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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14d;剥取5mm的茎尖,在含有0.5mol.L-1蔗糖的MS(无Ca2+)液体培养基预培养1d;25℃条件下经改良MS+2PVS2装载20min;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24h后取出冷冻管在40℃水浴中化冻1min;以含有1.2mol.L-1蔗糖的改良MS(无Ca2+)溶液洗涤2次,每次10min;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20d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。
For the long-term stable preservation of silver bar germplasm, established a vitrification method cryogenic preservation of the tip of the technical system. The sterilized seedlings of silver bars that were subcultured for 4 generations were acclimatized for 14 days at low temperature of 5 ° C. The stem tips of 5 mm were stripped and pre-cultured for 1 day in MS (Ca 2+ -free) liquid medium containing 0.5 mol·L -1 sucrose. The modified MS + 2PVS2 was loaded for 20min; dehydrated at 95% ethanol in PVS2 for 4h at -20 ℃; fresh PVS3 was added into liquid nitrogen and stored for 24 hours; then the frozen tube was thawed for 1min at 40 ℃; The modified MS (Ca2 + free) solution containing 1.2 mol.L-1 sucrose was washed twice for 10 min each; the relative viability after cryopreservation was more than 70%. The frozen shoot tips were transferred to regeneration medium, dark culture for 20 days and then transferred to normal light culture conditions, the survival rate of 63.7%; continue to cultivate normal differentiation and growth of regenerated plants, after rooting transplanting survival .