云杉属胚性组织超低温保存技术研究进展

Research Progress on Cryopreservation of Picea spp. Embryogenic Tissues

云杉属树种是重要经济和用材树种。随体细胞胚胎发生技术的不断发展,多个云杉属树种胚性愈伤组织(ETs)已经可以大量诱导生产,为维持其胚性、防止遗传变异、避免长期增殖,云杉属多数种实现了ETs超低温保存技术。超低温保存细胞时,细胞停止代谢和生长,不会发生遗传变异,其遗传特性可以长期保存下来。本文首先,简述了超低温保存植物细胞的基本原理;介绍了超低温保存植物材料的一般程序,包括材料选取、预处理、超低温冷冻、解冻复性培养的方法,重点介绍了两种常用超低温冷冻方法——程序降温冻存法和玻璃化法的操作步骤。其次,阐述了影响云杉属超低温保存的主要因素,包括:基因型、细胞年龄及生长状态,冷冻保护剂和各种预处理方法,不同超低温冷冻方法及降温速率。得出:推荐使用年轻、体积小、胞质密度大、含水量低的分生组织或细胞进行低温保存;不同类型保护剂配合使用,效果更佳;较低的降温速率有利于超低温保存,且玻璃化法逐渐成为超低温保存的首选方法。本文还简述了外源添加物(酶促和非酶促抗氧化反应物、新型碳纳米材料)在植物超低温保存中的应用,以期为改良云杉属ETs超低温保存提供参考。最后,总结了云杉属ETs超低温保存技术存在的问题,并提出思考。

Picea spp. is an important economic and timber species. With the continuous development of Picea spp. somatic embryogenesis technology,embryogenesis tissues(ETs) could be induced to produce in large quantities. In order to maintain their embryogenic,prevent genetic variation,and reduce the workload of long-term proliferation,cryopreservation of several species of Picea spp. ETs were realized. When ETs were cryopreserved,the cells stopped metabolism and growth,and genetic variation did not occur,and their genetic characteristics could be preserved for a long time. Firstly,this paper briefl y described the basic principles of plant cells cryopreservation. The general procedures of plant materials cryopreservation was introduced,including material selection,pretreatment,ultra-low temperature freezing,and thawing methods. There were two commonly cryopreservation methods:procedures cooling and vitrifi cation. Secondly,the main factors affecting the cryopreservation of Picea spp. were described. The differences among different genotypes of cryopreservation and their causes were studied. It was recommended to use young,small,high cytoplasmic density,low water content meristems/cells for ultra-low temperature storage. Comparing the effects of different ultra-low temperature freezing and cooling rate on the preservation of Picea spp. ETs,the slower cooling is better;and the vitrifi cation was becoming the preferred method for cryopreservation,recently. The application of exogenous additives(enzymatic and non-enzymatic antioxidants,new carbon nanomaterials) in plant cryopreservation were instituted,which might provide an idea to improve the survival of Picea spp. ETs in cryopreservation. Finally,the problems and thinking of the cryopreservation technology of Picea spp. ETs are summarized.