FHI Biotechnology Approaches

1. FHI Biotechnology Approaches Clonal testing New varieties Marker-aided breeding Transgenics Genome sequencing GE trees

2. UGA Group Personnel and CollaboratorsScott Merkle and Joe Nairn Dr. Lisheng Kong Assistant Research Scientist Transformation and SE technology Ray Sakowski Part-time Research Technician II Transgenic SE and SS production • Ethan Epps • Research Technician III • Vector construction • Gene cloning • Transgenic screening Paul Montello Research Professional III Cryopreservation Sam Bryson Part-time Research Technician II Transgenic SE and SS production • Louise Jacques • Part-time Assistant • DNA isolations • PCR reactions Christine Holtz Undergraduate Student Researcher SE culture initiation and bioreactor testing Ryan Tull Research Technician III SE culture initiation and screening Acknowledgments Forest Health Initiative Institute of Forest Biotechnology ArborGen LLC Consortium for Plant Biotechnology Research 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

3. 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

4. Summary of Year 1 Deliverables GUS intron pFHI-GUSi • Construct 1st generation vectors • Binary vectors • Agrobacterium-mediated transformation • Clone different genes and/or promoters • Constitutive promoters • ArabidopsisUbiquitin-10 (pFHI-03) • CaMV 35S (pFHI-04) • npt II gene for selection in chestnut • pFHI-03-based reporter vectors • Characterization of • Transformation efficiency • Promoter activity • Transcription • Translation • Destructive and non-destructive assays • GUS staining • Fluorescent protein visualization GUS intron-YFP fusion pFHI-GUSiYFP GFP pFHI-GFP

5. Progress with Supplement 1 DeliverablesMolecular 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

6. Progress with Supplement 1 DeliverablesMolecular characterization of transformed American Chestnut • pTACF6 plants • >150 plants, 3 genotypes • Transplanted to field for blight screening, May 2011 • Transferred to Clemson for Phytophthora screening, May 2011 1 2 3 4 19 20 21 22 23 24 Tag # 81 73 28 29 47 NPTII • Embryogenic cultures • Only need 10 mg of tissue • Screen tissues at 6 weeks post-transformation • Early identification of somatic seedling lines ESF39A CDlac Cd cystatin (control) Cm thaumatin

7. Progress with Supplement 1 DeliverablesIncrease 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

8. 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% • 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 B3F3 culture initiation “Captured” B3F3 culture

9. 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

10. 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 SUNY-ESF collaborators in February 2011 Geneticin-resistant colonies arise under liquid selection

11. Progress with Year 2 Deliverable: Transform Candidate Genes into American chestnut lines Progress through the “pipeline”

12. 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

13. 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