Establishment Of Leaf Regeneration System Of Pyrus Ussuriensis Maxim And Transformation

The leaves of aseptic plant were obtained through seed and stem culture in vitro and then were selected as explants, in order to establish the leave regeneration systems for Pyrus ussuriensis Maxim and transform gene roIb into it. The research result is as follows:1. The result showed that when the seeds of Pyrus ussuriensis Maxim were selected as inoculum. The optimal time of seed sterilization was 5 minutes, with the least pollution and highest germination rate. The seeds germinated in three days or so, and grew into plantlets in ten days. Leaves subcultured for 30 days were used as explants. The seeds can germinate in MS or V1 medium without cytokinins.2. The stems of Pyrus ussuriensis Maxim were taken as explants in MS medium. The results showed that the optimal time of stems taken from fruit orchard was in April. The optimal time of stems sterilization with 0. 1% Hgcl₂ was 7 minutes, with the highest germination rate of axillary buds, which was 90%. When stems were inoculated in MS medium containing 1. 0-2. 5 mg/L BA and 0. 4 mg/L IBA , new shoots grew out from axillae in 10 days, and the average height of shoots was 2. 4 cm in 20 days. Young shoots grew well and can be subcultured 30 days later. Plantlets grew well in MS medium containing 2. 0 mg/L BA and 0. 2 mg/L, and were induced to grow proiiferation shoots.3. Leaves cultured as explants showed that when leaves subcultured for 30 days were inoculated in MS medium containing 5.0 mg/L BA and 0.4 mg/L, adventitious shoots appeared 20-30 days later, with regeneration frequency being 48. 6% and buds No. per leave, 3. 83. The highest Regeneration frequency of leaves was obtained when leaves were cultured for 20 days in the dark. The regeneration frequency and Buds No. per leave of leaves were higher when abaxial side was placed downward onto medium than upward. Regeneration frequency of the basal part of leaves was higher than that of the middle part and top part, and reached68. 1%.4. Root promoting experiment of plantlets in vitro showed that adventitious roots were induced 10-14 days later. The optimal rooting medium for Pyrusussuriensis Maxim is 1/2MS rather than 1/3MS or 1/4MS. Although IBA was more effective than NAA, better effect was achieved when both were used in combination. Medium with 2% sucrose was effective than 3% sucrose. The conclusion is that the optimal rooting medium for Pyrus ussuriensis Maxim was 1/2MS+1. 0 mg/L IBA+0. 5 mg/L NAA+2% sucrose and the rooting rate reached 71. 9%.5. Pyrus ussuriensis Maxim leaves were transformed with Agrobacterium tumefaciens containing roIB transgene. Before transformation, Kanamycin selection test was made and 50⁷5 mg/L turned out to be good. But transformation plants were not achieved because of various kinds of reasons.