Constructed Regeneration System And Preliminary Genetic Transformation System Of ’Fuxuan 01’

Axillary buds of cassava variety ’Fuxuan 01’ was used to establish asexua propagation system. This experiment mainly studied sterilization method of explant, media component of primary culture, proliferation culture and rooting culture of explants, deal with sterile shoots vitrification method. Leaves in vitro materials, a genetic transformation system was preliminarily established by agrobacterium-mediated transformation. The results are listed as follow:1. Propagation system constructionSterilization:Appropriate sterilization time of stem from field culture, plastic house sand culture and hydroponic culture were 75.0% alcohol 30 s with 0.1% HgCl2:stem with pith from field culture was 13 min, stem with no pith was 10 min; stem with pith from plastic house sand culture was 11 min, stem with no pith was 9 min, stem from hydroponic culture was 6 min.Primary culture:The germination rate of adding KT was much higher than adding 6-BA and TDZ, but the growth of adventitious buds was weaker than 6-BA. The treatment with TDZ was increased the vitrification phenomenon. Comprehensive consideration of the germination rate and the bud growth, MS with 1.0 mg/L 6-BA were the best medium to primary culture.Proliferation culture:In single factor experiment, the treatment with 6-BA was better than treatment with 6-BA or TDZ, the proliferation rate of the bud was 2.5 at the concentration of the 6-BA was 0.5 mg/L. In the double factors experiment, the proliferation rate was 3.2 at 1.0 mg/L 6-BA with NAA 0.05 mg/L, and buds were strong and regular, this medium was suitable proliferation.Rooting culture:NAA, IBA, IAA, MET could induced adventitious buds generation roots. Consideration of the average root of per, MS with 0.1 mg/L IBA were the best medium in our experimental design.Transplant:After 30 days, the mortality rate of the plant-let in vitro was 75% in the mixed transplanted matrix of peat:perlite:vermiculite (1:1:1), which higher than other two treatments. This mixed transplanted matrix was suitable the transplant of’Fuxuan01’.2. Regeneration system constructionIn our study, the suitable medium to induce callus formation by leaves of ’Fuxuan 01’was MS adding 12.0 mg/L Picloram,0.2 mg/L CuSO4 The suitable medium for proliferation of callus was MS with lOmg/L Picloram and 0.2 mg/L GuSO4. The suitable medium for differentiation of callus were 0.5-1.0 mg/L 6-BA and 4.0 mg/L AgNO3 put in MS.3. Preliminarily established of genetic transformation systemMS+12.0 mg/L Picloram+0.2 mg/L GuSO4 as the basic media, which were added 10.0-200.0 mg/L Km to resistance selection for the leaves of buds in vitro. The experimental results showed that the Km suitable concentration was 150.0 mg/L.Leaves were impregnated by Agrobacterium with target gene and co-cultured with Agrobacterium, which observed by fluorescence microscopy, the results indicated that the suitable Agrobacterium impregnation time was 15 min at co-cultured 3 days, the impregnation rate was 50.0%. In co-cultured 4 days, the suitable impregnation time was 10 min, and the impregnation rate was 53.6%.