Studies on genetic transformation of banana for resistance to banana bunchy top virus using Agrobacterium tumefaciens

• Main Authors: Wen-Wen Yang, 楊文雯
• Other Authors: Pung-Ling Huang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/23987718051650668837

碩士 === 國立臺灣大學 === 園藝學研究所 === 90 === The development of a regeneration system from banana (Musa spp. cv. ‘Pei Chiao’, Cavendish, AAA group) hand primordia on male flowers is described. The most important factors affecting embryogenic callus initiation were the source of explant and the composition of the culture medium. The closer the hand primordia is situated to the inflorescence apical meristem, the stronger is the regenerative capability. Improved callus initiation was obtained on culture medium supplemented with 1 mg/l picloram and 4 mg/l 2,4-D. Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing 1 mg/l picloram, 4 mg/l 2,4-D, 1 mg/l IAA, and 1 mg/l NAA. Somatic embryo germination and plantlet development was obtained using established protocols. A protocol for the production of transgenic banana (Musa spp. cv. ‘Pei Chiao’, Cavendish, AAA group) was developed via Agrobacterium-mediated genetic transformation of hand primordia. Two disarmed Agrobacterium tumefaciens strains, A 281 and LBA 4404, both carrying the binary plasmid pBIRC1 with the nptⅡ gene were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While wounding was inhibitory, the pre-culture (12 days), acetosyringone treatment (200 μM), bacterial growth phase (optical density; OD600 = 1.0), co-cultivation period (2 days) had positive effects on transformation. Following co-cultivation, hand primordia were placed on multiplication medium with cefotaxime for 2 weeks then transfered onto the same medium and stressed with geneticin (50 mg/l) for 1 month. Further selection occurred in the medium at an elevated geneticin level (100 mg/l). A number of geneticin-resistant microadventitious buds were multiplied in vitro. Banana plants transformed with the banana bunchy top virus (BBTV) antisense replicase gene were generated and twenty-one independently transformed plant lines were analyzed for resistance to BBTV. Three different responses were obtained. Some of the transgenic plants showed a pre-established, complete, and highly resistant phenotype since no viral symptoms were observed, and no virus detected. Some of the transgenic plants showed no viral symptoms before winter, but showed a delayed viral symptom in the next spring. The remaining plants from these plants showed a rapid disease symptom. Furthermore, the transformants resistant to BBTV genotypeⅠwere secondary challenged with BBTV genotypeⅡ. Transformants exhibited two different responses. One transgenic plant still showed complete, highly resistant phenotype since no viral symptoms were observed, and no virus detected. The other two transformants showed disease symptom after 2 months of inoculation.