外源花粉诱导百合雌核单倍体胚胎形成的机理研究

Unfertilized ovary or ovule culture is an effective technology for the production of homozygous materials (doubled haploids) via gynogenesis. In commercial lily species which have a high level of heterozygosity, production of doubled haploids opens a way to obtain more information concerning the genetic base of characters, increase selection efficiency and create more novel germplasm. But one problem with the practical application of gynogenesis is the very low frequency of embryo germination in many lily genotypes. In our previous research, controlled alien pollination has been shown to significantly enhance embryo germination frequency in lily. In this study, in order to understand the mechanism on induction of embryogenesis and embryo germination from unfertilized ovules of lily, one lily genotype'Sorbonne' will be tested and cytological and molecular aspects of gynogenic embryogenesis will be investigated. Morphological characteristics and the origin of gynogenic embryos induced by controlled alien pollination will be studied under the microscope. In addition, to gain insight into the molecular events occurring in the late stage of lily gynogenic embryogenesis induced by controlled alien pollination, cDNA clones corresponding to genes differentially expressed during the late stage of gynogenic embryogenesis will be isolated, identified and characterized by constructing SSH-cDNA libraries. Some embryo-specific genes are expected to be found. The study will provide novel information on gynogenic embryogenesis and embryo germination, and also improve breeding efficiency and germplasm innovation in lily.

未受精子房离体培养是诱导雌核产生单倍体的技术之一，在百合上此项技术可快速简化遗传背景、提高新品种选育效率和加速种质创新等。目前，百合雌核单倍体胚胎萌发频率低是该技术上存在的一个亟待解决的问题。本实验室研究表明外源花粉诱导显著地提高了百合未受精子房培养胚胎萌发频率。本项目拟以百合'Sorbonne'为试材，采用组织切片和显微镜技术从细胞学水平研究外源花粉诱导对雌核胚胎起源、发育的影响；建立SSH-cDNA差减文库并结合RACE技术从mRNA水平分离外源花粉诱导及非诱导方式下雌核胚胎发育后期过程中差异表达的相关基因，预测基因的蛋白质序列、结构及功能，并研究其组织表达特性。通过研究将从细胞学和分子水平探明外源花粉诱导方式下百合雌核胚胎的起源与发育，以及雌核胚胎形成及萌发的分子机理，为提高百合未受精子房培养胚胎萌发频率提供理论依据和技术支撑，对加快百合育种、种质创新和遗传学基础研究具有重要意义。

百合未受精子房离体培养在快速简化遗传背景、加速种质创新和提高新品种选育效率方面就有重要作用。但是百合雌核单倍体胚胎萌发频率低是影响该技术有效利用的关键因素之一。本项目以‘Sorbonne’等基因型为试材，对培养条件进行了优化，研究了外源花粉对雌核胚胎起源和发育的影响，并探讨了外源花粉诱导雌核胚胎发生的机理。结果表明，成熟的胚囊适合于诱导雌核胚胎发生，开花后0~3d（柱头有粘液分泌）的中段子房为最佳取样；开花当天的外源花粉最适用于诱导处理，最佳诱导处理时间为开花后0~2d；外源花粉诱导了较高比例的胚囊细胞有丝分裂的发生，并能促进胚胎继续发育，相比，对照也能诱导胚囊细胞的发生，但是在后续的发育过程中珠被内表皮细胞增值较快而挤压胚囊，导致胚囊内细胞分裂停止而退化解体；但是在这2种处理方式下，大多数胚胎均起源于卵细胞，少数起源于助细胞或极核细胞；外源花粉可能诱导了一系列有利于胚胎发育的特异基因表达。本项目研究探明了外源花粉诱导雌核胚胎起源和发育的机理，为进一步的功能基因分离和提高百合未受精子房培养胚胎萌发频率提供理论依据和技术支撑，对加快百合育种、种质创新和遗传学基础研究具有重要意义。