|Authors: ||W. Borth, E. Perez, K. Cheah, Y. Chen, W.S. Xie, D. Gaskill, S. Khalil, D. Sether, M. Melzer, M. Wang, R. Manshardt, D. Gonsalves, J.S. Hu|
Embryogenic cell suspensions (ECS) initiated from immature male flowers of banana cultivar ‘Dwarf Brazilian’ (AAB, Pome subgroup) were transformed using Agrobacterium tumefaciens containing one of four constructs derived from the replicase-associated protein (Rep) gene of the Hawaiian isolate of Banana bunchy top virus (BBTV). Each construct was engineered under control of a double CaMV 35S promoter and the AMV enchancer sequence in the binary plasmid pBI121. Constructs were transferred into A. tumefaciens strain AGL0 and used to transform banana ECS. Plantlets that survived antibiotic selection were acclimated to greenhouse conditions and challenged with viruliferous banana aphids (Pentalonia nigronervosa). Ten adult or late instar aphids were allowed to feed for 2-4 weeks on test plants.
All test plants were kept in the greenhouse and monitored for symptom expression for a period of 6 months.
Control plants transformed with empty vector pBI121 only were included in all tests.
A total of 270 test plants and 63 control plants were screened for BBTV resistance using this approach.
One of 32 test plants transformed with the M1 (mutant Rep gene) construct, 5 of 74 test plants transformed with the AS1 (antisense Rep gene) construct, 5 of 38 test plants transformed with the PR1 (partial Rep gene) construct, and 10 of 126 test plants transformed with the R/PR1 (full-length Rep gene fused to antisepses partial Rep gene) construct were found to be resistant to BBTV challenge and showed no bunchy top symptoms.
All of the control plants became infected with BBTV under these experimental conditions.
Plants that survived BBTV challenge were analysed by quantitative PCR (per) and Southern hybridisations to determine the number of transgenes that were present in their genomes.
Results from these analyses indicated that the resistant plants contained from 2 to more than 9 copies of the NPTII (kanamycin resistance) transgene carried on the pBI121 plasmid.
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