Gm or not gm in vino veritas
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Transcript of Gm or not gm in vino veritas
“Trans-grafting”: A new paradigm for the regulatory framework
“GM or not GM: that is the question:In vino veritas”
Paulino, Haroldsen 2010
Source: UN
Population: Challenge of the 21st Century
Source: FAO
How will we feed the new generation?
Our basic needs compete with each other
Food/Feed
Fuel
Fiber
Water
Science
EconomicsSociology
Food Security a multi-layered issue
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
What drives GM development?
Profitability
Scientific curiosity
Disaster
What are the big hurdles?
Market Size
Intellectual Property
Regulatory
Public Acceptance
Commodity Crops in the Private Sector
R&D
Regulatory Affairs
Business Development/Marketing
Legal/IP
$Cost: $70 millionTime: 10 years
$
US Revenues from major GM crops (Carlson R., 2009)
22 x
Specialty Crops in the Public Sector
Research IP? Regulatory? Business Development?
Corn = $40B
Grape = $3BPIPRA
$$
45% (2004)
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.
Who will benefit from this technology?
• Fruit tree crops• Middle East/Europe (watermelons, tomatoes)
Papaya:
$13 Million
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
Regulatory agencies
USDA/APHIS/BRS
EPA
GM Crop
FDA
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.
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
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
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
•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?
Transgrafting experiments initiated here at UCD
•Aguerro- pPGIP grapeactivity detected, protein not shown
•Escobar- RNAi expressing walnuttomato 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
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
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
Tomato & Grape
•Propagated seeds or cuttings•Grafted and included “logical “controls
GMWT
fruit
scion
rootstock
(-)ctrl
ctrls
gDNA analysis- walnut vs. tomato
iaaM
GUS
Actin
GMWT
gDNA analysis-walnut vs. tomato
ipt
NPTII
GMWT
gDNA analysis- grape vs. tomato
pPGIP
NPTII
ACTIN
•Genotyping confirmed grafts generated correctlyGMWT
Certain types of RNA are mobile
Kudo, Harada 2007
Probing Scion material
WT ctrls
tomato
potatoDifferential splicing
Me tomato
•Bill Lucas @ UCDselective delivery of RNAs to scions through vascular system
• “Zip codes” in the 3’UTR•Targeting to shoots or roots
mRNA analysis-walnut vs. tomato
iaaM
GUS
Actin
GMWT
ipt
NPTII
mRNA analysis-walnut vs. tomato
GMWT
mRNA analysis- grape vs. tomato
NPTII
pPGIP
ACTIN
pPGIP
NPTII
ACTIN
•Transgenes active in GM portions; no mobility detectedGMWT
•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
(-) c
trl
pPGIP
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
NPTII analysis- Grape vs. Tomato
•5X loading in tissues where abundance may be low
•Obtaining purified NPTII for detection limit
NPTII analysis- Walnut vs. Tomato
Ongoing with GUS as well
•Obtaining purified GUS for detection limit
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 leaf5X “overloading” in red boxed regions
•Consider using dilutions of GM for GUS and NPTII
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 targetGFP-derived hairpin to silence
•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
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
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
•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)
•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
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? ?
“GM, or not GM?”In vino veritas