西藏卷丹地下鳞茎的组织培养和悬浮体系的建立
摘要
本论文主要探讨了西藏卷丹(Lilium lancifolium Thunb．)组织培养技术。实验以西藏卷丹的地下鳞茎作为外植体，一方面诱导芽点生根成苗，另一方面诱导形成愈伤组织。本研究为西藏卷丹的引种驯化和大规模工厂化育苗，以及对其药用成分研究打好了一个坚实的基础。通过实验得出以下结果：
1、鳞茎初代培养的最佳配方是：MS+NAA1.5mg／L+BA0.5mg／L，鳞片的分化能力依次为中层＞内层＞外层，鳞片下部形成小鳞茎能力最强，中部其次，上部最少；将鳞片背面紧贴培养基的接种方式比以基部斜插入培养基和腹面放入效果好，其诱导率高；而以腹面放入的方式诱导不出小鳞茎。
2、继代培养中的最佳配方是：MS+NAA2.0mg／L+BA1.0mg／L，平均增殖系数达到为8.2，且小鳞茎生长较强壮，糖的浓度以20g／L为最利于鳞茎的增殖。
3、小鳞茎生根的最佳实验组合为为MS+IBA1.0mg／L+活性炭1.0g／L，生根率最高，达到98.0％，且根生长粗壮。卷丹组培移栽以蛭石为最好，成活率最高，可以达到93.3％，移栽苗生长良好，叶片伸展。
4、当BA=1.5mg／L，NAA=0.5mg／L时愈伤诱导率最高，达到91％，暗环境下更有利于愈伤组织的形成；实验中还观察到，光培养下愈伤浅绿色，较硬，暗环境下的愈伤组织呈淡黄绿色，松散。
5、当BA=1.0mg／L，NAA=0.5mg／L时，愈伤增殖最快，愈伤组织淡黄绿色，疏松，含糖为20g／L的培养基中愈伤生长最快，加入0.5g／L活性炭利于愈伤组织的增殖，且有效抑制褐化现象。
6、培养基为MS+NAA1.5mg／L+BA0.1mg／L时，愈伤分化率较高，且芽生长情况最好。
7、pH5.8-6.2的MS+BA0.8mg／L+NAA0.3mg／L+20g／L蔗糖的液体培养基中愈伤组织增殖最快，颗粒大，结构疏松，颜色黄绿色。
The present study was carried out to establish a plant regeneration system of Lilium lancifolium and underground bulbs were selected as the explants. On one hand, the budding spots was induced from explants and then transferred into rooting media to induce plantlets. On the other hand, the explants was transferred onto media to induce calli. Results of the study would provide fundamental supports to induction, domestication and culture of seedlings at a factorial scale, and to studies on medicinal ingredients of Lilium lancifolium.
Results show that the best formula for primary culture of the bulbs was MS+ NAA1.5mg/L+BA0.5mg/L, that for regenerating culture was MS+NAA2.0 mg/L+ BA1.0 mg/L, and that for rooting culture was MS + IBA1.0 mg/L + AC 1.0 g/L. Upon transplantation of the in vitro plant seedlings, the survival rate in vermiculites was 93.3%, much higher than that transplanted in sandy soils.
It is found that the best formula for callus induced from the bulbs was MS+BA 1.5 mg/L+NAA 0.5 mg/L. The best formula for callus multiplication was MS+BA 1.0 mg/L + NAA 0.5 mg/L. At the same time, the results indicated that dark condition was favorable to the growth of calli and change the content of sugar in the medium had obvious influence on its growth. Supplemented with active carbon of 0.5 g/L can prevent serious brown. Rates of bud formation from well-grown calli was the highest in medium with a formula of MS + NAA1.5 mg/L+BA0.1 mg/L. Yields of growth of suspension culture cells were the highest with no serious brown when medium was MS +BA0.8mg/L+NAA0.3mg/L, adding sucrose of 20 g/L, pH5.8-6.2 was better than in the original medium.
1、鳞茎初代培养的最佳配方是：MS+NAA1.5mg／L+BA0.5mg／L，鳞片的分化能力依次为中层＞内层＞外层，鳞片下部形成小鳞茎能力最强，中部其次，上部最少；将鳞片背面紧贴培养基的接种方式比以基部斜插入培养基和腹面放入效果好，其诱导率高；而以腹面放入的方式诱导不出小鳞茎。
2、继代培养中的最佳配方是：MS+NAA2.0mg／L+BA1.0mg／L，平均增殖系数达到为8.2，且小鳞茎生长较强壮，糖的浓度以20g／L为最利于鳞茎的增殖。
3、小鳞茎生根的最佳实验组合为为MS+IBA1.0mg／L+活性炭1.0g／L，生根率最高，达到98.0％，且根生长粗壮。卷丹组培移栽以蛭石为最好，成活率最高，可以达到93.3％，移栽苗生长良好，叶片伸展。
4、当BA=1.5mg／L，NAA=0.5mg／L时愈伤诱导率最高，达到91％，暗环境下更有利于愈伤组织的形成；实验中还观察到，光培养下愈伤浅绿色，较硬，暗环境下的愈伤组织呈淡黄绿色，松散。
5、当BA=1.0mg／L，NAA=0.5mg／L时，愈伤增殖最快，愈伤组织淡黄绿色，疏松，含糖为20g／L的培养基中愈伤生长最快，加入0.5g／L活性炭利于愈伤组织的增殖，且有效抑制褐化现象。
6、培养基为MS+NAA1.5mg／L+BA0.1mg／L时，愈伤分化率较高，且芽生长情况最好。
7、pH5.8-6.2的MS+BA0.8mg／L+NAA0.3mg／L+20g／L蔗糖的液体培养基中愈伤组织增殖最快，颗粒大，结构疏松，颜色黄绿色。
The present study was carried out to establish a plant regeneration system of Lilium lancifolium and underground bulbs were selected as the explants. On one hand, the budding spots was induced from explants and then transferred into rooting media to induce plantlets. On the other hand, the explants was transferred onto media to induce calli. Results of the study would provide fundamental supports to induction, domestication and culture of seedlings at a factorial scale, and to studies on medicinal ingredients of Lilium lancifolium.
Results show that the best formula for primary culture of the bulbs was MS+ NAA1.5mg/L+BA0.5mg/L, that for regenerating culture was MS+NAA2.0 mg/L+ BA1.0 mg/L, and that for rooting culture was MS + IBA1.0 mg/L + AC 1.0 g/L. Upon transplantation of the in vitro plant seedlings, the survival rate in vermiculites was 93.3%, much higher than that transplanted in sandy soils.
It is found that the best formula for callus induced from the bulbs was MS+BA 1.5 mg/L+NAA 0.5 mg/L. The best formula for callus multiplication was MS+BA 1.0 mg/L + NAA 0.5 mg/L. At the same time, the results indicated that dark condition was favorable to the growth of calli and change the content of sugar in the medium had obvious influence on its growth. Supplemented with active carbon of 0.5 g/L can prevent serious brown. Rates of bud formation from well-grown calli was the highest in medium with a formula of MS + NAA1.5 mg/L+BA0.1 mg/L. Yields of growth of suspension culture cells were the highest with no serious brown when medium was MS +BA0.8mg/L+NAA0.3mg/L, adding sucrose of 20 g/L, pH5.8-6.2 was better than in the original medium.
引文
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[2] 丁兰．刘国安，田卫东．等．新铁炮百合组织培养和快速繁殖研究[J]．西北师范大学学报，2001．37(1)：80-82．
[3] 董志渊，郑思乡．百合的组织培养及在其育种中的应用．西部林业科学[J]，2004．33(2)：64-68．
[4] 傅玉兰，何风群．影响百合试管鳞茎增殖因素的研究[J]．安徽农业大学学报．2001，28(2)：179-181．
[5] 樊君．伊贝母细胞悬浮培养的研究[J]．西北大学学报(自然科学版)，1995，25(6)：40．
[6] 黄敏玲，陈诗林．透百合离体快速繁殖[J]．植物生理学通讯，1993，29(6)：437．
[7] 黄惠英．东方百合的离体培养[J]．甘肃农业大学学报．2000．35(4)：450～454．
[8] 韩磊．汪旭东，吴先军等．植物组织培养技术及其应用研究发展[J]．种子．2005．24(1)：38-43．
[9] 李云侠，王建华，刘思秀．兰州百合快速繁殖的研究[J]．国土与自然环境研究，1993(3)：68～69．
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