American Chestnut Research & Restoration A Biotechnology Approach to Resistance Complementary to the Breeding Program W.A. Powell (ESF), S.A. Merkle (UGA),

American Chestnut Research & Restoration

A Biotechnology Approach to ResistanceComplementary to the Breeding Program

W.A. Powell (ESF), S.A. Merkle (UGA), and C.A. Maynard (ESF)and many students, techs, and postdocs

Our first two transgenic American chestnut planted on 6/7/06

Wirsig Variety (LP-2V28 event)

Proof of transformation concept.

2006 2007

WB-275-27 Southern genotype

Note: TACF-NY meeting in Syracuse, NY next year. Everyone is invited!

Outline of presentation

• Short overview of gene constructs

• Current status of transgenic American chestnuts

• Next steps - research moves to the field

Testing transgenic American chestnutsto find the ideal tree to include in restoration

Optimum gene

Optimum promoter(genetic switch)

All transgenic plants to date (corn, soybean, cotton etc.) use a “constitutive” promoter

We are testing regulated promoters (wound-inducible and vascular)

Gene pyramids?(combining 2 or more genes)

Optimum event(different levels of expression)

An event represents the gene going into a specific location in the chromosomes.In the U.S. regulatory process, each even must be deregulated.

Process: Test many transgenic trees, but eventually choose only the best oneor two events to be deregulated. These trees would be out-cross to capture thesurviving chestnut’s genetic diversity and added to the restoration program.

The advantage of this technique is that new genes can be added to the programin less than two years, if the need arises (for example if new pests are introduced).

Genes & vectors

1. Oxalate oxidase (OxO) gene from wheatA. Detoxifies oxalic acid produced by the blight fungus & protects the lignin produced by the tree

2. ESF39 or ESF12 antimicrobial peptide

A. Kills the blight fungus, Cryphonectria parasiticaB. Might also be useful against Phytophthora cankers

3. Chitinase from TrichodermaA. Degrades the cell wall of C. parasitica

AmericanAmerican chestnutchestnutAmericanAmerican chestnutchestnut

Steven N. Jeffers

Clemson University

4. Ac-AMP1.2 antimicrobial peptide from Ameranth

Control vectors: pGFP & pWVK147

p∆VspB-OxO, pTACF3, pTACF7, p35S-CNO, p35S-OxO

pTACF6, pTACF7, pCWEA1

p35S-CNO

pCWEA1, pCA1

(8 currently being tested)J. Agric. Food Chem., 49 (6), 2799 -2803, 2001. 10.1021/jf010154d S0021-8561(01)00154-6

Not subject to U.S. Copyright. Published 2001 American Chemical Society

Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1

Kanniah Rajasekaran,* Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland

Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124

Received for review February 6, 2001. Revised manuscript received April 5, 2001. Accepted April 5, 2001.

Abstract:

Broad-spectrum antimicrobial activity of a synthetic peptide, D4E1, is documented in this paper. D4E1 inhibited the growth of several fungal phytopathogens belonging to four classes-Ascomycetes, Basidiomycetes, Deuteromycetes, and Oomycetes, and two bacterial pathogens, Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. malvacearum race 18. The minimum inhibitory concentration (MIC) of D4E1 required to completely inhibit the growth of all fungi studied ranged from 4.67 to 25 M. Fungal pathogens highly sensitive to D4E1 include Thielaviopsis basicola, Verticillium dahliae, Fusarium moniliforme, Phytophthora cinnamomi, and Phytophthora parasitica. Comparatively, the least sensitive fungal pathogens were Alternaria alternata, Colletotrichum destructivum, and Rhizoctonia solani. The two bacterial pathogens, P. syringae pv. tabaci and X. campestris pv. malvacearum race 18, were most sensitive to D4E1 with MIC values of 2.25 and 1.25 M, respectively. Microscopic analysis of D4E1 effects on fungal morphology of Aspergillus flavus and R. solani revealed abnormal hyphal growth and discontinuous cytoplasm. After 8 h of exposure to 25 M D4E1, A. flavus spore germination was reduced by 75%. The suitability of peptide D4E1 to enhance disease resistance in transgenic crop plants is discussed.

Keywords: Antifungal; antimicrobial; D4E1; disease resistance; phytopathogens; synthetic p

Possible source of future resistance-enhancing genes

Chinese chestnut• NSF Fagaceae genome mapping project• Many putative resistance genes have been identified

– Now need to narrow the field by linkage mapping and chestnut transformation

• Diphenol oxidase is involved in the oxidation of phenolic compounds and is associated with wound healing, lignification, and detoxification.

• Diphenol oxidase can be competitively inhibited by oxalic acid (Ferrar & Walker,1993, Mol. Plant Path. 43:415)

• If it maps to a resistance loci, transformation will be needed to confirm its function– Our lab - plants in ~ 18 months (multiplication from shoots 6 months)

– Dr. Merkle’s lab plants in ~ 12 months

7

Current status of transgenic American chestnut

Research is moving from the lab to the field

Transformation FieldCultures of single event

Shoot regeneration

Rooting(or nut grafting)

Acclimatization

Transgenic American chestnut transformation & regeneration pipeline (~18 months)

Transferring every 2 weeks and visually selecting spotted to fully fluorescent embryos

Extract DNA & test for genes using PCR

Multiply up the numbers of embryos

Extract DNA for Southern hybridization to determine insert copy number

Maintain cultures of every event, transferring every 2-3 weeks

Multiply up the numbers of shoots

Maintain cultures of every event, transferring shoots every 4 weeks

Watering, fertilizing,& watchingGrowth chamber & greenhouse

Site prep, fertilizing,Weeding, watering and pest control

RT-PCR, enzyme assays, resistance assays, & other exp.

Biggest bottleneck - acclimatization(~18 months to produce transgenic chestnut plants)

New Growth chambers with humidity, light intensity, & CO2 control

Nut Grafting

Testing for best commercial soil mix

Last year produced >400 potted plants, only 15 survived to the field

Two new growth chambers for improved acclimatization

Old growth chamber(the “dungeon”)

Replacement in Nov. 2007Two Conviron ATC60s

Roots come out of grafted nut

Transgenic shoot grafted onto a germinated chestnut. Tree was planted on June 7, 2007. Photo was taken 2 weeks after planting.

Cut a slit that bisects the two cotyledons

Slice a wedge at the bottom of a tissue culture shoot. Insert chestnut shoot

into the slit

Place graft in vessel with peat moss

Nut Grafting

Potting Mixes1. La Pierre Special (2 peat: 1 vermiculite: 1 pearlite)2. Fred Hebard’s Special (1 peat: 1 vermiculite: 1 pearlite)3. Faford Germinating Mix4. Faford #52 Mix5. Faford C1-P Growers Mix6. Faford Nursery Mix7. Sungro Metro Mix 3608. Sungro Metro Mix 3909. Sungro Metro Mix HP10. Sungro Metro Mix 560 Coir11. Standard Control – La Pierre Special

Ave. Total Dry Weight (g) by Potting Mix

3.86

4.95

6.37

5.55

5.91

5.38

5.87

3.21 3.21

7.23

4.24

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

1.La PierreSpecial

2.FredHebardÕsSpecial

3.FafordGerminating

Mix

4.Faford #52Mix

5.Faford C1-P Growers

Mix

6.FafordNursery Mix

7.SungroMetro Mix

360

8.SungroMetro Mix

390

9.SungroMetro Mix

HP

10.SungroMetro Mix560 Coir

11.StandardControl Š La

PierreSpecial

Potting Mix

Ave

rag

e D

ry W

eig

ht (

g)


Shoot regeneration


Acclimatization


WirsigWirsigWB275-27

p∆VspB-OxO 15 Wirsig trees in the field

Wirsig Variety (LP-2V28 event)2 growing seasons later (~15 months)

Stable transformation

Control WirsigWirsig ControlRT-PCROxO RNA

OxO assays

Transformation Field

WirsigWirsigWB275-27

p∆VspB-OxO

Cultures of single event

Shoot regeneration


Acclimatization

Transgenic American chestnut transformation events & regeneration pipeline (~18 months)

15 Wirsig trees in the field

RR-1V4 & 13Pond1-1

p∆VspB-OxO

LP-3V5330015-2

p∆VspB-OxO

LP-1V1 & 38Pond1-1

p∆VspB-OxO

LP-5V32WB348-5

p∆VspB-OxO


Shoot regeneration


Acclimatization


AN-2X(1-5)WB275-27

pTACF3 (OxO)

JM-1E1Pond1-1

pTACF6 (ESF39)

JM-4E2Ellis-1

pTACF6 (ESF39)

LN-1N(1-?)Pond1-1

p35S-CNO (Chitinase + OxO)

LN-3N(1-?)30015-2

p35S-CNO (Chitinase + OxO)

Shoot formation

GFP expressing embryoGFP spots

Transformation FieldCulture of

single eventShoot

regenerationRooting

(or nut grafting)Acclimatization

JH-1W(1-?)Pond1-1

pWCEA1 (ESF12 + AcAMP1.2)

JH-3W(1-?)30015-2

pWCEA1 (ESF12 + AcAMP1.2)

JH-1A(1-?)Pond1-1

pCA1 (AcAMP1.2)

JH-3A(1-?)30015-2

pCA1 (AcAMP1.2)

TR-3X(1-?)30015-2

pTACF3 (OxO)

TR-1X(1-?)Pond1-1

pTACF3 (OxO)


GFP expressing embryoGFP spots

Transformation FieldCulture of

single eventShoot

regenerationRooting

(or nut grafting)Acclimatization

LN-1P(1-?)Pond1-1pTACF7

(OxO + ESF39)


AZ-1C(1-?)Pond1-1

p35S-OxO(constitutive OxO)

AZ-1K(1-?)Pond1-1

pWVK147(empty vector control)


WirsigWirsig


Shoot regeneration


Acclimatization

AN-2X(1-5)JM-1E1

Summary of transgenic American chestnut transformation & regeneration pipeline (~18 months)

JM-4E2

LN-1N(1-?)LN-3N(1-?)JH-1W(1-?)JH-3W(1-?)JH-1A(1-?)JH-3A(1-?)TR-3X(1-?)TR-1X(1-?)

LN-1P(1-?)AZ-1C(1-?)AZ-1K(1-?)

Transformation started spring semester 2007(therefore estimated fall 2008 planting)

Transformation started summer 2007(therefore estimated spring 2009 planting)

Transformation will start fall semester 2007(therefore estimated spring 2009 planting)

LP-5V32LP-3V53

RR-1V4 & 13

LP-1V1 & 38

Transformation 2006(spring 2008 planting)


WirsigWirsig


Shoot regeneration


Acclimatization

AN-2X(1-5)JM-1E1

Number of trees(try for minimum of 10 trees per event)

JM-4E2

LN-1N(1-?)LN-3N(1-?)JH-1W(1-?)JH-3W(1-?)JH-1A(1-?)JH-3A(1-?)TR-3X(1-?)TR-1X(1-?)

LN-1P(1-?)AZ-1C(1-?)AZ-1K(1-?)

LP-5V32LP-3V53

RR-1V4 & 13

LP-1V1 & 38

Minimum of 110 - 550 transgenic trees for testing. If resources allow, we would like a goal of 1000 trees in the field by summer 2009. A total of 3000 transgenic American chestnuts by summer 2010. Equal number of control trees.

Minimum of 70 transgenic trees for testing, hopefully more for Fall 2008. Equal number of control trees.

Number unknown, because growthchamber optimization is being done.(Maybe 60 trees for Spring 2008?)

22

NextField testing

Beginning the road to deregulation:

USDA APHIS (benefit & risk assessment)

EPA (environmental impact)

FDA (GM food - substantial equivalence)

Controls & Standard Panel

• Used to compare transgenic trees to a population of similar trees

• Ideally the transgenic chestnut trees will fall within the variability of of the standard panel

• Control trees– Transgenic American chestnut of the same clone but without

the resistance enhancing construct - example: with pGFP only or pGFP and an empty vector only

– If effects are detected, this differentiates between transformation effects and resistance-enhancing gene effects

Controls & Standard Panel

• Standard panel for transgenic American chestnut– Non-transgenic American chestnut of the same

clonal line - example: Ellis1, Pond1-1, 30013-2, etc.– Seedlings of American chestnut trees from different

regions– BC3F2 or F3 American chestnut from backcross

program– Hybrids of American, Chinese, European, and/or

Japanese chestnut– Chinese chestnut as a related species

Blight-resistance

Increasingresistance

Non-transgenicStandard panel

American ChestnutTransgenic Events

In this case, looking for resistance as high or higher that Chinese chestnut

Transgenic with empty vector control

Mycorrhizal colonization

IncreasingMycorrhizalAssociation(type and abundance)

In this case, looking for normal mycorrhizal associations as compared to standard panel




(this has been done with our transgenic American elm)

Insect feeding

Incidence of insect feeding and growth & development of insects

In this case, looking for normal insect associations as compared to standard panel




(this has been done with our transgenic American elm)

28

Test many genes, vector constructs, and events

Only one or two events will be submitted for deregulation and added to the restoration

program(outcross breeding will increase genetic diversity)

Questions?

Experiment: hypovirulence inoculations using paintballs

Moon LibraryTransgenic American ElmAugust 2006 to August 2007

New transformation protocol

• Co-transformation (separating reporter gene and resistance-enhancing gene on two vectors) – new pGFP (GFP + Finale resistance)

– >40% of events should have both vectors

• Allows using the green fluorescent protein (GFP) for selection and

environmental studies, but for restoration it can be breed out.

GFP spots seen under UV light & filter with a microscope

Normal green fluorescence in all chestnuts

Brighter fluorescence with GFP

Questions?

Documents

American Chestnut Research & Restoration A Biotechnology Approach to Resistance Complementary to the Breeding Program W.A. Powell (ESF), S.A. Merkle (UGA),