Transgenic Banana Resistant to Banana Bunchy Top Virus

John Hu, et al.Plant & Environmental Protection Sciences

Transgenic Banana Project at UHM

This work is a result of contributions made by many individuals: Dr. Eden PerezDr. John Hu

Dr. Kheng Cheah Dr. Diane SetherDr. Said Khalil

Mr. Jason Lutz Dr. Wayne BorthMr. Doug Gaskill

Ms. Bethany Kimokeo (UH) Mr. Scott Henderson (UH) Dr. Douglas Becker (Aus)

Previous Work• ‘Dwarf Brazilian’ meristems were transformed• About 300 transformed plants were produced• These were screened for BBTV resistance in

bioassays using aphid inoculations• 100% of non-transformed control plants developed

symptoms within 3-4 weeks• 7 transformed plants did not develop BBTV

symptoms for one year after inoculation– BUT…all 7 eventually developed symptoms

Initiation of Cell Suspension Cultures

Bracts and ‘hands’Male inflorescence

Surface sterilization

Apical ~3cm

Production of EmbryogenicCell Suspensions

Male Inflorescence Male Flowers EmbryogenicCallus

2-4 weeks

EmbryogenicCell Suspension

Homogeneous Cell Suspension

1-6 months

Transformation of EmbryogenicCell Suspensions

Agrobacterium tumefaciens mediated

BBTV constructs:Coat Protein (CP)Coat Protein Mutation (M1)Replication Associated Protein (Rep)

What is BBTV?

100 nm ORF 1: Replication-associated protein (Rep)

ORF 3: Coat protein (CP)

Genome Component Functions

Regeneration of Transgenic Plants

CR

LP

RI

Establishment of Transgenic Plants

Establishment in greenhouse

Tissue culture

Screening for BBTV Resistance

Adults collected 5-10 adult aphids allowed 10 day

inoculation access

Aphids raised on BBTV-infected banana

Current Status of Transgenic Banana Lines

• # inoculated / # of lines– M1: 40 / 60– AS: 59 / 90– Rep∆2/5: 3 / 113– ∆2/5: 0 / 59– Control: 23 / 49

• Total # lines = 322• Total # inoculated = 102

Future Work

• Continue screening transgenic banana lines for BBTV resistance

• Further characterize any BBTV-resistant transgenic banana lines

• Establish and maintain new ‘Dwarf Brazilian’ embryogenic cell cultures

• Transform these embryogenic cell cultures with new constructs

Funding Support

USDA-ARSHawaii DOA

USDA-CSREES (T-STAR)