应用茎尖滴冻法超低温保存香蕉资源研究(英文)

Study on the cryopreservation of in vitro shoot tips of Musa germplasm by droplet vitrification

采用正交试验探讨了滴冻法中装载液处理时间、玻璃化溶液处理时间和过渡培养时间的最优组合：另外还研究了不同冰冻保护剂、茎尖大小、恢复培养基中大量元素浓度对滴冻法保存效果的影响。通过试验筛选出了最佳的滴冻法保存方案：剥取含有1～2个叶原基的茎尖，长约1．5～2．5mm，先在室温下用装载液预处理20～120min．再用玻璃化溶液PVS2于0℃下处理30～50min，接着将PVS2溶液滴于铝箔上，每个液滴中放一个茎尖，迅速投入液氮。保存1h后，将铝箔取出投入卸载液（Suc．1．2mol·L^-1＋MS）中快速解冻。15min后取出茎尖，用滤纸吸去水分，转入过渡培养基上（Suc 0．3mol^-1＋MS）暗培养24h。再转到恢复培养基上，暗培养1周后转移到光下常规培养。采用滴冻法成功保存了分属于5个基因组型的15份香蕉资源，每种基因组型的平均成活率为：AAA78．0％．AAB57．8％．ABB72．1％，AA43．3％，野生蕉42．2％。

Droplet vitrification method for cryopreservation of shoot-tips of AAA and ABB genotypes banana （Musa spp.） were optimized using orthogonal table of the time for loading of cryoprotective solution, dehydrating and recovery. The effects of different eryoprotectors and shoot tip types on cryopreservation were also studied. The appropriate procedure was as follows. Shoot-tip 1.5 to 2.0 mm in length carrying 1 or 2 leaf primordia was excised and then pretreated with loading solution for 120min at room temperature. After loading, the solution was replaced by ice-cooled vitrification solution （PVS2） for 30 to 50 min. Then the shoot-tip was transferred to a droplet of PVS2 solution on a strip of aluminum foil and plunged into liquid nitrogen and stayed in liquid nitrogen for at least one hour. The strips were rinsed in unloading solution at room temperature for 15 min. Then shoot-tips were placed onto a piece of sterilized filter paper on top of semi-solid MS medium containing 0.3 mol. L^-1 sucrose. After 24 h, the shoot-tips were transferred onto a regeneration medium without filter paper. Using this droplet vitrification protocol, we successfully cryopreserved 15 accessions belonging to 5 different genomic groups of Musa germplasm. The average survival rate was 78.0% for AAA group, 57.8% for AAB group, 72.1% for ABB group, 43.3% for AA group, and 42.2% for M. itinerans.