development of transgenic bananas for bbtv resistancesnelson/hbia/development_of... · development...
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Development of Transgenic Bananas for BBTV Resistance
John Hu956-7281 [email protected]
Plant and Environmental Protection Sciences
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Progress Report
1. Detection and characterization of BBTV2. Banana regeneration and transformation3. Transformation with BBTV genes 4. Current research and future plans
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Banana Bunchy Top Virus
DETECTION CHARACTERIZATION MANAGEMENT
ELISADot-blot
PCRReal-time PCR
Vector transmissionHost range
Virus diversityGenome organization
IndexingGenetic
engineering
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Real-Time PCR Assay
Advantages:- Sensitive- Robust- Easy- Large number- Quantitative
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Comparison of Chinese BBTV Isolate
Hawaii Philippinesn.t. a.a. n.t. a.a.
___________________________________REP 96 88 96 98CP 93 99 - 98MP 91 88 - -
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Previous Work
• Used the meristem system• Produced ~300 putative transformed plants• Examined for BBTV resistance in bioassays
using aphid inoculations• 100% of non-transformed control plants
developed symptoms within 3-4 weeks• 7 did not develop BBTV symptoms one year
after inoculation• All 7 were chimeras and eventually became
infected
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BBTV Titers in Leaves
1 2 3 4 5
#190 1.876 0.074 0.232 0.001 0.001# 8 0.603 0.066 1.101 0.001 0.002
-CK 0.002 0.003 0.001 0.002 0.001+CK 1.594 1.468 1.112 0.897 0.376
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‘Williams’ ‘Dwarf Brazilian’
Banana Cell Suspension Cultures
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Regeneration
AA BB C
EE FFD
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‘Brazilian’ in the nursery
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‘Brazilian’bunches
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Field Observations
• Uniform characteristics - no off-types - flowered at the same time
• Fruit shape similar to ‘Dwarf Brazilian/Apple’
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Transformation
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BBTV Gene ConstructsHawaiian BBTV genes
CP geneREP gene
- anti-sense (AS)- sense mutant (M1)
Chinese BBTV genes
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Transformation of Cell Suspensions
A B
CC DD EE
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Greenhouse Analyses for BBTV Resistance
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Results
1) Over 600 banana plants were produced and screened for BBTV resistance using aphid bioassay
2) More than 60 plants showed initial resistance to BBTV (delay in the expression of symptoms compared to non-transgenic banana plants)
3) 3 plants remained symptom-free after two challenges, but eventually became symptomatic
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Collaboration with Australia
• Potential BBTV-resistant banana plants• Field evaluation in Hawaii• IP issues• “tabled”, focus on our own efforts
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Current Work1. Established new cell suspension cultures
for both ‘Williams’ and ‘Apple’ cultivars2. Developed new BBTV gene constructs
using “gene silencing” technology3. Producing more transgenic banana
plants with NEW BBTV gene constructs4. Characterizing transgenic banana plants
using real-time PCR5. Evaluating BBTV-resistant banana plants
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Established ECS of ‘Williams’ and ‘Dwarf Brazilian’
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Established Lines of ECS
Cultivar Total No. No. of flasksof lines in culture
Old ECSWilliams 1 20 (125-ml)Dwarf Brazilian 1 10 (125-ml)
New ECSWilliams 4 8 (50-ml)Dwarf Brazilian 6 10 (125- & 50-ml)
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New BBTV Gene Constructs
RB LB1 P-NOS NPT II NOS-T 35S Rep∆2/5Rep NOS-T Rep∆2/5Rep
RB LB2 P-NOS NPT II NOS-T 35S 2/5Rep NOS-T 2/5Rep
RB3 P-NOS NPT II NOS-T 35S ∆2/5Rep NOS-T LB ∆2/5Rep
(Using similar gene constructs for TYLCV, Yang et al. obtained up to 60% immune tomato plants!)
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Progress on ECS Transformation with Different Rep Gene Constructs
Constructs T02/22 T04/22 T04/25 T05/01 T05/22 T05/24
W DB W DB W DB W DB W DB W DB
pBI121 - - 0/6 5/6 0/5 3/5 0/5 2/5 0/5 2/5 0/5 5/5
M1 9/12 7/12 0/6 3/6 0/5 0/5 - - 4/5 5/5 0/5 5/5
AS 6/12 12/12 0/6 2/6 0/5 3/5 - - 2/5 5/5 0/5 5/5
2/5Rep - - - - - - 0/5 5/5 0/5 5/5 0/5 5/5
∆2/5Rep - - - - - - 0/5 4/5 0/5 5/5 0/5 5/5
Rep ∆2/5Rep - - - - - - - 0/5 5/5 0/10 4/10
Data represents plates with Agro-infected ECS forming embryos/total plates with. Severe browning of ECS were observed on W and only slight on DB’s.
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Summary of Banana ECS Transformed with BBTV Rep Gene Constructs
Constructs Transformation No. of Plates with EmbryosExperiments Produced from Agro-infected
ECS/Total No. of Plates Williams Dwarf Brazilian
pBI 121 5 0/27 17/27
M1 5 13/33 20/33
AS 5 8/33 27/33
2/5 Rep 3 0/15 15/15
∆2/5 Rep 3 0/15 14/15
Rep ∆2/5 Rep 2 0/15 9/15
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Summary of ECS Transformation with Rep Gene Constructs
T02/22/06 M1 & AS in maturation media (M3Kan100)
T04/22/06 pBI121,M1,AS in maturation media (M3Kan100)
T04/25/06 pBI121,M1,AS due for transfer into maturation media
T05/01/06 pBI121, 2/5Rep, 84 days in selection (ModM2Cef200Kan100)
∆2/5Rep
T05/22/06 pBI121, 2/5Rep, 63 days in selection
∆2/5Rep,
Rep∆2/5Rep
M1, AS
T05/24/06 same constructs 61 days in selection
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Acknowledgments
Hawaii DOAUSDA-ARS PBARC
USDA-CSREES (T-STAR)
Kheng Cheah Said Khalil Doug Gaskill Yan Chen Eden Perez Wayne Borth
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Acknowledgments
Scot NelsonDiane SetherCerruti Hooks
Rodrigo AlmeidaDennis Gonsalves
Hawaii Banana Industry Association