【作者】 陈辉； 陈晓玲； 陈龙清； 卢新雄；

【Author】 Chen Hui,Chen Xiaoling, Chen Longqing, Lu Xinxiong Key Laboratory for Crop Germplasm and Biotechnology of Agricultural Ministry/The National Key Facility for Crop Gene Resources and Genetic Improvement,NFCRL Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China; College of Horticultural and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China

【机构】 中国农业科学院作物科学研究所／农业部作物种质资源与生物技术重点开放实验室／国家农作物基因资源与基因改良重大科学工程； 华中农业大学园艺林学学院；

【摘要】 以切花百合西伯利亚试管苗离体茎尖为试材,通过正交设计试验对预培养基中蔗糖浓度、预培养时间和PVS2处理时间等影响超低温保存存活率的主要因素进行了分析,初步建立了切花百合种质玻璃化法超低温保存的技术方案:将继代培养45-60d的试管苗置于4℃低温锻炼一周以上,剥取茎尖1-2mm用0．3mol．L-1蔗糖液体预培养基培养2-3d后用MS+0．4mol．L-蔗糖 +2mol．L-1甘油混合溶液装载20min,0℃下用冰冻保护剂PVS2处理60-120min后迅速投入液氮中保存。40℃水浴中快速化冻2min,用MS+1．2mol．L-1蔗糖溶液洗涤10min,接种在MS+ 0．5mg．L-1 BA+0．1mg．L,-1 NAA+0．3mg．L-1 GA3+30g．L-1蔗糖+7g．L-1琼脂的再生培养基中常温下暗培2周,之后转移至正常光照下培养,存活率可达到50％以上。通过形态观察、可溶性蛋白和同工酶检测,冻存前后材料的遗传稳定性没有发生改变。更多还原

【Abstract】 Shoot tips from in vitro grown cut flower of lily (Lilium L.),Lilium (?)siberia cultivar, were successfully cryopreserved by vitrification method.Dominant parameters.such as sucrose concentration of preculture medium,duration of preculture and duration of exposure to PVS2 were examined, respectively.Orthogonal design was utilized to analyze the effects of the dominant parameters on survival rate.Shoot dps with the size of 1-2mm excised from in vitro plants,which were subcultured for 45-60d and cold-hardened at 4℃ for over one week,were precultured in liquid MS medium supplemented with 0.3mol.L-1 sucrose for2-3d.And then it was loaded with the mixture consisted of MS medium, 2mol.L-1 glycerol and 0.4mol.L-1 sucrose for 20 min.After the loading,shoot tips were exposed to vitrification solution PVS2 for 60-120min at 0℃ and then directly stored in liquid nitrogen.Rapidly warmed shoot tips were diluted in MS medium supplemented with 1.2mol.L-1 sucrose for 10min.And then incubated on MS medium supplemented with 0.5mg.L-1 BA, 0.1mg.L-1 NAA, 0.5mg.L-1 GA3, 30g.L-1sucrose, +7g.L-1agar.The material was transferred to normal condition after incubation in dark for two weeks.The survival rate was over 50%.There was no variation in the regenerated plant on the level of morphology, protein and isoenzymic zymogram.This vitrification method appears promising for cryopreservation of cut flower of lily germplasm.更多还原