Conclusiones del Capítulo

Creates new genetic diversity for exploitation in cultivar development and provides opportunity to combine conventional resistance with nonconventional resistance harnessed from unrelated organism; Genetic transformation adopted only when a reliable tissue culture system to regenerate plants exists; Regeneration highly influenced by genotype, media, light, temperature and growth regulators; Now reliable regeneration protocols for groundnut available; Gene promoters are the most critical element for obtaining gene expression in groundnut; Till date (2014) no groundnut transgenic cultivar released in the world; Public resistance to transgenic food crops, particularly in Europe, heavy cost of meeting regulatory requirements for the release of transgenic cultivars including food safety and issues related to patented

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technologies come in the way of field testing and release of transgenic cultivars;

Product of all breeding approaches is a genetically modified genotype when compared to its predecessor genotype (GMO), the better term for product of genetic transformation is transgenics; The first transgenic food came in the market in 1996;

In 2012, transgenic crops covered 1.5 billion ha globally; Most of soybean, cotton, maize and canola in the USA and other countries are transgenic.

Cisgenesis: The process of engineering a genotype with desired genes transferred artificially between organisms that could otherwise be conventionally bred; Unlike transgenics, genes are transferred between closely related organisms; Gene belongs to conventional gene pool, a cis plant contains no foreign gene; Has been used to transfer natural resistance genes to blight in potato and scab in apple.

Binary vector: A pair of plasmids consisting of a binary plasmid and a helper plasmid – the two plasmids used together to produce genetically modified plants; Both artificial vectors created from T1 Plasmid found in Agrobacterium tumefaciens.

Agrobacterium - mediated transformation: Groundnut tissues susceptible to infection by A. tumefaciens include - leaf sections, cotyledonary nodes, longitudinal cotyledon halves, embryo axes, embryo leaflets, and hypocotyls; A target gene of interest engineered into the T-DNA region of a disarmed plasmid and introduced into A.

tumefaciens; Transgene within the T-DNA borders further transferred into plant cells by co-cultivation of A. tumefaciens and wounded plant tissue in appropriate medium;

PCR, which can amplify a few molecules of the gene from residual Agrobacterium because of its very sensitive nature, Southern Blot assay, where the digesting enzyme cuts at only one site or not at all within the introduced plasmid, should be used to confirm gene integration and stable transformation; It is an efficient and rapid technique for transformation due to circumvention of tissue culture step (4-5 months to obtain transgenic plants); However, technology is genotype specific and of limited use in groundnut.

Microprojectile bombardment – mediated transformation: Directly transfers target genes into plant cells by delivering coated microprojectiles at high velocity; Less dependent on host genotype; Microprojectile bombardment can be followed as long as groundnut can be generated from somatic tissues; It is low in efficiency, results in frequent infertility among tissue culture regenerants and takes longer (12-14 months) from induction of transformation event to plant maturity; Successfully used in transferring multiple genes for protein and RNAi-mediated gene silencing.

Steps involved and other requirements in field testing and release of transgenics in India: Department of Biotechnology, Ministry of Science and Technology, Government of India has issued two publications – Recombinants DNA Safety Guidelines and Regulations in 1990 and Revised Guidelines for Research in Transgenic Plants and Guidelines for Toxicity Evaluation of Transgenic Seeds, Plants and Plant Parts in 1998 – which give guidelines and regulatory requirements to be followed by all those engaged in recombinant DNA research and product development; Department of Biotechnology provides an oversight to all DNA recombinant research in the country;

There are different levels of transgenic research; Transgenic research can be carried Page 61

out only in laboratories following good practices and in specified greenhouse/glasshouse meeting prescribed standards of environmental safety; In addition to standard agronomic evaluation of transgenic material under controlled conditions on-station and in fields, biosafety tests are also required; Toxicity evaluation of transgenic seeds, vegetables and leaves involves oral toxicity tests using rats and goats and skin irritation tests involving rabbits and guinea pigs; Allergenicity tests are to be carried out in animal model.

Status of transgenic material: First successful transformation and accompanying plant generation using microprojectile bombardment technique in groundnut was achieved in 1993 in the USA; In most cases, the level of resistance achieved through transgenic is more or less similar to that achieved through conventional breeding; However, transgenics do provide opportunity to combine conventional resistance with that of nonconventional resistance to improve the level of protection against pathogen or stress factors.

Virus diseases: Received more attention than fungal diseases in transgenic research;

Coat protein-mediated resistance, in general, offered only moderate protection; RNAi-mediated resistance expected to offer higher levels of protection.

Peanut bud necrosis disease (PBND): Both A. tumefaciens- and microprojectile-mediated genetic transformation approaches using PBNV nucleocapsid gene encoding for viral coat protein pursued at ICRISAT; Transgenic events with PBNVnp gene showed lower incidence and delayed onset of disease and also recovery from disease suggesting only a modest tolerance to PBNV; Currently, RNAi-mediated approach being followed to counter the effect of non-structural silencing suppressor gene (NSs gene) in the PBNV genome.

Tomato spotted wilt virus disease(TSWV): Protection of transgenic plants against TSWV under control of both RNA - and protein-mediated control; Nucleocapsid protein gene (NP) introduced via microprojectile bombardment into New Mexico Valencia A cultivar and a runner cultivar; A. tumefaciens - mediated transformation also followed;

AT 120 (with antisense nucleocapsid gene) and Marc 1 (with coat protein gene) cultivars also transformed; Expression of sense or antisense NP gene from TSWV delayed expression of symptoms and prevented systemic virus infection but did not provide complete resistance to the disease; This single gene resistance may be short-lived because of highly heterogeneous population of the virus.

Peanut stripe virus disease (PStV): Transgenic plants of Gajah and NC 7 cultivars containing one of the two forms of PStV coat protein gene (cp 2 or cp 4) exhibited high levels of resistance to PStV; Mechanism of resistance appears to be RNA-mediated.

Peanut stem necrosis disease (PSND): Transgenics produced following A. tumefaciens - mediated transformation with TSV coat protein gene (TSVcp gene) showed three symptoms – blockage of systemic movement of TSV within the plants, recovery from an initial infection and subsequent new growth devoid of TSV symptoms and susceptible reaction; Transgenic lines cv. JL 24 containing sense and antisense coat

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protein gene of TSV using A. tumefaciens - mediated transformation developed;

However, these lines yet to be tested for disease reaction at hotspot locations under field conditions.

Peanut clump disease: Transgenic lines having IPCVcp and IPCVrep genes of Indian peanut clump following A. tumefaciens-mediated transformation produced and tested under containment facilities at ICRISAT; Some events showed resistant phenotype where the virus titre declined with maturity.

Groundnut rosette disease (GRD): Pathogen-derived resistance (introduction of GRAV or GRV genomic sequences or genes or SatRNA - derived sequences that down regulate GRV replication) a potential strategy for controlling GRD through the generation of transgenic plants; Groundnut transgenics having GRAVcp gene developed at ICRISAT and currently being tested in South Africa (as the disease does not occur in India).

Fungal diseases: Several genes (glucanase, chitinase, SniOLP and Rs-AFP2 for late and early leaf spots, chitinase for rust, oxalate, glucanase and chitinase for sclerotinia blight and Stilbene synthase, glucanase, chitinase, mod 1, anionicperoxidase, synthetic peptide D4E1, LOX 1, Nonheme chloroperoxidase (cpo) and Pn LOX 3 for A.

flavus infection and aflatoxin biosynthesis) used in genetic transformation; These genes suppressed the disease, delayed the onset of disease, enhanced resistance and decreased disease incidence; In the case of sclerotinia blight, reduced lesion area and in the case of A. flavus, reduced aflatoxin production also noticed.

Insect pests: Synthetic genes, cry1 EC against S. litura, cry1 X against H. armigera and S. litura, and cry1 Ac against lesser cornstalk borer showed good promise.

Drought and salinity tolerance: a. A cis-acting transcription factor that binds to dehydration responsive element (DRE) from Arabidopsis thaliana, AtDREB1 A, under the control of a stress inducible promoter from the rd29A gene used to develop transgenic lines with drought tolerance; One transgenic line showed 40% increase in transpiration efficiency. b. Transgenic lines of groundnut transformed with AtNHX1 showed drought and salt tolerance. c. Transgenic lines transformed with Isopentenyltransferase (IPT) gene driven by SARK promoter showed improved biomass retention and an average of 58% yield increase.

Nutritional quality:

Vitamin A: Zmpsy 1 gene from maize and beta-lycopene cyclase gene from tomato used to enrich groundnut seeds with vitamin A; Second generation transgenic events showed many fold increase in vitamin A content.

Oleic/Linoleic fatty acid ratio: Transgenic event with an FAD2 gene RNAi construct showed reduced content of linoleic acid and increased stability of groundnut oil.

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Allergens: Endogenous allergens, Ara h 2 and Ara h 6, silenced by introducing RNAi construct targeting homologous coding sequence; Human IgE binding to these proteins was greatly reduced.

Herbicide tolerance: Transgenic groundnut expressing human Bcl-xL gene showed improved tolerance to paraquat; Transgenic groundnut overexpressing pEGAD-EPSPS with altered kinetics of enzyme showed improved tolerance to glyphosate (round up).