In vitro plant regeneration of grapevine (Vitis sp.) hybrid cultivars and genetic transformation through microprojectile bombardment of in vitro-derived axillary buds.

摘要

In vitro plant regeneration experiments were conducted on grape hybrid cultivars adapted to Nebraska: 'Chancellor', 'Landot', 'Leon Millot', 'MN 1047', 'Valiant' and 'Vincent'. The effects of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) on axillary buds were investigated. The variation in responses regarding number of nodes, fully expanded leaves, and leaves measuring at least 0.5 cm diameter was highly genotype-dependent. 'Valiant' was the most responsive for the above variables, and 'Vincent' the least responsive. Murashige and Skoog (MS) medium, supplemented with 0.1 to 1.0 mg BA/l in combination with 0.01 mg NAA/l, proved to be the best medium for the explant establishment and shoot production. Multiple shoot proliferation and root formation were not favored with any of the BA and NAA treatment combinations. Thidiazuron (TDZ), a substituted urea with cytokinin-like activity, was effective in promoting shoot production and shoot proliferation from both intact and bisected in vitro-derived axillary buds. The optimal TDZ concentration was 1.0 mg/l for 'Landot', 'Leon Millot' and 'Valiant' and 0.5 mg/l for 'Chancellor' and 'MN 1047'. A low concentration of TDZ (0.01 mg/l) stimulated shoot elongation and root formation. The effect of kanamycin on the inhibition of shoot production was evaluated for intact and bisected in vitro axillary buds. Kanamycin at 100 mg/l completely inhibited shoot regeneration. Bisected buds were resistant to at least 50 mg/l of kanamycin. Differences in kanamycin sensitivity of grape cultivars were detected. Microprojectile bombardment (Biolistic PDS-1000/He) was used for the delivery of DNA into bisected axillary buds of 'Chancellor' and 'Valiant'. Transient {dollar}beta{dollar}-glucuronidase (GUS) and stable expression (neomycinphosphotransferase, NPTII) was visualized after bombardment with gold microprojectiles coated with a gene construct containing the fusion of GUS::NPTII (pBI426). Highest frequencies of transient transformation in both cultivars were achieved using microprojectiles of 1.6 {dollar}mu{dollar}m diameter, vacuum pressure of 68.6 cm Hg, 0.3 M mannitol/sorbitol in the medium as osmoticum for preconditioning, and a target distance of 6 cm. There were not significant differences in transient transformation between 'Chancellor' and 'Valiant'. Only a few plantlets were demonstrated to be putative stable transformants. At present, it is not possible to indicate that they are true transformants.