【作者】 李琰；

【导师】 马希汉； 蔚芹；

【作者基本信息】 西北农林科技大学 ， 野生动植物保护与利用， 2003， 硕士

【摘要】 本试验探讨了基本培养基、接种方式、外植体种类、采样时间、培养条件、植物生长调节剂等因素对杜仲愈伤组织诱导、继代培养的影响以及培养基成分、培养条件等对愈伤组织中的药用有效成分绿原酸、总黄酮的积累进行了研究，主要取得了以下研究结果： 1．比较了MS，LS，B₅，N₆，H，Nitsch，White和WPM8种基本培养基，发现MS培养基适合于杜仲幼茎愈伤组织诱导，B₅培养基不仅对叶的愈伤组织诱导有利，对茎和叶愈伤组织的继代培养也是比较理想的。 2．2,4-D、NAA和IBA三种生长素均可100％诱导出愈伤组织，以0.5mg／L 2,4-D、0.5-1.0mg／L NAA、1.0mg／L IBA效果最好；2,4-D和BA及KT的组合均不利于愈伤组织诱导，低浓度的BA与NAA和IBA组合能促进愈伤组织的诱导和生长，其中1.0mg／L NAA+0.3-0.5mg／L BA、1.0mg／L IBA+0.5mg／L BA诱导出的愈伤组织生长旺盛，富有光泽，适合进一步继代培养，而高浓度则起抑制作用。在继代培养中单独使用NAA时，以0.5mg／L效果最好。NAA与细胞分裂素配合使用时，以0.5mg／L NAA+0.5mg／L BA的组合最有利于愈伤组织的生长，其愈伤组织生长量明显大于单独使用NAA。KT的加入对愈伤组织的诱导及生长均有抑制作用。 3．田间材料取样时间幼叶以3月中旬为好，幼茎以4月中旬以前最佳；其余时间以无菌苗的下胚轴、子叶、真叶诱导效果最好。愈伤组织的诱导和生长培养基pH值以6.3比较适宜；培养室的光照条件以12h光照、12h暗培养有利于愈伤组织诱导；茎段的大小和接种方式以0.7cm长度横放诱导效果最好。 4．糖以分析纯的蔗糖比葡萄糖和市售普通白糖更有利于愈伤组织的诱导和生长。当蔗糖浓度为40mg／L时不仅有利于愈伤组织的增长，也有利于总黄酮的积累。 5．系统研究了B₅培养基中大量元素、微量元素、蔗糖浓度对杜仲愈伤组织生长和次生代谢产物积累的影响。实验证明：增加培养基中KNO₃、（NH₄）₂SO₄、ZnSO₄和FeSO₄的含量有利于愈伤组织生长，而不利于绿原酸和总黄酮的积累；MgSO₄对细胞的生长和绿原酸、总黄酮的积累均起抑制作用；增加培养基中NaH₂PO₄、CaCl₂的浓度不仅有利于愈伤组织的生长也有利于绿原酸、总黄酮的积累。MnSO₄有利于愈伤组织的生长和绿原酸的积累，而显著抑制黄酮的形成。蔗糖对愈伤组织生长和次生代谢产物的积累多有显著的促进作用。更多还原

【Abstract】 The callus induction and subculture of Eucommia ulmoides Oliv. are studied from the aspects of basic media, installing method, type of explant, sampling time, culturing conditions, phytohormones etc. The influence of the condition of tissue culture on the contents of metabolites contained in the callus is also studied preliminarily. The results are as follows:1. Among the 8 media tested, MS is suitable for the callus induction of immature shoots of E. ulmoides Oliv. 65 medium however is suitable not only for callus induction of leaves, but also immature shoots.2. The callus are induced 100 per cent when explants are cultured on their medium supplemented with 0.5 mg/L 2,4-D, 0.5-1.0 mg/L NAA, 1.0 mg/L IBA is the best one for the induction and growth of callus. Combination of 2,4-D and BA or KT is worse than 2,4-D used singly. The combinations of low concentrations of BA and NAA or IBA have better effect, but high concentration of BA is not suitable. The best combination of the media for callus induction and growth is 1.0 mg/L NAA+0.3-0.5 mg/LBA or 1.0 mg/L IBA+0.5 mg/L BA. And calluses grow quickly with glossy appearance under this combination, and suitable for subculture. 0.5 mg/L NAA is the best one for callus subculture. The best combination is 0.5 mg/L NAA+0.5 mg/L BA. Their callus yield was higher obviously than using NAA singly. KT contains callus induction and growth.3. Middle of March is a favorable period for the collection of immature leaves as explant. Early April however is suitable for the collection of shoots. Other times are suitable for hypocotyl , cotyledon and leaf to culture sterile seedlings. The best pH value for the callus induction and subculture is 6.3. 12 hour light and 12 hour dark are benefit for the callus induction ,subculture and the production of secondary metabolites. 0.7 cm shoot explants implanted horizontally in the medium recommended.4. Sucrose as carbon source is better than glucose and refined sugar for callus induction. 40 mg/L sucrose is not only for callus subculture but also for the accumulation of flavonoids.5. Applications of KNO3, (NFL;) 2804, ZnSO4and FeSO4 can increase the growth of callus contains the of accumulation of chlorognic acid and flavonoids. MgSO4 is unprofitable for the callus growth or the accumulation of chlorognic acid and flavonoids. The applications of NaH2P04 and CaCl2 not only increase the callus growth, but also for the accumulation ofchlorognic acid and flavonoids. The applications of MnSO4 not only increase the callus growth and hlorognic acid accumlation, but not beneficial for the accumlation of flavonoids. The applications of sucrose can increase the growth of callus contains the of accumulation of chlorognic acid and flavonoids.更多还原