【作者】 陈肖英；

【导师】 叶庆生；

【作者基本信息】 华南师范大学 ， 植物学， 2003， 硕士

【摘要】 霍山石斛（Dendrobium huoshanness）在自然条件下，从播种到开花需3年左右。研究霍山石斛的开花过程，缩短其开花年限；在理论上或是在应用上都具有重要意义。用组织培养方法研究诱导霍山石斛花芽发端并发育成开花植株，由于试管兰花一年四季都有花蕾形成，这样有助于进一步了解花的发育过程。 本文研究了TDZ、BA、糖浓度、低温15/5℃（日/夜）、氮含量、转瓶时间对霍山石斛组培苗花芽发生的影响。材料为4个月苗龄的组培苗和来自于4个月苗龄的组培苗的假鳞茎。筛选出诱导开花的适宜培养基后，定期取材（茎尖），进行石蜡切片，试图找出花芽分化的起始时间；使用HPLC测定植物中三种内源激素IAA、ABA、GA₃含量的变化；通过SDS-聚丙烯酰胺凝胶电泳进行蛋白组份分析。 研究发现，在改良的MS基本培养基（1/6氮、5倍磷），糖浓度提高到40g/L，并加入适宜浓度（0.02mg/L）的TDZ可诱导霍山石斛小苗在2～5个月开花，5个月内统计诱导花芽发生率为50％左右。这种诱导提早开花现象，是发生在小苗基部新萌发的幼苗。花芽顶生，在叶腋间也有花发生。低浓度TDZ诱导的花芽多为顶生单花。高浓度TDZ诱导的花芽多为总状花序。BA诱导霍山石斛开花率较低，而且小苗褐化严重。减少培养基中氮含量和低温有利于霍山石斛花芽发生。由于所诱导的花芽正常开放率较低，尝试着将小苗在诱导花芽发生的培养基中培养一段时间后，转移到无激素的MS培养基中，发现大多花芽能正常开放。 处理组为MS（1/6N，5P）+TDZ0.02，附加蔗糖40g/L，琼脂8g/L，pH5.6～5.8。同时设置对照组：MS，附加蔗糖30g/L，琼脂8g/L，pH5.6～5.8。通过制作石蜡切片，观察到处理组在90天时叶腋间有花芽发生。处理组内源激素IAA含量在90～120d明显减少，GA₃与诱导花芽发生关系不明显，由于某种原因，未检测到ABA。处理组在90～120d出现一特异蛋白。更多还原

【Abstract】 The time for wild Dendrobium huoshanness to flower is about 3years . Flowering is a unique developmental event in the life cycle of a higher plant . Studying D. huoshanness flower bud formation and development and decreasing the juvenile phases is important . In vitro flowering by plantlets produced through tissue culture was studied . And in vitro flowering was induced by many ways in the whole year. It is helpful to further our understanding of flowering regulated.The objective of this research was to study flowering may have been induced by many factors, such as TDZ, BA, sucrose, lower temperature 15/5 C nitrogen, and time of transferring to new medium(MS medium lacking plant growth regulator. Flowering was induced in 4-month-old D. huoshanness plantlets and pseudobulbs in an in vitro system. After having screened the optimal medium which induces flower bud initiation on D. huoshanness plantlets, levels of endogenous IAA,GA3 and ABA were studied by HPLC. Soluble proteins were studied by SDS-PAGE. At the same time morphological differentiation of floral buds were observed, and the time of flower bud initiation was found out.The results of our research show that a larger number of floral buds were produced in the MS(1/6N,5P) medium added 40 g/L sucrose and 0.02g/L TDZ. Plantlets, which were new stem growth from subcultured plantlets, were induced to form floral buds in 2-5 months. The rate of flower induction was about fifty percent. Terminal or axillary flowers were formed on plantlets. TDZ at lower concentration was suitable for inducing terminal flowers but higher concentration induced botryose inflorescence . BA was less effective in the induction of floral buds. MS containing reduced concentrations of nitrogen sources, and lower temperature pretreatment promoted the formation of floral buds. In order to make it flowering normally, Plantlets were transferred to hormone-free MS medium after cultured in medium with TDZ.The optimal medium for inducing floral buds is MS medium added 0.02 mg/L TDZ, 40 g/L sucrose and 8g/L agar. The inefficient medium for inducing floral buds isMS medium added 40 g/L sucrose and 8g/L agar.The pH of all media used in this study was adjusted to 5.6-5.8 with IN NaOH. Physiological characteristic during flower induction were studied by culturing the plantlets in two kinds of medium . Results showed that axillary floral buds were induced after 90 days of culture. And there was a notable increase on the level of endogenous IAA during 90-120 days contrast to CK. The level of endogenous GA3 wasn’t related to the formation of floral buds. A special soluble protein was also found during 90-120 days when plantlets cultured in the optimal medium.更多还原