| A transformation system of Hc-Pro gene mediated by Agrobacterium was established in the present study. Tuber mustard (Brassica juncea Coss. var. tumida Tsen et Lee) was used as the material. Cotyledon and its induced callus of tuber mustard were served as the explants, and transformed plants were successfully obtained. This study provided theoretical and technical basis for obtaining virus-resistant plants by modern molecular biology and plant genetic engineering, and technical platform for gene transformation in mustard and Cruciferae as well. The main results were listed as follows:1. Establish of transformation system using Cotyledon as explantAdventitious buds could be efficiently induced on MS medium supplemented with 3 mg/L BA and 0.5 mg/L NAA. The effect of pre-culture time(2d, 3d, 4d), co-culture time(2d, 3d, 4d) and co-culture temperature(20℃, 24℃, 28℃) on transformation efficiency of cotyledon was investigated in the present study. Meanwhile, the effect of Agrobacterium and antibiotics on regeneration of cotyledons was studied. The best transformation frequency was found in 3-day pre-cultured explants after 3-day co-culture at 28℃, it reached 16.34%. Higher concentration of Agrobacterium had an obvious effect on regeneration of cotyledons, and it showed a declination step by step with the concentration improvement of Agrobacterium. We also found that Carbenicillin and cefotaxime had adverse effect on regeneration of cotyledons.2. Establish of transformation system using Callus as explantCallus could be induced on MS medium supplemented with 0.25 mg/L NAA, 0.25 mg/L 2,4-D and 0.5 mg/L BA. The effect of sub-culture time(0, 1, 2, 3), co-culture temperature(20℃, 24℃, 28℃) and co-culture time(ld, 2d, 3d) on transformation efficiency of callus was researched in the present study. Meanwhile, the influence of antibiotics on regeneration of callus and genetic diversity of transformed plants were investigated. The result showed that after being co-cultured for 2 days at 24 ℃, 2-sub-culrured explants got the best transformation frequency, itwas 3.95%. At the same time, carbenicillin and cefotaxime showed positive effect on regeneration of callus. After RAPD analysis of transformed shoots, genetic diversity was found. 3. Molecular and virus-resistant identification of transformed shootsBy PCR and Southern blot analysis, we found that Hc-Pro gene and Bar gene have inserted into the tuber mustard genome. ELISA assay showed that lower density of TuMV was found in transformed shoots than non-transformed one. Delayed symptoms of TuMV were found in transformed plants.
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