银条茎尖玻璃化法超低温保存及其植株再生

 为长期稳定保存银条种质, 建立了玻璃化法超低温保存其茎尖的技术体系。选择继代4次的 银条无菌壮苗, 5 ℃低温驯化14 d; 剥取5 mm的茎尖, 在含有0.5 mol·L ^{- 1}蔗糖的MS (无Ca^{2 +} ) 液体培养基预培养1 d; 25 ℃条件下经改良MS + 2PVS₂装载20 min; 在- 20 ℃, 95%乙醇浴中以PVS₂脱水处理4h; 更换新鲜的PVS₃后投入液氮, 保存24 h后取出冷冻管在40 ℃水浴中化冻1 min; 以含有1.2 mol·L ^{- 1}蔗糖的改良MS (无Ca^{2 +} ) 溶液洗涤2 次, 每次10 min; 冻存后的茎尖相对存活率超过70%。将冻存后的茎尖转接到再生培养基上, 暗培养20 d后转入正常光照条件下培养, 存活率达63.7%; 继续培养得到正常分化和生长的再生植株, 生根后可移栽成活。

Cryopreservation of Shoot-tips of Stachys floridana Schuttl. ex Benth. by Vitrification and Its Regeneration

In vitro-grown shoot tips of Stachys floridana Schuttl.ex Benth.were successfully cryopre sened by the encapsulation-vitrification method.Excised 5 mm shoot tips,subcuhured 4 times and domesticated at 5 ℃ for 14 d,were precultured for 1d in liquid MS medium supplemented with 0.5 mol·L~(-1) sncrose.PrecuItured shoot tips loaded with a mixture of improved MS and 2PVS_2 for 20 min at 25℃ were directly dehvdrated with a highly concentrated vitrification solution(PVS2)for 4 h at-20℃ and exchanged fresh PVS_3 prior to immersion in liquid nitrogen for 24 h.After rewarming at 40 ℃ for 1min,the cryopreserved tips were washed twice in improved MS liquid medium supplemented with 1.2 mol·L~(-1) sucrose for 2×10 min and then post-cultured on regeneration medium in dark for 20 d,the rate of root recovery amounted to about 63.7％.