Cryopreservation Of In Vitro-grown Shoot-tips Of Vitis By Vitrification And The Study On Genetic Stability Of Regenerative Plantlets

Plant genetic resources are one of the most important substance bases for human to survive and develop, and to keep their diversity is the important guarantee for sustainable development. In vitro conservation has been becoming the important complementary conservation technique for its advantages of economy, safety and saving room and money. Studies on cryopreservation technology of shoot tips of Vitis by vitrification were conducted with "Atlantic",and the analysis of genetic stability ofregenerative plantlets.The major results are as follows:1 Obtained a system to suit cryopreservation of Vitis shoot-tipsThe Vitis shoot-tips of 5mm in length were pre-cultured on MS medium with 0.3mol/L sucrose for 48 hours.Then the excised shoot tips of 3mm were pre-treated with 60% PVS₂ for 20-30 minutes at 25℃,and treated with PVS₂ at 0℃for 70 minutes. Followed by changing the solution with fresh PVS₂,the shoot tips were immersed into LN directly, and kept for 24 h. After rapid thawing in a water bath at 38℃for 90s, the shoot tips were washed with 1.2mol/L sucrose solution for twice and transferred onto MS medium supplemented with 6-BA 0.5mg/L+NAA 0.1 mg/L. The cultures were kept in poor light at 25℃for 7 days prior to exposure to the light. Survival rate and regeneration rate of shoot tips of Vitis was separately 50.0% and13.3%.2 Established a system and procedure to suit the Vitis SRAP analysisWe established a system and procedure to suit the Vitis SRAP analysis. The condition was in a 40μL volume with 3.0μLMg²⁺(20mmol/L),1.0μLdNTPs (10mmol/L),forward primers(1mmol/L) 9.0μL,reverse primers(1mmol/L) 9.0μL4.0μL template DNA(50ng/L),1.0μL Taq DNA polymerase(2U/μL), ddH₂O9.0μL and 4.0μL PCR Buffer(10X).Amplification was carried out in two steps:4 minutes of denaturing at 94℃,five cycles of three steps:1 minutes of denaturing at 94℃,1 minutes of annealing at 35℃and 2 minutes of elongation at 72℃.In the following 30 cycles the annealing temprerature was increased to 55.5℃,with a final elongation step of 5min at 72℃.3 The genetic stability of regenerated plantlets examined on SRAP-based moledular levels. As a result,there were no variations SRAP patterns.The result provided a feasible proof of conservation Vitis. Through cryopreservation of in vitro shoot tips by vitrification for long term.