豫产道地中药材金银花离体培养技术研究
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摘要
金银花(Lonicera japonica Thunb.)是忍冬科忍冬属多年生木质藤本,其干燥花蕾和初开的花是临床上常用的药物,主要药用成分是绿原酸和异绿原酸,具有抑菌消炎、抗病毒、抗肿瘤、解热、保肝、止血、降脂、抗生育、抗氧化、免疫调节等多种功效。主产于河南省封丘县的金银花是我国著名的道地中药材,但在生产中,其品种单一、退化严重且繁殖速度较慢,无法满足大田规模化种植和市场的需求。针对以上问题,我们首次对金银花中的优良品种“金丰一号”的离体培养进行了系统的研究。取得了如下研究成果:
1.建立了无菌培养技术体系。将金银花顶芽剥离成两叶一心后,在75 %酒精中灭菌30 s,再用0.1 %升汞处理15 min,然后无菌水冲洗5-6次,无菌苗得率达78.90 %,接种于MS+6-BA 1.0 mg·L~(-1) +NAA 0.2 mg·L~(-1)培养基上进行培养,30 d即可获得无菌试管苗。
2.建立了快繁技术体系。适于试管苗快繁的培养基是MS+6-BA 0.5 mg·L~(-1)+NAA 0.1 mg·L~(-1)+生物素-D 2 mg·L~(-1),繁殖周期为28 d,繁殖系数达到7.37;适于生根的培养基为1/2MS+IBA3 mg·L~(-1)+0.2 g·L~(-1)活性炭+15 g·L~(-1)蔗糖,配以合适的栽后管理,移栽成活率达100 %。
3.探讨了愈伤组织诱导与继代的最佳条件。最佳外植体是花蕾,其次是叶片,最佳培养基是MS+NAA 2 mg·L~(-1)+6-BA 0.01 mg·L~(-1)+2,4-D 0.5 mg·L~(-1),诱导率达100 %;适宜的继代培养基是MS+6-BA 2 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.25 mg·L~(-1),在此继代培养基上,经过3-5次继代后可以得到四种不同类型的愈伤组织。
4.建立了细胞悬浮培养技术体系。选择黄白色、小颗粒、结构疏松、生长迅速的愈伤组织转入MS+6-BA 1.5 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.5 mg·L~(-1)的液体培养基中,建立细胞悬浮培养体系。在该体系中细胞生长曲线呈“S”型,可分为延迟期(0~(-1)2 d)、指数生长期(12~(-1)8 d)和缓慢生长期(18-24 d)三个阶段,培养过程中细胞干重呈现上升趋势,并在18 d时达到最大,为8 g·L~(-1)。
5.研究了细胞悬浮培养过程中生理生化变化规律。(1)可溶性糖含量在培养过程中先下降后上升,3~(-1)8 d时,可溶性糖含量迅速上升,并于18 d时达到峰值;(2)可溶性蛋白质含量呈降升降趋势,0-6 d时缓慢下降,6-9 d时明显增长,9~(-1)5 d时相对含量变化不大,于12 d时可溶性蛋白质含量达到峰值,比细胞生长高峰提前6 d,之后呈下降趋势;(3)POD活性大致呈先升后降的趋势,0-6 d时先升后降,3 d时是一小高峰,6~(-1)2 d时伴随着细胞旺盛生长,开始迅速上升并在12 d时达到高峰,之后持续下降;(4)SOD活性呈先升后降的趋势,0-9 d时缓慢增长,9~(-1)2 d时迅速升高,12 d时达到峰值,之后呈持续下降趋势;(5)悬浮培养液的pH值先下降、后略微上升、再渐趋于平缓,维持在5.14-4.89之间;(6)悬浮培养液的电导率呈先缓慢上升后急剧下降再稍上升的趋势,0~(-1)2 d时,培养液的电导率一直呈极缓慢上升趋势,在12 d时升至最高点(0.53×104μs·cm~(-1)),随即呈下降趋势,与细胞生长呈现一定的相关性;(7)培养周期中绿原酸含量呈升降升降的趋势,0-6 d,绿原酸呈急剧上升趋势,在第6 d时升至最高点,随即呈下降趋势,15-21 d时再次呈上升趋势,直至在第21 d时达到另一峰值,只是比第一峰值(6 d时)低,21 d之后绿原酸含量又急剧下降。
6.初步研究了金银花染色体制片技术。以根尖为材料,用0.1 %的秋水仙素室温下避光预处理4 h,在1 mol·L~(-1)盐酸65℃恒温水浴中解离3 min,改良卡宝品红染液室温下染色4 h后,能取得良好的制片效果。
本文的研究结果,为进一步通过植物生物技术进行新品种的培育、良种的离体快繁以及有效药用成分的工厂化生产奠定了技术基础。
Lonicera japonica Thunb. is one of perennial woody vine and belonging to Lonicera genus, Caprifoliaceae family, its main medicinal ingredients are chlorogenic acid and isochlorogenic acid, which have effects on anti-bacterium, anti-inflammation, anti-virus, anti-tumor, immunoregulation and so on. Lonicera japonica Thunb. is a famous authentic Chinese herbal medicine, which is major in Fengqiu city of Henan province. Because of the single species, serious degradation and the low speed of propagation, there is no ways to satisfy the need of large scale planting and market. In order to solve the above problems, we studied in vitro culture of improved variety“Jin Feng No.1”for the first time systemicly. The results were as followed:
1. The technological system of asepsis culture had been established. Firstly, to dip the apical buds with two leaves from farmland into the ethanol of 75 % for 30 s, secondly, to put them in the HgCl2 of 0.1 % for 15 minutes, thirdly, to wash them in the germfree water for five to six, finally, to inoculate them on MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1). According to these steps, we could get plantlets after they were cultured for 30 days, and the yield of vaccine is up to 78.90 %.
2. The techology system of rapid propagation had been established. The best medium for rapid propagation of Lonicerajaponica Thunb.is MS+6-BA 0.5 mg·L~(-1)+NAA 0.1 mg·L~(-1)+biotin-D 2 mg·L~(-1) with agar in can bottle of 500 ml, and the propagation coefficient could reach 7.37 by 28 days. The best rooting medium was 1/2 MS+IBA 3 mg·L~(-1)+ AC 0.2 g·L~(-1)+ sugar 15 g·L~(-1), and with reasonable managements the survival rate of transplantation could reach to 100 %.
3. The callus induction and the best conditions of their subculture had been probed into. The best explants were buds, secondly were leaves, and the best induction medium was MS+NAA 2 mg·L~(-1)+6-BA 0.01 mg·L~(-1)+2,4-D 0.5 mg·L~(-1), the inducing ratio could reach 100 %; The best subculture medium for callus is MS+6-BA 2 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.25 mg·L~(-1), in which the callus could be classified into four types after subcultured for three to five generations.
4. The techology system of cell suspension culture had been established. TypeⅠwhich is yellowy, loosen grain and rapid growth was used to establish the suspension culture system in the liquid medium MS+6-BA 1.5 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.5 mg·L~(-1): The S - like growth curve of suspension culturing cell was divided into three phases: lag phase (0~(-1)2 d ), exponential phase (12~(-1)8 d ) and lentamente phase (18-24 d ), cell dry weight was upward trend during the incubation period and it reached a maximum of 8 g·L~(-1) at 18 days.
5. The rules of physiology and biochemistry had been researched during cell suspension culture. (1) During the incubation period the content of soluble sugar declined, raised, then declined, raised, the period of reaching peak was 18 days, it was the same as that of growth cruve doing; (2) The content of soluble protein declined firstly, then raised, and at last declined. Soluble proteins contents reached its peak value at 12 days, 6 days earlier than that of the cell growth peak; (3) POD and SOD activities all increased firstly, and then decreased. 6 days earlier than that of the cell growth peak; (4) pH of the medium dropped firstly and then raise slightly, and at last gradually leveled off and maintained about 5.14 after being cultured for 12 days; (5) Conductivity of the medium presented a very slow upward trend, then rapidly declined and at last raised slightly, it presented certain relativity with cell growth; (6) The content of chlorogenic acid had change of two peaks, that is, raised firstly, then declined, raised followly and at last declined. The period of two peaks was 6 days, 21 days respectively.
6. Lonicera japonica Thunb. chromosome preparation technology has been primary Studied: the root was used for the material, at the room temperature, they were treated with 0.1 % colchicine for 4 h in the dark, then, they were dissociated for 3 min under the conditions of 65℃in a water bath with 1 mol·L~(-1) HCl, at last, they were stained for 4 h in Carbol fuchsine, and the effect was better.
The results of this paper were with a view to further carry out new varieties generalizing and rapid propagation in vitro through biotechnology and industrial production based of the effective officinal ingredients.
1.建立了无菌培养技术体系。将金银花顶芽剥离成两叶一心后,在75 %酒精中灭菌30 s,再用0.1 %升汞处理15 min,然后无菌水冲洗5-6次,无菌苗得率达78.90 %,接种于MS+6-BA 1.0 mg·L~(-1) +NAA 0.2 mg·L~(-1)培养基上进行培养,30 d即可获得无菌试管苗。
2.建立了快繁技术体系。适于试管苗快繁的培养基是MS+6-BA 0.5 mg·L~(-1)+NAA 0.1 mg·L~(-1)+生物素-D 2 mg·L~(-1),繁殖周期为28 d,繁殖系数达到7.37;适于生根的培养基为1/2MS+IBA3 mg·L~(-1)+0.2 g·L~(-1)活性炭+15 g·L~(-1)蔗糖,配以合适的栽后管理,移栽成活率达100 %。
3.探讨了愈伤组织诱导与继代的最佳条件。最佳外植体是花蕾,其次是叶片,最佳培养基是MS+NAA 2 mg·L~(-1)+6-BA 0.01 mg·L~(-1)+2,4-D 0.5 mg·L~(-1),诱导率达100 %;适宜的继代培养基是MS+6-BA 2 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.25 mg·L~(-1),在此继代培养基上,经过3-5次继代后可以得到四种不同类型的愈伤组织。
4.建立了细胞悬浮培养技术体系。选择黄白色、小颗粒、结构疏松、生长迅速的愈伤组织转入MS+6-BA 1.5 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.5 mg·L~(-1)的液体培养基中,建立细胞悬浮培养体系。在该体系中细胞生长曲线呈“S”型,可分为延迟期(0~(-1)2 d)、指数生长期(12~(-1)8 d)和缓慢生长期(18-24 d)三个阶段,培养过程中细胞干重呈现上升趋势,并在18 d时达到最大,为8 g·L~(-1)。
5.研究了细胞悬浮培养过程中生理生化变化规律。(1)可溶性糖含量在培养过程中先下降后上升,3~(-1)8 d时,可溶性糖含量迅速上升,并于18 d时达到峰值;(2)可溶性蛋白质含量呈降升降趋势,0-6 d时缓慢下降,6-9 d时明显增长,9~(-1)5 d时相对含量变化不大,于12 d时可溶性蛋白质含量达到峰值,比细胞生长高峰提前6 d,之后呈下降趋势;(3)POD活性大致呈先升后降的趋势,0-6 d时先升后降,3 d时是一小高峰,6~(-1)2 d时伴随着细胞旺盛生长,开始迅速上升并在12 d时达到高峰,之后持续下降;(4)SOD活性呈先升后降的趋势,0-9 d时缓慢增长,9~(-1)2 d时迅速升高,12 d时达到峰值,之后呈持续下降趋势;(5)悬浮培养液的pH值先下降、后略微上升、再渐趋于平缓,维持在5.14-4.89之间;(6)悬浮培养液的电导率呈先缓慢上升后急剧下降再稍上升的趋势,0~(-1)2 d时,培养液的电导率一直呈极缓慢上升趋势,在12 d时升至最高点(0.53×104μs·cm~(-1)),随即呈下降趋势,与细胞生长呈现一定的相关性;(7)培养周期中绿原酸含量呈升降升降的趋势,0-6 d,绿原酸呈急剧上升趋势,在第6 d时升至最高点,随即呈下降趋势,15-21 d时再次呈上升趋势,直至在第21 d时达到另一峰值,只是比第一峰值(6 d时)低,21 d之后绿原酸含量又急剧下降。
6.初步研究了金银花染色体制片技术。以根尖为材料,用0.1 %的秋水仙素室温下避光预处理4 h,在1 mol·L~(-1)盐酸65℃恒温水浴中解离3 min,改良卡宝品红染液室温下染色4 h后,能取得良好的制片效果。
本文的研究结果,为进一步通过植物生物技术进行新品种的培育、良种的离体快繁以及有效药用成分的工厂化生产奠定了技术基础。
Lonicera japonica Thunb. is one of perennial woody vine and belonging to Lonicera genus, Caprifoliaceae family, its main medicinal ingredients are chlorogenic acid and isochlorogenic acid, which have effects on anti-bacterium, anti-inflammation, anti-virus, anti-tumor, immunoregulation and so on. Lonicera japonica Thunb. is a famous authentic Chinese herbal medicine, which is major in Fengqiu city of Henan province. Because of the single species, serious degradation and the low speed of propagation, there is no ways to satisfy the need of large scale planting and market. In order to solve the above problems, we studied in vitro culture of improved variety“Jin Feng No.1”for the first time systemicly. The results were as followed:
1. The technological system of asepsis culture had been established. Firstly, to dip the apical buds with two leaves from farmland into the ethanol of 75 % for 30 s, secondly, to put them in the HgCl2 of 0.1 % for 15 minutes, thirdly, to wash them in the germfree water for five to six, finally, to inoculate them on MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1). According to these steps, we could get plantlets after they were cultured for 30 days, and the yield of vaccine is up to 78.90 %.
2. The techology system of rapid propagation had been established. The best medium for rapid propagation of Lonicerajaponica Thunb.is MS+6-BA 0.5 mg·L~(-1)+NAA 0.1 mg·L~(-1)+biotin-D 2 mg·L~(-1) with agar in can bottle of 500 ml, and the propagation coefficient could reach 7.37 by 28 days. The best rooting medium was 1/2 MS+IBA 3 mg·L~(-1)+ AC 0.2 g·L~(-1)+ sugar 15 g·L~(-1), and with reasonable managements the survival rate of transplantation could reach to 100 %.
3. The callus induction and the best conditions of their subculture had been probed into. The best explants were buds, secondly were leaves, and the best induction medium was MS+NAA 2 mg·L~(-1)+6-BA 0.01 mg·L~(-1)+2,4-D 0.5 mg·L~(-1), the inducing ratio could reach 100 %; The best subculture medium for callus is MS+6-BA 2 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.25 mg·L~(-1), in which the callus could be classified into four types after subcultured for three to five generations.
4. The techology system of cell suspension culture had been established. TypeⅠwhich is yellowy, loosen grain and rapid growth was used to establish the suspension culture system in the liquid medium MS+6-BA 1.5 mg·L~(-1)+KT 0.75 mg·L~(-1)+NAA 0.5 mg·L~(-1): The S - like growth curve of suspension culturing cell was divided into three phases: lag phase (0~(-1)2 d ), exponential phase (12~(-1)8 d ) and lentamente phase (18-24 d ), cell dry weight was upward trend during the incubation period and it reached a maximum of 8 g·L~(-1) at 18 days.
5. The rules of physiology and biochemistry had been researched during cell suspension culture. (1) During the incubation period the content of soluble sugar declined, raised, then declined, raised, the period of reaching peak was 18 days, it was the same as that of growth cruve doing; (2) The content of soluble protein declined firstly, then raised, and at last declined. Soluble proteins contents reached its peak value at 12 days, 6 days earlier than that of the cell growth peak; (3) POD and SOD activities all increased firstly, and then decreased. 6 days earlier than that of the cell growth peak; (4) pH of the medium dropped firstly and then raise slightly, and at last gradually leveled off and maintained about 5.14 after being cultured for 12 days; (5) Conductivity of the medium presented a very slow upward trend, then rapidly declined and at last raised slightly, it presented certain relativity with cell growth; (6) The content of chlorogenic acid had change of two peaks, that is, raised firstly, then declined, raised followly and at last declined. The period of two peaks was 6 days, 21 days respectively.
6. Lonicera japonica Thunb. chromosome preparation technology has been primary Studied: the root was used for the material, at the room temperature, they were treated with 0.1 % colchicine for 4 h in the dark, then, they were dissociated for 3 min under the conditions of 65℃in a water bath with 1 mol·L~(-1) HCl, at last, they were stained for 4 h in Carbol fuchsine, and the effect was better.
The results of this paper were with a view to further carry out new varieties generalizing and rapid propagation in vitro through biotechnology and industrial production based of the effective officinal ingredients.
引文
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