Cryopreservation Of In Vitro-grown Shoot-tips Of Potato(Solanum Tuberosum L.) By Vitrification And The Analysis Of Genetic Stability Of Regenerative Plantlets

Plant germplasm is the material base of species evolution ,genetic research and plant breeding. It is very important to cryopreserve for plant germplasm. Studies on cryopreservation technology of shoot tips of potato by vitrification were conducted with "Atlantic",and the analysis of genetic stability of regenerative plantlets.The main results were as follows:1 Obtained a system to suit cryopreservation of potato shoot-tipsThe vitrification method involves removing apical shoot tips(3mm)from plantlets grown in vitro for 40d,The shoot tips consist of 4-5 leaf primordial and the apical dome. The shoot tips were loaded in a solution containing 60%PVS2 for 40 minutes at room temperature,The shoot tips were sufficiently dehydrated with a highly concentrated vitrification solution(PVS2)for 50 minutes at 0℃.after changing the solution with fresh PVS2,the shoots were immersed into liquid nitrogen directly and conservated for 24 hours.After rapidly thawing in a water bath at 38℃for 90 seconds, the shoot tips were washed twice with MS medium with 1.2 mol.L^-1sucrose,2g.L^-1V_c, 400mg.L^-1PVP for 10 minutes each time.and then transferred onto MS medium with 0.04mg.L^-1KT,0.5mg.L^-1GA,0.5mg.L^-1IAA,2g.L^-1V_c,400mg.L^-1PVP,2%sucrose,0.4% agar for 5 days in dark prior to exposure to the light.2 Established a system and procedure to suit the potato SRAP analysisWe established a system and procedure to suit the potato SRAP analysis. The condition was in a 30μL volume with 2.4μLMg²⁺(25mmol.L^-1), 0.6μLdNTPs (10mmol.L^-1), forward primers(1mmol.L^-1) 1.5μL,reverse primers(1mmol.L^-1) 1.5μL 1μL template DNA(50ng.L^-1), 1.7μL Taq DNApolymerase(1U.μL^-1), ddH₂O18.3μL and 3.0μL PCR Buffer(10X). Amplification was carried out in two steps:5 minutes of denaturing at 94℃,five cycles of three steps: 1 minutes of denaturing at 94℃,1minute of annealing at 35℃and 2minutes of elongation at 72℃. In the following 40 cycles the annealing temprerature was increased to 50℃,with a final elongation step of 5minutes at 72℃.3 The genetic stability of regenerated plantlets examined on SRAP-based moledular levels. As a result,there were no variations SRAP pattems.The result provided a feasible proof of conservation potato. Through cryopreservation of in vitro shoot tips by vitrification for long term.