| Cassava is planted in Guangxi, the largest province in cassava planting in our county, and form a series of processing industry chain based on cassava starch processing, which is an important part of the economy of Guangxi. "Xixuan 03", a fine variety of cassava and can resist to drought, bred by Guangxi University, have the higher economic value and application prospects. It is the effective way to improve cassava quality to add the excellent resistance gene into cassava varieties through agrobacterium-mediated to obtain new varieties with higher resistance. This experiment focused on three aspects that establish rapid propagation system taken tender stem segments as materials, regeneration system based on young leaves of aseptic seedlings and genetic transformation system Agrobacterium-mediated, the results were as follows:1. Sterilization of explants:The best sterilization effect with 0.1% HgCl2 for explants taken from field garden, indoor and greenhouse respectively was 10 min,5 min,6 min after the same pretrement of 0.1% KMnO4 10 min and 75% alcohol 20 s. The contamination rate was 30.8%,11.1% and 17.1%, and the survival rate was up to 61.5%, 80.0% and 74.4%.2. Primary culture:The germination rate of explants with medium added low concentration of 6-BA was better than that in medium added KT and TDZ, and the most suitable medium for inducing buds was MS+6-BA 0.05 mg/L. The explants were induced to appear within a week, and the germination rate was 90.0%. The new buds were robust.3. Subculture:The suitable medium for single bud proliferation was MS+6-BA 0.1 mg/L, and the proliferation rate could reach 4.6. The suitable medium for the dense cluster buds was MS+6-BA 0.3 mg/L+GA3 0.5 mg/L. It was better to use MS+ PP333 3.0 mg/L as medium to make thin seedlings grow slower and stronger. The medium added with AC and low concentration of 6-BA could be used to make buds stronger in some extents.4. Root induced:The medium added NAA, PP333 alone or nothing added all could induce roots appear, of which MS+PP333 0.3 mg/L had the best roots induced effect. The rooting rate reached 100%, and the average root number was 6.9.The roots short and stout.5. Transplant:The robust seedlings that the number of roots was around 3 and the height were 3-4 cm should be choose for transplanting to the matrix of peat:vermiculite: garden soil=1:1:1, and the survival rate was up to 55.1% after 30 days later.6. Callus induction:MS+picloram 12 mg/L+CuSO4 0.5 mg/L suit as the medium of inducing sterile leaves to form callus, and the callus induction rate was up to 100%. The callus produced presented pale yellow and viscous soft, transparent, and part of the callus had the granular tower.7. Callus subculture:The best medium for callus subculture was 1/2 MS+ picloram 12 mg/L, and the proliferation rate was 86.7%. Took this medium to subculture callus could make the viscous soft to be hardened and prepare for transformated the normal callus into embryogenic callus.8. Differentiation induction of callus:The differentiation rate is very low if took the medium with the use of auxin and cytokinin plant growth regulators to induce callus to produce adventitious buds. Using MS+6-BA 1.0 mg/L+IAA 0.5 mg/L+AgNO3 0.5 mg/L+CuSO4 0.5 mg/L as differentiation induction medium could slightly improve the conversion rate of adventitious buds, but it was still less than 15%.9. Antibiotic selection pressure test:Stems with axillary buds, sterile leaves and callus were used to determine the minimum concentration of antibiotic caused all death. The results indicated that the medium for three explants respectively was MS+6-BA 0.05 mg/L+Km 400 mg/L, MS+Picloram 12 mg/L+Km 200 mg/L, MS+Picloram 12 mg/L+Km 150 mg/L.10. Infection and detection of GFP:Sterile leaves used as materials, were infected by agrobacterium tumefaciens-mediated which carried the target gene, and the suitable infection time was 20 min, the expression of GFP gene was 63.3%. And the test of different co-culture time showed that 4 d was the optimum time, up to 56.7%. |
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