NaCl和Ce~(3+)对银杏悬浮细胞生长和黄酮代谢的影响
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摘要
银杏是我国传统的药源植物，本研究以春季的银杏幼叶为外植体，通过建立悬浮细胞培养体系，探讨不同浓度的NaCl和稀土铈（Ce~(3+)）对细胞生长生理及黄酮代谢的影响，主要结论如下：
1、以春季的银杏幼叶为外植体，建立无菌培养体系。经过正交设计和叶块生长情况的综合分析，表明以70%酒精30s+0.1%升汞8min的消毒灭菌方案最佳。该灭菌组合处理下叶片的成活率高而污染率、褐化率相对较低，利于后代的继代培养。
2、愈伤组织诱导率最高的组合为：MS+NAA1.0mg/L+KT1.0mg/L；细胞悬浮培养成活率最高的激素组合为：NAA1.0mg/L+KT0.5mg/L。
3、不同浓度NaCl对细胞的生长生理和黄酮含量的影响表明：当NaCl浓度为5~50mmol/L时，利于细胞生长，生物量和蛋白质的积累，含水量高，而膜透性低；100mmol/L时，MDA含量和PAL活性达到最大值，125mmol/L时，脯氨酸含量达到最大值；当NaCl浓度为25和175mmol/L时，黄酮含量分别出现峰值，前者是促进细胞生长，积累生物量的浓度值，后者是形成胁迫，细胞转入次生代谢途径时的浓度值。
4、不同浓度NaCl对细胞生长和黄酮含量动态变化的影响表明：培养基中添加一定浓度的NaCl可以缩短合成黄酮的时间，这种效应以较低浓度NaCl为佳（25、75mmol/L），培养第4d时黄酮合成量明显提高，高浓度NaCl（150mmol/L）处理组在培养第16d时，出现了褐化，总黄酮含量明显高于培养期间其他处理组，可能与此时的细胞受到严重胁迫，次生代谢增强有关。
5、不同浓度Ce~(3+)对细胞的生长生理和黄酮含量的影响表明：当Ce~(3+)浓度为0.5mmol/L时，细胞生长旺盛，活力较强，生物量和可溶性蛋白质含量最高；Ce~(3+)浓度为1mmol/L时，对细胞膜造成一定伤害，细胞生长变慢，刺激了次生代谢的增强，黄酮含量最高。
6、不同浓度Ce~(3+)对细胞生长和黄酮含量动态变化的影响表明：第12d时，对照、0.1、0.5、1mmol/L处理组的细胞干鲜重均达到了最大值，0.5mmol/L处理组在第9d时干鲜重高于其他处理组，第12d时干重高于其他处理组。表明低浓度Ce~(3+)可以显著促进细胞的生长，加速代谢、生物量的积累。黄酮含量的动态分析表明，培养第6d时低浓度（0.1、0.5mmol/L）Ce~(3+)可以显著提高黄酮含量，高浓度（1mmol/L）处理组第12d时含量明显高于其他处理组，可能由于此时细胞受到重度胁迫，次生代谢明显增强。
Ginkgo biloba is the traditional medicinal plant in our country. In this research, cellsuspension culture system was set up and used to study the effects of different concentrations ofNaCl and cerium(Ce~(3+)) on the cell growth physiology and flavonoid metabolism using youngleaves of Ginkgo biloba as experimental materials in the spring. The results are as follows
1) A sterile system was established by using young leaves of Ginkgo biloba as explant inspring, and comprehensive analysis of orthogonal design treatment and leaves blocks growthcondition indicated that70%alcohol30s+0.1%mercuric chloride8min was the best treatmentof disinfection and sterilization for spring ginkgo leaves. Under this treatment leaves blocks hadhigher survival rate, lower disinfection and browning rate relatively, and beneficial to furthersubculture.
2) The best treatment of callus induction was MS+NAA1.0mg/L+KT1.0mg/L, and the besttreatment for high survival rate of cell suspension culture was NAA1.0mg/L+KT0.5mg/L.
3) Effect of different concentrations of NaCl on the growth physiology of cell andflavonoids content indicated that at concentration range of5~50mmol/L, cell grew well,biomass and protein accumulated more quickly, water content was higher and membranepermeability was lowwer; at100mmol/L concentration of NaCl, MDA content and PALactivity reached the maximum;at125mmol/L concentration of NaCl, proline content reachedmaximum. Flavonoids content got peak under25and175mmol/L respectively, the formertreatment concentration was to promote cell growth and accumulate biomass, the latter was theformation of stress, forced cells transfer to secondary metabolic pathways.
4) Dynamic change of cell growth and flavonoids content under different NaCl treatmentindicated that certain concentration of NaCl in the medium could shorten the time offlavonoids synthesis, this effect was better at lower concentration of NaCl (25,75mmol/L);flavonoid synthesis significantly improved at4d; high concentration of NaCl (150mmol/L)treatment turned brown at16d, total flavonoids content was significantly higher than othertreatment, which maybe ralated to the the pathways of secondary metabolism when cellsustained salt stress.
5) Effect of different Ce~(3+)concentration on cell growth,physiology and flavonoid contentindicated that cell growth was exuberant, biomass and soluble protein content got highest at0.5mmol/L concentration of Ce~(3+)treatment; cell membrane caused certain damdge, cell growthslowed down and stimulated secondary metabolites synthesis at1mmol/L concentration of Ce~(3+),thus, got the highest flavonoid content.
6) Dynamic change of cell growth and flavonoids content under different Ce~(3+)treatment indicated that cell’s fresh and dry weight of the control,0.1,0.5,1mmol/L treatment reached amaximum value at12d.Fresh and dry weight of0.5mmol/L treatment was higher than othertreatment at9d, and dry weight at0.5mmol/L concentration of Ce~(3+)was higher than othertreatment at12d, indicated that low concentrations of Ce~(3+)could significantly promote cellgrowth, accelerate metabolism and accumulate biomass. Dynamic analysis of flavonoidsindicated that, low concentrations of Ce~(3+)(0.1,0.5mmol/L)could significantly increaseflavonoids content at6d, the high concentration treatment (1mmol/L) was significantly higherthan other treatments at12d, probably due to the cells subjected to severe stress, enhancedsecondary metabolites.
1、以春季的银杏幼叶为外植体，建立无菌培养体系。经过正交设计和叶块生长情况的综合分析，表明以70%酒精30s+0.1%升汞8min的消毒灭菌方案最佳。该灭菌组合处理下叶片的成活率高而污染率、褐化率相对较低，利于后代的继代培养。
2、愈伤组织诱导率最高的组合为：MS+NAA1.0mg/L+KT1.0mg/L；细胞悬浮培养成活率最高的激素组合为：NAA1.0mg/L+KT0.5mg/L。
3、不同浓度NaCl对细胞的生长生理和黄酮含量的影响表明：当NaCl浓度为5~50mmol/L时，利于细胞生长，生物量和蛋白质的积累，含水量高，而膜透性低；100mmol/L时，MDA含量和PAL活性达到最大值，125mmol/L时，脯氨酸含量达到最大值；当NaCl浓度为25和175mmol/L时，黄酮含量分别出现峰值，前者是促进细胞生长，积累生物量的浓度值，后者是形成胁迫，细胞转入次生代谢途径时的浓度值。
4、不同浓度NaCl对细胞生长和黄酮含量动态变化的影响表明：培养基中添加一定浓度的NaCl可以缩短合成黄酮的时间，这种效应以较低浓度NaCl为佳（25、75mmol/L），培养第4d时黄酮合成量明显提高，高浓度NaCl（150mmol/L）处理组在培养第16d时，出现了褐化，总黄酮含量明显高于培养期间其他处理组，可能与此时的细胞受到严重胁迫，次生代谢增强有关。
5、不同浓度Ce~(3+)对细胞的生长生理和黄酮含量的影响表明：当Ce~(3+)浓度为0.5mmol/L时，细胞生长旺盛，活力较强，生物量和可溶性蛋白质含量最高；Ce~(3+)浓度为1mmol/L时，对细胞膜造成一定伤害，细胞生长变慢，刺激了次生代谢的增强，黄酮含量最高。
6、不同浓度Ce~(3+)对细胞生长和黄酮含量动态变化的影响表明：第12d时，对照、0.1、0.5、1mmol/L处理组的细胞干鲜重均达到了最大值，0.5mmol/L处理组在第9d时干鲜重高于其他处理组，第12d时干重高于其他处理组。表明低浓度Ce~(3+)可以显著促进细胞的生长，加速代谢、生物量的积累。黄酮含量的动态分析表明，培养第6d时低浓度（0.1、0.5mmol/L）Ce~(3+)可以显著提高黄酮含量，高浓度（1mmol/L）处理组第12d时含量明显高于其他处理组，可能由于此时细胞受到重度胁迫，次生代谢明显增强。
Ginkgo biloba is the traditional medicinal plant in our country. In this research, cellsuspension culture system was set up and used to study the effects of different concentrations ofNaCl and cerium(Ce~(3+)) on the cell growth physiology and flavonoid metabolism using youngleaves of Ginkgo biloba as experimental materials in the spring. The results are as follows
1) A sterile system was established by using young leaves of Ginkgo biloba as explant inspring, and comprehensive analysis of orthogonal design treatment and leaves blocks growthcondition indicated that70%alcohol30s+0.1%mercuric chloride8min was the best treatmentof disinfection and sterilization for spring ginkgo leaves. Under this treatment leaves blocks hadhigher survival rate, lower disinfection and browning rate relatively, and beneficial to furthersubculture.
2) The best treatment of callus induction was MS+NAA1.0mg/L+KT1.0mg/L, and the besttreatment for high survival rate of cell suspension culture was NAA1.0mg/L+KT0.5mg/L.
3) Effect of different concentrations of NaCl on the growth physiology of cell andflavonoids content indicated that at concentration range of5~50mmol/L, cell grew well,biomass and protein accumulated more quickly, water content was higher and membranepermeability was lowwer; at100mmol/L concentration of NaCl, MDA content and PALactivity reached the maximum;at125mmol/L concentration of NaCl, proline content reachedmaximum. Flavonoids content got peak under25and175mmol/L respectively, the formertreatment concentration was to promote cell growth and accumulate biomass, the latter was theformation of stress, forced cells transfer to secondary metabolic pathways.
4) Dynamic change of cell growth and flavonoids content under different NaCl treatmentindicated that certain concentration of NaCl in the medium could shorten the time offlavonoids synthesis, this effect was better at lower concentration of NaCl (25,75mmol/L);flavonoid synthesis significantly improved at4d; high concentration of NaCl (150mmol/L)treatment turned brown at16d, total flavonoids content was significantly higher than othertreatment, which maybe ralated to the the pathways of secondary metabolism when cellsustained salt stress.
5) Effect of different Ce~(3+)concentration on cell growth,physiology and flavonoid contentindicated that cell growth was exuberant, biomass and soluble protein content got highest at0.5mmol/L concentration of Ce~(3+)treatment; cell membrane caused certain damdge, cell growthslowed down and stimulated secondary metabolites synthesis at1mmol/L concentration of Ce~(3+),thus, got the highest flavonoid content.
6) Dynamic change of cell growth and flavonoids content under different Ce~(3+)treatment indicated that cell’s fresh and dry weight of the control,0.1,0.5,1mmol/L treatment reached amaximum value at12d.Fresh and dry weight of0.5mmol/L treatment was higher than othertreatment at9d, and dry weight at0.5mmol/L concentration of Ce~(3+)was higher than othertreatment at12d, indicated that low concentrations of Ce~(3+)could significantly promote cellgrowth, accelerate metabolism and accumulate biomass. Dynamic analysis of flavonoidsindicated that, low concentrations of Ce~(3+)(0.1,0.5mmol/L)could significantly increaseflavonoids content at6d, the high concentration treatment (1mmol/L) was significantly higherthan other treatments at12d, probably due to the cells subjected to severe stress, enhancedsecondary metabolites.
引文
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