CN104542303A - 一组通关藤快速繁殖培养基 - Google Patents
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Abstract
一组通关藤快速繁殖培养基。该组培养基的主要配方为:诱导培养基:MS+6-BA 2.0mg/L+NAA 0.5mg/L;增殖培养基:MS+6-BA 1.0mg/L+NAA 0.3mg/L;壮苗培养基:MS+6-BA 1.0mg/L+NAA 0.3mg/L+NH4NO30.66g/L+NaH2PO40.56g/L+K2SO41.78g/L+香蕉100g/L;生根培养基:1/2MS+6-BA 0.5mg/L+IAA0.2mg/L;本发明的诱导率高达88%以上,增殖倍数高,生根率高达95%以上,组培繁殖周期短,仅需64天~72天,实现了通关藤有效快速的无性繁殖。
Description
技术领域
本发明属于植物无性繁殖技术领域,具体涉及一种中药材通关藤的快速繁殖方法。
背景技术
通关藤(Marsdenia tenocissima(Roxb)Wight et Arm)为萝藦科牛奶菜属藤本植物,又名乌骨藤。始载于《滇南本草》,广泛分布于亚洲的热带和亚热带地区。其全株都具有清热解毒、消炎止痛和止咳平喘之功效。通关藤所含的乌骨消癌素(C21-甾体甙和乌骨总碱)对癌细胞分裂的根源-微管排列有很强的破坏作用,可阻断癌细胞的有丝分裂,从而有效切断癌细胞扩散、转移,癌症因此萎缩直至完全消失。可用于晚期肺癌、急性白血病、食管癌、胃癌、肝癌、喉癌、鼻咽癌等各种晚期恶性肿瘤的治疗。以通关藤为主要原料制备而成的中药消癌平制剂,疗效良好。
目前,通关藤主要依靠自然繁育和有限的野生资源供给。由于通关藤野生资源遭到严重破坏,而药材需求量仍逐年提高,通关藤自身花多果少的生物特性,其植物自然繁殖力低。加之,高强度的砍伐采收,致使当前通关藤野生资源量急剧萎缩,人工繁育势在必行。优质种苗的快速获取是通关藤栽培最为关键的基础环节。而种子繁殖得到的实生苗,品种容易退化,并且生长周期长,结实率低;其次扦插繁殖虽然成活率高,但后期感病严重,很难满足市场的需求。因此,探索一种具有快速的、适宜规模化生产优质种苗的通关藤组培培养基配方,对保证通关藤原料药材的持续供应具有重要意义。
发明内容
本发明的目的是要解决通关藤种子繁殖率低、生长周期长,变异性较大,扦插繁殖苗容易感病的技术问题,提供一组适宜规模化生产、繁殖快、成活率高的通关藤快速繁殖培养基配方。
本发明所提供的一组通关藤快速繁殖培养基配方由如下诱导培养基、增殖培养基、壮苗培养基和生根培养基组成:
所述的诱导培养基为:MS+6-BA 2.0mg/L+NAA0.5mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
所述的增殖培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
所述的壮苗培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L++NH4NO30.66g/L+NaH2PO40.56g/L+K2SO41.78g/L+香蕉100g/L+蔗糖25g/L+琼脂7.1g/L,pH5.8;
所述的生根培养基为:1/2MS+6-BA 0.5mg/L+IAA 0.2mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8。
本发明的有益效果是:
1、本发明为通关藤人工繁育提供了新的途径。
本发明所提供的一组通关藤组织培养基对通关藤进行组培快繁,使通关藤能保持亲本的优良性状,缩短繁育周期,提高繁殖系数,提高产量和质量,缓解了市场用药压力,解决了通关藤由于花多果少而致的结实率低,种子繁育周期长,易变异的缺陷。
2、本发明通过大量试验和研究,筛选出一组通关藤各个生理时期生长所需营养的培养基配方,达到诱导率高(88%以上),增殖倍数高(每月可增殖2~2.5倍),生根率高,可达95%以上,组培繁殖周期短,繁殖率高,整个繁殖周期仅需64天~72天,实现了通关藤有效快速的无性繁殖,达到了适宜规模化生产、繁殖快、有抗性、成活率高的目的,对加快通关藤优良无性系的推广和保证抗癌原料药持续稳定供应具有非常重要的作用和意义。
3、采用本发明一组通关藤快速繁殖培养基还能提高通关藤苗木对病虫害的抗性,解决了由于通关藤植株藤茎疏松多孔,内含大量乳汁,扦插繁殖后容易感病的缺点,为通关藤人工栽培提供了优质种苗。
具体实施方式
以下结合实施例对本发明作进一步的说明。以下各实施例按常规组培要求对各种玻璃器皿、培养基进行灭菌,并在常规的组培无菌条件下进行快速繁殖。各实施例无特殊说明为常规方法。
实施例1
(1)外植体的选择与处理
外植体来源于云南省蒙自县野外的通关藤植株,选取无病虫害通关藤的当年新生梢,剪成带一个芽的单芽茎段为外植体,按如下顺序进行初步灭菌:用流水冲洗2.5h,放入质量分数为5%洗衣粉水中浸泡5min,流水条件下冲洗25min;将经过初步灭菌处理过的通关藤茎段用体积分数为75%乙醇溶液浸泡1min,然后用质量分数为0.1%氯化汞溶液浸泡12min后,再用无菌水洗涤3~5次,用无菌纸吸干通关藤茎段表面水分备用。
由于通关藤嫩枝密被淡黄色绒毛,上面会粘着许多的尘埃物,采用一般方法不能达到好的灭菌效果,甚至造成整个组培的失败,本发明经过大量试验(见表1),得出必须用本发明所述的灭菌方法才能达到的污染率最低,成活率是最高的状况,再用无菌水洗涤3~5次,用无菌纸吸干通关藤茎段表面水分,放在已经灭过菌的培养皿上备用。
表1用0.1%氯化汞在不同消毒时间
对外植体成活率及培养基污染率的影响
试验表明:用质量分数为0.1%氯化汞溶液在不同消毒时间浸泡外植体8~13min,随着消毒时间的增加,污染率是逐渐降低的,其原因是随着消毒时间的增加,对外植体上所携带的病菌杀死就越彻底。而成活率从消毒8min至12min是逐渐增加的,到13min分时突然减少,其原因是消毒时间增至13min时,消毒液已严重损害了外植体的组织细胞,所以成活率突然降低。因此最为理想的消毒时间为11min~12min。
(2)诱导培养
在无菌条件下,将步骤(1)经灭菌处理的外植体切成长度为0.5cm~1.0cm带有一个芽的茎段接种于诱导培养基中,培养条件为:温度25℃士2℃,光照强度2000-2500Lx,光周期为12h光期、12h暗期,空气相对湿度保持在70%~80%;所述的诱导培养基为:MS+6-BA 2.0mg/L+NAA 0.5mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
(3)增殖培养
步骤(2)诱导培养形成的芽长到2~3cm时分割下来,剪成带有一个芽的茎段,接种于增殖培养基中,培养条件与步骤(2)相同;所述的增殖培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L+蔗糖25g/L+琼脂7.1g/L,pH值5.8。
(4)壮苗培养
将步骤(3)增殖培养形成的增殖苗分切成带一个芽的茎段,接种于壮苗培养基中,培养条件与步骤(2)相同;所述的壮苗培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L++NH4NO30.66g/L+NaH2PO40.56g/L+K2SO41.78g/L+香蕉100g/L+蔗糖25g/L+琼脂7.1g/L,pH值5.8;
(5)生根培养
步骤(4)的苗长到1.5cm以上时,剪切成长为0.8~1.2cm、至少带一个芽的茎段接种于生根培养基中,培养条件与步骤(2)相同;所述的生根培养基为:1/2MS+6-BA0.5mg/L+IAA 0.2mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8。待生根并长出4~5片新叶时炼苗移栽。
移栽前,在温室内去掉瓶盖炼苗3~5天,使幼苗接触自然环境,增强对外界不良因素的抵抗力,可以提高移栽成活率。移栽时用镊子将小苗从培养瓶内取出,并在水中洗去黏附在根上的培养基,再栽入加有少量珍珠岩与腐叶土的混合基质内(珍珠岩:腐叶土的体积比为1:5),移栽后浇透水,要注意保温保湿,湿度为60%左右,温度在20~25℃,移栽前期(移栽后25天以内)用遮光率为75%的遮阳网遮阳,后期(移栽生长40天之后)适当增加光照,遮光率为35%,成活率达到95%以上。
实施例2
实施例2除以下步骤操作不同外,其余各步骤操作与实施例1相同,不再赘述。
(1)外植体的选择与灭菌处理
外植体来自云南省蒙自县野外的通关藤植株,移栽到云南农业大学温室驯化后当年长出的新梢。其它消毒处理步骤与实施例1外植体处理方法一致,不同的是用质量分数为0.1%氯化汞溶液消毒时间为11min,就可达到最低污染率同时成活率高。
实施例3
实施例3除以下步骤操作不同外,其余各步骤操作与实施例1相同,不再赘述。
(1)外植体的选择与灭菌处理
外植体来源于云南省马关县野外的通关藤植株,选取无病虫害通关藤的当年新生梢,剪成带一个芽的单芽茎段为外植体,用质量分数为0.1%氯化汞消毒时间为12min时,污染率达到最低同时成活率高。它的消毒时间与实施例1外植体来自于云南省蒙自县的一致,这与都是来自于野外,植株上带有很多的灰尘与病菌有关。
实施例1至实施例3的试验结果详见表2。
表2实施例1-实施例3的试验效果
本发明通过大量试验和研究,筛选出通关藤各个生理时期生长所需营养的培养基配方及各参数,使其来自于不同地区的通关藤外植体都能达到优良的灭菌效果和高成活率的效果,都能繁殖出健壮的种苗,诱导率高(88%以上),增殖倍数高(每月可增殖2~2.5倍),繁殖周期短,整个繁殖周期64天~72天,生根率高,可达95%以上,产生了特别显著的技术效果。
Claims (1)
1.一组通关藤快速繁殖培养基,其特征在于由如下诱导培养基、增殖培养基、壮苗培养基和生根培养基组成:
所述的诱导培养基为:MS+6-BA 2.0mg/L+NAA 0.5mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
所述的增殖培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
所述的壮苗培养基为:MS+6-BA 1.0mg/L+NAA 0.3mg/L+NH4NO30.66g/L+NaH2PO40.56g/L+K2SO41.78g/L+香蕉100g/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8;
所述的生根培养基为:1/2MS+6-BA 0.5mg/L+IAA 0.2mg/L+蔗糖25g/L+琼脂7.1g/L,pH 5.8。
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CN107333550A (zh) * | 2017-08-03 | 2017-11-10 | 云南信通植物药业有限公司 | 一种通关藤种子育苗方法 |
CN108207504A (zh) * | 2018-01-04 | 2018-06-29 | 云南农业大学 | 一种营养袋繁育通关藤种苗的方法 |
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CN108207504A (zh) * | 2018-01-04 | 2018-06-29 | 云南农业大学 | 一种营养袋繁育通关藤种苗的方法 |
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