| Cassava is an important dryland crops and energy plants, Guangxi is the main producing area of cassava in China. As one of the major varieties of cassava,"Xinxuan048" has features of drought resistance and high yield and high economic value. An effective way of cassava breeding is to transfer resistance genes into the fine varieties of cassava, by Agrobacterium tumefaciens-mediated to obtain new varieties of cassava with high yield, good quality and resistance. In this study, resistance genes were transferred into "Xinxuan048" by Agrobacterium tumefaciens-mediated, main results were obtained as following:1. Establishment of regeneration system that the axillary buds were used as explantsSterilization:the explants should be taken in spring or autumn, the best effect of sterilization was to deal with0.1%HgCl2(Mercuric chloride) treatment for12min, which the contamination rate could be controlled within30%,while survival rate was56.7%.As a result, the more suitable culture medium for germination were found as added KT, which germination rate came to85%, the buds sturdy and had3.5cm high. the buds group had more severe bud germination glass in medium added TDZ.Induction culture of initial generation:explants germination of medium that added KT was higher than which added6-BA and TDZ, and the optimum medium was MS+0.5mg/L KT, which the germination rate of explants was up to85.0%.Multiplication culture:Combination of Cytokinins with NAA payed a better effect on buds proliferation than used Cytokinins only or combined with GA3, and the optimum medium for proliferation of axillary buds were found as MS-media+1.0mg/L6-BA+0.1mg/L NAA, which showed high germination rate, the highest average height (up to5.0cm).Rooting culture:the rooting rate could be100%in proper concentration that added NAA, IBA, IAA in medium only. The average number of roots in MS+0.6mg/L NAA medium was most, which was6.9piece.Transplanting:Controlled the air humidity was very important in transplanted tissue culture seedlings. While the relative humidity was too high, the plant stems were mildew and rot easily, and affected the survival rate. The result in this test showed a below10%survival rate after transplanted18d, technology of transplanting should be further studied.2、Establishment of regeneration system of sterile leavesSterile leaves were used to induce callus, and the results indicated that MS+5.0mg/L2,4-D was the optimum induction medium, which the induction rate was up to90%, and the callus was pale yellow, transparent and soft that could be easily transformed into embryonic callus by subculture.The appropriate multiplication medium for callus was MS+3.0mg/L2,4-D, which the proliferation rate was up to90%6-BA and NAA together or CuSO4were used to induce adventitious buds from callus differentiation, the results indicated that there were no differentiation of adventitious buds when used6-BA and NAA together, while MS+200mg/LCuSO4could differentiate adventitious buds, but the differentiation rate was low.3. Establish of genetic transformation systemSterile leaves and callus were used as materials of antibiotic selection pressure test to determine the minimum concentration of antibiotic that caused all cells of culture materials death. The results indicated that the antibiotic selection pressure medium for leaves was MS+3.0mg/L2,4-D+2.3g/L Km, while for the callus was MS+3.0mg/L2,4-D+2.5g/LKm.Sterile leaves were infected by Agrobacterium tumefaciens-mediated which carried the target gene,and the optimum infection time was5min, the transient expression of GUS gene increased at first and then decreased, up to83.3%. For callus, the optimum time was10min, and the transient expression of GUS gene was85%. And tested about the the transformation efficiency in different co-culture time showed that2d was the optimum time, up to77.8%.Thus, genetic transformation system of cassava variety "Xinxuan048" was initially established. |
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