Tissue Culture Of Sophora Japonica F. Pensula And Robinia Hispida

Using different explants of cotyledon, anther and leaf as- materials, studied the tissue culture and plant regeneration of Sophora japonica f. pensula hort and Robinia hispida. 1. Tissue culture of Sophora japonica f. pensulaUsing the cotyledon, anther and leaf as materials, studied the tissue culture of Sophora japonica f. pensula and the results showed that:(1)Cotyledon was the best explant for the rapid propagation of Sophora japonica f. pensula. Using the cotyledon as explant, a large number of regenerated plantlets were obtained from somatic embryogenesis and organogenesis. Calli formed from cotyledons cultured on MS basal medium supplemented with BA 0.5-5mg/L for 20 days and then a lot of adventitious buds formed from the callus. The plantlets could be obtained when the buds were cut and continually cultured on the root-induced medium. Embryoids could be induced when the cotyledons were placed on the MS basal medium supplanted with 2,4-D 0.1-40mg/L or NAA 20mg/L for two weeks. The induction of somatic embryogenesis was influenced by the explants size and the orientation on the medium. The embryoids gradually matured and could develop into plantlets when they were transferred to MS medium supplemented with BA 1 mg/L, but many embryoids were morphologically abnormal.( 2 ) In the course of anther culture, the effects of hormone, pretreatment of low temperature, sucrose concentration and pH on the anther culture were studied and the results showed that: The combination of BA and 2,4-D could obviously improved the induction rate of callus; pretreatment of low temperature at 4癈 could not improve the callus induction efficiency; 9% sucrose and pH6.5 was beneficial for the anther culture. Though the induction rate of anther callus could reach as high as 90%, the buds couldn't be obtained from the callus.(3)When the leaves were cultured on MS medium with different hormones, a little white and green callus formed from the incision and the induction rate were all 100%. The differentiation rate of callus was about 10% and the callus formed adventitious buds when they were cultured on the MS medium supplemented with BA 3mg/L forin2-3 months subculture.2. Tissue culture of Robinia hispidaUsing the anther and leaf as explants, studied the tissue culture of Robinia hispida and the results showed that:(l) The highest induction rate of callus was 41.5% when the anthers were cultured on MS medium supplemented with 2,4-D 0. Img/L and BA 3mg/L for 20 days. When the callus were subcultured for twice or three times on MS medium supplemented with BA 5.0mg/L, green buds formed from the callus. When the shoots grew to 2-3 cm long, they were detached and transferred to MS medium supplemented with IBA Img/L for rooting. The shoots produced roots within 3 weeks of culture and formed plantlets. In the course of anther culture, Pretreatment of low temperature couldn't improve the induction rate of callus; The callus induction efficiency improved when the concentration of sucrose improved from 3% to 6% and 9%, but it obviously reduced when the concentration come up to 12%.(2)A little white and green callus formed from the incision when the leaves of Robinia hispida were cultured. After subculture for three months, some green buds formed from the callus and further developed into plantlets. Using the leaves as explants, the differentiation rate of callus was about 12%. 3.The primary study of gene transformationSome work about gene transformation were did. Using the anther callus as transgenic receptor, the CHI8 gene were tried to transfer to Sophora japonica f. pensula by Agrobacterium-medialed method. The antibiotic resistant callus were obtained. In the course of gene transformation, the time of pre-culture, immersion, co-culture and the concentration of antibiotic were studied and the results showed that: 2-3 days pre-culture, 5-10 minutes immersion, 2 days co-culture, 50mg/L kanamycin and 500mg/L cefotaxime in the selection medium were suitable.The study of tissue culture and plant regeneration not on...