应用改良滴冻法超低温保存两种柿属植物的研究

以君迁子（Diospyros lotus L．）和柿（D．kaki Thunb．）组培苗茎尖为试材，对影响超低温保存效果的主要因素，如低温锻炼方式、预培养条件、PVS：（30％甘油＋15％乙二醇＋15％二甲基亚砜＋0．4mol／L蔗糖）处理时间等进行了研究。建立了2种柿属植物的超低温保存程序：（1）切取1cm左右试管苗梢段继代到1／2MS（KNO3和NH4NO3减半）培养基中，交替低温[昼（25±1）℃、夜（4±1）℃]锻炼6周；在含0．5mol／L蔗糖的1／2MS培养基上预培养5d，20℃下装载液（2．0mol／L甘油＋0．4mol／L蔗糖）过渡10min，0℃下PVS2处理1．5h；（2）投入液氮保存；（3）40℃水浴化冻，洗涤5～6次后接种于含1．0mg／LTDZ、0．6g／L可溶性PVP、30g／L蔗糖和7．0g／L琼脂的培养基（作者在优化柿属植物离体培养体系试验中获得）上暗培养1周，转入25℃，1500lx培养室。按照上述程序培养，‘鄂柿1号’、‘湘西甜柿’和君迁子的成活率分别为79．6％、67．4％和60．9％。

Studies on Cryopreservation of Two Diospyros spp.Germplasm by Modified Droplet Vitrification

The factors were studied that effected the cryopreservation of Diospyros lotus L. and D. kaki Thunb. in vitro shoot-tips by modified droplet vitrification, such as cold hardening method, preculture time, PVS2 treatment time etc. A suitable procedure was established：shoot-tips of in vitro axillaries were cultured on 1/2MS medium and followed by alternating temperatures（25 ± 1 ）℃ in daytime/（4 ± 1 ）℃ at night cold-hardening for 6 weeks, precultured with 0. 5 mol/L sucrose for 5 days, which were loaded in loading solution for 20 min at 20℃, and incubated in PVS2 for 1.5 h at 0℃ prior to direct plunge into liquid nitrogen. After rapid thawing at 40℃ warm water bath and rinsed five to six times, the shoot-tips were plated on the regeneration medium supplemented with 1.0 mg/L TDZ ,0. 6 g/L PVP,30 g/L sucrose and 7 g/L agar for one week in dark prior to exposure to the normal condition（The result is found in optimizing of regeneration system of Diospyros spp. by tissue culture）. Using this procedure, the survival rate of ‘Eshi 1＇ ,‘ Xiangxi-tianshi＇ and D. lotus L. in vitro shoot-tips was 79. 6% ,67. 4% and 60. 9% ,respectively.