洋桔梗双单倍体胚胎发生的关键影响因子的研究

Lisianthus (Eustoma grandiflorum) is the worldwide No.1 cut flower crop which propagated by F1 seeds. Productions in China are fully dependent on imported seeds, which of annual value reached 20 million CNY. The great breakthrough of F1 hybrid varieties breeding in abroad is probably contributed by the efficiently Double Haploid Technology (DHT), however, there is no published paper indicated. Our experiment results showed anther culture is suffered from genotype and environmental restriction, while cell proliferation was still temporally not observed in microspore culture and ovule culture; interestedly the pollens of Gentiana rigescens and Gentiana expansa can stimulate Ovary expansion of lisianthus and harvesting aborted seeds. For gentiana plants, it was proved by complete research in abroad that anther culture is highly genotype restrict, while unfertilized ovule culture is the much more efficient method for doubled-haploid production. Till now it is still very unclear for in vitro doubled haploid embryogenesis of lisianthus which are the key restricts factors, which is the best pathway. So four strategies will be taken to search for efficient method for in vitro doubled-haploid production:1). Pseudogamy induction via inter-genus pollens & X-ray radiated pollens combined with embryo saving culture; 2). Quantified artificial low temperature treatment dawrf pot-plant in blooming stage; 3).Chromsome doubling of anther & ovary before in vitro culture；4). Develop molecular markers for doubled haploid identification. To find out the key factors that affect doubled haploid embryogenesis, the effects of low temperature treatment and chromsome doubling before culture will be quantitative analyzed; the different embryogenesis between marcrospore and zygotic embryo will be characterized with microscope.

洋桔梗是世界第一的F1种子繁殖切花作物，我国用种全靠每年约2000万元的进口。国外育种突破可能得益于高效的双单倍体技术，但尚无文献证明。试验发现洋桔梗花药培养易受基因型和培养环境制约，而小孢子培养和胚珠培养则暂未发现细胞能够增殖; 滇龙胆和盐丰龙胆花粉能诱导洋桔梗子房膨大并获得败育种子。在其同科异属植物上，国外有完整的研究证明了花药培养受基因型限制，而未受精胚培养才是获得双单倍体植株的高效方法。至今洋桔梗(双)单倍体胚诱导培养的可行性、关键制约因素和最佳途径仍不清楚。计划采取远缘花粉和X-射线辐射花粉诱导假受精结合胚挽救培养；人工定量低温处理盆栽矮化的花期植株；大小孢子先进行体内染色体加倍后培养；双单倍体的分子标记早期鉴定四种策略，来寻求高效的诱导培养方法。通过大孢子胚和合子胚发育差异的显微观察，低温处理与染色体加倍对胚诱导培养效果影响的定量分析，确定（双）单倍体胚胎发生的关键影响因子

洋桔梗是世界排名第一的种子繁殖切花作物，生产用种完全依赖进口。创建高效的双单倍体技术有助于加快新品种选育进程。项目采用花药培养、小孢子培养、辐照花粉和远缘花粉诱导大孢子孤雌生殖四种方式，探索了洋桔梗双单倍体胚胎发生的关键影响因子和适合路径。结果发现：在花药培养中因体细胞竞争极易出现非目标培养植株，小孢子液体培养获得了单倍体愈伤组织，但发生频率极低且愈伤组织诱导植株过程漫长。测试了11种的低温诱导栽培环境，依然不能抑制小孢子体内提前萌发和提高胚性小孢子发生频率。体内诱导阶段如何获得高比例胚性小孢子是开展花药和小孢子培养的前提和基础，培养过程中小孢子的可持续分裂和极化是胚胎发生的关键。利用能够实现全生态因子精准调控的人工气候箱或温室来提高胚性小孢子的发生频率非常重要。为此项目申请了2件相关发明专利。在花粉辐照诱变中，X-射线使用方便但难以获得高的辐射剂量率而使花粉败育。后来采用γ射线把辐射剂量从50Gy增加到1800Gy，但仍然获得充分结籽，表明洋桔梗花粉抗辐射能力很强，洋桔梗结实量很大，如何快速鉴定真假受精结籽问题是辐照花粉诱导孤雌生殖的难点。反复多年试验发现，龙胆远缘花粉诱导能够获得无融合生殖结实，并通过瓶内播种后，可以大幅提高出芽率，获得无融合合生殖的植株。这是下一步应当聚焦的研究重点。