| In this research, a stable and high-efficiency plant regeneration system was established via cotyledonary node explants of’Chengjiang 7’’Guagndong jinguan’ ’Baisha 17’’Zhaibuwan’ and ’Hongda’. The tomato disease-resistant transcription factor Gene Pti4 was translated into’Chengjiang 7’through Agrobacterium-mediated cotyledonary node infection and ovarian in planta transformation. Moreover, the transformed material was detected by PCR, the transformation steps were screened and optimum parameters of each factor was selected. The results can provide some reference and technical support for cowpea transgenosis. The main results were as following:1. We investigated the effect of different concentration of 6-B A on the whole process of regeneration of cowpea cotyledonary node. At the stage of germination, adding 6-B A will facilitate the formation of strong seedlings and promote the induction of adventitious buds. The optimal concentration of this stage was 3.0mg/L. While at the induction stage and the elongation stage, the optimal concentration were 1.0 mg/L and 0.5 mg/L, respectively. The number of induced adventitious buds was 5.37.2. To solve the problem in elongation of adventitious buds, we add KT in the induction and elongation stages. As a result, adventitious buds were significantly elongated, and the optimal concentration of KT was 0.06mg/L. Compared with the control group, both the number and the length of induced adventitious buds were increased. After four weeks of culture, the average number of buds was 5.97 and the average length of buds was 2.62cm.3. Simultaneously, the effect of pre-treatment of TDZ was also studied. The best result of induction was obtained with the adding of 0.01 mg/L TDZ at aseptic seedling stage. The number of induced adventitious buds was 5.56.4. The adventitious buds of cowpea can root well in culture medium without any hormone or with 0.5mg/L IBA. The rooting rate could reach 100%. The adding of IBA can cut down the rooting time from 7.5d to 6.3d.5. There existed some differences in adventitious buds induction rate among five cowpea genotypes. Chengjiang 7th and Guangdong-Jinguan were superior to the other three varieties, the induction rate of which were 95.45% and 92.75% and the number of induced buds was 5.40 and 5.91, respectively.6. The effect of Kan and Cef on regeneration of cotyledonary nodes from cowpea was discussed, as well as the influence of pretreatment of TDZ and 6-BA, pre-cultivation time, infection time, co-cultivation time and pH. The suitable concentration of Kan should be 110mg/L, explants transferred to medium contained Kan after 1 week of excising from seedlings followed by 3 weeks of screening was choose as the best scheme. The Cef was used as antibiotic. When the concentration of Cef was 200mg/L, the growth of Agrobacterium was suppressed. So the suitable concentration of Cef was selected with 200mg/L. In the process of genetic transformation system, pretreatment with TDZ was superior to that with 6-BA. Pre-cultivated with 24h, infected with 20 min, co-cultivated with 2d and infected at pH 5.8, were the optimal conditions respectively.7. In the experimental field-site, we used ovarian in planta transformation. The flower bloomed in the same day were chosen. Style was completely removed and the ovarian Incision was infected by Agrobacterium in three periods of one day. The result showed that the flowers treated before twelve o’clock were almost completely fallen off, whereas the flowers treated between 3 pm and 6 pm can continue to develop until seeds were harvested. |
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