Gm or not gm in vino veritas

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“Trans-grafting”: A new paradigm for the regulatory framework “GM or not GM: that is the question: In vino veritasPaulino, Haroldsen 2010

description

This presentation introduces a new paradigm for the regulatory framework of genetically modified organisms or GMO. By only genetically engineering the rootstock of a plant and not it scion, it is possible to induces the benefit of the modification to the top part of the plant without modifying its genetic code. This introduces a new paradigm shift in the GMO regulation that we have named trans-grafting.

Transcript of Gm or not gm in vino veritas

Page 1: 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

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Source: UN

Population: Challenge of the 21st Century

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Source: FAO

How will we feed the new generation?

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Our basic needs compete with each other

Food/Feed

Fuel

Fiber

Water

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Science

EconomicsSociology

Food Security a multi-layered issue

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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

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What drives GM development?

Profitability

Scientific curiosity

Disaster

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What are the big hurdles?

Market Size

Intellectual Property

Regulatory

Public Acceptance

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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

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Specialty Crops in the Public Sector

Research IP? Regulatory? Business Development?

Corn = $40B

Grape = $3BPIPRA

$$

45% (2004)

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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.

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Who will benefit from this technology?

• Fruit tree crops• Middle East/Europe (watermelons, tomatoes)

Papaya:

$13 Million

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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

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Regulatory agencies

USDA/APHIS/BRS

EPA

GM Crop

FDA

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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.

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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

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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

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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

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•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?

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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

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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

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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

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Tomato & Grape

•Propagated seeds or cuttings•Grafted and included “logical “controls

GMWT

fruit

scion

rootstock

(-)ctrl

ctrls

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gDNA analysis- walnut vs. tomato

iaaM

GUS

Actin

GMWT

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gDNA analysis-walnut vs. tomato

ipt

NPTII

GMWT

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gDNA analysis- grape vs. tomato

pPGIP

NPTII

ACTIN

•Genotyping confirmed grafts generated correctlyGMWT

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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

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mRNA analysis-walnut vs. tomato

iaaM

GUS

Actin

GMWT

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ipt

NPTII

mRNA analysis-walnut vs. tomato

GMWT

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mRNA analysis- grape vs. tomato

NPTII

pPGIP

ACTIN

pPGIP

NPTII

ACTIN

•Transgenes active in GM portions; no mobility detectedGMWT

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•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

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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

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NPTII analysis- Grape vs. Tomato

•5X loading in tissues where abundance may be low

•Obtaining purified NPTII for detection limit

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NPTII analysis- Walnut vs. Tomato

Ongoing with GUS as well

•Obtaining purified GUS for detection limit

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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

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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

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•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

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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

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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

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•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)

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•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