Establishment Of Efficient Regeneration System Of Common Bean In Vitro

In this experiment,the sterile test-tube plantlets of three bean cultivars’Chao Ji Jiu Li Bai’,’Chao Kuan Sai Guan Wang’and’Jia Zhou Wang Si Ji Dou of America’were used as materials to get the influence factors of regeneration process,Which were genotypes and types of explants,media and hormone types,concentrations of various stages and other factors.In order to establish a regeneration system with high efficiency and low browning rate of common bean,provide a reference of genetic transformation and the breeding of new varieties,we studied the measures to reduce the browning of adventitious buds,the main results of this study are as follows:1.Establishment of efficient regeneration system of common bean in vitro（1）The explants of cotyledon（upper and lower parts）,stem segments and true leaves（upper part and lower part）could only induce callus.Cotyledon explants were extremely browning during subculture,and died after three generations due to browning,The best medium for callus induction was MS+2.0 mg/L 6-BA/2.4-D,with the induction rate of more than 96%.The stem explants callus died as browning after subcultured three generations,the optimum medium for callus induction was MS+1.0～1.5mg/L 6-BA/2.4-D,with the induction rate as high as 100%.The true leaves were more difficult to be induced than the other two explants,and extremely easy to brown death in the subculture.The best callus induction medium of true leaves was MS+1.5mg/L 6-BA/2.4-D,and the highest callus induction rate was 86%.In summary,the stems were the best explants for callus induction of common bean.（2）There were significant differences in the induction of common bean among different genotypes.Under the same induction conditions,the order of induction effect was as follows:’Chao Ji Jiu Li Bai’>’Jia Zhou Wang Si Ji Dou of America’>’Chao Kuan Sai Guan Wang’,and the rates of buds were 100%,85%and 81.67%,respectively.The average number of induced shoots was 3.98,2.51 and 2.35,respectively.Therefore,the best genotype in this experiment was’Chao Ji Jiu Li Bai’.（3）Adventitious buds can be induced directly and rapidly from cotyledon explant,which in the MS、MSB5 and B5 medium even without added any hormone,and showed the best growth of MS medium.The result was better that added low concentration of6-BA,and the best medium for adventitious bud induction was MS+1.5～2.0mg/L 6-BA with the highest induction rate of adventitious buds of 100%and the highest number of adventitious buds of 3.94.0.2mg/L IBA had no significant effect on the induction of adventitious buds in MS medium.（4）The induced adventitious buds were transferred to MS,MSB5 and B5 three medium supplemented with different concentrations of KT,can obtain good elongation effect,and it was better that contain KT of low concentration.Otherwise,it would dead owing to browning,the best medium for elongation was B5+0.3mg/LKT,and the average adventitious bud elongation was 4.35 cm.（5）The elongation of adventitious buds were transferred to MS,MSB5 and B5three kinds of supplemented with different concentrations of IBA,which all can induce rooting,and the MSB5+1.0mg/LIBA medium was best,the average root number of root was 11.51,average length was 7.43cm,and the survival rate reached 80%after transplantation.2.Optimization of browning factors on adventitious buds of common beanIn the optimization experiment of adventitious buds browning,the culture temperature and photoperiod were kept stable.The orthogonal design was used to explore the effects of 6-BA,inorganic salt concentration,agar,ascorbic acid and Ag NO₃on adventitious buds.The results showed that it can significantly reduce the in vitro seedling browning rate,adding 6-BA 1.5mg/L,agar 9g/L,ascorbic acid 4mg/L,Ag NO₃30mg/L and reducing the inorganic salt content in the medium to the 1/2 standard MS medium,it was far lower than that of the contrast of 38.2%.