【作者】 吴双秀；

【导师】 祖元刚； 吴汪黔生；

【作者基本信息】 东北林业大学 ， 植物学， 2001， 博士

【摘要】 珍贵中药植物高山红景天（Rhodiola sachalinensis A.Bor）的根和根茎中含有以红景天甙（salidroside）为主的次生代谢物质，具有抗疲劳、抗衰老、抗缺氧、抗微波辐射等作用，是非常有开发前途的环境适应药物。本实验利用植物组织和细胞培养技术，以诱导和建立较高红景天甙产量的高山红景天愈伤组织悬浮培养体系为目的，系统地对高山红景天愈伤组织的诱导和状态调控、分化了的愈伤组织颗粒（CCA）悬浮培养体系的特点和生长规律、红景天甙的积累规律、培养基优化和生产条件优化、利用诱导因子和添加前体方法提高红景天甙产量、以及红景天甙生物合成途径几个方面做了调查。 利用高山红景天无菌苗的子叶、幼叶、幼茎和根成功诱导出了7种愈伤组织。以其子叶最容易诱导产生愈伤组织，其次是幼叶、幼茎和根。对上述愈伤组织的状态进行调控，得到红景天甙含量高、生长速度快、初步分化了的CCA型愈伤组织颗粒。 CCA悬浮培养的生长周期为25～30天。在继代后的0～15天左右为指数生长期，15～20天以后生长速度减慢。最大红景天甙积累是在继代后的3～5天内。 外源8.27mg/L的BA与4.14mg/L的IBA的组合时，CCA悬浮培养的生长最好；外源2,4-D抑制CCA悬浮培养的生长，但是对红景天甙积累的刺激作用最大。以MS基本培养基的成分和含量为标准，总氮量不变、总糖量增至45g/L、pH值为5.7利于CCA悬浮培养的生长，但抑制其红景天甙的积累；总氮量减少1/2、总糖量减少1/2、pH值为4.2利于CCA悬浮培养细胞内红景天甙的积累，但抑制其生长。利于CCA悬浮培养生长的培养条件为较低的温度（15℃）和较低的摇床转速（100rpm/min）；但利于红景天甙积累的培养条件则为24℃和较高的摇床转速（150rpm/min）。 在继代培养后的第15天进行200W/m~2的254nm的紫外线照射20min对提高红景天甙产量效果最好；牛皮杜鹃根的水提液的诱导效率大于高山凤毛菊根的水提液。前体甙元酪醇（tyrosol）、苯丙氨酸（Phe）、水杨酸（SA）、色氨酸（Trp）和酪氨酸（Tyr）对提高CCA细胞内红景天甙含量的效果依次减小。 建议生产上可以采用“两步法”培养来提高红景天甙产量。首先在利于CCA悬浮培养生长的培养基上培养15～20天左右，使生物量积累达到最大，此时进行紫外线诱导处理，或添加约4mg/L的高浓度2,4-D，或更换到生产培养基中培养3～5天再收获红景天甙。CCA悬浮培养的生长培养基为总氮含量与基本MS培养基的含量相同、总糖含量为45g/L、pH值为5.7并附加5mg/L的BA和2.5mg/L的IBA的外源激素；相应培养条件为15～20℃、正常光照、摇床转速100rpm/min。红景天甙的生产培养基为总氮含量减少1/2、总糖含量为15g/L、pH值为4.2并附加4mg/L的2,4-D；相应培养条件为24℃、正常光照、摇床转速150rpm/min。更多还原

【Abstract】 Rhodio/a sachalinensis A. Bor is a kind of precious herb of the traditional Chinese medicine. Its root and rhizome contains salidroside, which has been shown to possess the medical functions such as resisting anoxia, microwave radiation and fatigue. in order to establish the differentiated callus suspension culture of Rh. Sachahnen.cis with high salidroside production. the induction of callus of Rh. sachalinensis, the regulation of the morphas of the callus, the establishment of the compact callus aggregates (CCA) suspension culture. the characteristics of the growth of CCA suspension culture and the accumulation of salidroside. the improvement of the culture media and culture conditions, the strategies of elicitation and precursors feeding, and the biosynthesis pathway of salidroside were studied in this experiments. 7 types of callus were induced from the cotyledon, the spire, the caulicle and the root of Rh. Sachalinen.cis. The induction of callus from the cotyledon is easiest, from spire is easier and from the root is very difficult. By regulating the morphas of the callus, the preIirninaril~ differentiated compact callus aggregates (CCA) with higher saLidroside content and higher giowth rate was obtained. The growth c~ cle of the CCA suspension culture is about 25 ?--3(1 days. The period from 0 to 1 5 days after subculture is the exponential stage of the gro~vth of the culture. From 15 to 20 days after subculture. the growth of the culture begins to decrease. The maximum of the accumulation of salidroside appears on the day 3 ?? after subculture. The basic MS liquid media with 5 nig/L BA and 2.5 mg/L IBA benefit the growth of the culture. Exogenous 2,4-D inhibited the growth of the culture greatly but stimulated the accumulation of salidroside significantly. If the basic MS medium is the standard. maintaining the quantity of total nitrogen and pH value and increasing the quantity of total sugar to 45 g/L improved the growth of the culture but inhibited the accumulation of salidroside. While. decreasing quantity of both total nitrogen and total sugar to half level and decreasing pH ~ alue to 4.2 separately improved the accumulation of salidroside but inhibited the growth of the culture. Low temperature at 15?C and low shaking speed with 100 rprn/miii benefit the growth of the culture, while temperature at 24~?C and high shaking speed with 150 rpm/rn in benefit the accumulation of salidroside. The ideal induction efficiency of UV irradiation (200 W/m2) on improvement of salidroside production of the culture is carried out for 20 mm at day 15 after subculture. The induction efficiency of the extract of the root of Rhododendron chrysanthum is higher than that of Saussurea alpicola. The improvement efficiency of possible precursor of salidroside such as Iv I / tvrosol, phenylalanine, salicylic acid, tryptophan, and tyrosine become less. ~Two-step?culture method is suggested to improve the salidroside production in mass CCA suspension culture of Rh. Sachalinensis. In order to gain the maximum biomass of the culture first. the CCA is cultivated in the growth-medium for 15~?O days. Then the CCA is transferred into production-medium to incubate for 3 5 days before cultivated. The growth-medium has the same amount of total nitrogen, the same pH value and 1.5 folds of total sugar as the basic MS medium with 5 mg/L BA and 2.5 mg更多还原