CPC – without Inbreeding depression (Onion, Carrot, Cole crops). Effective selection is possible because they contain additive component of variance only. Explant- pollen

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Pollen on media Callus formation Shoot initiation Root initiation Whole plant Source: TNAU portal

Crop species………:

Crop species……… Potato, Brinjal, Chilli, Sweet pepper, Tomato, Asparagus, French bean, Pea, Cowpea, Broad bean, Musk melon, Winter squash, Turnip, Broccoli, Brussels sprout, Cabbage, Cauliflower, Cassava (Kalloo 1994). Embryos developing from microspores of potato

ANTHER CULTURE:

ANTHER CULTURE CROP ACHIEVEMENT REF. Tomato MS purelines Schereva et al. 1990 Chilli Haihua 3 Variety Li & Jiang 1990 Homozygous resistant line for PV Y Selassie et al. 2006

EMBRYO RESCUE:

EMBRYO RESCUE Explant – immature embryo Used for overcoming post fertilization barriers ( EMBRYO ABORTION ) in distant hybridization.

Achievements of Embryo Rescue in various vegetables (Singh et al. 2001; Swaroop 2006; Jhakar & Sastry 2000):

Achievements of Embryo Rescue in various vegetables (Singh et al . 2001; Swaroop 2006; Jhakar & Sastry 2000) Cross Purpose for which cross was made L.esculentum X L.peruvianum Resistant to viral diseases L.esculentum X L.chilense Cold and heat tolerance P.retensis X P.vulgaris Low temperature tolerance

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C.chinense X C.pubescence Powdery mildew resistance C.annuum X C.baccatum var pendulum PM, CMV, Drought & High temp. tolerance C.melo X C.metuliferous RKN tolerance C.melo X C.anguira RKN tolerance

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Triploid seedless watermelon fruit produced by embryo rescue at IIHR, Bengaluru.

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Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures such as (typically) 77 K or −196 °C (the boiling point of  liquid nitrogen). To conserve the germplasm for future needs To prevent genetic erosion- loss of important wild spp and landraces (huge sources of resistant genes) during the process of domestication of cultivated types and HYV Cryopreservation

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A tank of liquid nitrogen, used to supply a cryogenic freezer for storing laboratory samples at a temperature of about −196 0 C Preservation of tissue inside the cylinder

NBPGR is the premier institute in India, which works on collection and preservation of plant genetic resources. It is having 3,805 accessions of Brinjal, 1,997 accessions of chilli and 15,754 accessions of other vegetables, which are cryopreserved.:

NBPGR is the premier institute in India, which works on collection and preservation of plant genetic resources. It is having 3,805 accessions of Brinjal , 1,997 accessions of chilli and 15,754 accessions of other vegetables , which are cryopreserved .

SOMATIC HYBRIDIZATION:

SOMATIC HYBRIDIZATION The result of the fusion of the two somatic cells of different species, genera or family are considered as somatic hybridization (Rai &Rai 2006). Also known as Protoplast fusion or Para sexual hybridization Used to produce Cybrids and Somatic Hybrids. Used to overcome pre fertilization barriers in distant hybridization Possible to transfer of cytoplasmic organelles like mitochondria, chloroplast. E.g. mt of R.sativus and cp of B.compestris have been transferred to B.oleracea to exploit organelle heterosis for yield (Pelletier et al. 1988).

CYBRID:

CYBRID Hybrid N1 N2 N1 N1 N1 N2 N1+N2 Contains the two protoplasm and nucleus- Allopolyploids N1 N2 Hetero karyon SOMATIC HYBRIDIZATION

Procedure :

Procedure Fusing agents : NANO3 Poly ethylene glycol Ca++ at high pH Electric treatment (Rai and Rai 2006)

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ISOLATION OF PROTOPLAST

Fusion of protoplasts:

Fusion of protoplasts

Brinjal…..:

Brinjal….. S.melongena + S.integrifolium Resistant to Pseudomonas Kameya et al. 1990 S.melongena + S.saintwongsesi Resistant to Pseudomonas Asao et al . 1994

Brassica……..:

Brassica …….. B.oleracea + B.compestris Black rot resistance Itoh et al . 1991 B.oleracea (CMS) + B.compestris (Atrazine resistant) Atrazine resistant CMS line Jourdan 1989 B.napus var polimakarat + Broccoli greencomet Transfer of CMS- napus Stephen et al . 1990 Broccoli + Cauliflower Transfer of CMS- ogura ( R.sativus ) Sigareva , Earle 1997

Potato…….:

Potato……. S.tuberosum + S.brevidans Resistant to PLCV, Lateblight Helgeson et al. 1989

Tomato…….:

Tomato……. L.esculentum + L.peruvianum Resistance to viral diseases Scala et al . 1984 L.esculentum + S.muricatum ( pepino ) For various desirable characters Sokomoto , Taguchi 1991

SOMACLONAL VARIATIONS (SCV):

SOMACLONAL VARIATIONS (SCV) The generation of considerable variation, which is heritable during tissue culture is known as SCV (Rai & Rai 2006). Variations can be created for : Higher yield Herbicide resistance Fungal toxin resistance Temperature tolerance Salinity tolerance

CREATI0N…….:

CREATI0N……. Chromosome structural changes (duplications, deletions) Gene mutations Plasmid gene mutations (Streptomycin rsst – mutation in cp DNA) Gene amplification ( Some Herbicide rsst) Chromosome rearrangements

Screening for SCV:

Screening for SCV Selection at Field level after Regenerated plants are transferred to field Selection at cellular level by exposing the cultured cells to stress environments Not possible for yield characters More reliable Need further field screening

SCV IN TOMATO :

SCV IN TOMATO Tolerant to Fusarium oxysporum culture filtrate Scala et al. 1984 Resistant to Fusarium oxysporum Sachin , Spivey 1986 Tolerance to TMV Cassells et al. 1986 Resistant to bacterial wilt Toyoda et al. 1989

SYNTHETIC SEEDS:

SYNTHETIC SEEDS Encapsulated Somatic embryo which functionally mimic seeds and can develop into seedlings under suitable conditions In broader sense, it refers to encapsulated buds, bulbs any type of meristem, which can develop into plantlets. These are analogous to seeds (Rai & Rai 2006).

Potentialities…….:

Potentialities……. Direct delivery of tissue cultured plants to the field avoiding laborious and time consuming steps of transplantations Propagation of desirable genotypes with genetic uniformity (outstanding hybrids can be multiplied through tissue culture and propagated through synthetic seeds). Reduction in cost of vegetatively propagated elite plants Effective handling of fragile somatic embryos or other invitro propagules Preservation of present germplasm and convenience in germplasm exchange.

Steps…..:

Steps….. CANDIDATE CROP SOMATIC EMBRYOGENESIS INVITRO PROPAGULES SOMATIC EMBRYOS SHOOT APICES, AXILLARY BUDS, BULBLETS ENCAPSULATION GREEN HOUSE PLANTING DELIVERY UNITS

Encapsulation agents:

Encapsulation agents Sodium alginate Sodium alginate with gelatin Potassium alginate Guar gum Carboxy methyl cellulose Agar Sodium pectate Germinating “Synthetic seeds” of potato derived from somatic embryos

Properties of good encapsulation agent:

Give physical protection and play the role of artificial endosperm Non toxic to propagule Allow proper growth and development of embryo Properties of good encapsulation agent

Nourishment & Protection:

After coating it is necessary to add nutrients, fungicides and pesticides to enhance the survival of encapsulents. Ogunwolu and Idowu (1994) powdered root bark and seed extract of neem for the control of insects and considered that these could be alternatives for insecticides Nourishment & Protection

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Crop Parts used Ref. Asparagus Somatic embryos Ghosh, Sen 1994 Carrot Somatic embryos Kitto, Janick 1982 Solanum spp Shoot tips Fabre, Dereuddre 1990 Brinjal Somatic embryos Rao, Singh 1991 Tomato seeds Garret et al. 1991 Reports on Synthetic seeds in vegetables

Genetic engineering:

Genetic engineering

Transgenics…..:

Transgenics….. A plant in which a foreign has been transferred through genetic engineering is called a transgenic plant and the gene so transferred is called transgene (Singh 2001).

recombinant DNA technology:

recombinant DNA technology The R-DNA technique involves breaking of a DNA molecule at two desired places to isolate a specific DNA segment and then inserting it into another DNA molecule at a desired position. The product thus obtained is called Recombinant DNA and this technology is known as R-DNA technology (Jakhar & Sastry 2002).

STEPS…….:

STEPS……. Cloning of DNA Transfer of DNA

Transfer of DNA:

Transfer of DNA Agrobacterium mediated gene transfer Micro injections – Micro pippets Micro projectiles – Gene gun Electroporation Poly ethylene glycol mediated direct DNA uptake

Ti Plasmid:

Ti Plasmid Tumor- producing genes Virulence region Opine catabolism ORI T-DNA region DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes tms 1, tms 2 genes induce the production of auxin or indole-3-acetic acid via the IAM pathway and ipt gene activates the cytokinin production. This stimulates cell proliferation and gall formation. T-DNA contains genes for encoding enzymes that cause the plant to create specialized amino acids which the bacteria can metabolize called opines

Will the bacteria cause disease?:

Will the bacteria cause disease? -No Because --- The disease causing gene has been removed before its use as vector. This process is known as disarming of Ti-plasmid

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Agrobacterium tumifaciens plasmid

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Agrobacterium tumefaciens mediated gene transfer Agrobacterium genome Ti Plasmid a: Gene of interest b: Vir genes c: Replication origin d: Opines catabolism genes Plant cell Nucleus Bacteria is attracted towards the Acetosyringone phenolic compound present in the plant cells. VirD1/D2 complex nicks the R-DNA at the left and right border sequences. Vir D2 motif causes moment of desired DNA fragment to point of attachment Vir E2 & VIP 1 proteins helps in the transfer of DNA across the membrane VIP 2 helps in insertion of foreign DNA into Plant genome

Engineering for Resistance:

Engineering for Resistance

Mechanism of Viral Resistance:

Mechanism of Viral Resistance Coat proteins synthesized in plants block the receptor proteins, which are essential for the uncoating of viral nucleic acid. They interfere with the viral replication or expression of viral genes. Anti sense RNA technique- which inhibits viral protein synthesis.

Viral resistance vegetables engineered against viruses (Singh et al., 2001). :

Viral resistance vegetables engineered against viruses (Singh et al., 2001). virus Transgenic product Tranformed plant Origin of transgene Ref. Potato virus X Viral coat protein Potato PV X Homenway et al . 1988 Potato virus Y Viral coat protein Potato Kufri jyothi PV Y CPRI, Shimla TYLCV Viral coat protein Tomato TYLCV Kunik et al. 1994 Cl- gene Tomato TYLCV Brunetti et al. 1997 TSWV N-gene Tomato TSWV Pang et al . 1992

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virus Transgenic product Tranformed plant Origin of transgene Ref. Tomato Mosaic Virus Anti sense RNA Tomato ToMV Beachy et al. 1997 Cucumber Mosaic Virus Satellite RNA Tomato CMV Sotmmel et al. 1998 Cucumber Mosaic Virus Viral coat protein Cucumber CMV Gonsalves et al. 1992

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Tomato plants infected with tobacco mosaic virus (which attacks tomato plants as well as tobacco). The plants in the back row carry an introduced gene conferring resistance to the virus. The resistant plants produced three times as much fruit as the sensitive plants (front row) and the same as control plants. ( Courtesy Monsanto Company. )

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The transgenic summer squash plant (upper left) is resistant while the conventional plants are susceptible to single infection by CMV (bottom left), ZYMV (bottom right) and WMV (upper right) (Photo by M. Fuchs 2008).

Fungal resistance resistance genes against various fungal diseases (Singh et al 2001). Mechanism :Degradation of fungal cell wall and proteins:

Fungal resistance resistance genes against various fungal diseases (Singh et al 2001). Mechanism :Degradation of fungal cell wall and proteins Disease Casual organism Gene Transferred Downy mildew Perenospora parasitica Chitinase Wire stem Rhizoctinia solani Glucanase Leaf spot Alternaria brassicae Thionin Cabbage yellows Fusarium oxysporum RIP Damping off Phythium spp . Permatins White rust Albugo candida Osmotin Black rot Xanthomonas compestris Chitinase Soft rot Erwinia caratovora Chitinase

Bacterial resistance (Singh et al 2001). Mechanism: Detoxification of Bacteriotoxins:

Bacterial resistance (Singh et al 2001). Mechanism: Detoxification of Bacteriotoxins Resistance against Transgenic product Origin of transgene Crop Erwinia caratovora Lysozyme T4 bacteriophase Potato Erwinia caratovora Tachypelsin Horseshoe crab Potato Erwinia caratovora Pestate lyase Erwinia caratovora Potato Erwinia caratovora Glucose oxidase Aspergillus niger Potato Pseudomonas syringae var phaseocola Phaselotoxin insensitive OCTase Pseudomonas syringae var phaseocola Bean

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Bt denotes Bacillus thuringiensis , a soil borne gram positive bacteria, whose genes produce delta endotoxins that are toxic to insect pest. These toxins interfere with ion uptake in alkaline insect midgut. They are not toxic to humans and animals because it works in the alkaline digestive track of insects. Bt- insect toxin

Bt genes transferred for insect resistance in vegetables:

Bt genes transferred for insect resistance in vegetables Transgenic product Organism of transgene Target insect Transformed plant Ref. Cry 1 AB Bacillus thuringiensis Helicoverpa armigera Tomato Kumar et al. 1998 Cry 1 H Bacillus thuringiensis Helicoverpa armigera Corn Duck et al. 1997 Cry 3 A Bacillus thuringiensis Leptinotarsa decimlineata Brinjal Jelenkovic et al . 1998 Cry 1 A Bacillus thuringiensis DBM Plutella xylostella Cabbage Sharma 1999 Cry 1 A Bacillus thuringiensis Butterfly Pieries brassicae Cabbage Sharma 1999

Synthetic Bt (NRC Pl. Bio Tech.):

Synthetic Bt (NRC Pl. Bio Tech.) GENE CROP / PEST VARIETY REMARK Gy 1Ab Brinjal Leucinodes orbonaslis Pusa Purple Long 70% resistance Gy 1AC Tomato Helicoverpa armigera Pusa Ruby 100% rsst in Lab Gy 1Ac, Gy 113 Cabbage Plutella xylostella Under trail

Genes transferred other than Bt Some plant anti nutritional factors act against insect pest Mechanism: degradation of insect digestive enzymes:

Genes transferred other than Bt Some plant anti nutritional factors act against insect pest Mechanism: degradation of insect digestive enzymes Transgenic product Organism of transgene Target insect Transformed plant Ref. CpT (Cowpea Trypsin Inhibitor) Cowpea Coleoptera Lepidoptera Tomato Gatehouse et al 1992 Tomato protease inhibitor I & II Tomato Lepidoptera Tomato Mc Gurl et al 1994 Alpha amylase inhibitor Common bean Coleoptera Pea, Potato, Common bean Scgroeder et al 1995 Snowdrop lectin ( GNA ) Snowdrop Lepidoptera Tomato Gatehouse et al 1992 Pea lectin Pea Aphid Potato Bovine pancreatic trypsin inhibitor Bovine Lepidoptera Lettuce Schuler et al 1998

Engineering for herbicide tolerance (Rai &Rai 2006):

Engineering for herbicide tolerance (Rai &Rai 2006) Transgene Source Resistant to Transferred plant Basis of resistance Glutathione-S- transferase Atrazine Tomato Detoxification of herbicide bxn (nitrilase) Klebsiella pneumoniae Bromoxynil Tomato ,, bar (phosphinothricin acetyl transferase) Streptomyces spp. L- phosphinothricin Tomato, Sugarbeet ,,

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Effect of the herbicide bromoxynil on tobacco plants transformed with a bacterial gene. Transformed plants - top row and control plants - bottom row. (Courtesy of Calgene , Davis, CA.)

Engineering for Abiotic stress resistance:

Engineering for Abiotic stress resistance Heat shock protein encoding gene HSP17.7 is found in carrot (Malik et al. 1989) which can be transferred to other species for inducing thermal stress. Anti freezing protein gene has been transferred from winter flounder fish to tomato and soyabean (Hightower et al. 1999).

Engineering for parthenocarpy (Rai &Rai 2006):

Engineering for parthenocarpy (Rai &Rai 2006) Transgene Origin of transgene Transfor-med plant Ref. Rol B gene A. rhizogens Tomato Carmi et al. 1997 IaaM gene Pseudomonas syringae var savas Brinjal Rotino et al. 1997 Seedless fruits – consumer preference But lower size of fruit- reduce consumer preference Bio technology provided the solution indolacetamide monoxygenase tryptophan indolacetamide Precursor of Auxin iaaM gene

Engineering for post harvest traits :

Engineering for post harvest traits Purpose Transgene product Origin of transgene Transformed plant Ref. Improved shelf life Antisense polygalacturonase Tomato Tomato Sheehy et al. 1988 Inhibition of ripening Antisense ACC synthase Tomato Tomato Oeller et al . 1991 Antisense ACC oxidase Tomato Tomato Hamilton et al . 1990 Fruit pigmentation Phtoene synthase gene Tomato Tomato Bird et al . 1991

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Antisense technology - Gene silencing Attacks the cell walls of mature fruit & softens the skin of the fruits by degrading the cell wall pectin POLY GALACTURONASE SILENCING OF POLY GALACTURONASE GENE EXPRESSION STOPS THE SOFTENING OF CELL WALL &PREVENTS BRUISING.

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Flavr Savr Supermarket Ripe and Flavoured. Traditional The traditional tomato must be harvested while it is mature green (before they attain the flavour) to facilitate long distance transport, so that it is not crushed on the way to the supermarket. The traditional tomato is sprayed with ethylene after transport to induce ripening. Supermarket Ripe but doesnot contain original flavour We can harvest the tomato fruits for long distance transport after attaining full flavour in breaker stage . Due to inhibiting the cell wall pectin breakdown after breaker stage it remains firm during transport and don’t bruise & don’t loss the flavour

Modification in Ethylene production:

Modification in Ethylene production Methionine S- Adenosyl methionine (SAM) 1-Amino Cyclopropane – 1 -Carboxylic acid (ACC) Ethylene Ripening Modification Modification ) Methylthio adenosine & homoserine S- adenosylmethionine hydrolase ( SAMase ) ACC deaminase , ketoglutarate

M a l e s t e r I l I t y:

M a l e s t e r I l I t y Male sterility is a tool for economic hybrid seed production In crops like Okra, where malesterility hasn’t been found in cultivated as well as wild relatives we can transfer it from micro organisms by using R-DNA technology Already used in Brassicas and Tomato Engineering for

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TA 29 barnase bar Bacillus amyloliquefaciens Degenarates tapetum layer Malesterile Phosphinithrycin resistant Male sterile plants are alive barster Inhibits RNAase of barnase Produces RNAase Undegenerated tapetum layer Malefertile plants Restoration of fertility Mariani et al. 1990; Mariani et al. 1992

Engineering for Quality & Nutrition:

Engineering for Quality & Nutrition Grain Amaranthus - A. hypochondriacus contain higher storage grain protein content. This protein contain 2-4 folds more essential amino acids – Methionine, Lysine, Leucine & Threonine than normal ones The gene responsible for this protein AmA1 has been transferred successfully into Potato (Chakraborty et al. 2000 JNU, ND)

Engineering for Quality & Nutrition:

Engineering for Quality & Nutrition Potato var. with high sugar content leads to charring of sugar while chips making Potato varieties with high starch content are needed by Food industry. ADPGPP- ADP Glucose Pyro Phosphorylase, the first enzyme in the pathway of starch biosynthesis. This gene has been transferred to potato cultivars from E.coli, which resulted in dramatic increase in starch content in potato tubers. (Monsanto, USA)

COMMERCIALLY RELEASED TRANSGENIC VEGETABLE VARIETIES IN PRIVATE SECTOR (Rai & Rai 2006):

COMMERCIALLY RELEASED TRANSGENIC VEGETABLE VARIETIES IN PRIVATE SECTOR (Rai & Rai 2006) TRAIT CROP NAME COMPANY PRODUCT STATUS QUALITY (vine ripen flavour- shelflife) Tomato Flavr-savr Calgene Released 1994 QUALITY (vine ripen flavour- shelflife) Tomato Endless summer DNA plant technology Blocked by patent cliams QUALITY (paste consistancy) Tomato - Zenca Released 1995

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Viral resistance Tomato Capsicum Released in china 1993-94 CPB resistance Potato New leaf Monsanto Released 1996 CPB resistance ( cry 3B ) Potato Black jacket Monsanto Expression of gene is not sufficient Viral resistance Squash Freedom II Asgrow Released in 1995

Scenario….. of Transgenic vegetables:

Scenario….. of Transgenic vegetables China- first country to commercialize transgenic vegetables with the introduction of Viral resistant transgenic tomato. Total area under transgenic vegetables increased is 12.8 million ha. In 2006. Proagro- PGS (Guargaon) company got permission to do research on ms cauliflower & Bt brinjal, tomato, cauliflower and cabbage. Rallis India got permission to do research on pepper vein binding virus with coat protein gene At NRC – Plant biotech. Bt brinjal evaluation is going on.

On going programmes on development of transgenic vegetables in Govt. funding laboratories (Rai & Rai 2006):

On going programmes on development of transgenic vegetables in Govt. funding laboratories (Rai & Rai 2006) Programme Aim Development of insect pest resistance with Bt gene Brinjal, Tomato, Cauliflower, Cabbage Development of transgenic Tomato Resistant to TCLV ,CMV, TSWV, Early blight Genetic manipulation of ripening Tomato Development of transgenic Amaranthus Low oxalate & nitrate level

Genetic markers:

Genetic markers Markers are just tags Genetic markers are tags for genes An entity helps to identify some thing….. Where, genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait.

Advantages of molecular marker:

Advantages of molecular marker Identification of genotype that is having a specific trait (GENE TAGGING) More effective, reliable and repeatable than phenotypic marker No influence of environment unlike phenotypic marker Uniform expression in all plants Advantageous to select parent for F 1 hybrid production. Assessment of relationship between parent and hybrid.

Tagging of disease resistant gene in vegetable crops (Singh 2001):

Tagging of disease resistant gene in vegetable crops (Singh 2001) Crop Disease Marker type Resistance gene Ref. Common bean CBMV RAPD I Melatto et al 1996 Lettuce Downey mildew ( B. lactucae ) RAPD Dm 17/18 Maisonneuve et al 1994 Tomato Pseudomonas syringae pv. tomato RFLP Pto Wiiliamson et al 1994 Tomato Meloidigynae incognita (RKN) RAPD Mi Wiiliamson et al 1994 Potato Phytophthora infestans RFLP R1 and R3 El-kharbotly et al 1994 Water melon Fusarium wilt RAPD - Rebing et al . 2004

Tagging of male sterility:

Tagging of male sterility Crop Marker Study Reference Water melon RAPD MS gene linked with glabrous leaf Watts (1992) Cauliflower RFLP MAS for male sterility Gueguen (1995) Tomato RAPD Identified two CMS lines Nakajma et al. (1997) Chinese cabbage AFLP, STS To map GMS Ying et al. (2002) Radish AFLP, STS Fertility restorer gene for CMS Marayama et al. (2002)

Drawbacks :

Drawbacks High Cost involved in research (Equipment & Lab) The stability of transgene expression has not always been assessed. Requirement of tissue specific promoters (MS, Parthenocarpy, Ripening) Controlled gene expression –(E.g. Cpt ) Ecological disturbance Culture contamination problems Pathogen, Insect biotype Most of them are of academic purpose only

Terminator gene Technology:

Terminator gene Technology LEA Gene A TOXIN Gene B PB PC Gene C tetracycline “ Control of Plant Expression ”- US patent issued to MELVIN OLIVER & CO-WORKERS 1998 Incase of pureline varieties we can use the harvested seed for next season sowing, as they are genetically true to type. So farmer has no need to purchase the seed every year. This is disadvantageous to Pvt. seed agencies.

Verminator Gene Technology:

Verminator Gene Technology In second season sowing of harvested transgenic seeds, the plant growth is abnormal as it is hooked by the RFP (rat fat protein gene). Until and unless spraying of specific chemicals the plant growth will not be normal. This was developed by “Giant Geneca”, UK based Transgenic company

Triator Gene Technology:

Triator Gene Technology In this technology the transgene expression in the plant is tied to a specific chemical. To get the expression of desirable transgene the plants are to be sprayed with that particular chemical or the seeds are to be soaked in that chemical before sowing. In these Verminator & Triator cases the chemical to be used are known only by that particular company and the farmer has to purchase from them .

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This technology is disadvantageous to poor farmers, who generally uses the some part of harvested seed for next season sowing (Purelines / hybrids) DEVILISH SEEDS / SUICIDAL SEEDS

Undesirable gene flow (E.g. Super weeds):

Undesirable gene flow (E.g. Super weeds ) I am a “ SUPER WEED”

IPR (Patent) related issues:

IPR (Patent) related issues CALGENE

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CONSUMER INACCEPTANCE HARMFUL ALLERGIC POISION

Future thrust:

Future thrust The improvement of more efficient transformation protocols The search and development of new genes of agronomical interest The development of strategies to better meet with public acceptability of transgenic plants Improvement of secondary or under-exploited species through Trangenics Multi disciplinary approach to develop multiple resistant varieties with agronomical interest Discouraging the suicidal / devil seed technoilogies

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THANK YOU WELL BEING OF MANKIND IS POSSIBLE ONLY WITH THE WELL BEING OF FARMER

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