| Somatic embryogenesis and plant regeneration were induced by higher concentrations of auxins with immature cotyledon, leaf, cotyledonary node and hypocotyl of fifty-five genotypes from Northeast area and abroad in soybean. The factors affecting somatic embryogenesis were investigated such as genotypes, category of explants, kinds and concentrations of anxins, and time of pretreatment with lower temperature. The secondary embryos of soybean were induced and correlative factors with proliferation, germination and regeneration of embryogenic tissue were discussed. The system of tissue culture and regeneration were optimized.By PCR and PCR-Southem detection, bivalent insect resistant genes (Bt gene and CpTI gene) were transformed into immature cotyledon and embryogenic tissue of soybean via Agrobacterium-mediated and particle bombardment. The factors affecting genetic transformation were investigated. The main results were summarized as followed:1. Somatic embryogenesis of fifty-four genotypes with immature cotyledon was induced successfully. Eight genotypes that possessed high frequency of somatic embryogenesis (more than 50%) were selected from soybeans planted in Northeast area. The best explant was immature cotyledon for inducing somatic embryogenesis in soybean. Induction of somatic embryogenesis with high concentration of 2,4-D was better than with NAA. The frequencies of somatic embryogenesis varied with genotypes of soybean (0-85.83%).2. The system of tissue culture was established for somatic embryogenesis for varieties in Northeast area. The frequency of somatic embryogenesis was influenced by components of medium, kinds and concentrations of anxins and time of pretreatment with lower temperature. The frequency of somatic embryogenesis of immature cotyledon could be improved by following factors: (1) medium plus a high concentration of 2,4-D, with or without organic element (proline and adenine); (2) the young pods treated with 4℃ lower temperature before immature cotyledons culture and (3) immature cotyledons 3-5mm in length, sampling pods 16-20 days and 20-24 days after flowering for early- and medium-maturing varieties and late-maturing varieties, respectively.3.The new system of inducing secondary embryo formation, proliferation, regeneration and seeds setting from embryogenic tissue in soybean was constructed for varieties cultured in Northeast area. The proliferation of embryogenic tissue was associated with the concentration of 2,4-D and light condition.4. The inhibiting role of antibiotics to soybean varieties was investigated. Various concentration of kanamycine (25-100 mg/L), a selecting marker, was applied according todifferent varieties, kinds of exlpants, and development period of explants. Different lines of Agrobacteriwn needed different kinds and concentrations of antibiotics for inhibiting Agrobacterium growth. Cefotaxime sodium was the best for inhibiting LBA4404 and the concentration varied 200-500 mg/L with different explants.5. Immature cotyledons of soybean varieties cultured in Northeast area (Jilin 35, Jilin 43 and Hefeng 35) were inoculated with LBA4404 (including pGBI121S4ABC plasmid). The resistant calli were induced and regenerated plantlets were obtained. Among the 15 transplanted survival T0 plants, 12 plants gave positive PCR reaction. PCR-Southern blot was conducted with probe of Bt gene. All the 12 plants were positive and indicated that the Bt gene had been integrated into the soybean genome. The frequency of transformation varied from 0 to 2.00% according to varieties of soybean, the concentration of Agrobacterium, time of inoculation and co-culture. The mean frequency of transformation was 0.56%. The transformation by Agrobacterium- mediated was influenced by genotypes of soybean and time of co-culture. Jilin 43 co-cultured 1-2 days showed a high frequency of transformation. Embryogenic tissue transformed with Agrobacterium gave GUS gene stable expression. PCR detection indicated that the bivalent insect resistant genes (Bt gene and CpTI gene) were...
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