藥用植物青脆枝玻璃化法超低溫保存之研究：玻璃化程序及培養基對存活率的影響

青脆枝（Nothapodytes nimmoniana）為臺灣植物紅皮書列名接近威脅的物種，也是極具價值的抗癌藥用植物。本研究利用玻璃化技術，探討超低溫處理程序及回復培養基，對保存後莖頂存活之影響。首先分別比較培植體在0.3 M ~ 0.7 M蔗糖濃度下及0.7 M蔗糖濃度1~7天兩種預處理，對存活率產生之影響；除此也比較抗凍劑PVS2的處理時間和2種不同回復培養基對保存結果產生的差異。結果顯示，完整培植體經0.7 M蔗糖浸泡或預處理3天，接著在25℃下切下莖頂浸入含有2.0 M glycerol及0.4 M蔗糖溶液60分鐘後在0℃下透過PVS2脫水90分鐘，未放入液態氮的存活率分別為72.6%和73.3%，如經超低溫保存實驗，最佳的的抗凍脫水處裡時間為90分鐘，莖頂存活率可達68.8%。除此，解凍程序則是將冷凍管置於40℃水浴30秒後吸出PVS2，在25℃條件下加入1.2 M蔗糖溶液，靜置30分鐘以置換PVS2，再將莖頂轉入含1.0 mg⋅L–1 BA and 0.1 mg⋅L–1 IBA的WPM最佳回復培養基，4周後存活率可達73.3%，本結果提供青脆枝種原保存的一種高存活率方法。

Nothapodytes nimmoniana (J. Graham) Mebb. is a highly valuable anticancer medicinal plant and a near-threatened species on the Red List of Taiwan Plants. This study aims to study the influence of the vitrification technique’s treatment procedures and recovery media on the survival of in vitro shoot tips in cryopreservation. To prepare the explants for a suitable state before cryopreservation, the pretreatment involved comparing sucrose concentrations ranging from 0.3 M to 0.7 M and durations ranging from one to seven days following the 0.7 M concentration. Additionally, to ensure reliable cryopreserved results, we tested the cryoprotectant (PVS2) dehydration period and the recovery medium after post-thawing. The results of sucrose pretreatment after non-cryopreservation showed that the highest survival rates, 72.6% and 73.3%, were achieved either with a 0.7 M sucrose concentration or after a three-day sucrose pretreatment. These results are based on the following procedure: intact plantlets underwent different pretreatment processes, in which dissected shoot tips were treated with a loading solution containing 2.0 M glycerol and 0.4 M sucrose for 60 minutes at 25°C, and the shoot tips were subsequently dehydrated in PVS2 for 90 minutes at 0°C before being plunged into liquid nitrogen. The optimum cryoprotectant dehydration for 90 minutes resulted in a 68.8% survival rate after cryopreservation. Rewarming was conducted in a water bath for 30 seconds at 40°C, and PVS2 was replaced with a 1.2 M sucrose solution for 30 minutes at 25°C. Shoot tips transferred to WPM medium supplemented with 1.0 mg⋅L–1 BA and 0.1 mg⋅L–1 IBA achieved a 73.3% survival rate after four weeks of recovery. The procedure provided another high survival rate of in vitro preservation of N. nimmoniana.