Transgenic Crop Research at ICRISAT From product development to deployment. Kiran K. Sharma

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1 2nd Annual South Asia Biosafety Conference, Colombo, Sri Lanka September 15-16, 2014 Transgenic Crop Research at ICRISAT From product development to deployment Kiran K. Sharma & Pooja Bhatnagar-Mathur

2 Challenges in agriculture Need for increased and efficient agricultural production to provide sufficient food for the growing population, estimated to increase by 3 bn in next 50 years. Find solutions for important constraints to crop productivity. Develop new technologies that raise the yield in low potential areas. Create opportunities for diversification in agricultural value-chains & Develop sustainable models.

3 Constraints to crop improvement 1. Crop improvement a major challenge. 2. Available germplasm may lack genes for major disease and pest resistance. 3. Limited variability in the available germplasm. 4. Future breakthroughs will depend on creating additional variability and inflow of desirable genes from related or unrelated species through biotechnology. Modern Biotechnology has the potential to provide new ideas and techniques to complement agricultural research

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5 Applications of Plant Genetic Engineering Herbicide resistant plants (glyphosate, sulfonylureas, basta). Virus resistant plants (viral coat protein genes). Insect resistant plants (Bt genes; trypsin inhibitors). Alteration of seed storage proteins and lipids. Biofortification of nutrients & micronutrients in foods. Antisense/RNAi for the suppression of gene expression. Production of secondary metabolites. Studies on gene function via transient gene expression. Creation of mutants by transposable elements. Bioremediation and environmental clean-up. Basic studies on plant biology, functional genomics. Biofuels.

6 Producing Transgenic Plants Efficient tissue culture system for regenerating shoots Introduction of gene into plant cells Selection of transformed cells or tissues Regeneration of putatively transformed whole plants Transfer to greenhouse and advancement of generations Molecular and genetic characterization Selection of events under greenhouse conditions Contained field testing under natural conditions Open field testing for agronomic performance Food and Environmental safety Release, introgression into new varieties, & Commercialization

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11 and traits In 63 Countries: 57 Fruits, Vegetables, Field crops and other plantsranging from lab trials to commercial production Source: Runge 2004

12 Transgenic crops research in India Public Private 6 technologies (cotton) already approved (5 private & 1 public) 4 technologies waiting for commercial approval (all private) 29 technologies approved for field testing in 2010 (4 in BRL stages and all belong to private sector) The figures include basic research Source: IGMORIS and public domain

13 Status of ICRISAT ICRISAT uses the transgenic technology as need-based for crop improvement when other biotechnological and plant breeding options do not exist

14 Efficient Transformation Systems for legumes Groundnut Chickpea Pigeonpea

15 Efficient Transformation System for Sorghum 30% success with genetic transformation in sorghum

16 Compatible gene transfer methods Agrobacterium-mediated transformation Agrobacterium-based transformation methods High frequencies of regeneration and transformation (>65%) Use of mature tissues as explants Genotype independent methods High reproducibility Protocols for markerfree transgenics available

17 Biotic Constraints Insect resistance: Pigeonpea Chickpea Sorghum

18 Constructs with Bt genes cry1ac cry2aa cry1f T R T-DNA Border CaMV35S OCS-T Promoter Cry2Aa T L T-DNA Border T R CaMV35S TEV NOS-T Promoter Cry1Ac T L T R Ara SSU Cry2Aa T L T-DNA Border T-DNA Border T-DNA Border T-DNA Border T R PsTP Promoter Cry1Ac 2A Cry1F CaMV35S Cam35S PolyA Cam35S Promoter nptii Cam35S PolyA T L T-DNA Border T-DNA Border T R CaMV35S PsTP Promoter Cry1Ac 2A Cry2Aa Cam35S PolyA Cam35S Promoter nptii Cam35S PolyA T L T-DNA Border T-DNA Border

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20 Pigeonpea transgenics for insect resistance

21 Pupal growth & development

22 Current status Over 170 transgenic events of pigeonpea produced using 6 different constructs. Transgenics analyzed and selected based on PCR, RT-PCR, qpcr, ELISA & Western blots. A considerable reduction in larval weights and survival observed in detached leaf and flower twig bioassays. Pod bioassays indicate a significant larval mortality (up to 95%) and reduction in larval weights (up to 45-fold). Promising events being advanced to further generations (embryo rescued) till T6.

23 Chickpea transgenics for insect resistance +ve sample UT Control

24 Sorghum for Stem borer resistance (Chilo partellus)

25 Abiotic Constraints Drought tolerance Groundnut Chickpea

26 A holistic approach for enhancing drought tolerance Elite genotypes Molecular Markers Applied Genomics Drought Responsive Elements (rd29a :DREB1A) Transformation -Anti-oxidative enzymes (Annexin) -Ion homeostasis (Na + -H + antiport) MAS Osmoregulation (coda, P5CF-129A, mtld) Transgenics Plant Breeding Proper Phenotyping is critical for a multigenic trait like drought!

27 Regulation of drought-responsive gene expression rd29a:dreb1a CaMV 35S:DREB1A Drought Plant Cell Signal Perception rd29a-p DREB1A Transcription/Translation Signal transduction DRE Regulatory protein [Transcriptional activator] Promotor A Gene A Promotor B Promotor C Promotor D Gene B Gene C Gene D Gene Expression Stress Response and Tolerance rd29a:dreb1a Stress Response rd29a:dreb1a Stress Response

28 Groundnut transgenics for drought tolerance GN RD2 Control

29 Phenotyping for drought tolerance Groundnut JL24 Series of green house trials for different component traits conducted ( ) RD19 Strip trials for event selection under confined field conditions In lysimeters: Rabi , In-soil strip trial: Rabi RD12 Y=T*TE*HI (Passioura, 1977) RD20 RD2 RD11 A range of events that ranged from very similar to very different from JL 24 were further tested

30 HI Yield (Kg -1 hectare) Yield and HI under drought stress (contained field trial) JL24 ICGV86031 RD11 RD12 RD19 RD2 RD20 RD *18% *26% Untransformed Parent Harvest Index (HI) 0.6 * 34% *20%*17*% *17% JL24 ICGV86031 RD11 RD12 RD19 RD2 RD20 RD33

31 Seed yield and seed filling under drought stress Fig. 5 Bhatnagar-Mathur et al., 2013, Mol. Breeding

32 Drought Tolerance in Peanut Drought Plant Cell DRE rd29a-p Promoter A Signal Perception Signal Transduction DREB1A Regulatory Protein (Transcriptional Activator) Gene A Transcription / Translation Promoter B Promoter C Promoter D Gene B Gene C Gene D W W DS Gene Expression Stress response & tolerance Yield evaluations under field conditions 18-26% increased yield under drought stress Trait based evaluations Transgenic development & characterization

33 Way Forward (Product development) Translational activities on groundnut DREB: Biosafety dossier development. Generating biosafety data. Conduct BRL1 & BRL2 field trials (regulatory approval). Introgressing rd29a:dreb1a from the selected transgenic events to different backgrounds, and their molecular characterization. Phenotying of the introgressed lines for their drought tolerance.

34 Food safety Combating Aspergillus flavus & aflatoxin Groundnut

35 Transgenic resistance to Aspergillus flavus & Aflatoxin Aflatoxin B 1 Genes Chitinase Lipoxygenase (13 LOX) RNAi (9 LOX) Defensins

36 Screening of transgenics for A. flavus and aflatoxin Screening transgenic events for sources of resistance and studying A. flavus drought interactions under micro sick plot conditions Transgenic events RCGN- 12 & RCGN-30 carrying rice chitinase gene (Rchit), Checks J11, and JL 24

37 Phenotyping for A. flavus and aflatoxin contamination A Transgenic Untransformed control B 6 T8 events overexpressing Rchit gene identified for <10% infection. Validation in confined field carried out. 12 transgenic events overexpressing 13 LOX gene being evaluated in greenhouse

38 Event seletion trial (Kharif 2010 & Rabi ) 7 events showed significantly lower A. flavus infection. 2 transgenic events showed significantly lower aflatoxin content following seed plating assays for six days.

39 Health & nutrition Biofortification for combating VAD Groundnut Pigeonpea

40 Strategies to address VAD Dietary diversification Supplementation Fortification Biofortification

41 Golden Crops Source: Gerard Barry Source: Torbert Rocheford

42 Why biofortify Groundnut & Pigeonpea? Grown and consumed worldwide Excellent sources of wholesome nutrition, but very poor in provitamin A; negligible genetic variability Contribute for ~ % total energy and protein intake: Groundnut: Oil: 48%; Protein: 30%; Carbohydrates: 15%. Pigeonpea: Oil: 3.8%; Protein: 22%; Carbohydrates: 64%. Biofortification targets Goal: 500 µg RDA; Assumption: 100 g seed intake Groundnut seed [bioconversion 4:1]: (500 X 4)/100 = 20 µg/g Pigeonpea seed [bioconversion 6:1]: (500 X 6)/100 = 30 µg/g Delivery/Consumption modes: Raw, Boiled, Dry roasted, confectionary, cooked, oil cooking, vegetable, dal etc.

43 Isoprenoid pathway -Psy1 ß-lycopene cyclase

44 Pro-vitamin A enrichment in groundnut Oleo:psy ß-carotene in GN oil Standards ( µg/ well) GN Transgenic events

45 Carotenoid profiling Lutein Zeaxanthin 1.7 µg/gm B-carotene β-carotene levels increased up to 25- folds (0.75 μg/g) in the first generation transgenic events carrying psy1 gene The second generation transgenic events carrying the Zmpsy1 and Le β-lycopene cyclase gene showed multi-folds increase in β-carotene levels ( μg/g) 5.5 µg/gm

46 Transgenic events for enhanced ß-carotene Crop/ basal levels Construct Genotype Total events T0 T1 ß-carotene (Groundnut) (not detectable) oleo:psy1 JL µg/g JL /55 tested μg/g CS Being tested oleo:psy1:35s:ßlcy ICGV Being tested High Fe/Zn lines* ICGV Being tested Pigeonpea 0.5 μg/g oleo:psy1 ICPL >10 µg/g (pooled 6 seeds) oleo:psy1:35s:ßlcy Oleo:psy1 ICPL ; under development Being tested

47 Product development time-line 1-2 years 2-3 years 2-4 years 2-4 years 3-6 T0 s Event Selection (GH) Event Selection (Fld) Regulatory Release New Tools RNAi Proteomics Pigeonpea & Chickpea Bt Groundnut GRAV PBNV Pigeonpea & Groundnut ß-carotene Chickpea DREB Pigeonpea & Chickpea Bt Groundnut TSV Groundnut A, flavus Translational Activities (PTTC) Groundnut DREB Pigeonpea pod borer Groundnut ß-carotene Groundnut drought Chickpea pod borer Groundnut viral diseases

48 Bottlenecks in GM technology development & deployment Failure to test the concepts on a large scale Failure to translate proof-of-concept into commercial products Barriers of IP/Trade/interest of private sector Biosafety regulations/food safety Risk assessment, risk management & stewardship Training & Capacity building Partnerships for product development & deployment Commercialization limitations, a serious barrier

49 Problems in commercialization Lack of proper understanding of issues related to: Intellectual property rights Regulatory aspects Roadmap for lab to farmers field Market demands Public-private partnerships in R&D Priority setting for Transgenic Plants

50 Bottlenecks in Technology Commercialization Lack of awareness on commercialization process Market orientation of technology Valuation of technology Lack of tech transfer / facilitating agencies Monitoring and tracking (Stewardship) From Excelence Through Stewardship (ETS)

51 Developing new Biotechnology is not cheap! Most current research is being done by private sector companies in the developed world Distribution of Total R&D Expenditure in Science and Technology Public Percentage Private India Mexico Indonesia Zimbabwe USA Switzerland In most of the developing countries, public sector invests in research and development. Private sector investment is beginning to increase due to globalization and reforms in some of them. Source: S.R. Rao-DBT

52 Timeline & Costs Indian Context Basic Research Translational Research R&D Event Selection BRL 1 BRL 2 Introgression 205, , , , , Years 3 5 Years BRL 1 Biosafety Research Level 1 BRL 2 Biosafety Research Level 2

53 Platform for Translational Research on Transgenic Crops An ICRISAT-DBT Initiative (PTTC) Mission To translate transgenic technology and harness its products to meet the needs of agricultural growth Principles 1.Create and charter an entity with express purpose of translating genetic engineering research into a practical, value adding technology 2.The entity would embody the requisite scientific and business skills that are appropriately balanced

54 The concept

55 Objectives To develop and deploy state-of-the-art infrastructure for conduct of transgenic research. To act as a clearinghouse for technology inputs, transgenic research leads/ prototypes with proof of concept derived from Indian research institutes, universities, and other likely sources. To evaluate specific concepts, ideas and technologies, and advance the promising transgenic events through a development cycle with adequate safety assessments. To evolve the technology to a point where a practical application can be demonstrated, and transfer this evolved technology for product development and distribution to appropriate agencies

56 Activities Independent evaluation of trait-specific transgenic events Examine IPR issues in transgenic product development Develop product dossiers for commercialization Create specific projects with defined milestones and end-points Coordinate and conduct the evaluation of transgenic events for biosafety studies Introgression of the desirable transgenic events Identify partnerships for sharing mechanisms for marketing of the final product

57 Infrastructure & Facilities State-of-the-art infrastructure for transgenic crop development on an area of 50,000 sq. ft. High-throughput transformation facility Well equipped molecular biology laboratory Analytical laboratory & Instrumentation facility Plant pathology & virology laboratory Insect rearing facility P2 level contained and regular greenhouses Contained Fields

58 The PTTC Network Project Partners ICRISAT & DBT Activity Partners - MoA with NARS like NIN, ITRI, SAUs, etc. and private sector organisations on need basis Preliminary discussion with some institutions already completed MoU with Associates / Consultants

59 Outreach Flyers, FAQs, Posters, Website, etc. Stakeholder meetings, Media Workshops, Biosafety workshops etc. Crop biology documents/biosafety Policies (e.g., Regulation of RNAi technology & Access to Biological Diversity)

60 Public Awareness

61 Way Forward Emphasis on public sector product development Strengthening capacities for monitoring & evaluation Advanced testing & detection mechanisms Increased communication & awareness Partnerships (Public-Public & Public- Private) Resource Pooling & Consortium Approach

62 Benefits to Public Sector Tech Transfer Public sector technology developers find an outlet for their technologies. Small & Medium scale seed companies have access to products of biotechnologies. Technologies available to the stakeholders (resource-poor farmers) at an affordable cost. Products developed in the PTTC can be easily transferred to the participating countries and need only to be evaluated for their local agronomic performance.

63 Biotechnology is a valuable tool in eliminating global hunger, poverty and malnutrition! It is a strategic weapon in winning the next Green Revolution! Let us harness biotechnology with due regard for consumer and environmental safety!

64 Thank you