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Gm or not gm in vino veritas
- 1. “Trans-grafting”: A new paradigm for the regulatory framework “GM or not GM: that is the question: In vino veritas” Paulino, Haroldsen 2010
- 2. Source: UN Population: Challenge of the 21st Century
- 3. Source: FAO How will we feed the new generation?
- 4. Our basic needs compete with each other Food/Feed Fuel Fiber Water
- 5. Science EconomicsSociology Food Security a multi-layered issue
- 6. GM will be part of the solution 1973: First recombinant organism 1975: Asilomar Conference 1978: Genentech founded 1986: - First field trial with Ice-Minus by Lindow S. - Coordinated Regulatory Framework 1994-97: First commercially available GM crop: Flavr Savr Tomato 1998: Transgenic Papaya in HI FAO definition: Genetically engineered/modified organisms, and products thereof, are produced through techniques in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination. (Definition not agreed by the Codex Alimentarius Commision). EFSA: genetically modified organism (GMO)" means an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination
- 7. What drives GM development? Profitability Scientific curiosity Disaster
- 8. What are the big hurdles? Market Size Intellectual Property Regulatory Public Acceptance
- 9. Commodity Crops in the Private Sector R&D Regulatory Affairs Business Development/Marketing Legal/IP $Cost: $70 million Time: 10 years $ US Revenues from major GM crops (Carlson R., 2009) 22 x
- 10. Specialty Crops in the Public Sector Research IP? Regulatory? Business Development? Corn = $40B Grape = $3BPIPRA $ $ 45% (2004)
- 11. Transgrafting: Hope in deregulation? “GM, or not GM?” In vino veritasBenefits: • Reduction in cost/time for regulatory approval • Pollen containment • Public acceptance Trans-grafting: a wild type scion grafted onto transgenic rootstock . May confer a benefit to rootstock only or both rootstock and scion.
- 12. Who will benefit from this technology? • Fruit tree crops • Middle East/Europe (watermelons, tomatoes) Papaya: $13 Million
- 13. Industry Threats vs. Public Research Fruit/Nut Crop Disease of Interest No. GM articles on disease Total No. Pest- related GM strategies Grape Pierce's Disease 4 24 Powdery Mildew 4 Eutypa Dieback 1 Mealybug 0 Nematode 0 Citrus Canker 10 43 Greening (HLB) 2 Phytophthera Root Rot 2 Asian Citrus Psyllid 0 Thrips 0 Almond Anthracnose 0 1 Brown Rot 0 Rust 0 Scab 0 Shothole 0 Apple Fireblight 6 18 Scab 6 Codling Moth 2 Oblique Banded Leaf Roller 0 Powdery Mildew 0
- 15. USDA/APHIS/BRS “Protecting American agriculture” Notification and Permit for Interstate Movement and Release into the Environment Petition for Deregulation of Regulated Article the Animal and Plant Health Inspection Service (APHIS) is responsible for protecting agriculture from pests and diseases.
- 16. EPA • The EPA through a registration process regulates the sale, distribution and use of pesticides in order to protect health, and the environment, regardless of how the pesticide was made or its mode of action • IR-4 can help in the process
- 17. FDA • The FDA is responsible for ensuring the safety and proper labeling of all plant-derived foods and feeds, including those developed through bioengineering. • Voluntary consultation process: nutritional composition & allergenicity
- 18. How will agencies consider transgrafting? • No official opinion on this paradigm • Depends on the trait/species combination • If movement to the scion: risk assessment on rootstock + scion combination • Case by case
- 19. •Grafted plants traditionally thought of as maintaining own genetic identity •What defines identity? •DNA, RNA, proteins, miRNAs? •Epigenetic changes? ? DNA RNA protein miRNA GM or non-GM?
- 20. Transgrafting experiments initiated here at UCD •Aguerro- pPGIP grape activity detected, protein not shown •Escobar- RNAi expressing walnut tomato scion tested for microRNA, walnut not examined •Comprehensive follow up •Establishing if tomato is a good model system •Analysis of genetic components: •iaaM, ipt: smRNA from hairpin •GUS, NPTII: cytosolic proteins •pPGIP: apoplast targeted protein •mRNA and gDNA component for all
- 21. Plastid DNA can “cross” the graft junction WT Pt-spec:gfp Nuc-kan:yfp YG-29 Stegemann 2009 Stegemann 2003 •Chloroplast transformed with nuc:nptii and recombination sites present in cassette •1 in 5 million gene transfer events •Incorporation of transgene and segments of flanking pDNA
- 22. Walnut •Utilized available grafted saplings •2-4 nuts/plant •Only Wt/Wt control available •4 unique transformation events -greenhouse inoculation data -field testing GM WT WT (+)ctrl (-)ctrl
- 23. Tomato & Grape •Propagated seeds or cuttings •Grafted and included “logical “controls GM WT fruit scion rootstock (-)ctrl ctrls
- 24. gDNA analysis- walnut vs. tomato iaaM GUS Actin GM WT
- 25. gDNA analysis-walnut vs. tomato ipt NPTII GM WT
- 26. gDNA analysis- grape vs. tomato pPGIP NPTII ACTIN •Genotyping confirmed grafts generated correctlyGM WT
- 27. Certain types of RNA are mobile Kudo, Harada 2007 Probing Scion material WT ctrls tomato potato Differential splicing Me tomato •Bill Lucas @ UCD selective delivery of RNAs to scions through vascular system • “Zip codes” in the 3’UTR •Targeting to shoots or roots
- 28. mRNA analysis-walnut vs. tomato iaaM GUS Actin GM WT
- 29. ipt NPTII mRNA analysis-walnut vs. tomato GM WT
- 30. mRNA analysis- grape vs. tomato NPTII pPGIP ACTIN pPGIP NPTII ACTIN •Transgenes active in GM portions; no mobility detected GM WT
- 31. •5 fold serial dilutions starting with ~10,000/rxn -30 cycles •Background of 100ng of WT DNA -walnut -grape -tomato •Similar ranges of 4-22 copies of gene/rxn PCR-based Detection Limits: 4-22 copies nptII ipt iiaM gus 105 104 2740 548 109 22 4.4 0.8 (-)ctrl pPGIP
- 32. Proteins can mobile •100’s…some larger than 100kDA •GFP expressed (CC-promoter) found in sink tissues •GFP fusions at least 50kdA, <67kDA can diffuse into the SE “Non specific macromolecular trafficking a general feature of plasmodesmata in sink tissues”—Oparka 1999 7 hours and 2 days post bombardment Imlau 1999
- 33. NPTII analysis- Grape vs. Tomato •5X loading in tissues where abundance may be low •Obtaining purified NPTII for detection limit
- 34. NPTII analysis- Walnut vs. Tomato Ongoing with GUS as well •Obtaining purified GUS for detection limit
- 35. 1ug 333ng 111ng 37ng 12ng 4ng •Dilution of GM in WT background •Purified pPGIP would be ideal pPGIP analysis- Tomato & Grape Preliminary blots in tomato and grape leaf 5X “overloading” in red boxed regions •Consider using dilutions of GM for GUS and NPTII
- 36. Molnar, 2010 •21-24 nt smRNAs mobile across graft junctions in Arabidopsis •Some associated with epigenetic effects •Used deep sequencing Small RNAs can be mobile •Most miRNA are probably cell-autonomous or localized (Voinnet 2009) •Nutrient signaling, defense probably are systemic (Pant 2008) GFP target GFP-derived hairpin to silence
- 37. •Sense/co-suppression was graft transmissible but antisense was not Palauqui 1996, Crete 2001 Shaharuddin 2006 •Antisense silencing can be transmitted to scions but at slower rate • Target is necessary Transmissibility of sense, antisense, and co-suppressed signal
- 38. smRNA detection in walnut and tomato •Tomato grafts >12 weeks old •Walnut grafts ~7 years old •Ribonuclease protection assay (RPA) Ambion’s mirVana series reagents •Can be up to 100X more sensitive than Northern blot •Deep-sequencing as an alternative
- 39. Summarizing Mobility •DNA can cross organelle and even cell boundaries •low frequencies •probably not relevant to grafting scenarios •RNA can cross cell boundaries and enter vascular system •correct “zip codes” •unless designed a priori, not likely to happen •Proteins can “leak” into the vascular system • Especially if expressed in companion cells •Dependant on size, may degrade •smRNAs can enter vascular system •sense, antisense, or hairpin origin •likely requires a target to obtain high levels •may be time dependant •Sensitivity is key to detection, case-by-case scenario
- 40. •Single Molecule ELISA (Nature Biotech 2010) Single Molecule Detection •Single Molecule Real Time Sequencing -Pacific Bioscience -Helicos •RNAseq •Direct Real-Time RNA sequencing (Nature, 2009)
- 41. •RNAs, protein present in scion? •What if microRNAs are present in scion? •What about epigenetic changes? •How will this affect commercialization? •Would you grant deregulation? Transgrafting Considerations ?? ? ?
- 42. “GM, or not GM?” In vino veritas