佛手茎尖玻璃化超低温保存和植株再生

Vitrified cryopreservation and regeneration of shoot-tips for Citrus medica var.sarcodactylis

目的为佛手种质资源的保存提供一条新途径。方法对佛手茎尖进行了玻璃化超低温保存,并对保存后的茎尖进行微嫁接试验,获得再生植株。结果佛手茎尖在含5%DMSO和5%蔗糖的MS培养基上预培养48h后,切取3～4 mm的茎尖,室温(25℃)下装载液(60%PVS_2)预处理30 min,0℃下玻璃化液PVS_2处理80 min,投入液氮保存24 h后取出,在40℃水浴快速化冻,室温下分别用1.2 mol/L蔗糖和MT基本培养基洗涤2次,每次10 min。保存后的茎尖经TTC法检测,成活率达81.25%;将茎尖嫁接到黑暗培养基的砧木上,再生率可达52.94%,且再生植株生长和分化正常,经PCR检测不含黄龙病病原。结论玻璃化超低温保存方法可用于佛手种质资源保存。

Objective To provide a new approach of preservation for germplasm resources of Citrus medica var. sarcodactylis （CMS）. Methods A micrografting test was made on CMS shoot-tips after vitrified eryopreservation, resulting in living shoot-tip. Results First the CMS shoot-tip is inoculated in medium consisting of MS, 5% DMSO, and 5% sucrose for 48 h preincubation. Then the shoot-tips were cut off 3--4 mm long and fore-treated by 60%PVS2 at room temperature （25 ℃） for 30 rain. After that, they were treated by PVS2 at 0℃ for another 80 rain and conserved in liquid nitrogen for 24 h. Next the shoottips were defrosted by aqueous bath at 40 ℃ and cleaned twice at room temperature （25 ℃） by MT minimal medium with additive 1.2 mol/L sucrose, 10 rain once. Finally data collected recorded a higher survival rate of 81.25% by TTC inspectation and a better regeneration rate of 52.94% if the CMS shoot-tips had been grafted on darkly-cultured parental stock, growing, and differentiating normally afterwards. The pla ntlets detected by polymerase chain reaction （PCR） showed that the pathogens of Huanglong disease had been removed. Conclusion Therefore a conclusion is made that the approach of vitrified cryopreservation may be applied to the preservation of CMS germplasm resources.