基于芽苗砧嫁接技术油茶砧穗愈合机制研究

Camellia oleifera is an important woody oil tree in south china , which there are high value of nutrition in camellia oil. With the increasing of planting area, the demand of grafting seedlings are huge. For improved the efficiency of grafting and growth ability in cell and molecular level, it is necessarily research formation mechanism of grafting union. We are systematic study regularities of of secondary metabolites after plant defense response in different development status of root and scion and analyze affection to the formation of callus. Meanwhile,we will observed development of vascular and express of related genes in this research; Through observing grafting of rootstock and scion in the whole process of Camellia oleifera by paraffin section, histochemistry, transmission electron microscopy, it is research the mechanism of callus in formation, development and differenation; They are help to understand the he cellular changes mechanism and characteristics of Camellia grafting healing, and analyzing reason of growth. Meantime. It will analyse the expression of related gene by transcriptome sequencing technology in the process of grafting union healing, and clarify the feature of gene expression in terms of time and space. It will be comprehensive analysis of callus changes in cell and molecular level, which reveal the camellia grafting healing mechanism, and determine the healing and growth effects. Some efficient strategy of breeding seedling will be proposed based on the mechanism of grafting heal.

油茶是我国重要的木本油料树种，油茶籽压榨后的茶油具有较高的食用价值；随着种植面积的增大，苗木需求量巨大；为充分提高油茶嫁接育苗效率及其生长量，亟需了解其嫁接愈合机制。本研究拟系统地研究油茶砧穗各发育状态下，接触面受创前木质素含量变化和受创后防御应激反应下次生代谢产物变化及对愈伤组织形成的影响、维管束发育及相关基因表达；通过对油茶砧穗嫁接的全过程观察，采用石蜡切片、透射电镜等技术对愈合过程进行研究，了解油茶嫁接愈合的细胞变化机制及特点，分析可能存在影响生长的因素。同时采用转录组测序技术对嫁接愈合过程中相关基因的表达情况进行测序分析，阐明相关基因在时间和空间上表达的特征。综合分析维管束形成过程在细胞和分子水平上的变化，揭示油茶的嫁接愈合机制，确定影响愈合和生长的因素，提出油茶高效育苗策略。

培育优质种苗是实践良种良法，是推动油茶产业快速发展，实现脱贫致富的重要基础，因此掌握和分析影响种苗生长的各类因子，对提高繁殖效率和质量具有重要意义。通过本项目工作：1）探明矿质元素随穗条芽位萌发生长的含量逐渐降低或持平，茎段内富含磷、钾元素，且18号除锌和锰外，均优于53号，揭示芽位萌发20-30d时穗条综合营养状态最佳。2）探明品种间穗条纤维素、半纤维素和木质素含量及木质素/纤维素比值变化趋势相似，揭示53号纤维素和木质素含量较18号受到更多矿质元素如氮、锰、镁和磷的显著或极显著影响，明确芽位萌发后30d左右为穗条韧性最佳。3）探明砧穗受创后53号创伤面有较高的防御激素应激反应，SA、JA分别与细胞分裂素呈显著或极显著负相关，18号创伤面激素快速促进愈伤组织形成；揭示穗条受创后18号应10min，53号40min，及两品种砧木10min的激素含量对嫁接有利。4）探明嫁接愈合各阶段内源激素虽变化趋同，但含量及比值差异明显，揭示嫁接初期18/18激素含量与比值利于愈伤组织快速发育，后期18/53更有利于根系生长。5）石蜡切片和透射电镜观察揭示嫁接部位形态重建各阶段的细胞增殖为18号快于53号，砧穗嫁接后创伤面细胞质变的浓密，细胞骨架逐渐变的松散，愈合后细胞间连接密切。6）揭示嫁接愈合中大量激素相关基因和转录因子参与调控细胞增殖、分化和形态建成，并发现激素Karrin相关基因，探明18/53嫁接2d后显著表达的WRKY75等12个基因，以及18/53各阶段负表达调控的20个显著表达基因，显示嫁接组合间基因表达差异。7）揭示18/53根系内存在调控根系构型的的特异miRNA，探明靶基因调控植物激素信号转导通路的miRNA为gma-miR5037b、ptc-miR156l。以上研究结果为解析53号迟滞生长机理，阐明油茶砧穗嫁接愈合规律和互作机制提供了依据。