Objective Cryopreservation is an important method for long-term preservation of plant germplasm. This paper aims to explore the effects of gradient preconditioning on the survival rate of cryopreservation to preserve the developmental potential of embryonic tissues of Larix gmelinii var. principis-rupprechtii.
Method In this study, the embryonic tissues of Larix gmelinii var. principis-rupprechtii were used as materials to conduct research on the key links of pre-cultivation, freezing treatment methods, thawing methods and recovery culture in the cryopreservation procedure. The pre-cultivation and cryoprotection were designed into 4 treatments, and the direct cryopreservation without pre-cultivation and cryoprotection was used as the control, and each treatment was repeated 3 times.
Result Although there was no significant difference between treatment combinations 1, 2, and 3, the combination of cryoprotectant DMSO had a certain toxic effect on cells. High concentration of DMSO will affect the recovery of subsequent embryonic callus and even cause cell death, so the effective cryopreservation method we selected was: the combination of 0.2 mol/L and 0.4 mol/L sorbitol gradient pretreatment and cryopreserve with 0.4 mol/L sorbitol and 5% DMSO as a cryoprotectant for cryoprotection; the best thawing method was 37 ℃ water bath, and the activity of embryonic callus was up to 78% after thawing. There was no significant difference in appearance and microstructure between the embryogenic tissue after cryopreservation and that of normal proliferation.
Conclusion The results will establish good base for long-time cryopreservation for embryogenic cell lines in Larix gmelinii var. principis-rupprechtii and even some other conifers.