Micropropagation And Genetic Stability Analysis Of PthA-nls Transgenic Sweet Orange

This research use tissue culture methods to propagate the trans pthA-nls sweet orange, and explore the trans pthA-nls sweet orange in the process of embryonic tissue culture cultivation, value-added culture, rooting and purpose of the stability of gene identification pthA-nls. The results as follows:1,Found the best method of sweet orange sterilization combination of pretreatment:20s with 75% ethanol,10% sodium hypochlorite disinfection 20min, after Sterile water rinse 4 times 2d at room temperature, then 10% sodium hypochlorite sterilization 20min。Washed four times with sterile water, reduce the contamination rate of sugar orange, germination average success rate of 76.66%.2,Initial establishment of the micropropagation system of common sweet orange. Explore the optimum medium. Best germination medium:MS+ sucrose30g/L+Agar 4.5g/L +6-BA3mg/L, the best medium for proliferation:MS+ME1.5g/L+sucrose30g/ L+ Agar4.5g/L+NAA0.05mg/L+6-BA0.5mg/L, the best rooting medium was:1/2MS +AC2.0g/L+sucrose20g/L+Agar5.0g/L+IBA4mg/L. Propagation by seedlings of different hardening time of treatment, hardening time obtained 14d, the highest survival of 65%.3,Established a transfer pthA-nls Orange's micropropagation system. Found its germination, proliferation, rooting hormone is relatively more suitable for the size ratio and concentration. the optimal germination medium:MS+sucrose30g/L+Agar 4.5g/L+6-BA3mg/L; optimal proliferation medium for:MS+ME1.5g/L+sucrose30g/ L+Agar4.5g/L+6-BA1.0mg/L+NAA0.10 mg/L; best rooting medium was:1/2MS+ AC2.0g/L+sucrose20g/L+Agar5.0g/L+IBA3.0mg/L.Now we have encouraged a group of micropropagation seedlings.4,Randomly selected nine of the rapid propagation of seedlings of PCR, results are able to design specific primers pthA-nls target band. Further confirmed by pthA-nls orange seedlings were transgenic plants propagation.5,Using SRAP markers on the transfer pthA-nls sweet orange genetic stability analysis, the results show that propagation of transgenic seedlings did not mutate, can stabilize genetic maternal traits.