马铃薯超低温保存茎尖不同部位细胞存活和胁迫应答模式研究

Potato (Solanum tuberosum L.) is very important crop germplasm in the world, and the germplasm conservation system consists of field genebank, in vitro genebank and cryopreservation genebank. In China, the potato germplasm are conserved mostly in field genebank and in vitro genebank, while the numbers of the cryopreserved accessions are small. The cryopreservation techniques of potato are used widely in many cultivars succesfully, and the potato germplasm would be the important materials for cryopreservation genebank construction. However, the survival and regrowth rates of different potato cultivars recovered from nitrogen are different and the ability of survival and regrowth is closely correlated to the location and the quantity of survival cells in the shoot tip meristems. In addition, we found that the cells in different region of the shoot tip meristems suffered from different extents of stress during the cryopreservation. Thus, it is urgent to carry on the study of the mechanism of cryopreservation of potato shoot tips, especially focusing on the patterns of survival and stress reponse patterns of different cells in the regions of the shoot tip meristems. The aim of this study is to find fundamental aspects in ultrastructure, histological, thermal and non-invasive micro-test analysis which can explain the cryopreservation process and results. The present work will investigate the effect of preconditioning, cryoprotection, rewarming and regeneration of cryopreserved shoot tips. Comparison between cold-tolerant and cold-sensitive cultivated potatos and the analysis of survival and the stress of cells in different region will be performed. Results should give answer to the question, whether the genotype and the survival cells in different regions can be correlated to cryopreservation results. The synthesis of these analysis can help to find the causes for genotype effects and to improve reactions of accessions with low regeneration. Moreover, it is intended to apply the results for methodical and/or technical improvement of cryopreservation for potato.

马铃薯是重要的作物种质资源，目前国际上形成了圃位、试管苗和超低温保存的立体安全保存体系。我国马铃薯资源主要以田间圃和试管苗库进行保存，超低温保存规模尚未达到国际先进水平。马铃薯茎尖超低温保存技术相对成熟，可作为我国超低温保存重点对象。研究发现马铃薯茎尖经超低温保存处理不同基因型成活率相差较大，而存活潜力与茎尖存活细胞分布和数量紧密相关，我们研究发现茎尖不同部位细胞可能产生不同的应答胁迫，因此亟需开展超低温保存机理研究，对茎尖不同位置细胞进行定位研究。本项目以不同超低温耐性品种的马铃薯茎尖为材料，观察超温保存关键步骤茎尖不同部位的存活和胁迫应答行为，采用细胞学方法定位存活部位，利用热力学方法分析玻璃化状态，无损伤微测技术、显微和透射电镜等方法观察茎尖不同位置自由基积累和细胞超显微结构。本项目将进一步深入对马铃薯茎尖超低温保存的机理研究，为马铃薯种质资源安全保存提供理论指导。

马铃薯栽培种是继水稻、小麦和玉米之后的第4大粮食作物，为避免种质资源丧失，国内外通过建立种质圃、试管苗库和超低温保存库保存马铃薯种质资源。超低温保存，作为马铃薯种质资源长期备份保存方式而备受各国种质资源保存专家重视。研究发现，马铃薯不同种质茎尖经超低温保存处理后存活率和再生率相差较大，而存活潜力与茎尖存活细胞分布紧密相关。主要研究内容：本项目以不同耐性种质马铃薯茎尖为研究对象，开展超低温保存关键步骤茎尖不同部位存活和胁迫应答行为研究，包括马铃薯茎尖超低温保存技术优化及确定超低温保存关键点，以及分析超低温保存关键点茎尖不同部位细胞存活状态和氧化胁迫组织细胞学染色变化、细胞超显微结构和离子流流速和方向变化。重要结果：① 差示热量扫描研究确定了马铃薯茎尖超低温保存技术关键处理为预培养、PVS2脱水处理和化冻恢复培养阶段，且揭示了在PVS2处理阶段茎尖平均含水量和吸热焓值最低，并发生玻璃化状态改变；② TTC、NBT和DAB组织细胞学研究揭示了不同种质马铃薯茎尖M1-M3区域细胞存活，而M3区域细胞在PVS2处理后发生强烈的氧化胁迫；③ 超显微结构和非损伤测定研究揭示了PVS2处理后，不同种质马铃薯茎尖M和L区域O2内流流速显著减弱，细胞未发生明显质壁分离；经LN和化冻处理后，M区域细胞Ca2+内流和H+外流流速减弱，且M与L区域细胞显微结构响应不同。科学意义：① 本项目首次以不同基因型马铃薯种质为材料，经过不同超低温保存处理，研究茎尖不同部位在超低温保存过程中活力和受到的氧化胁迫响应模式，为进一步阐明超低温保存机制，实现马铃薯种质资源安全长期保存提供理论依据；② 本项目创新地利用非损伤微测新技术，对超低温保存关键步骤的茎尖进行活体实时观察，开展重要离子流流速和方向变化研究，拓宽了超低温保存机理研究技术类型，实现了技术突破。