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作 者 ：张桂芳,徐鸿华,贺红

期 刊 ：中草药 2009年

关键词：佛手；玻璃化法；超低温保存；茎尖微嫁接；

Keywords:Citrus medica.var.sarcodactylis Swingle, vitrification, cryopreservation, shoot-tip micrografting,

摘 要 ：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%PVS₂）预处理30 min,0℃下玻璃化液PVS₂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。

Abstract: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 cryopreservation, 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% PVSz at room temperature (25℃)for 30 min.After that,they were treated by PVS₂ at 0℃ for another 80 min 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 min 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 plantlets 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.

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