American Chestnut Research & Restoration
A Biotechnology Approach to Resistance
Complementary 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)
WB-275-27
Southern genotype
2006
2007
Proof of transformation concept.
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 chestnuts
to find the ideal tree to include in
restoration
Optimum gene
Optimum promoter
(genetic switch)
Gene pyramids?
(combining 2 or
more genes)
Optimum event
(different levels
of expression)
Process: Test many transgenic trees, but eventually choose only the best one
or two events to be deregulated. These trees would be out-cross to capture the
An event represents the gene going into a specific location in the chromosomes.
surviving chestnut’s genetic diversity and added to the restoration program.
In the U.S. regulatory process, each even must be deregulated.
All transgenic plants to date (corn, soybean, cotton etc.) use a “constitutive” promoter
The advantage of this technique is that new genes can be added to the program
We
are than
testing
(wound-inducible
vascular)
in less
tworegulated
years, if promoters
the need arises
(for exampleand
if new
pests are introduced).
Genes & vectors
(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
1.Broad-Spectrum
Oxalate oxidase
(OxO)
from
wheatPeptide D4E1
Antimicrobial
Activity gene
in vitro of
the Synthetic
A.
Detoxifies oxalic acid produced by the blight fungus
& protects the lignin produced by the tree
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.
p∆VspB-OxO,
pTACF3,
p35S-CNO, p35S-OxO
Lee Boulevard, New Orleans,
LouisianapTACF7,
70124
for review
February 6,antimicrobial
2001. Revised manuscript
received April 5, 2001. Accepted April 5, 2001.
2.Received
ESF39
or ESF12
peptide
Abstract:
A.
Kills the blight fungus, Cryphonectria parasitica
B. Might also be useful against Phytophthora cankers
Broad-spectrum antimicrobial activity of a synthetic peptide, D4E1, is documented in this paper. D4E1 inhibited the
American
growth of several fungal phytopathogens belonging to four classes-Ascomycetes, Basidiomycetes, Deuteromycetes,
chestnut
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
3.growth
Chitinase
fromranged
Trichoderma
of all fungi studied
from 4.67 to 25 M. Fungal pathogens highly sensitive to D4E1 include
Thielaviopsis
basicola, Verticillium
Fusarium
Phytophthora cinnamomi, and Phytophthora
A. Degrades
the dahliae,
cell wall
of moniliforme,
C. parasitica
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 Steven N. Jeffers
analysis of D4E1 effects on fungal morphology of Aspergillus flavus and R. solani revealed abnormal hyphal growth Clemson University
4.
Ac-AMP1.2 antimicrobial peptide from Ameranth
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.
pTACF6, pTACF7, pCWEA1
p35S-CNO
pCWEA1, pCA1
Control vectors: pGFP & pWVK147
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
Current status of transgenic
American chestnut
Research is moving from the lab
to the field
7
Transgenic American chestnut transformation &
regeneration pipeline (~18 months)
Transformation
Transferring
every 2 weeks
and visually
selecting
spotted to fully
fluorescent
embryos
Cultures of
single event
Shoot
regeneration
Multiply up
the
numbers of
embryos
Maintain cultures
of every event,
transferring every
2-3 weeks
Extract
DNA &
test for
genes
using
PCR
Rooting
(or nut grafting)
Multiply up
the
numbers of
shoots
Extract DNA for
Southern
hybridization to
determine
insert copy
number
Acclimatization
Field
Watering,
fertilizing,
& watching
Growth
chamber &
greenhouse
Site prep,
fertilizing,
Weeding,
watering
and pest
control
Maintain
cultures of
every event,
transferring
shoots every
4 weeks
RT-PCR,
enzyme
assays,
resistance
assays, &
other exp.
Biggest bottleneck - acclimatization
(~18 months to produce transgenic chestnut plants)
Last year produced >400 potted plants,
only 15 survived to the field
New Growth chambers with
humidity, light intensity, &
CO2 control
Nut Grafting
Testing for best commercial
soil mix
Two new growth chambers for improved
acclimatization
Old growth chamber
(the “dungeon”)
Replacement in Nov. 2007
Two Conviron ATC60s
Nut Grafting
Cut a slit that bisects
the two cotyledons
Place graft in vessel
with peat moss
Slice a wedge at the bottom
of a tissue culture shoot.
Roots come out of
grafted nut
Insert chestnut shoot
into the slit
Transgenic shoot grafted onto a
germinated chestnut. Tree was
planted on June 7, 2007. Photo
was taken 2 weeks after planting.
Potting Mixes
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
La Pierre Special (2 peat: 1 vermiculite: 1 pearlite)
Fred Hebard’s Special (1 peat: 1 vermiculite: 1 pearlite)
Faford Germinating Mix
Faford #52 Mix
Faford C1-P Growers Mix
Faford Nursery Mix
Sungro Metro Mix 360
Sungro Metro Mix 390
Sungro Metro Mix HP
Sungro Metro Mix 560 Coir
Standard Control – La Pierre Special
Ave. Total Dry Weight (g) by Potting Mix
8.00
7.23
7.00
6.37
5.91
6.00
Average Dry Weight (g)
5.55
5.87
5.38
4.95
5.00
4.24
4.00
3.86
3.21
3.21
8.Sungro
Metro Mix
390
9.Sungro
Metro Mix
HP
3.00
2.00
1.00
0.00
1.La Pierre
Special
2.Fred
HebardÕs
Special
3.Faford
4.Faford #52 5.Faford C16.Faford
Germinating
Mix
P Growers Nursery Mix
Mix
Mix
Potting Mix
7.Sungro
Metro Mix
360
10.Sungro
Metro Mix
560 Coir
11.Standard
Control Š La
Pierre
Special
Transgenic American chestnut transformation &
regeneration pipeline (~18 months)
Transformation
Cultures of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
Wirsig
WB275-27
p∆VspB-OxO
15 Wirsig trees
in the field
Wirsig Variety (LP-2V28 event)
2 growing seasons later (~15 months)
RT-PCR
OxO RNA
Wirsig
Control
Control Wirsig
OxO assays
Stable transformation
Transgenic American chestnut transformation events &
regeneration pipeline (~18 months)
Transformation
Cultures of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
Wirsig
WB275-27
p∆VspB-OxO
LP-1V1 & 38
Pond1-1
p∆VspB-OxO
LP-5V32
WB348-5
p∆VspB-OxO
LP-3V53
30015-2
p∆VspB-OxO
RR-1V4 & 13
Pond1-1
p∆VspB-OxO
15 Wirsig trees
in the field
Transgenic American chestnut transformation &
regeneration pipeline (~18 months)
Transformation
Cultures of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
AN-2X(1-5)
WB275-27
pTACF3 (OxO)
JM-1E1
Pond1-1
pTACF6 (ESF39)
JM-4E2
Ellis-1
pTACF6 (ESF39)
LN-1N(1-?)
Pond1-1
p35S-CNO
(Chitinase + OxO)
Shoot formation
LN-3N(1-?)
30015-2
p35S-CNO
(Chitinase + OxO)
GFP spots
GFP expressing embryo
Transgenic American chestnut transformation &
regeneration pipeline (~18 months)
Transformation
Culture of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
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 spots
GFP expressing embryo
Transgenic American chestnut transformation &
regeneration pipeline (~18 months)
Transformation
LN-1P(1-?)
Pond1-1
pTACF7
(OxO + ESF39)
AZ-1C(1-?)
Pond1-1
p35S-OxO
(constitutive OxO)
AZ-1K(1-?)
Pond1-1
pWVK147
(empty vector control)
Culture of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
Summary of transgenic American chestnut transformation
& regeneration pipeline (~18 months)
Transformation
Cultures of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
Wirsig
LP-1V1 & 38
LP-5V32
LP-3V53
RR-1V4 & 13
AN-2X(1-5)
JM-1E1
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 2006
(spring 2008 planting)
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)
Number of trees
(try for minimum of 10 trees per event)
Transformation
Cultures of
single event
Shoot
regeneration
Rooting
(or nut grafting)
Acclimatization
Field
Wirsig
LP-1V1 & 38
LP-5V32
LP-3V53
RR-1V4 & 13
AN-2X(1-5)
JM-1E1
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-?)
Number unknown, because growth
chamber optimization is being done.
(Maybe 60 trees for Spring 2008?)
Minimum of 70 transgenic trees for testing,
hopefully more for Fall 2008. Equal number
of control trees.
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.
Next
Field testing
Beginning the road to deregulation:
USDA APHIS (benefit & risk assessment)
EPA (environmental impact)
FDA (GM food - substantial equivalence)
22
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
In this case, looking for
resistance as high or
higher that Chinese
chestnut
Increasing
resistance
Non-transgenic
Standard panel
Transgenic
with empty
vector
control
American Chestnut
Transgenic Events
Mycorrhizal colonization
(this has been done with our transgenic American elm)
In this case, looking for
normal mycorrhizal
associations as
compared to standard
panel
Increasing
Mycorrhizal
Association
(type and
abundance)
Non-transgenic
Standard panel
Transgenic
with empty
vector
control
American Chestnut
Transgenic Events
Insect feeding
(this has been done with our transgenic American elm)
In this case, looking for
normal insect
associations as
compared to standard
panel
Incidence of
insect feeding
and growth &
development
of insects
Non-transgenic
Standard panel
Transgenic
with empty
vector
control
American Chestnut
Transgenic Events
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)
28
Questions?
Experiment: hypovirulence inoculations using paintballs
Moon Library
Transgenic American Elm
August 2006 to August 2007
New transformation protocol
• Co-transformation (separating reporter gene and resistanceenhancing 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.
Brighter fluorescence with GFP
Normal green fluorescence in all chestnuts
GFP spots seen under UV
light & filter with a microscope
Questions?