american chestnut research & restoration a biotechnology approach to resistance complementary to...
TRANSCRIPT
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
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)
Transformation FieldCultures of single event
Shoot regeneration
Rooting(or nut grafting)
Acclimatization
Transgenic American chestnut transformation & regeneration pipeline (~18 months)
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
Rooting(or nut grafting)
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
Transformation FieldCultures of single event
Shoot regeneration
Rooting(or nut grafting)
Acclimatization
Transgenic American chestnut transformation & regeneration pipeline (~18 months)
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)
Transgenic American chestnut transformation & regeneration pipeline (~18 months)
GFP expressing embryoGFP spots
Transformation FieldCulture of
single eventShoot
regenerationRooting
(or nut grafting)Acclimatization
LN-1P(1-?)Pond1-1pTACF7
(OxO + ESF39)
Transgenic American chestnut transformation & regeneration pipeline (~18 months)
AZ-1C(1-?)Pond1-1
p35S-OxO(constitutive OxO)
AZ-1K(1-?)Pond1-1
pWVK147(empty vector control)
Transformation Field
WirsigWirsig
Cultures of single event
Shoot regeneration
Rooting(or nut grafting)
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)
Transformation Field
WirsigWirsig
Cultures of single event
Shoot regeneration
Rooting(or nut grafting)
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
Non-transgenicStandard panel
American ChestnutTransgenic Events
Transgenic with empty vector control
(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
Non-transgenicStandard panel
American ChestnutTransgenic Events
Transgenic with empty vector control
(this has been done with our transgenic American elm)
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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)
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