诱导子对霍山石斛悬浮培养原球茎生长和生物碱的影响
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摘要
霍山石斛是安徽省的名贵中草药,国家重点保护的名贵药用植物,为药用石斛珍品;富含石斛多糖和生物碱等多种生物活性成分,具有滋阴清热、益胃生津、抗衰老、提高机体免疫力等功效。由于霍山石斛生境狭窄、繁殖困难、产量低等原因,加之过度采挖利用和生态环境的恶化,野生种质资源严重匮乏。目前,生产上主要是通过快繁技术提供试管苗,但由于试管苗生物碱含量较低,已不能满足市场的需求;而悬浮培养技术可以实现在较短周期内生产生物碱,但细胞生长缓慢、生物碱产量低,始终是其工业化生产的制约因素。
本试验通过60Co-γ射线以及添加诱导子SNP、PA、SA和直流磁场等因素对霍山石斛原球茎进行诱导调控,并对霍山石斛原球茎生长及生物碱积累规律进行研究,以期为大规模悬浮培养生产高含量生物碱提供技术参数,同时也为其合成机理的研究提供理论依据。
主要研究结果如下:
1. 60Co-γ辐射处理霍山石斛原球茎的适宜剂量为10Gy。10Gy处理的原球茎在44d的培养周期中生长曲线近似“S”型,原球茎在36d左右达到生长高峰,为26.54g/瓶,比对照最大生长量提高了22.47%;生物碱在40d左右达到最大值,为0.035g/(DW·flask),比对照提高了81.76%。此时培养液pH和原球茎电导率变化较小,培养液环境适合原球茎继续培养。60Co-γ辐射处理促进了原球茎POD、SOD、CAT和PAL酶活性,抑制PPO酶活性,从而促进石斛原球茎生长和生物碱合成。
2.高浓度的SNP对原球茎生长产生明显抑制作用,但促进了原球茎生物碱的合成积累,适宜浓度为0.1mmol·L-1。0.1mmol·L-1浓度SNP处理后的原球茎,经44d的周期培养,在32d左右达到生长高峰,为14.51g/瓶,低于对照最大生长量;生物碱含量在36d左右达到最大值,为0.0395g/(DW·flask),比对照提高了101.53%。SNP的加入导致培养液酸化,原球茎相对电导率上升,可见,高剂量SNP对原球茎有一定损伤作用,但提高了POD和PAL酶活性,使生物碱含量大幅度提高。
3. PA促进霍山石斛原球茎生长和生物碱的合成,适宜浓度为7.5g·L-1。原球茎鲜重为17.26g/瓶,比对照提高了39.31%。生物碱含量达到0.026g/(DW·flask),比对照提高了83.8%。PA处理促进了PAL酶活性,抑制POD酶活性,防止细胞衰老,从而促进细胞生长和生物碱的合成。
4. SA的较适添加浓度为100μmol·L-1,原球茎鲜重为13.60g/瓶,比对照降低了17.18%;虽然原球茎生长量有所下降,但生物碱含量达到最高,0.022g/(DW·flask),为对照的1.62倍。SA的加入导致培养液酸化,原球茎相对电导率上升,使得培养液环境对原球茎继续培养有一定影响。POD和PAL酶活性得到显著提高,促进了生物碱的积累。
5.磁场作用下,霍山石斛原球茎生长量显著提高,但对生物碱积累影响不大。磁场与SA协同作用下,鲜重量达到8.65g/瓶,比对照提高了41.57%;生物碱的最大产量为0.0135g/(DW·flask),仅为SA单独处理组的88.82%,表明磁场和SA共同作用并没有增加生物碱的积累。原球茎POD和PAL酶活性有所降低,磁场处理使原球茎次生代谢表达强度有所减弱。
6.通过对霍山石斛原球茎进行石蜡切片染色观察,发现原球茎中生物碱主要分布在细胞质中。
It is one of the most famous and valuable Chinese herbal medicine in Anhui Province of China, and it is regarded and protected for its value by government. It has the advantage of diminishing inflammation, resisting senescence and improving immunity because of its content of amylose and alkaloid. But the wild resources of D. Huoshanense are reducing as the reason of the poor living conditions, low productivity, overexploitation and so on. At present, the main breeding method using in D. Huoshanense is vitro propagation technology, but that cannot satisfy with the needs of market because of the low content of alkaloid in plants. However, the liquid culture can produce alkaloid in a short period with slow development of cells except for low output, that is the very reason for restricting industrial produce.
The experiments were used 60Co-γ、SNP、PA、SA and direct current magnetic field on D.huoshanense protocorm like-bodies(PLB) to research the living situation of PLB and the accumulating of alkaloids, so that we can provide technical parameters for establishing large-scale liquid culture and producing higher content alkaloids, and finally provide theoretical basis for the synthesis of alkaloids.
The main results were following:
1. The optimum irradiation dose for PLBs was 10Gy, the growth curve was similar as“S”type in the 44days subculture period. And the biomass reached peak at the 36th day, which was 26.54g/DW·flask, 22.47% higher than the CK; the alkaloids quantity reached peak at the 40th day, which was 0.035g/DW·flask, 81.76% higher than the CK. The pH of culture and the electrical conductivity changed slightly, the medium environment fit PLBs to be cultured continuously. 60Co-γirradiation could increase the activities of POD, SOD, CAT, PAL and decrease the activities of PPO to improve cell growth and the accumulation of alkaloid of PLBs.
2. High concentration of SNP decreased the biomass of PLBs, but enhanced alkaloids production. The optimum concentration was 0.1mmol·L-1, the biomass reached peak at the 32th day in the 44 days subculture period, which was 14.51g/DW·flask, lower than the CK; the alkaloids production reached peak at the 36th day, which was 0.0395 g/DW·flask, 101.53% higher than the CK. By adding SNP could cause the medium acidification and increase the relative conductivity of PLBs. So, high concentration of SNP hurt PLBs but could increase the activities of POD、PAL to improve the accumulation of alkaloid of PLBs.
3. PA could improve the biomass and alkaloid content of suspension-cultured D.huoshanense PLBs, 7.5g·L-1 was the optimum concentration, the fresh weight was 17.26g/flask, 39.31% higher than the CK; the alkaloid content was 0.026g/DW·flask, 83.8% higher than the CK. PA could increase the activities of PAL and decrease the activities of POD to improve cell growth and the accumulation of alkaloid of PLBs.
4. The optimum concentration of SA for PLBs was 100μmol·L-1, the fresh weight was 13.60g/flask, 17.18% lower than the CK; the alkaloid content was 0.022g/DW·flask, 1.62-fold higher than the CK. By adding SA could cause the medium acidification, and increase the relative conductivity of PLBs, but increase the activities of POD、PAL to improve the accumulation of alkaloid of PLBs.
5. With the direct current magnetic field on D.huoshanense PLBs, the biomass was obviously enhanced. Under the condition of magnetic field and SA on PLBs, the fresh weight was 8.65g/flask, 41.57% higher than the CK; the alkaloid content was 0.0135g/DW·flask, 11.18% lower than the CK. The results showed that Magnetic field associated with SA could not increase the accumulation of alkaloid, decrease the activities of POD and PAL and the secondary metabolites of PLBs.
6. Through observing of PLBs by paraffin slice technique, we found that alkaloid of PLBs mainly distribute in the cytoplasm.
本试验通过60Co-γ射线以及添加诱导子SNP、PA、SA和直流磁场等因素对霍山石斛原球茎进行诱导调控,并对霍山石斛原球茎生长及生物碱积累规律进行研究,以期为大规模悬浮培养生产高含量生物碱提供技术参数,同时也为其合成机理的研究提供理论依据。
主要研究结果如下:
1. 60Co-γ辐射处理霍山石斛原球茎的适宜剂量为10Gy。10Gy处理的原球茎在44d的培养周期中生长曲线近似“S”型,原球茎在36d左右达到生长高峰,为26.54g/瓶,比对照最大生长量提高了22.47%;生物碱在40d左右达到最大值,为0.035g/(DW·flask),比对照提高了81.76%。此时培养液pH和原球茎电导率变化较小,培养液环境适合原球茎继续培养。60Co-γ辐射处理促进了原球茎POD、SOD、CAT和PAL酶活性,抑制PPO酶活性,从而促进石斛原球茎生长和生物碱合成。
2.高浓度的SNP对原球茎生长产生明显抑制作用,但促进了原球茎生物碱的合成积累,适宜浓度为0.1mmol·L-1。0.1mmol·L-1浓度SNP处理后的原球茎,经44d的周期培养,在32d左右达到生长高峰,为14.51g/瓶,低于对照最大生长量;生物碱含量在36d左右达到最大值,为0.0395g/(DW·flask),比对照提高了101.53%。SNP的加入导致培养液酸化,原球茎相对电导率上升,可见,高剂量SNP对原球茎有一定损伤作用,但提高了POD和PAL酶活性,使生物碱含量大幅度提高。
3. PA促进霍山石斛原球茎生长和生物碱的合成,适宜浓度为7.5g·L-1。原球茎鲜重为17.26g/瓶,比对照提高了39.31%。生物碱含量达到0.026g/(DW·flask),比对照提高了83.8%。PA处理促进了PAL酶活性,抑制POD酶活性,防止细胞衰老,从而促进细胞生长和生物碱的合成。
4. SA的较适添加浓度为100μmol·L-1,原球茎鲜重为13.60g/瓶,比对照降低了17.18%;虽然原球茎生长量有所下降,但生物碱含量达到最高,0.022g/(DW·flask),为对照的1.62倍。SA的加入导致培养液酸化,原球茎相对电导率上升,使得培养液环境对原球茎继续培养有一定影响。POD和PAL酶活性得到显著提高,促进了生物碱的积累。
5.磁场作用下,霍山石斛原球茎生长量显著提高,但对生物碱积累影响不大。磁场与SA协同作用下,鲜重量达到8.65g/瓶,比对照提高了41.57%;生物碱的最大产量为0.0135g/(DW·flask),仅为SA单独处理组的88.82%,表明磁场和SA共同作用并没有增加生物碱的积累。原球茎POD和PAL酶活性有所降低,磁场处理使原球茎次生代谢表达强度有所减弱。
6.通过对霍山石斛原球茎进行石蜡切片染色观察,发现原球茎中生物碱主要分布在细胞质中。
It is one of the most famous and valuable Chinese herbal medicine in Anhui Province of China, and it is regarded and protected for its value by government. It has the advantage of diminishing inflammation, resisting senescence and improving immunity because of its content of amylose and alkaloid. But the wild resources of D. Huoshanense are reducing as the reason of the poor living conditions, low productivity, overexploitation and so on. At present, the main breeding method using in D. Huoshanense is vitro propagation technology, but that cannot satisfy with the needs of market because of the low content of alkaloid in plants. However, the liquid culture can produce alkaloid in a short period with slow development of cells except for low output, that is the very reason for restricting industrial produce.
The experiments were used 60Co-γ、SNP、PA、SA and direct current magnetic field on D.huoshanense protocorm like-bodies(PLB) to research the living situation of PLB and the accumulating of alkaloids, so that we can provide technical parameters for establishing large-scale liquid culture and producing higher content alkaloids, and finally provide theoretical basis for the synthesis of alkaloids.
The main results were following:
1. The optimum irradiation dose for PLBs was 10Gy, the growth curve was similar as“S”type in the 44days subculture period. And the biomass reached peak at the 36th day, which was 26.54g/DW·flask, 22.47% higher than the CK; the alkaloids quantity reached peak at the 40th day, which was 0.035g/DW·flask, 81.76% higher than the CK. The pH of culture and the electrical conductivity changed slightly, the medium environment fit PLBs to be cultured continuously. 60Co-γirradiation could increase the activities of POD, SOD, CAT, PAL and decrease the activities of PPO to improve cell growth and the accumulation of alkaloid of PLBs.
2. High concentration of SNP decreased the biomass of PLBs, but enhanced alkaloids production. The optimum concentration was 0.1mmol·L-1, the biomass reached peak at the 32th day in the 44 days subculture period, which was 14.51g/DW·flask, lower than the CK; the alkaloids production reached peak at the 36th day, which was 0.0395 g/DW·flask, 101.53% higher than the CK. By adding SNP could cause the medium acidification and increase the relative conductivity of PLBs. So, high concentration of SNP hurt PLBs but could increase the activities of POD、PAL to improve the accumulation of alkaloid of PLBs.
3. PA could improve the biomass and alkaloid content of suspension-cultured D.huoshanense PLBs, 7.5g·L-1 was the optimum concentration, the fresh weight was 17.26g/flask, 39.31% higher than the CK; the alkaloid content was 0.026g/DW·flask, 83.8% higher than the CK. PA could increase the activities of PAL and decrease the activities of POD to improve cell growth and the accumulation of alkaloid of PLBs.
4. The optimum concentration of SA for PLBs was 100μmol·L-1, the fresh weight was 13.60g/flask, 17.18% lower than the CK; the alkaloid content was 0.022g/DW·flask, 1.62-fold higher than the CK. By adding SA could cause the medium acidification, and increase the relative conductivity of PLBs, but increase the activities of POD、PAL to improve the accumulation of alkaloid of PLBs.
5. With the direct current magnetic field on D.huoshanense PLBs, the biomass was obviously enhanced. Under the condition of magnetic field and SA on PLBs, the fresh weight was 8.65g/flask, 41.57% higher than the CK; the alkaloid content was 0.0135g/DW·flask, 11.18% lower than the CK. The results showed that Magnetic field associated with SA could not increase the accumulation of alkaloid, decrease the activities of POD and PAL and the secondary metabolites of PLBs.
6. Through observing of PLBs by paraffin slice technique, we found that alkaloid of PLBs mainly distribute in the cytoplasm.
引文
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