苹果小孢子/花药培养诱导胚状体机理及纯合基因型种质创制

Abstract: Apple is a temperate fruit species which is characterized by a long reproductive cycle with several years of a juvenile phase, a large tree size and a high degree of heterozygosity derived from self-incompatibility. For these reason, to obtain homozygous pure lines is almost impossible only by crossbreeding. Therefore, the production of doubled haploid (DH) by anther and microspore culture offers new possibilities for genetic studies and breeding. Research on doubled haploids production in apple via anther and microspore culture started at the beginning of the 1970s and most technical aspects of anther culture in apple are described. However the embryos induction rate and the regeneration of shoot is rather little and limited in genotypes and there was no literature about the origin and regular of embryogenesis from pollen in apple. In the present study, experiment will be carried out with the cultivars ?American Summer Pearmain? and ?senshu? and aims to obtain more than 50 lines of homozygous plants by changing the media component and condition of culture, to study the origin and regular of embryogenesis from pollen by morphological and anatomical observations of embryos at different stages, to analysis the key gene concerned with hormone synthesis in the formation of embryos by using transcriptone sequencing and expression profiling and clone the full-length sequence of the gene, to analysis the structure of protein by using bioinformatics soft and predict the function, to verify its function via transgenic Arabidopsis Thaliana and analysis the regulation mechansim in the development of embryos form pollen. This study would be help to improve the embryos induction rate in apple anther culture and reveal the mechanism of embryogenesis from pollen in the level of cytology and molecular biology.

利用小孢子/花药培养诱导产生胚状体获得纯合基因型株系，对基因型高度杂合的苹果育种及遗传分析有重要意义。本研究用两个不同基因型苹果品种 "祝"、"千秋"的花药/小孢子为试材，通过调整培养基成分及培养条件，诱导花药/小孢子形成胚状体并再生成植株，获得50株以上的纯合基因型群体；从细胞学水平明确胚状体的起源、发生规律，以及胚状体形成的各个细胞形态学时期；通过转录组及表达谱测序、生物信息学分析，筛选与胚状体形成有关的特异大量上调表达关键基因，克隆其全长cDNA，利用大肠杆菌进行体外原核表达，分离、纯化蛋白，进行蛋白的结构和功能预测与分析；通过转化拟南芥鉴定基因功能，分析基因在胚、花粉发育过程中的调控机制。研究结果将进一步指导优化苹果花药/小孢子培养条件、提高胚状体诱导效率，同时为揭示苹果雄性生殖细胞诱导形成胚状体的细胞形态学和分子生物学机理提供理论依据。

2013年度本研究首先以“祝光”、“千秋”和“红星”三个品种的花蕾为试材，4℃低温预处理后取出花药进行培养，证明低温预处理促进了胚状体和愈伤组织的形成，随着低温处理时间的延长，胚状体诱导率呈不断上升的趋势，愈伤组织诱导率则在胚状体诱导率开始快速上升时呈现下降趋势。观察上述3个品种花蕾外观发育与小孢子发育的对应关系，并培养了各个时期的花药表明，绿蕾期的花药培养能够获得较高的胚状体诱导率，这个时期小孢子发育处于单核后期或双核早期。选用“丹霞”、“富士”、“嘎啦”、“红星”、“红玉”、“津轻”、“硕红”、“千秋”、“珊夏”、“王林”、“祝光”和“wijcik”12个品种的花药培养显示，基因型影响了胚状体的形成，“祝光”的胚状体诱导率最高达到了34.9%，与其他11个品种存在显著差异；其次为“红星”的11.9%和“千秋”5.1%，与“祝光”以外的其它品种间不存在差异。利用流式细胞仪分析了7个株系，其中2倍体有5个，非整倍体2株，没有检测到单倍体植株，说明花药培养得到的植株在培养过程中自然加倍。目前我们获得了5个品种来源的26个纯合基因型株系，全部株系已在试管内成功扩繁、生根，部分株系移植到大田。