【作者】 蔡永萍；

【导师】 骆炳山； 李合生；

【作者基本信息】 华中农业大学 ， 植物学， 2005， 博士

【摘要】 石斛（Dendrobium）为兰科多年生附生草本植物,一种兼性的CAM植物,是国家重点保护的药用植物和名贵中药材,由于长期的人为破坏和滥采滥拔,其野生资源已近枯竭或濒临绝迹。为了保护野生资源或满足药用的需求,目前,许多珍贵石斛主要依赖人工栽培,但石斛人工栽培技术依然不成熟,石斛对光强的适应机理鲜见报道。本课题以安徽省霍山县3种石斛为材料,系统研究了石斛生长的物候期、有益伴生植物、茎高生长与生态因子和内源激素的关系、光合生理特性及其与环境因子的关系等;在理论上探讨兼性的CAM植物对不同光环境的适应机理,在应用上可为石斛人工栽培提供科学依据。此外,石斛的种质改良还未见报道,本课题通过比较研究霍山2种石斛亲本及其杂交F₁代的生长、生理特性、有效成分的含量、石斛多糖复合物的结构特征和体外清除自由基的作用等,探讨了通过人工杂交进行石斛种质改良的可能性。研究结果如下: 1.总结了霍山3种石斛生长发育的物候期,探明了其生长的生态因子、生长与内源激素的相关性以及有益伴生植物种类。 霍山3种石斛物候期有明显的差别,米斛、铁皮石斛、铜皮石斛新茎生长期分别为150d、154d和160d左右。霍山3种石斛新茎的纵、横向生长过程为S—型曲线,米斛生长只有5月～7月快速生长的一个生长高峰;铁皮石斛、铜皮石斛的生长有双峰现象,5月～8月初快速生长,8月下旬温度渐低后还有一次小的生长过程。 在霍山3种石斛野生群落中,发现紫金牛、卷柏、瓦韦、石韦、有柄石韦、狭叶缩叶藓、大羽藓、大麻羽藓、地衣等都是其有益伴生植物种类。 适宜霍山3种石斛生长环境的相对湿度为80%左右,高光强、高温和低湿度抑制石斛生长。米斛、铁皮石斛、铜皮石斛生长的年有效积温分别为2070℃、2256℃和2248℃。米斛和铜皮石斛生长的最适温度为16℃～20℃,铁皮石斛生长的最适温度为19℃～22℃。适宜的降水也是石斛生长的必要条件。 霍山3种石斛茎高生长与内源GA₁₊₃含量呈显著正相关,与内源ABA含量呈显著负相关;合适的GA₁₊₃/ABA比值是影响石斛茎高生长的重要因素。米斛的内源GA₁₊₃/ABA含量比值低,可能是其茎高生长缓慢的原因之一。 2.从叶绿素荧光参数与光强的关系方面探讨了石斛易受强光伤害的机理,系统研究了光强对石斛光合作用的影响,为石斛适宜栽种光强的调控提供了依据。 霍山3种石斛的光饱和点低、光补偿点和CO₂补偿点较高,净光合速率、表观量子效率、羧化效率、RuBP再生速率都较低,属于阴生植物光响应型。栽培林下,石斛的光合速率较低,呈日下降趋势;自然光下,石斛的光合速率日变化呈V型,12:00时光合速率最低。9:00到17:00时之间,当光强高于800μmol photons·m^-2·s^-1的光合速率为负值,处于光下呼吸状态,没有光合产物积累;只有早晚光强较低时更多还原

【Abstract】 Dendrobiums, perennial herbages in orchidaceae family, normally grow in shadow environment as facultative CAM plants. Wild resources of many species in dendrobium genus, as medicinal plants and rare traditional Chinese medicines, are approached in exhausted or endanger because of manmade effect and non-limited utilization. Although artificial cultivation of dendrobium is useful for protecting wild resources and satisfying medical demand, artificial cultivation techniques are still no success because there are no systemic research on cultural physiology, adaptive mechanism for light intensity and genesis improvement of dendrobium. In this research, phenological phase, companion plants, relation between stem growth and ecosystem factors are conducted to study light adaptive mechanism of facultative CAM plants and physiological bases of artificial cultivation in 3 dendrobium species in Huoshan County, Anhui Province. Genesis improvement of dendrobium is also researched through comparison study of growth, physiological characteristics, available components, structure and function of polysaccharide complex between F₁ generation and parents in dendrobium. The results are as follows:1. Phenological phase of 3 dendrobiums in Huoshan is illustrated. Ecosystem factors affecting growth, correlations between growth and endogenous hormones and available companion plants for dendrobium had been indicated.There are different in phenological phase of growth and development among three dendrobiums in Huoshan. Sustainable growth time of new stem for D. huoshanense, D. candidum and D. moniliforme is 150 days, 154 days and 160 days respectively, and growth curve of stem height and diameter is ’S’ type. Growth procedure of D.candidum and D. moniliforme has two growth peaks from May to August and late August while D. huoshanense has only one from May to July.Ardisia japonica, Selaginella tamariscina, Lepisorus thunbergianus, Pyrrosia lingua, Pyrrosia petiolosa, Ptychomitrium linearifolium, Thuidium cymbifolium, Claopodium assurgens and Lichen are available companion plants for dendrobium cultivation, which were found through wild dendrobium community investigation.Environment of RH 80% is available for dendrobiums growing, and high light intensity, high temperature and low RH will influence growth of dendrobium. The annual accumulated temperature is 2070’C, 2256 °C and 2248 *C respectively for effective growth of D.Huoshanense, D.candidum and D.moniliforme. The most optimal temperature for growth of D. Huoshanense and D. candidum is 16°C²0"C as well as 19 °C ²2"C for D. moniliforme. Feasible precipitation is necessary for dendrobium growth.There is a significant positive correlation between content of endogenous GAi+3 and stem growth of dentrobiums in Huoshan, as well as a significant negative correlation between content of endogenous ABA and stem growth of dentrobiums in Huoshan. Suitable GA1+3/ABA is important factor affecting stem growth of dentrobiums in Huoshan. Low GAi+yABA in D. huoshanense may be one of reasons to grow slowly.2. Mechanism easily damaged by intensive light for dendrobium is illustrated through studying relation between parameters of chlorophyll fluorescence and light intensity; and effects of light intensity on dendrobium photosynthesis are systemically studied, which provide the foundation for selecting optimal light intensity during dendrobium cultivation.Their light saturation point, net photosynthesis rate, apparent quantum yield, carboxylation efficiency and RuBP recovering rate point are low during photosynthesis, light compensative point and CO2 compensative point are high during photosynthesis, which is meaning that dendrobium is a plant for shadow responsibility on light intensity. Under cultivation environment of forest shadow, photosynthesis rate of dendrobium is reduced gradually. Daily change of photosynthesis for dendrobium shows V type and there is the lowest photosynthesis at noon under natural light. Photosynthesis rate is negative value and there is no photosynthesis accumulation from 9:00 A.M. to 17:00 P.M. because light intensity is higher than 800 u mol photons-m^s"1, as well as there is photosynthesis accumulation only during low light intensity on morning or night. Suitable tight intensity for photosynthesis of dendrobiums in Huoshan is 400 u mol photons-m"2s"1⁶00n mol photonsm^s*1 normally.3. Dendrobim genesis improvement is achieved through dendrobium cross. The researching results primarily illuminate that Fi generation of cross dendrobium has not only hybrid advantage and cultivation significance, but also significant economic benefit.D. Huoshanense （male） has better quality and lower yield while D. moniliforme （female） reverse, so they are selected as parents for interspecific hybridization. Fresh weight of annual stem of Fi generation is as 4.23,1.08 times as ones of A Huoshanense and D. moniliforme respectively; yield of it is as 3.91,1.11 times as ones of D. Huoshanense and D. moniliforme respectively. Available medical and nutritive components of Fi generation are higher than D. moniliforme and almost similar with D. Huoshanense. Physiological characters of Fi generation are greatly improved with wide adaptivecapacity and high photosynthesis for Fi generation has more photosynthetic area, higher content of chlorophyll, lower chla/b rate, higher light saturation point, lower light compensative point, higher carboxylation efficiency and net photosynthesis rate than its’ parents. Content of endogenous hormones in Fj generation is intervened parents. Rate of GAi+3/ABA and ZR+GA1+.3 /ABA in Fi generation is almost similar with D. moniliforme, which is one of reasons why growth quantity of Fi generation approaches D. moniliforme. Not only does Fi generation integrate characteristic enzyme spectrum of parents and express hybrid characters from their POD and EST isoenzyme spectrum, but it form itself specific isoenzyme spectrum. All results indicate that Fi generation expresses advantages on physiology.4. Composition and effects cleaning up oxygen free radical of polysaccharide from Fi and parents are similar, which further proofs dendrobium improved through hybrid.Polysaccharide extracted and purified from Fi and parents is a neutral polysaccharide or a complex of polysaccharide and protein containing glucuronic acid and protein without reductive sugar. The monosacchride composition of polysaccharide in D, Huoshanens and in Fi generation is similar mainly including L-Rha, L-Ara, D-Man and D-Glc, but their molar ratio is 0.087 : 1.000 : 102.000 : 79.500 in D. Huoshanens as well as 0.170 : 1.000 : 81.900 : 52.100 in Fi generation. Polysaccharide in D.moniliforme is mainly composed of L-Rha, L-Ara, D-Xyl, D-Man and D-Glc, and their molar ration is 0.400: 1.000: 1.510: 63.000: 35.400. The results mean that composition characters of polysaccharide complex in Fi generation have no big variation and are similar with parents.Dendrobium polysaccharides have certain inhibitory action on OH and O2’ within experimental concentration range, and their inhibitory actions increase with the raising of concentration. The inhibitory concentration of 50% （IC50） on OH of polysaccharides in D. Huoshanens, D. moniliforme and Fi generation is 6.79 mgml"1, 6.75 mg-ml"1, 7.09 mgml"1 respectively and their IC50 on O2 * is 3.04 mg-ml"1, 3.44 mg-ml"1, 3.02mg-ml"1 respectively. Moreover, dendrobium polysaccharides have inhibitory action on lipid peroxidation of mice liver homogents in vitro and can alleviate oxidation damage of mice liver mitochondria induced by Vc-Fe2+ system. The results means that polysaccharide function for oxygen free radical clearance in Fi generation is similar with parents and hybrid has no damage on capacity for polysaccharide cleaning oxygen free radical.更多还原