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    陈晓艺, 江帅菲, 代剑峰, 袁德水, LishengKong, 张金凤, 赵健. 华北落叶松胚性组织超低温保存技术研究[J]. 北京林业大学学报, 2021, 43(10): 47-53. DOI: 10.12171/j.1000-1522.20200251
    引用本文: 陈晓艺, 江帅菲, 代剑峰, 袁德水, LishengKong, 张金凤, 赵健. 华北落叶松胚性组织超低温保存技术研究[J]. 北京林业大学学报, 2021, 43(10): 47-53. DOI: 10.12171/j.1000-1522.20200251
    Chen Xiaoyi, Jiang Shuaifei, Dai Jianfeng, Yuan Deshui, Lisheng Kong, Zhang Jinfeng, Zhao Jian. Cryopreservation of embryogenic callus for Larix gmelinii var. principis-rupprechtii[J]. Journal of Beijing Forestry University, 2021, 43(10): 47-53. DOI: 10.12171/j.1000-1522.20200251
    Citation: Chen Xiaoyi, Jiang Shuaifei, Dai Jianfeng, Yuan Deshui, Lisheng Kong, Zhang Jinfeng, Zhao Jian. Cryopreservation of embryogenic callus for Larix gmelinii var. principis-rupprechtii[J]. Journal of Beijing Forestry University, 2021, 43(10): 47-53. DOI: 10.12171/j.1000-1522.20200251

    华北落叶松胚性组织超低温保存技术研究

    Cryopreservation of embryogenic callus for Larix gmelinii var. principis-rupprechtii

    • 摘要:
        目的  超低温保存是植物优良种质长期保存的重要方法。本文为探究梯度预处理对超低温保存存活率的影响,以期有效保存华北落叶松胚性组织的发育潜能。
        方法  本研究以华北落叶松胚性组织为材料,对超低温冷冻保存程序中预培养、冷冻处理方式、解冻方式和恢复培养等关键环节开展研究。预培养和冷冻保护共设计成4种处理,以不经过预培养和冷冻保护直接冷冻保存为对照,每个处理重复3次。
        结果  结果表明:处理组合1、2、3之间虽无显著差异,但结合冷冻保护剂二甲基亚砜( DMSO)对细胞具有一定毒害作用,高浓度的DMSO会影响后续胚性组织的恢复甚至造成细胞死亡,因此选出效果较好的超低温保存方法为: 0.2、0.4 mol/L山梨醇液体梯度预处理与0.4 mol/L山梨醇 + 5%DMSO为冷冻保护剂进行冷冻保护,最佳解冻方式为37 ℃水浴,解冻后的胚性愈伤组织细胞活性最高达78%;超低温保存后的胚性组织与正常增殖的胚性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保持分化形成体细胞胚的能力。
        结论  研究结果为华北落叶松乃至其他针叶树胚性组织的超低温保存提供了参考。

       

      Abstract:
        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.

       

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