板栗后期自交不亲和性细胞学机理研究

Chestnut is an important woody grain tree species in China, but exhibiting the characters of high empty cupule and the low seed set being very low. We found that the reason for the low yield was late-acting self-incompatibility (LSI) in chestnut. However, we poorly understood the mechanism of LSI in chestnut. We still did not konow that it was pre-zygotic LSI or post-zygotic LSI in chestnut? Based on our preliminary results, after self-pollination 15d, pollen tubes could pass through the style, 20d entering into the embryo sac, and the central cell was fertilized in 22d. Although the free nuclei of the endosperm were produced in 25d, we didn’t observe the proembryo. We observed a large number of aborted ovules in 30d after self-pollination. However, we didn’t sure whether the egg was fertilized, and ovule death of the physiological factors were unclear. Thus, to further reveal the mechanism of LSI, in our present project, we will performe on the changing patterns in each stage of the fertilizing process after self-pollination and cross-pollination in Castamea mollissima, via the tissue sections, organization chemical positioning and immune technology etc. The results will be compared with the contemporaneous physiological metabolic substances (IAA, ABA, Ca2+, polysaccharides, lipids and proteins) for the correlation analysis, to clarify whether the egg cell is fertilized and the reason why the embryo can not be formed after self-pollination. The project will break through technology bottleneck of the low yield in Castamea mollissima.

板栗是我国重要的木本粮食树种，但存在空苞率高、结实率低的问题。研究发现，其低产的一个重要原因是它存在后期自交不亲和性。然而目前对于板栗后期自交不亲和的认识仅停留在现象表面，其后期自交不亲和是合子前还是合子后作用机制尚不清楚？前期研究表明，自交15d花粉管正常穿过花柱基部，20d进入胚囊；自交22d后中央细胞发生了受精，25d有胚乳游离核产生而未见原胚的出现，30d观察到大量胚珠死亡，但对其卵细胞是否受精、胚珠死亡的生理因素等仍不清楚。因此，为进一步揭示其后期自交不亲和性机制，本项目采用组织切片、组织化学和免疫化学等技术，重点比较研究板栗自交和异交传粉后受精动态过程和变化规律；并将这些规律与其生理和营养物质（IAA、ABA、Ca2+、多糖、淀粉、脂类和蛋白质）进行关联分析，以期探明板栗自交传粉后卵细胞是否受精以及不能形成胚的生理因子。本项目可为解决板栗低产技术瓶颈提供理论依据。

项目针对我国板栗生产中存在的空苞率高、结实率低的生殖生物学问题，为探明其生殖机制，采用组织切片、光学显微技术、电子显微技术、组织化学定位等技术手段，通过对比分析板栗自交与异交的一系列生殖生物学事件的差异，较为系统研究了板栗后期自交不亲和生殖机制。主要结果：（1）揭示了板栗结实率低的根本原因是其存在自交不亲和性，并鉴定板栗为合子前的后期自交不亲和类型，不亲和反应位置在子房，时间在合子前；（2）比较了自交与异交授粉花粉管进入胚囊发生受精的生物学差异，发现了自交受精比例2.58%显著低于异交18.09%，确定了大多数自交胚珠存在受精障碍而败育；（3）田间验证发现人工授粉结实率自交（13.3%）显著低于异交（79.5%），明确了板栗生产中必须科学合理配置授粉树，并筛选出了供试品种的最佳配置授粉的组合；（4）探明了授粉后花柱形态结构变化和生理变化特征，揭示了板栗花和果实生殖发育规律，发现了胚珠败育与淀粉等营养物质积累有关，并建立了外部形态和内部结构的对应关系，为板栗花果发育调控提供了理论依据；（5）建立了板栗不同授粉阶段雌蕊的转录组数据库，筛选出与自交不亲和相关的候选基因36个。结论：阐明了板栗后期自交不亲和性细胞解剖学基础，这为生产中板栗花期调控克服自交不亲和、提高座果率具有重要的实践指导意义。