The Optimization Research On Regeneration System In Sweet Sorghum

Sweet sorghum was widely considered as the most outstanding energy crop, its photosynthetic efficiency is more 2 or 3 times than that of wheat and other crops, it can be used as sugar crop or grain crop for its high comprehensive value。But so far, there are no comprehensive superior traits in the varieties of sweet sorghum. Transgenic technology may be utilizied to purposefully improving the varieties of sweet sorghum, then the varieties of high-biomass and strong anti-adversity would be expected in the short run。The stability and efficiency of regeneration system in sweet sorghum plays a key role in its genetic transformation.In this study, the mature seeds of two varieties of sweet sorghum (BT3 and LT3) which were introducted to Nanning, Guangxi Province were used as experimental materials, the test purpose is to obtain convenient, efficient and stable regeneration system of sweet sorghum and lay a good foundation for establishing genetic transformation system in the next. So far, the main experimental results obtained were as follows:1. The optimization on callus inductionFirstly, the optimal combinations were concluded through optimizing the concentration of different plant hormones(2,4-D, NAA,6-BA, KT), BT3 is 2.0mg/L 2,4-D+0.1mg/L NAA, LT3 is 2.0mg/L 2,4-D+0.2mg/L NAA.Secondly,the best callus induction formula of two varieties were selected out by testing on different basic medium(MS,1/2MS, B5, N6), different carbohydrate sources(sucrose, glucose, glucose:fructose=3:2), different pH value(5.3,5.8,6.3) and different concentrations of AgNO3(four levels:2mg/L,3mg/L,4mg/L,5mg/L), BT3: MS+2.0mg/L 2,4-D+0.1mg/L NAA, LT3:MS+2.0mg/L 2,4-D+0.2mg/L NAA, pH value was 5.8, sugar source was sucrose. AgNO3 added in the callus induction medium can not promote the callus formation of BT3 and LT3, but instead it inhibited the induction of embryogenic callus.In addition, the best callus proliferation medium was explored through constantly testing and observing embryogenic callus subculture, and was MS+2.0mg/L 2,4-D+0.5～1.0mg/L NAA. The callus of two varieties on this medium was browned lowly, and also added quickly.2. The effects of Hygromycin on callus growth of sweet sorghumWe have added four concentration of Hygromycin (25,50,75 and 100mg/L) on the callus proliferation medium, the result showed two Hygromycin concentrations (75 and 100mg/L) treatments could make embryogentic callus of BT3 and LT3 dead and their colors were changed into dark and brown. If we adopted the resistance callus of sweet sorghum by the two Hygromycin concentrations 75 and 100mg/L treatments, the two treatments may influence normal growth of cells transformed. The 25mg/L treatment of Hygromycin concentration can not completely restrain the growth of embryogentic callus of sweet sorghum, the concentration was used to screen transformed callus and could not make many non-transformed cells die. The 50mg/L treatment of Hygromycin concentration could completely restrain the growth of embryogentic callus of sweet sorghum and finally make it die, but the treatment can not make lots of cells die fast, so the 50mg/L treatment of Hygromycin can be used as an ideal method of the sceening of transformed cells in two breeds of sweet sorghum.In addition, the two Hygromycin concentration (25 and 50 mg/L) treatments have different influence on the growth of the callus between two breeds of sweet sorghum.The result showed the callus survival rate of LT3 was higher than BT3. The result told us that the effect of Hygromycin on the callus has some difference in different genotypes of sweet sorghum.3.The optimization research on shoot differentiationThrough the optimization test of the different concentration ratios of plant hormones(6-BA, NAA and 2,4-D), it was concluded that the best hormone ratio on shoot differentiation in two varieties of sweet sorghum were both MS+1.0mg/L 6-BA+0.2mg/L NAA+0.5 mg/L 2,4-D, but there were some difference in its induction rates. In addition, comparing many results in the test, we have found the best proliferation formula of bud in two varieties of sweet sorghum were as follows: MS+1.0mg/L NAA+1.0mg/L 6-BA, and the suitable proliferation formula of strong seedlings of two varieties of sweet sorghum were as follows:MS+0.1mg/L NAA+1.0mg/L 6-BA.4. The optimization research on root inductionFirst, through the test of different basic media (MS,1/2MS) on promoting root induction of seedlings, it was concluded that adding hormone to the media 1/2MS was more beneficial for promoting rooting. Next, adding different concentrations of plant hormone to the media 1/2MS, and integrating various factors such as the quality of root, rooting rate, rooting time, the growth of rooted seedlings and so on, after analysising and finding the optimum rooting medias were as follows:the optimum rooting media of BT3 was 1/2MS+2.0mg/L IBA, the optimum rooting media of LT3 was 1/2MS+3.0mg/L IBA. All seedlings have not tanken root in the media MS and 1/2MS which didn't add any hormone.