宝莲灯组培褐变的发生机制及解决对策研究

The project is performed to explore the comprehensive mechanism of tissue-culture browning using the aseptic plantlets obtained by ovary cluture of Medinilla magnifica, which is a kind of rare ornamental plant and easily browns during tissue culture. Effects of browning after cutting on their growth and development will be studied by analysizing the spatial and temporal changes of cellular morphology and phenolics accumulation. And the molecular mechanism of browning will be revealed by screening browning-related genes via expression profiling analysis. Furthermore, available inhibitor of browning also will be searched based on the preceding results on browning-mechanism studies, to solve the browning response of tissue culture by comparing the effects of different types and concentrations of inhibitors. Finally, the optimal medium for proliferation and rooting will be explored in order to establish tissue culture system of Medinilla magnifica from shoot tip explants. This study will help roundly elucidate the mechanism of tissue-culture browning and provide theoretical support to preclude their jeopardizing, which also has important significance for the progress of plant tissue culture research.

本项目以极易褐变的珍稀花卉植物宝莲灯为研究对象，通过子房培养获得其无菌植株材料后，观察并分析切伤对宝莲灯叶片、茎段的显微形态结构及酚类氧化物积累的时空变化分析，研究组培褐变对宝莲灯生长发育的影响方式；同时，通过对褐变前后宝莲灯表达谱的差示分析，筛选与褐变相关的基因并从分子水平上深入揭示褐变的基因调控机制；进一步地，以褐变机制研究结果为基础，通过观察和比较不同抑制剂类型及浓度对宝莲灯组培褐化的抑制效果，寻找解决宝莲灯组培褐变的对策；最后，通过对宝莲灯组培最佳增殖及生根培养基的探索，以茎尖为外植体建立组培体系。本研究将有助于全面阐明植物组培褐变的发生机制，为解决褐变危害并获得组培成功提供理论支持，对于植物组织培养研究的发展具有重要的意义。

本项目以极易褐变的酸脚杆属植物宝莲灯为研究对象，对其组培褐变的发生特性进行了系统研究。连续的形态学及解剖学观察发现：切伤是导致宝莲灯发生褐变的直接原因，但褐变危害对植物不具有致死性。研究首次对宝莲灯的转录组进行重头测序，并对宝莲灯切伤后的表达谱进行时间序列分析。对获得的6431条差异表达基因（DEGs）的聚类分析显示：持续上调的DEGs主要参与酚类物质的合成及氧化、转录调控和糖代谢过程；持续下调的DEGs主要参与营养物质运输、碳水化合物代谢及负调控转录等；而先下调后上调的DEGs主要调控光合作用、能量代谢及胁迫反应等过程。分子生物学研究结果支持组培褐变是植物对切伤胁迫的一种适应反应。此外，项目提出了抑制宝莲灯褐变的方案并成功建立其组培快繁体系，但是褐变的彻底消除还有待于对褐变发生信号转导及基因调控的进一步研究。通过该项目的资助，目前已发表与该课题直接相关的科技论文3篇，其中SCI论文2篇，授权国家发明专利1项，培养硕士研究生1名。