抗松材线虫病马尾松胚性愈伤组织超低温保存

Cryopreservation of Nematode-resistant Pinus massoniana Embryogenic Callus

为使抗松材线虫病马尾松(Pinusmassoniana Lamb.)胚性细胞保留幼年特征,阻止长期继代发生遗传变异,以抗松材线虫病马尾松马7-6及全172-1-1细胞系胚性愈伤组织为材料,通过正交试验及单因素试验探讨超低温保存(冷冻保护剂、预处理时间、解冻温度)对愈伤组织存活的影响,并对恢复生长的愈伤胚性维持状况进行评估。结果表明:预处理36~48h,以2.5%~10.0%DMSO、0.4~0.5mol·L-1蔗糖和15%PEG作为冷冻保护剂,经30~35℃解冻后,两细胞系的胚性愈伤组织细胞活性较高,愈伤恢复生长率达100%,所需恢复生长的时间也相对较短(马7-6细胞系23d;全172-1-1细胞系15~19d)。超低温保存后的愈伤与正常增殖的愈伤组织在外观及显微结构上无明显差异,且低温保存后的愈伤组织仍具有分化的能力。

The embryogenic callus of nematode-resistant Pinus massoniana was used to explore the effects of various factors of cryopreservation on the ultra-low temperature preservation. The three factors were determined by orthogonal design test and single factor test using the embryogenic callus of Ma7-6 and Quan172-1-1 cell lines of resistant P. massoniana . The situation of callus survival after cryopreservation was analyzed, and the maintenance of callus recovery of growth was evaluated. The optimal cryopreservation conditions for the embryogenic callus of nematode-resistant P. massoniana were screened out: pretreatment for 36-48 h, with 2.5%-10% DMSO, 0.4-0.5 mol/L sucrose and 15% PEG as cryoprotectant, and rewarming at 30 ℃-35 ℃. Under this condition, the embryogenic callus of the two lines had higher activity after rewarming, the rate of recovered embryogenic callus was also the highest (both the two cell lines were 100%), while the callus need less time to recovery (Ma7-6 need 23 d, Quan 172-1-1 need 15-19 d). Compared with the normal proliferation of callus, the callus after cryopreservation had no significant difference in appearance and microstructure. The result also showed that the callus after cryopreservation still had the ability to differentiate.