CN105325292A - 一种减缓长期继代蝴蝶兰类原球茎衰老的方法 - Google Patents
一种减缓长期继代蝴蝶兰类原球茎衰老的方法 Download PDFInfo
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Abstract
一种减缓长期继代蝴蝶兰类原球茎衰老的方法，在继代培养基添加抗氧化剂、DNA甲基化抑制剂和DNA结构稳定剂，减缓类原球茎生理衰老和维持遗传物质DNA稳定，三类调节剂可同时混用，也可两两组合应用。抗氧化剂主要有抗坏血酸、半胱氨酸盐、谷胱甘肽、亚硫酸氢钠、亚硒酸钠、硫脲。DNA甲基化抑制剂主要有5-氮杂胞嘧啶核苷、甜菜碱、胆碱、茶多酚、甘露醇。DNA结构稳定剂主要有精胺、亚精胺、腐胺。本发明能减缓长期继代蝴蝶兰类原球茎增殖、分化能力降低，对类原球茎的某些生理、分子指标有一定修复作用，采用的化学调节剂对植物材料没有毒副作用，使用无须特别仪器，操作简便。
Description
技术领域
本发明属于农林领域的园艺植物生物技术，具体涉及一种采用化学方法减缓长期继代蝴蝶兰类原球茎衰老的方法。
背景技术
在著名的兰花工业中，真正形成专业化、规模化、商品化生产的只有蝴蝶兰属（Phalaenopsis），蝴蝶兰也是我国最重要的年宵花卉，组培蝴蝶兰类原球茎（PLB）在快速育苗、基因转化、人工种子研制、种质保存等方面具有相当应用潜力。虽然类原球茎扩繁系数大，但在生产过程中，为提高生产效率（扩大繁殖倍率）及受市场需求、供应时间等多种因素影响，不可避免的会出现频繁继代培养，经长期继代培养，常常出现类原球茎的形态正常，但生长减缓，分化率低、甚至再生苗畸形等衰老现象，无法达到获得更多组培无性系或种质保存的目的。目前长期继代培养（保存）组培材料专利或申请有，“高产原花青素刺葡萄愈伤组织的获取与继代保持方法”（申请号201410319042.1），采用剥去所述愈伤组织紫红色表层细胞，选里层愈伤组织接种的方式，而且在两种继代保持培养基交替继代培养。“快速获得葡萄松散型愈伤组织及其长期继代保持的方法（申请号：201210246117.9）”主要采用继代保持在两种培养基交替继代培养的方法。“大豆未成熟子叶体细胞胚增殖、继代保存和植株再生方法”（申请号02144578.8）挑选没有被周围的愈伤所吞食掉（或去掉周边愈伤组织）球形胚，转接到新鲜培养基增殖。其他申请如申请号201310157607.6、201010514729.2、201010249615.X、201410166898.X、200710084622.7主要在常规培养基稍改的常规培养对愈伤组织进行继代保存。
植物培养物继代保持的方法报道不多，几乎都以愈伤组织或体细胞胚为材料，上述申请培养基配方复杂，或者操作繁琐，常温下长期继代保存效果也不明确。
发明内容
植物组织培养物随着继代培养时间（次数）的增加，培养材料发生衰老或退化，其机理包括生理衰老和遗传物质变异。活性氧自由基在植物生理衰老和遗传物质变异上均有重要的效应。活性氧可使类脂中的不饱和脂肪酸发生过氧化反应，破坏酶等生物大分子和细胞膜的结构，引发代谢紊乱；活性氧攻击核酸分子，引起DNA单链破坏与断裂等损伤；此外，DNA胞嘧啶甲基化水平随着植物材料继代代数和时间的增加而增加，所以遗传和表观遗传变异也是引起长时间培养材料丧失分化能力的重要原因。抗氧化剂维生素C、半胱氨酸、类胡萝卜素、谷胱甘肽、硫脲等能提高抗氧化酶（SOD、POD、CAT等）活性，清除活性氧，降低活性氧对细胞毒害，抑制或延缓植物衰老。减少活性氧积累的的因素都能减轻或消除DNA损伤，槲皮素、维生素C、亚硒酸钠等对细胞DNA氧化损伤修复研究具有悠久的历史和丰硕的成果。一些植物材料在组织培养过程中的表观遗传变化有可能通过甲基化抑制剂5-azaC（5-氮杂胞嘧啶核苷）修复而得以维持正常化（聂丽娟王子成.2007.DNA甲基化抑制剂作用机理及其在植物发育生物学研究中的应用.核农学报，21(4)：362-365），有报道葫芦巴碱、甜菜碱和胆碱（BerglundT．Nicotinamide，amissinglinkintheearlystressresponseineukaryoticcells：ahypothesiswithspecialreferencetooxidativestressinplants．FEBSLett，1994，351：145—149）、茶多酚类（李培坤，耿小平，朱立新.DNA甲基化抑制剂研究进展.国际药学研究杂志，2010，37（3）：198-202）、一定浓度甘露醇（HardingK.ThemethylationstatusofDNAderivedfrompotatoplantsrecoveredfromslowgrowth.PlantCellTissueOrganCult,1994,37,31–38.杜亚琼，王子成.甘露醇对拟南芥基因组DNA甲基化的影响.植物学报,2011,46(3):285-292）也具有去甲基化效应。
外源多胺不仅能调节清除活性氧和抗氧化酶的活性，抑制或消除胁迫损伤，而且多胺能中和DNA的负电荷，对改善DNA的稳定性及损伤修复起到重要作用（崔凯荣，戴若兰,主编,2000.植物体细胞胚发生的分子生物学.北京：科学出版社,pp.75-76），有学者提出精胺稳定DNA双链结构的“多胺-DNA复合”模型（BenAR,LuricS,Mattoo,AK.1982.Temperature-denpendentinhibitoryeffectsofcalciumandsperineonethylenebiosynthesisinapplediscscorrelatewithchangesinmicrosomalmembranemicroviscosity.PlantSci.lett.24:239-247）。
蝴蝶兰组培类原球茎随继代次数增加（培养时间延续），出现扩繁增殖能力下降，分化（出芽长根）能力降低的衰老或退化现象。本发明在培养基添加抗氧化剂、DNA甲基化水平抑制剂和DNA结构稳定剂，从生理调节和遗传物质稳定性上共同作用，减轻长期继代类原球茎的衰老现象。
本发明提供的技术方案如下：
一种减缓长期继代蝴蝶兰类原球茎衰老的方法，综合应用如下方法减缓长期继代蝴蝶兰类原球茎退化或衰老，即在继代培养基添加三类调节剂，抗氧化剂、DNA甲基化抑制剂和DNA结构稳定剂。本发明所述三类调节剂可同时混用，也可两两组合应用。
本发明所述的调节剂在培养基的终浓度（工作浓度）及配制方法，抗氧化剂抗坏血酸10-300mg/L、半胱氨酸盐10-300mg/L、谷胱甘肽10-300mg/L、亚硫酸氢钠10-300mg/L、亚硒酸钠10-300mg/L、硫脲10-300mg/L，一种或几种组合，配制时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基。DNA甲基化抑制剂在培养基的终浓度及配制方法，5-氮杂胞嘧啶核苷5-50mg/L、甜菜碱10-100mg/L、胆碱10-100mg/L、茶多酚10-200mg/L、甘露醇5-10g/L，一种或几种组合，其中甜菜碱、胆碱、茶多酚配置时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基，甘露醇可加入培养基直接高压灭菌。DNA结构稳定剂在培养基的终浓度及配制方法，精胺10-100mg/L，亚精胺10-100mg/L，腐胺10-100mg/L，一种或几种组合，配置时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基。综上所述，除了甘露醇可与培养基一起高压灭菌外，三类调节剂可混合溶解于水，一起过滤除菌加入已经高压灭菌未冷凝培养基。
本发明能减缓长期继代蝴蝶兰类原球茎增殖、分化能力降低，对类原球茎的某些生理、分子指标有一定修复作用，采用的化学调节剂对植物材料没有毒副作用，使用无须特别仪器，操作简便。
具体实施方式
实施例1.
蝴蝶兰初代（0个月）类原球茎接种于继代培养基1/2MS+椰子汁20%（v/v）+蔗糖2%（w/v）。处理组为继代培养基添加抗氧化剂半胱氨酸100mg/L培养基，DNA甲基化抑制剂甘露醇8g/L培养基，甘露醇加入培养基直接高压灭菌，半胱氨酸先混溶于水，过滤除菌注入已经高压灭菌未冷凝培养基，分装到培养瓶，冷凝后接种蝴蝶兰类原球茎。培养条件均为光照1500lx，12h/d，温度24-26℃。在培养的第24个月（期间分瓶扩繁12次），检验类原球茎增殖能力（每瓶鲜重），成活率和分化率，见表1。
表1.类原球茎继代培养24个月的鲜重、成活率、萌发率
初代（0个月）类原球茎培养2个月每瓶鲜重10.75g，经数理统计，对照组类原球茎培养第24个月9.38g，显著降低，处理组培养第24个月鲜重与0个月没有显著差异，第24个月处理组较对照显著提高，即抗氧化剂与DNA甲基化抑制剂组合显著地抑制了长期继代类原球茎增殖能力下降。类原球茎成活率指标，对照组、处理组在培养第24个月均与0个月的差异不显著，即继代24个月并不显著影响类原球茎成活。类原球茎萌发率变化，对照（0个月）类原球茎萌发率72.22%，对照和处理组都在第24个月极显著降低，处理组较对照组极显著提高，所以在培养基添加抗氧化剂与DNA甲基化抑制剂组合能极显著延缓继代类原球茎的萌发率下降。
类原球茎继代培养24个月总抗氧化能力与DNA胞嘧啶甲基化水平变化见表2。
表2.类原球茎继代培养24个月的总抗氧化能力和DNA胞嘧啶甲基化水平
初代（0个月）类原球茎总抗氧化能力29.6U·min-1·g-1FW，经数理统计，对照和处理组都在第24个月极显著降低，处理组较对照组极显著提高，所以在培养基添加抗氧化剂与DNA甲基化抑制剂组合能极显著减少继代类原球茎总抗氧化能力下降。初代（0个月）类原球茎DNA甲基化水平17.7%，经数理统计，对照和处理组都在第24个月极显著提高，处理组较对照组有所下降，达显著水平，所以在培养基添加抗氧化剂与DNA甲基化抑制剂组合能显著降低继代类原球茎DNA甲基化水平。
实施例2.
蝴蝶兰初代（0个月）类原球茎接种于继代培养基1/2MS+6-BA3mg/L+椰子汁20%（v/v）+蔗糖2%（w/v）。处理组为继代培养基添加抗氧化剂半胱氨酸100mg/L培养基，DNA结构稳定剂精胺40mg/L培养基，两者先混溶于水，过滤除菌注入已经高压灭菌未冷凝培养基，分装到培养瓶，冷凝后接种蝴蝶兰类原球茎。培养条件均为光照1500lx，12h/d，温度24-26℃。在培养的第24个月（期间分瓶扩繁12次），检验类原球茎增殖能力（每瓶鲜重），成活率和分化率，见表3。
表3.类原球茎继代继代培养24个月的鲜重、成活率、萌发率
初代（0个月）类原球茎培养2个月每瓶鲜重10.75g，经数理统计，对照组类原球茎培养第24个月极显著降低，处理组培养第24个月鲜重与0个月没有显著差异，第24个月处理组较对照极显著提高，即抗氧化剂与DNA结构稳定剂组合显著地抑制了长期继代类原球茎增殖能力下降。类原球茎成活率指标，对照组、处理组在培养第24个月均与0个月差异不显著，即继代24个月并不显著影响类原球茎成活。类原球茎萌发率变化，对照（0个月）类原球茎萌发率72.22%，对照和处理组都在第24个月极显著降低，处理组较对照组极显著提高，所以在培养基添加抗氧化剂与DNA结构稳定剂组合能极显著延缓继代类原球茎的萌发率下降。
类原球茎继代培养24个月总抗氧化能力与DNA增色效应变化见表4。
表4.类原球茎继代培养24个月的总抗氧化能力和DNA增色效应变化
初代（0个月）类原球茎总抗氧化能力29.6U·min-1·g-1FW，经数理统计，对照和处理组都在第24个月都极显著降低，处理组较对照组极显著提高，所以在培养基添加抗氧化剂与DNA结构稳定剂组合能极显著减少继代类原球茎总抗氧化能力下降。DNA增色效应反映DNA的解链程度，而DNA链间交联则使增色效应下降，初代（0个月）类原球茎增色效应指标0.105，经数理统计，对照组在第24个月极显著降低，即DNA结构损伤，处理组在第24个月没显著变化，第24个月处理组较对照组极显著提高，所以在培养基添加抗氧化剂与DNA结构稳定剂能显著抑制继代类原球茎DNA结构损伤（链间交联）。
Claims (2)
1.一种减缓长期继代蝴蝶兰类原球茎衰老的方法，其特征在于，在继代培养基添加三类调节剂，三类调节剂可同时组合混用，或可两两组合应用；
所述三类调节剂及使用浓度分别为：（1）抗氧化剂，抗坏血酸10-300mg/L、半胱氨酸盐10-300mg/L、谷胱甘肽10-300mg/L、亚硫酸氢钠10-300mg/L、亚硒酸钠10-300mg/L、硫脲10-300mg/L，一种或几种组合；（2）DNA甲基化抑制剂，5-氮杂胞嘧啶核苷5-50mg/L、甜菜碱10-100mg/L、胆碱10-100mg/L、茶多酚10-200mg/L、甘露醇5-10g/L，一种或几种组合；（3）DNA结构稳定剂，精胺10-100mg/L，亚精胺10-100mg/L，腐胺10-100mg/L，一种或几种组合。
2.如权利要求1所述的一种减缓长期继代蝴蝶兰类原球茎衰老的方法，其特征在于，所述的（1）抗氧化剂抗坏血酸、半胱氨酸盐、谷胱甘肽、亚硫酸氢钠、亚硒酸钠、硫脲配制时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基；（2）DNA甲基化抑制剂5-氮杂胞嘧啶核苷、甜菜碱、胆碱、茶多酚配制时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基，甘露醇可在配制培养基时添加，一起高压消毒；（3）DNA结构稳定剂精胺、亚精胺、腐胺配制时可直接溶解于水，过滤除菌加入已经高压灭菌未冷凝培养基。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105815216A (zh) * | 2016-03-22 | 2016-08-03 | 蒋凡 | 一种红花荷组培苗快速繁育方法 |
| CN106069786A (zh) * | 2016-08-11 | 2016-11-09 | 岭南师范学院 | 一种在桉树组织培养中抗褐化和促进不定芽分化的方法 |
| CN106417026A (zh) * | 2016-10-14 | 2017-02-22 | 屏南县惠荣农业科技有限公司 | 一种多肉植物的组培快繁方法 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103782911A (zh) * | 2014-02-07 | 2014-05-14 | 福建省亚热带植物研究所 | 一种蝴蝶兰体细胞胚发生同步化调控方法 |
| CN104099287A (zh) * | 2014-07-07 | 2014-10-15 | 福建省农业科学院农业工程技术研究所 | 高产原花青素刺葡萄愈伤组织的获取与继代保持方法 |
| CN104521763A (zh) * | 2015-01-23 | 2015-04-22 | 福建省亚热带植物研究所 | 一种减轻植物组培外植体褐化的衣膜包囊培养法 |
-
2015
- 2015-09-30 CN CN201510635778.4A patent/CN105325292B/zh not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103782911A (zh) * | 2014-02-07 | 2014-05-14 | 福建省亚热带植物研究所 | 一种蝴蝶兰体细胞胚发生同步化调控方法 |
| CN104099287A (zh) * | 2014-07-07 | 2014-10-15 | 福建省农业科学院农业工程技术研究所 | 高产原花青素刺葡萄愈伤组织的获取与继代保持方法 |
| CN104521763A (zh) * | 2015-01-23 | 2015-04-22 | 福建省亚热带植物研究所 | 一种减轻植物组培外植体褐化的衣膜包囊培养法 |
Non-Patent Citations (6)
| Title |
|---|
| 叶秀仙等: "甘露醇对蝴蝶兰种质离体保存的影响", 《中国园艺学会观赏园艺专业委员会2011年学术年会论文集》 * |
| 杜亚琼等: "甘露醇对拟南芥基因组DNA甲基化的影响", 《植物学报》 * |
| 杨淑慎等: "活性氧、自由基与植物的衰老", 《西北植物学报》 * |
| 聂丽娟等: "DNA甲基化抑制剂作用机理及其在植物发育生物学研究中的应用", 《核农学报》 * |
| 聂卫卓等: "蝴蝶兰叶片组织培养中抗褐化的研究", 《黑龙江八一农垦大学学报》 * |
| 顾德峰等: "蝴蝶兰无性快繁规模化生产的研究", 《园艺学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105815216A (zh) * | 2016-03-22 | 2016-08-03 | 蒋凡 | 一种红花荷组培苗快速繁育方法 |
| CN106069786A (zh) * | 2016-08-11 | 2016-11-09 | 岭南师范学院 | 一种在桉树组织培养中抗褐化和促进不定芽分化的方法 |
| CN106069786B (zh) * | 2016-08-11 | 2019-03-22 | 岭南师范学院 | 一种在桉树组织培养中抗褐化和促进不定芽分化的方法 |
| CN106417026A (zh) * | 2016-10-14 | 2017-02-22 | 屏南县惠荣农业科技有限公司 | 一种多肉植物的组培快繁方法 |
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