白菜小孢子超低温保存及复苏后胚状体诱导

Cryopreservation of Chinese Cabbage(Brassica rapa)Microspore and Its Embryoid Induction

随着白菜游离小孢子培养技术的不断优化,通过诱导小孢子胚状体获得大量DH系在白菜育种等科研领域得到了广泛应用。但小孢子供体植株受生长季节、生长期等影响,无法长期持续获取高生活力白菜小孢子,成为目前阻碍白菜细胞工程相关研究顺利推进的重要问题之一。本试验利用超低温技术对10份白菜材料小孢子开展冻存复苏研究,结果表明:利用改良B5液体培养基作为冷冻保护剂,白菜小孢子生活力明显高于传统冷冻保护剂二甲基亚砜处理的白菜小孢子;小孢子-80℃(冰箱)和-196℃(液氮)冻存180 d后生活力仍保持在85%以上,且两个温度处理间差异不显著,实际应用中可选择-80℃冰箱冻存替代传统-196℃液氮冻存;利用0.5 mL冻存管保存的白菜小孢子最适复苏条件为37℃恒温水浴40 s,在-80℃冰箱冻存90 d后复苏的小孢子平均生活力达87.9%。复苏冻存30、60、90 d后易出胚材料的白菜小孢子诱导获得了胚状体及再生植株;复苏冻存180 d的白菜小孢子在高温热激诱导条件下仍可感应胁迫,表现为体积膨大,且具胚胎发生能力的小孢子能够进行细胞分裂。本试验为国内首次开展的白菜小孢子超低温冻存研究,建立了白菜小孢子超低温冻存复苏体系,对白菜小孢子培养以及细胞工程相关研究中试验样品的长期保存提供技术支持,并为芸薹属植物小孢子的长期冻存研究提供参考。

With continuous optimization of isolated microspore culture technology of Chinese cabbage(Brassica rapa),large number DH lines were obtained and widely applied in the fields of breeding and studying Chinese cabbage by inducing microspore embryoid.But,the donor plant of microspore,affected by growing season and growth period,could not continuously obtain microspores for a long period of time.This has become one of the important issues hindering the smooth advance of cell engineering research related to Chinese cabbage.This experiment studied cryopreservation and resuscitation of microspore for 10 Chinese cabbage materials by ultra-low temperature technology.The results showed that taking improved B5 liquid medium as cryoprotectant,the microspore viability of Chinese cabbage was significantly higher than the microspore of Chinese cabbage treated by traditional cryoprotectant dimethyl sulfoxide.The viability of microspores could still retain above 85%after being preserved in-80℃refrigerator and-196℃liquid nitrogen for 180 days.And there was no significant difference between these 2 temperature treatments.In practical application,-80℃refrigerator could replace the traditional-196℃liquid nitrogen in freezing microspores.The optimum resuscitation condition for Chinese cabbage microspores retained in 0.5 mL frozen pipe was constant temperature water bath at 37℃for 40 s.The average viability of resuscitated microspores could reach 87.9%after freezing in-80℃refrigerator for 90 days.After resuscitated freezing storage for 30,60,90 days,induction of Chinese cabbage microspores easy for germ extraction obtained embryoid and regenerated plant.After 180 days of cryopreservation and recovery,Chinese cabbage microspores could still keep the embryogenesis ability under the induction of high temperature treatment,showing expand volume.And the microspores with embryogenic ability could conduct cell division.This experiment carried out studies on cryopreservation of Chinese cabbage microspore for the first time in China;and had established cryopreservation and reviving system for Chinese cabbage microspore.This experiment provided technical support for long time preservation of test samples in studies related to Chinese cabbage microspore culture and cell engineering;and also references for studying long-term cryopreservation of Brassica microspore.