刺槐不去雄条件下实施控制授粉的花粉竞争与子代选择机制

The previous studies of Robinia pseudoacacia L. suggest that it is a kind of high level outcross plants. And for Robinia pseudoacacia L.,emasculation has been found to be a difficult task because it not only takes long time to finish, but also does some serious damage in its pistil which can lead to low setting rate,only 3.6%. The researches in recent years have found that pollination without emasculating in Robinia pseudoacacia L. can get larger group of offspring in this way,it can get high setting rate about 30.0%-40.7%. And these offspring may probably varied because of their different male parents. In this situation, it also has found that the recession in growth of embryo and offspring seedling and stepping out some offspring have occured. The outcross rate about 20 weeks old seedlings is 95.8%. All above can suggest that by the pollination without emasculating, the inner relationship among stigma compatibility, pollen competition, selfing recession and the genetic relationship in mating parents have been proved.To explore the causes, under the condition of pollination without emasculating in Robinia pseudoacacia L.and the pollination with emasculating as contrast, the natural pollination has been imitated by using male parents which have known their background. Then this research has been divided into three parts: using the fluorescence microscopy, scanning electron microscopy（SEM）and ISSR genetic relationship analysis to study the stigma compatibility and fertilization competition mechanism; using the paraffin section and the resin semi-thin section technology to observe the growth process of zygotic embryo resulted from selfing and outcross; using the SSR paternity analysis abortion by outcross choice and ovule abortion by selfing, as well as the influence mechanism in outcross and selfing caused by genetic relationship in mating parents. All these experiments under the condition of pollination without emasculating in Robinia pseudoacacia L. are used for screening excellent parents to control pollination, optimizing the pollination technology, maximizing the elimination of selfing offspring, realizing large groups of strong selection, breeding elite hybrids and providing evidence for new hybridization breeding method and strategy.

前人研究认为刺槐高度异交；去雄难且费工；去雄操作对雌蕊伤害重、结实率仅3.6%；不去雄自然授粉结实率高达30.0%-40.7%，能获较大的且父本来源丰富的子代群体，不同父本所产子代差异大；从种胚发育到子代苗生长中均逐级淘汰自交子代，20周龄苗的异交率达95.8%。说明不去雄自然授粉与柱头亲和力、花粉竞争、亲本亲缘关系及异交、自交比率变化规律之间有内在联系。为探究起因，在不去雄条件下，用已知的多父本模拟自然授粉，以去雄的为对照，用荧光显微、扫描电镜和ISSR亲缘关系分析技术研究不同花粉柱头亲和差异、受精竞争机制，用石蜡切片和半薄切片技术观察异交、自交合子胚发育过程，用SSR父本分析法研究异交选择和自交胚珠败育规律、亲本亲缘关系对异交、自交的影响机制等。为在不去雄条件下，选优良亲本，优化授粉技术，实施控制授粉，淘汰自交子代，实现大群体强选择，探索杂交新方法和育种新策略提供依据。

前人研究表明，刺槐本身的树种特性以及去雄后产生的大量落花落果现象，是阻碍刺槐杂交育种进程的主要原因。考虑到刺槐去雄产生的严重伤害，自然状态下的高异交率，以及人工收集花粉和授粉等耗时耗力的情况，本研究在刺槐不去雄条件下主要开展了以下几个方面的研究工作：①在刺槐不去雄条件下利用单一或混合父本实施人工控制授粉，对不同发育阶段的子代进行发育形态学观察，并综合选用EST-SSR和Genomic-SSR分子标记进行子代父本来源分析，发现花粉竞争和子代选择在子代发育早期就已经开始并存在于整个过程中，刺槐在不去雄条件下通过人工控制授粉可以产生杂种子代，但会导致子代大量败育；②通过嫁接建立可用于刺槐在不去雄条件下模拟天然授粉的杂交育种体系，并利用该体系进行刺槐不去雄条件下借助蜜蜂模拟天然授粉的试验，最终从三株母本上收获到成熟种子，并育出幼苗，三个子代苗群体异交率分别为95.40%，91.67%和100.00%，这表明在封闭环境内引入蜜蜂进行刺槐不去雄条件下的模拟天然授粉，可用于刺槐免于人工操作的杂交育种工作；③利用刺槐不去雄条件下人工控制授粉试验中所得子代的父本分析结果计算各个控制授粉组合中每个父本在子代中的贡献率以及各个亲本之间的遗传相似度，并对计算所得到的各父本贡献率和各个亲本之间的遗传相似度进行了相关性分析，结果发现在刺槐子代三个不同发育阶段，父母本的交配亲和力和遗传相似度都呈显著相关，这表明刺槐亲本之间的遗传关系会影响杂交亲和力。本研究的结果将为刺槐杂交育种中亲本的选配，以及不去雄条件下利用蜜粉授粉的杂交育种等相关研究提供技术支撑和参考依据。