Development of Transgenic Bananas for BBTV Resistance

John Hu956-7281 johnhu@hawaii.edu

Plant and Environmental Protection Sciences

Progress Report

1. Detection and characterization of BBTV2. Banana regeneration and transformation3. Transformation with BBTV genes 4. Current research and future plans

Banana Bunchy Top Virus

DETECTION CHARACTERIZATION MANAGEMENT

ELISADot-blot

PCRReal-time PCR

Vector transmissionHost range

Virus diversityGenome organization

IndexingGenetic

engineering

Real-Time PCR Assay

Advantages:- Sensitive- Robust- Easy- Large number- Quantitative

Comparison of Chinese BBTV Isolate

Hawaii Philippinesn.t. a.a. n.t. a.a.

___________________________________REP 96 88 96 98CP 93 99 - 98MP 91 88 - -

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

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

‘Williams’ ‘Dwarf Brazilian’

Banana Cell Suspension Cultures

Regeneration

AA BB C

EE FFD

‘Brazilian’ in the nursery

‘Brazilian’bunches

Field Observations

• Uniform characteristics - no off-types - flowered at the same time

• Fruit shape similar to ‘Dwarf Brazilian/Apple’

Transformation

BBTV Gene ConstructsHawaiian BBTV genes

CP geneREP gene

- anti-sense (AS)- sense mutant (M1)

Chinese BBTV genes

Transformation of Cell Suspensions

A B

CC DD EE

Greenhouse Analyses for BBTV Resistance

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

Collaboration with Australia

• Potential BBTV-resistant banana plants• Field evaluation in Hawaii• IP issues• “tabled”, focus on our own efforts

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

Established ECS of ‘Williams’ and ‘Dwarf Brazilian’

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)

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!)

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.

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

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

Acknowledgments

Hawaii DOAUSDA-ARS PBARC

USDA-CSREES (T-STAR)

Kheng Cheah Said Khalil Doug Gaskill Yan Chen Eden Perez Wayne Borth

Acknowledgments

Scot NelsonDiane SetherCerruti Hooks

Rodrigo AlmeidaDennis Gonsalves

Hawaii Banana Industry Association