| An effective system regeneration system of grape rootstock 99R was developed in this study. Agrobacterium-mediated transformation was adopted to introduce the salt-tolerant gene AtNHX1 into 99R, and new transgenetic salt-tolerant grape germplasm was obtained. In this research, factors that can increase the efficiency of grape plant regeneration and transformation were studied systematically, and the major technique parameters of agrobacterium-mediated transformation in grape were optimized, which finally led to the development of an effective transformation system. The detailed results were listed as follows:1. Adventitious buds were induced from leaves, petioles and stems of grape rootstock 99R. Optimum bud regeneration from leaf and petiole explants was obtained in MS medium containing BA5.0mg·L-1 and IBA0.1mg·L-1, and the regeneration rates were 40.54% and 50.00% respectively; optimum bud regeneration from stem explants was obtained in MS medium containing BA2.0mg·L-1 and IBA0.02mg·L-1, and the regeneration rate was up to 62.50%. In the combination of TDZ and IBA, the highest regeneration rate was 7.69%. Among different levels of the 6-BA and 2,4-D combinations, the level of MS+ BA8mg·L-1+2,4-D0.05mg·L-1 resulted in the highest regeneration rate and the rate for petioles could reach 42.11%. Darkness treatment was beneficial to regeneration of adventitious buds in 99R, and darkness treatment for 3 weeks was optimum. The regeneration rate of adventitious buds decreased with the decline of node position.2. Transgenic plants were generated from leaves, petioles and stems of grape rootstock 99R by agrobacterium-mediated transformation, and the percentages of GUS transient expression were analyzed to study the optimum parameters of this transformation system. The results showed that the highest percentage of GUS transient expression could be realized under the following condition, namely explants being dipped into agrobacterium suspension for 4 minutes; two days of co-cultivation on MS with Km3.0mg·L-1 , AS75mg·L-1 and Cef 250mg·L-1. Resistant buds were obtained after maintainable culture on MS supplemented with BA5.0mg·L-1 + IBA0.1mg·L-1 + Cef250mg·L-1 + Km3.0mg·L-1. |
PDF Full Text Request