Transcript Genetic Transformation Methods for Southern Pine

FHI Biotechnology Approaches
Clonal testing
New varieties
Marker-aided
breeding
Transgenics
Genome sequencing
GE trees
UGA Group Personnel and Collaborators
Scott Merkle and Joe Nairn
Dr. Lisheng
Kong
Ray Sakowski
Assistant
Research Scientist
Part-time
Research
Technician II
Transformation and
SE technology
Transgenic SE and
SS production
Ethan Epps
Research Technician
III
•Vector construction
•Gene cloning
•Transgenic screening
Paul Montello
Sam Bryson
Research
Professional III
Part-time Research
Technician II
Louise Jacques
Cryopreservation
Transgenic SE and
SS production
•DNA isolations
•PCR reactions
Ryan Tull
Part-time Assistant
Christine Holtz
Acknowledgments
Research
Technician III
Undergraduate
Student
Researcher
SE culture initiation
and screening
SE culture initiation
and bioreactor testing
Consortium for Plant
Biotechnology Research
Forest Health Initiative
Institute of Forest Biotechnology
ArborGen LLC
The American Chestnut
Foundation
The American Chestnut
Cooperators Foundation
Virginia Department of Forestry
Fred Hebard
Sara Fitzsimmons
Gary and Lucille Griffin
Sandy Anagnostakis
Herb Darling
Bryan Burhans
Jerre Creighton
Wayne Bowman
Bob Leffel
James Donowick
Gary Micsky
Kendra Gurney
Bill Powell
Chuck Maynard
Summary of Year 1 Deliverables
• Embryogenic culture initiation from
full-sib AC & hybrid germplasm
• >9000 seeds cultured
• 64 new embryogenic cultures initiated
•from 2 CP families & 1 OP family from ACCF and VDF
• Embryogenic culture capture rate = 0.7%
• 38 (1.5%) ACCF LSA cultures
• 26 (5.2%) VDF OP hybrid (ACxCCxJC)
cultures
• Screening for SE & SS production
• Some 2009 lines produced > 100 well-formed
embryos per 0.5 g of starting material
• Very high (>50%) conversion in some lines
• Cryostorage of embryogenic cultures
• > 3 copies of each line
• All (64) 2009 embryogenic lines cryostored
• All (276 so far) FHI transclones cryostored
Summary of Year 1 Deliverables
• Construct 1st generation vectors
–
–
–
–
Binary vectors
Agrobacterium-mediated transformation
Clone different genes and/or promoters
Constitutive promoters
• Arabidopsis Ubiquitin-10 (pFHI-03)
• CaMV 35S (pFHI-04)
• npt II gene for selection in chestnut
GUS intron
pFHI-GUSi
GUS intron-YFP fusion
pFHI-GUSiYFP
• pFHI-03-based reporter vectors
– Characterization of
•
•
•
•
Transformation efficiency
Promoter activity
Transcription
Translation
– Destructive and non-destructive assays
• GUS staining
• Fluorescent protein visualization
GFP
pFHI-GFP
Progress with Supplement 1 Deliverables
Molecular characterization of American Chestnut lines
•Evaluating pFHI vectors for American Chestnut
•High transformation efficiencies in multiple genotypes:
• pFHI vectors
• hyper-virulent Agrobacterium line AGL1
•Stringent Selection:
• < 0.8% escapes
•High levels of gene expression:
• Transcription & Translation
• GUS assays & in vivo YFP imaging
AM54
CD322
WB484
Embryo
pFHI-GUSiYFP
Progress with Supplement 1 Deliverables
Molecular characterization of transformed American Chestnut
Tag #
pTACF6 plants
81
73
28
29
47
NPTII
• >150 plants, 3 genotypes
• Transplanted to field for blight
screening, May 2011
TAG#
81
73
28
29
47
ESF39A
•Transferred to Clemson for
Phytophthora screening, May 2011
GENOTYPE NPTII ESF39A
WB484-3
+
+
WB484-3
+
+
WB484-3
+
+
WB484-3
+
+
AM54-1
-
Cdlac
+
+
+
+
+
CDlac
Embryogenic cultures
Lane
Sample #
Primers
Result
1
20
Thaum
+
• Screen tissues at 6 weeks post-transformation
2
20
CdCyst
+
• Early identification of somatic seedling lines
3
22
Thaum
+
4
22
CdCyst
+
19
Wt-1
Thaum
-
20
Wt-1
CdCyst
+
-
• Only need 10 mg of tissue
1
Cd cystatin (control)
Cm thaumatin
2
3
4
19 20 21 22 23 24
21
Wt-2
Thaum
22
Wt-2
CdCyst
+
23
ddH2O
Thaum
-
24
ddH2O
CdCyst
-
Progress with Supplement 1 Deliverables
Increase chestnut transformation productivity 4-fold,
from 2 CGs to 8 CGs per year
• Transformation rate boosted to
between 12 and 24 CGs built
into vectors and transformed
into chestnut cells per year
– Efficient vector construction
– Air-lift bioreactor-based
generation of embryogenic target
material makes a new batch
available for transformation with
CGs every 2 weeks
Progress with Year 2 Deliverables:
Initiate new embryogenic cultures from full-sib AC and
B3F3 germplasm and screen for SE and SS production
(second round)
• Over 8500 seeds cultured
– 107 new embryogenic cultures representing 19 different
AC and B3F3 families
• Overall capture frequency = 1.23%
– Best family capture frequency = 6%
B3F3 culture initiation
• New Germplasm Agreement with TACF!!!
– Allowed us to culture 10 TACF B3F3 families
– Captured B3F3 families representing both Graves (W)
and Clapper (D) lines of resistance
• All 2010 lines now being screened for SE and SS
production potential
“Captured” B3F3
Progress with Year 2 Deliverables:
Regenerate and establish somatic seedlings from ACCF
(or other American chestnut) lines and TACF B3F3 lines
• Two 2009 ACCF lines and four 2009 VDF ACxCCxJC
hybrid lines generated at least the minimum 10 somatic
seedlings required for clonal testing
Somatic seedlings from 2009 culture lines
Progress with Year 2 Deliverables:
Screen embryogenic lines for transformation competence
to identify best “workhorse” lines
• Based on screens of existing and new
lines for SE & SS productivity:
– One 2007 CP AC line chosen as current
lead “workhorse” line for transformation
• For each CG, 40 events 20 plant
forming transclones 10 SS each for
screening
– Second 2007 CP AC line chosen as first
back-up workhorse line for transformation
– One 2009 VDF ACxCCxJC line chosen as
second back-up workhorse line
• Copies of these lines sent to SUNYESF collaborators in February 2011
Geneticin-resistant
colonies arise under
liquid selection
Progress with Year 2 Deliverable:
Transform Candidate Genes into American chestnut lines
Progress through the “pipeline”
Construct
Target Transformed Geneticin Transclones Transclones Transclones in Somatic embryos
lines
lines
resistant
on plates
in flasks
SE production
harvested
pFHI-GUSi
4
3
>65
65
42
35
142
pFHI-GUSiYFP
6
4
360
360
16
7
NA
pFHI-NPR1
4
4
132
132
59
47
1,514
pFHI-Thaum
8
5
> 1,360
1,360
212
125
1,406
pFHI-ACPHOS
4
4
>708
708
pFHI-UDPGTI
4
3
pFHI-cmPRP
4
4
>1,307
1,307
pFHI-cmLac
3
2
>500
367
pFHI-B-Gluc
4
3
>109
109
pFHI-CBS1
4
4
>600
312
pFHI-ETF1
4
4
>600
366
pFHI-GAFP1
5
>3
> 42
42
pFHI-Cyst1
4
>2
NA
pFHI-LTP1
4
>2
NA
pFHI-GFP
4
NA
NA
329
214
3062
Totals
5128
Progress with Year 1 & 2 Deliverables:
Establish field (nursery) site in Georgia with current
"first generation" transgenic lines
• Field planting permit issued by
APHIS-BRS on March 4, 2011
• Planting of 100 trees
representing 10 different
pTACF6 (ESF39A) events and
10 transgenic controls in May
2011
• Trees should reach standard
inoculation diameter for
Cryphonectria screening by
summer 2012
Major milestones on the way to
“the plantable tree”
• Over 170 new embryogenic chestnut cultures
initiated for clonal testing and transformation
• Germplasm agreements allowed initiation of
the first ever TACF B3F3 embryogenic cultures
for clonal testing
• Bioreactor technology has accelerated
candidate genes through the “pipeline” by 12 X
• 12 candidate genes and 3 reporter genes
transformed into multiple chestnut genotypes
• Chestnut cultures can be screened for stable
transformation only 6 weeks after
transformation
• First “southern” field test of transgenic
chestnuts established