基于胚挽救技术的无核抗寒葡萄畸形苗发生机制及其种质创新

Embryo rescue technique is the primary means for genetic improvement of cold-tolerance in seedless grapes. However, a great proportion of abnormal seedlings, produced in the process of embryo rescue, have become a bottleneck restricting the efficiency of seedless grape breeding. The occurrence mechanism of deformity seedlings during embryo rescue in seedless and cold-tolerant grapes is as yet unknown. This project intends to interspecifically hybridize between pre-collected six diploid wild Shanxi Vitis spp. as the male parents and.prescreened Vitis vinifera, ie six diploid seedless and four tetraploid seeded grape cultivars, as the female parents. And then the zygotic seedlings will be obtained by means of embryo rescue. Hereafter we will attempt to dynamic track the each link which might cause the formation of abnormal seedlings in the process of zygotic embryo rescue. Accordingly, we will comprehensively analyze the reasons for the occurrence of abnormal seedlings in aspects of cellular and genetic factors and culture conditions. Furthermore, a scientific prevention or recovery and reuse program for abnormal seedlings during embryo rescue will be established. Meanwhile, the target characters of seedlessness and cold-tolerance of embryo rescue seedling progenies will be identified. The project aims to explore the occurrence mechanism of abnormal seedlings during embryo rescue and create the novel germplasms of seedless and cold-tolerant (under -15 ℃) grapes. Thereby it will provide the theoretical basis for improving the embryo rescue technique and lay the scientific foundation for above-ground overwintering style of seedless and cold-tolerant grapes in cold regions of northern China.

胚挽救技术是欧亚种无核葡萄抗寒性遗传改良的主要手段，而高比例畸形苗的出现，成为制约无核抗寒葡萄育种效率的瓶颈，其发生机制尚未明确。本项目拟利用课题组搜集到的6份山西野生二倍体抗寒葡萄种质资源为父本，前期筛选的欧亚种可挽救性优良的6个二倍体无核葡萄和4个四倍体有核葡萄品种为母本，通过常规杂交和现代胚挽救技术相结合的手段得到合子苗；动态追踪胚挽救过程中可能导致畸形合子苗发生的各个环节，在细胞学水平、遗传因素、培养条件等方面综合分析合子胚挽救过程中畸形苗的形成原因；建立胚挽救畸形苗的科学预防和分类恢复再利用方案；同时，对胚挽救杂种后代群体进行目标性状（无核、抗寒）的筛选。本项目旨在探索无核抗寒葡萄合子胚挽救过程中畸形苗的发生机制并创制具有山西特色的无核抗寒（可耐-15 ℃）葡萄新种质，为完善胚挽救技术体系提供理论依据，同时为我国北方寒区葡萄露地越冬奠定科学基础。

加快培育无核抗寒葡萄新品种，是全球葡萄育种工作者的共同目标之一。无核葡萄胚挽救畸形苗是制约其育种效率的瓶颈，而关于其发生机制的研究还未见报道。本研究通过常规大田种间杂交和现代胚挽救技术结合的手段，利用野生葡萄种质资源为父本材料，对栽培欧亚种二倍体或四倍体葡萄进行抗寒性遗传改良，并探索合子胚挽救过程中畸形苗的发生机制。研究结果表明，无核葡萄胚挽救操作第二阶段（胚萌发培养）中的无菌剥胚环节，可能是导致胚挽救畸形苗发生的重要环节。显微条件下无菌剥胚繁琐困难，切损的不完整胚可能是发育成单子叶畸形苗、有根无叶畸形苗、有叶无根畸形苗、无叶无根畸形苗的重要原因。而利用自愈培养基转接培养三次，可有效将单子叶（78.3%）和子叶扭曲褶皱型（42.1%）这两类胚挽救畸形苗成功转化恢复为正常试管苗；选择亲和性良好的砧木品种，采用锡箔纸为绑缚材料对有叶无根胚挽救畸形苗进行试管内微嫁接再培养，也可使其成功转化恢复（最高20%）；且这三类胚挽救畸形苗成功转化后，扩繁移栽定植的成活率与正常胚挽救苗差异不显著。另外，不同葡萄杂交配置的合子胚败育时期不同，其与胚乳败育时期呈正相关；在球形胚、心型胚和鱼雷型胚三个发育时期均可进行采样接种；且相同培养条件下，这三个时期的葡萄胚挽救成活率差异不显著。现有的葡萄分子标记，在合子胚挽救苗无核或抗寒性状早期辅助筛选鉴定应用中，其适用性还存在局限或缺陷，需结合流式细胞技术、去壁低渗染色体压片计数法、电解质渗透法等多种方法，综合对其进行再选择或重鉴定。同样是离体胚挽救的方法，杂种三倍体葡萄种子的发芽率（0~22.8 %）远低于自花授粉（50.9~61.2 %），而所有杂交种子离体胚挽救的发芽率（0~61.2 %）均高于播种的方法（0~42.0 %）。符合预期的胚挽救苗扩繁炼苗后，现于山西农业大学葡萄种植资源圃保存86份二倍体无核和35份三倍体葡萄种质资源。本研究在理论和技术上完善了无核葡萄胚挽救育种体系，同时为我国北方寒区葡萄露地越冬奠定了科学基础。