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本文建立了适合中国菊花种质资源长期保存的玻璃化超低温保存技术体系。在4℃下,把1~2mm的菊花茎尖放在含0.4mol/L蔗糖的MS培养基上暗培养2~3d,用预处理液在25℃下处理30min,再用玻璃化试剂PVS2在冰浴条件下处理15min,换新鲜的PVS2试剂并迅速投入液氮。液氮保存24h后,40℃水浴解冻2min,用含蔗糖1.2mol/L的MS液体培养基洗涤20min,滤纸吸干后接种到恢复培养基中,在25℃条件下弱光培养1~3d转入正常光照培养条件下培养,2周后成活率可达86%以上,成活的茎尖均可再生。
This paper established a suitable cryopreservation technology system for the long-term preservation of germplasm resources of chrysanthemum in China. The 1 ~ 2mm chrysanthemum shoots were placed on MS medium containing 0.4mol / L sucrose for 2 ~ 3d at 4 ℃ for 2 ~ 3d, pretreated at 25 ℃ for 30min and then vitrified PVS2 Ice bath treatment 15min, fresh PVS2 reagent and quickly put into liquid nitrogen. After being stored in liquid nitrogen for 24h, the cells were thawed in a water bath at 40 ° C for 2 min, washed with 1.2 mol / L MS liquid medium containing sucrose for 20 min, filtered and dried, and then inoculated into the recovery medium and cultured under weak light at 25 ° C. for 1 to 3 d Under normal light culture conditions, the survival rate can reach over 86% after 2 weeks, and the surviving shoot tips can be regenerated.