TAN Shuxin，ZHANG Shujiang，ZHANG Shifan，ZHANG Hui，LI Guoliang，LIU Xitong，LIU Yujia，SUN Rifei，LI Fei
China Vegetables. 2022, 1(7): 52-61.
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．
