丹参组织培养的初步研究
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摘要
丹参(Salvia miltiorrhiza)是我国传统的名贵中药。近年来，其新的应用价值不断被发现，需求不断增大，呈现出广阔的市场前景。
本文以丹参初春萌发的幼茎、叶、叶柄为外植体，探讨了不同的外植体、不同的植物生长调节物质、不同的理化因子对愈伤组织的诱导和增殖效应，研究了黑暗和光照条件下愈伤组织细胞培养生长周期以及生长周期中生理生化指标的变化特征，摸索了一套快速、高效、稳定的丹参快速繁殖技术，为丹参组织培养、次生代谢物的生产提供理论依据，为丹参快速繁殖应用于生产提供技术支持。
实验结果如下：
1、愈伤组织的诱导和增殖效应
不同种类、不同浓度的植物生长调节物质、不同理化因子对愈伤组织具有不同的诱导和增殖效应。综合考虑：MS+6-BA_(1.0mg／L)+2,4-D_(0.5mg／L)是诱导愈伤组织的最佳培养基，MS+6-BA_(1.5mg／L)+2，4-D_(0.5mg／L)是最佳的愈伤组织增殖培养基。
葡萄糖的浓度为20-40g／L时愈伤组织增殖效果均较好，蔗糖的浓度为30g／L时，愈伤组织的增殖效应最好；MS是比较适合愈伤组织增殖的基本培养基。
2、愈伤组织生理生化特性的研究
黑暗和光照条件下愈伤组织的生长周期均为27天，生长曲线呈“S”形，具有生长大周期特性，基本可以分为3个时期：延迟期(0-6天)，指数期(6-24天)，静止期(24-27天)。
愈伤组织中可溶性蛋白、POD、SOD的变化与生长周期密切相关，在光照和黑暗条件下培养，有不同的变化曲线。
3、丹参快速繁殖技术的研究
MS+6-BA 1.0mg／L为诱导丛生芽的最佳培养基，MS+6-BA_(1.0mg／L)+NAA_(0.05mg／L)为
蘸
硕士学位论文
八tASTER’S’l’l正515
最佳丛生芽增殖培养基，增殖倍数可达15倍。
MS十6一BA 0.5一2.Omg/L均可较好诱导不定芽，MS+卜BA，.、、。为最佳不定芽
增殖培养基，增殖倍数可达24倍。
月一参幼苗生根较好的培养基为1/ZMS+I BAO.;、L。;活性炭0.1%以下促进生
根，0.1%以上抑制生根。
Salvia miltiorrhiza Bunge is a kind of traditional and rare medicinal herbs. In these years, its new value of application is found unceasingly and its needs are increasing. So it should have widely applied prospect.
This paper reports something about it .The callus was induced and proliferated by using young stem , leaf and leafstalk as explants .which came out in early spring . In this paper , its effects on induction and proliferation were discussed by using different kinds of explants and hormone and different physical and chemical factors.
The growth circle and some physiological and biochemical factors were studied during the culture of callus, a set of rapid propagation techniques were founded, which was rapid, efficient and steady.
All this provided scientific basis for tissue culture and secondary metabolism matters yield, and provided technical support for application of rapid propagation to practical production. The results are as follows:
1. Effects on induction and proliferation of callus
Medium MS+6-BA1. Omg/L+2,4-D0.5mg/L was the optimum medium for callus induction , and MS+6-BA1. 5mg/L+NAA0. 5mg/L was the optimum medium for callus proliferation. The optimum concentration of glucose was 20-40g/L and that of sucrose was 30g/L for callus proliferation.
Medium MS did good to callus proliferation.
2. Study on physiological and biochemical characters
The growth circle of callus was 27 days and the growth curves looked like "S" under the condition cultured in light and dark
The growth circle could be divided in 3 parts: Delay(0-6d), Index (6-24d) and Steady (24-27d).
During the growth circle , the tendency of soluble protein contents , SOD actives and POD actives were different during the 3 parts. They showed some physiology and biochemical characters during callus proliferation and cell actives. It was different between the growth curve of callus cultured in bright and that cultured in dark. 3. Study on rapid propagation technique
Medium MS+6-BA1. Omg/L was the optimum medium for tufted bud induction and medium MS+6-BA1. 0mg/L+NAA0.05mg/L was the optimum medium for tufted bud proliferation with the 24 times breeding. Medium MS+6-BA0.5-2. 0mg/L was the optimum medium for adventitious bud induction and medium MS+6-BA1. Omg/L was the optimum medium for adventitious bud proliferation with the 15 times breeding.
The optimum medium for rooting induction was 1/2 MS +IBA0. lmg/L
本文以丹参初春萌发的幼茎、叶、叶柄为外植体，探讨了不同的外植体、不同的植物生长调节物质、不同的理化因子对愈伤组织的诱导和增殖效应，研究了黑暗和光照条件下愈伤组织细胞培养生长周期以及生长周期中生理生化指标的变化特征，摸索了一套快速、高效、稳定的丹参快速繁殖技术，为丹参组织培养、次生代谢物的生产提供理论依据，为丹参快速繁殖应用于生产提供技术支持。
实验结果如下：
1、愈伤组织的诱导和增殖效应
不同种类、不同浓度的植物生长调节物质、不同理化因子对愈伤组织具有不同的诱导和增殖效应。综合考虑：MS+6-BA_(1.0mg／L)+2,4-D_(0.5mg／L)是诱导愈伤组织的最佳培养基，MS+6-BA_(1.5mg／L)+2，4-D_(0.5mg／L)是最佳的愈伤组织增殖培养基。
葡萄糖的浓度为20-40g／L时愈伤组织增殖效果均较好，蔗糖的浓度为30g／L时，愈伤组织的增殖效应最好；MS是比较适合愈伤组织增殖的基本培养基。
2、愈伤组织生理生化特性的研究
黑暗和光照条件下愈伤组织的生长周期均为27天，生长曲线呈“S”形，具有生长大周期特性，基本可以分为3个时期：延迟期(0-6天)，指数期(6-24天)，静止期(24-27天)。
愈伤组织中可溶性蛋白、POD、SOD的变化与生长周期密切相关，在光照和黑暗条件下培养，有不同的变化曲线。
3、丹参快速繁殖技术的研究
MS+6-BA 1.0mg／L为诱导丛生芽的最佳培养基，MS+6-BA_(1.0mg／L)+NAA_(0.05mg／L)为
蘸
硕士学位论文
八tASTER’S’l’l正515
最佳丛生芽增殖培养基，增殖倍数可达15倍。
MS十6一BA 0.5一2.Omg/L均可较好诱导不定芽，MS+卜BA，.、、。为最佳不定芽
增殖培养基，增殖倍数可达24倍。
月一参幼苗生根较好的培养基为1/ZMS+I BAO.;、L。;活性炭0.1%以下促进生
根，0.1%以上抑制生根。
Salvia miltiorrhiza Bunge is a kind of traditional and rare medicinal herbs. In these years, its new value of application is found unceasingly and its needs are increasing. So it should have widely applied prospect.
This paper reports something about it .The callus was induced and proliferated by using young stem , leaf and leafstalk as explants .which came out in early spring . In this paper , its effects on induction and proliferation were discussed by using different kinds of explants and hormone and different physical and chemical factors.
The growth circle and some physiological and biochemical factors were studied during the culture of callus, a set of rapid propagation techniques were founded, which was rapid, efficient and steady.
All this provided scientific basis for tissue culture and secondary metabolism matters yield, and provided technical support for application of rapid propagation to practical production. The results are as follows:
1. Effects on induction and proliferation of callus
Medium MS+6-BA1. Omg/L+2,4-D0.5mg/L was the optimum medium for callus induction , and MS+6-BA1. 5mg/L+NAA0. 5mg/L was the optimum medium for callus proliferation. The optimum concentration of glucose was 20-40g/L and that of sucrose was 30g/L for callus proliferation.
Medium MS did good to callus proliferation.
2. Study on physiological and biochemical characters
The growth circle of callus was 27 days and the growth curves looked like "S" under the condition cultured in light and dark
The growth circle could be divided in 3 parts: Delay(0-6d), Index (6-24d) and Steady (24-27d).
During the growth circle , the tendency of soluble protein contents , SOD actives and POD actives were different during the 3 parts. They showed some physiology and biochemical characters during callus proliferation and cell actives. It was different between the growth curve of callus cultured in bright and that cultured in dark. 3. Study on rapid propagation technique
Medium MS+6-BA1. Omg/L was the optimum medium for tufted bud induction and medium MS+6-BA1. 0mg/L+NAA0.05mg/L was the optimum medium for tufted bud proliferation with the 24 times breeding. Medium MS+6-BA0.5-2. 0mg/L was the optimum medium for adventitious bud induction and medium MS+6-BA1. Omg/L was the optimum medium for adventitious bud proliferation with the 15 times breeding.
The optimum medium for rooting induction was 1/2 MS +IBA0. lmg/L
引文
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2. Hase K, R Kasimu, P Basnet et al. Preventive effect of lithospermate B from Salvia mitiorrhiza on experimental hepatitis induced by carbon tetrachloride or D-galactosamine/lipopolysaccharide[J]. Planta Med, 1997, 63: 22-26
3. Tanaka T, S Morimoto, G Nonaka et al. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia-preventive effect from Salvia mitiorrhiza[J]. Chem Pharm Bull, 1989, 37: 340-344
4. Arda N, N Goren, A Kuru et al. Saniculoside N from Sanicula europaea L[J]. J Nat Prod 1997, 60: 1170-1173
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