FHI Biotechnology Approaches

Genome sequencing

Clonal testing

Transgenics

GE trees

New varieties

Marker-aidedbreeding

UGA Group Personnel and CollaboratorsUGA Group Personnel and CollaboratorsScott Merkle and Joe NairnScott Merkle and Joe Nairn

Dr. Lisheng KongAssistant Research ScientistTransformation and SE technology

Paul MontelloResearch Professional IIICryopreservation

Ryan TullResearch Technician IIISE culture initiation and screening

Ray SakowskiPart-time Research Technician IITransgenic SE and SS production

Sam BrysonPart-time Research Technician IITransgenic SE and SS production

Christine HoltzUndergraduate Student ResearcherSE culture initiation and bioreactor testing

Ethan EppsResearch Technician III•Vector construction•Gene cloning•Transgenic screening

Louise JacquesPart-time Assistant•DNA isolations•PCR reactions

AcknowledgmentsForest Health Initiative

Institute of Forest Biotechnology

ArborGen LLC

Consortium for Plant Biotechnology Research

The American Chestnut Foundation

The American Chestnut Cooperators FoundationVirginia Department of Forestry

Fred HebardSara FitzsimmonsGary and Lucille GriffinSandy AnagnostakisHerb DarlingBryan BurhansJerre CreightonWayne BowmanBob LeffelJames DonowickGary MicskyKendra GurneyBill PowellChuck Maynard

Summary of Year 1 DeliverablesSummary 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 DeliverablesSummary of Year 1 Deliverables

• Construct 1Construct 1stst generation vectors 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

• pFHI-03-pFHI-03-based reporter vectorsbased reporter vectors– Characterization of

• Transformation efficiency

• Promoter activity

• Transcription

• Translation

– Destructive and non-destructive assays• GUS staining

• Fluorescent protein visualization

GUS intron

pFHI-GUSi

GUS intron-YFP fusion

pFHI-GUSiYFP

GFP

pFHI-GFP

Progress with Supplement 1 DeliverablesProgress with Supplement 1 DeliverablesMolecular characterization of American Chestnut linesMolecular 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 WB484

CD322

AM54

Embryo

pFHI-GUSiYFP

Tag # 81 73 28 29 47

NPTII

ESF39A

CDlac

TAG# GENOTYPE NPTII ESF39A Cdlac81 WB484-3 + + +73 WB484-3 + + +28 WB484-3 + + +29 WB484-3 + + +47 AM54-1 - - +

Progress with Supplement 1 DeliverablesProgress with Supplement 1 DeliverablesMolecular characterization of transformed American ChestnutMolecular 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

Embryogenic cultures• Only need 10 mg of tissue

• Screen tissues at 6 weeks post-transformation

• Early identification of somatic seedling lines

1 2 3 4 19 20 21 22 23 24

Lane Sample # Primers Result

1 20 Thaum +

2 20 CdCyst +

3 22 Thaum +

4 22 CdCyst +

19 Wt-1 Thaum -

20 Wt-1 CdCyst +

21 Wt-2 Thaum -

22 Wt-2 CdCyst +

23 ddH2O Thaum -

24 ddH2O CdCyst -

Cd cystatin (control)

Cm thaumatin

Progress with Supplement 1 DeliverablesProgress with Supplement 1 DeliverablesIncrease chestnut transformation productivity 4-fold, Increase chestnut transformation productivity 4-fold,

from 2 CGs to 8 CGs per yearfrom 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:Progress with Year 2 Deliverables:Initiate new embryogenic cultures from full-sib AC and Initiate new embryogenic cultures from full-sib AC and B3F3 germplasm and screen for SE and SS productionB3F3 germplasm and screen for SE and SS production

(second round) (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

Progress with Year 2 Deliverables:Progress with Year 2 Deliverables:Regenerate and establish somatic seedlings from ACCF Regenerate and establish somatic seedlings from ACCF (or other American chestnut) lines and TACF B3F3 lines (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:Progress with Year 2 Deliverables:Screen embryogenic lines for transformation competence Screen embryogenic lines for transformation competence

to identify best to identify best ““workhorseworkhorse”” lines 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

Progress with Year 2 Deliverable:Progress with Year 2 Deliverable: Transform Candidate Genes into American chestnut lines Transform Candidate Genes into American chestnut lines

Progress through the “pipeline”

ConstructTarget lines

Transformed lines

Geneticin resistant

Transclones on plates

Transclones in flasks

Transclones in SE production

Somatic embryos 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        

Totals       5128 329 214 3062

Progress with Year 1 & 2 Deliverables:Progress with Year 1 & 2 Deliverables:Establish field (nursery) site in Georgia with current Establish field (nursery) site in Georgia with current

"first generation" transgenic lines "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 Major milestones on the way to ““the plantable treethe 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