CN105724250B - 一种扁桃斑鸠菊的简化组培快繁方法 - Google Patents
一种扁桃斑鸠菊的简化组培快繁方法 Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
本发明公开了一种扁桃斑鸠菊的组培快繁育苗方法,包括以下步骤:1)外植体采集:采集幼嫩的茎段,去除叶片,清水冲洗后作为外植体备用;2)外植体消毒:分别用75%酒精和0.1%升汞溶液进行浸泡消毒,并用无菌水冲洗;3)无菌芽诱导:将消毒处理好的外植体切成小段接种于芽诱导培养基中;4)继代‑生根培养:将诱导培养基中形成的不定芽转入附加有特定植物生长调节剂的改良MS培养基(下称MS‑z培养基)中,进行继代和生根培养;5)移至炼苗棚中炼苗10天,洗去基部培养基,移栽入备用的基质中,成活率可达100%。
Description
技术领域
本发明涉及植物组织培养技术领域,特别是涉及一种扁桃斑鸠菊的简化组培快繁方法。
背景技术
扁桃斑鸠菊(Vernonia amygdalina Del.)又名桃叶斑鸠菊、杏叶斑鸠菊、神奇树、苦树、南非树,为菊科斑鸠菊属植物,原产于非洲。其叶片可以安全食用,在尼日利亚等地被当作一种蔬菜,可入药,在治疗肿瘤尤其是防治乳腺癌、降血压方面极具潜力。扁桃斑鸠菊在东南亚及台湾等地民间应用较多,而中国大陆则相对比较陌生,近年来两广地区陆续有引进,但相关研究较少。
采用组织培养技术生产种苗,可以实现周年生产、保障种苗的品质,加快推进种苗的标准化生产,但生产工艺繁琐、需要经过诱导无菌芽、丛生芽诱导继代和生根培养等环节。因此建立并优化组培快繁技术体系,不断改进培养基配方、简化生产流程,对加快产业化、降低生产成本具有重要意义。目前,尚无扁桃斑鸠菊组培快繁技术的相关报道。
发明内容
本发明的目的是提供一种扁桃斑鸠菊的简化组培快繁方法,该方法在同一种培养基上进行扁桃斑鸠菊的继代培养和生根培养,实现了继代培养和生根培养同时进行的一步成苗,大大简化了生产流程,节约了物料成本和人工成本,为扁桃斑鸠菊的规模化组培快繁提供了工艺方法。
本发明所述的一种扁桃斑鸠菊的简化组培快繁方法,包括以下步骤:
1)外植体采集:选取生长健壮、无病虫害的优良植株,剪取木质化程度较轻的当年生嫩枝为外植体;
2)外植体消毒:对步骤1)得到的外植体进行浸泡消毒,并用无菌水冲洗;
3)无菌芽诱导:将步骤2)完成消毒的外植体茎段接种于诱导培养基上进行培养;
4)继代-生根培养:将步骤3)培养得到无菌芽分段切割,转入附加有特定植物生长调节剂的MS-z培养基进行培养;
5)移栽:待根系发育完成,洗净基部培养基,移栽到备用的基质中。
本发明步骤2)所述的外植体消毒,优选将步骤1)得到的外植体截成长1.2-1.5cm的带节小段,剪去叶片,只保留叶柄基部(长约0.5cm),流水冲洗20min备用;在超净工作台上,用75%(v/v)酒精浸泡10s,0.1%升汞溶液振荡消毒8min,再用无菌水冲洗5次洗去残留药液,用无菌滤纸吸干水分后,切去茎段两端和叶柄上部少许(保留长度约0.2cm),备用。
步骤3)所述的诱导培养基为MS基本培养基附加6-苄氨基腺嘌呤(6-BA)0.5mg/L和萘乙酸(NAA)0.05mg/L,即MS+6-BA0.5mg/L+NAA0.05mg/L,蔗糖和琼脂粉添加量分别为每L培养基30g、6g,灭菌前培养基pH调整为6.0,培养室温度为(23±2)℃,光照强度1500-2500lx,光照时间为12h/d。
在诱导培养基中,侧芽易于诱导且生长迅速,诱导率可达70%,以春夏季消毒效果更佳,以茎段消毒诱导的无菌芽多为腋芽,即“以芽繁芽”的方式获得无菌芽,虽有愈伤组织产生,但愈伤组织很少成苗,降低了变异率,保证了种苗的品质。
步骤4)所述的继代-生根培养,优选待无菌芽生长至2cm时,将其分段切割,转入附加有特定植物生长调节剂的继代-生根培养基进行培养,培养周期为35天,培养室光照时间为16h/d,光照强度2000-3500lx,培养室温度控制在(21±2)℃以减少玻璃化、促使组培苗健壮;
所用继代-生根培养基即MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP333(多效唑)0.1mg/L,所述的MS-z培养基每升中所含的组分及浓度(mg/L)如下:
大量元素:硝酸钾1900、硝酸铵412.5、二水氯化钙880、七水硫酸镁370、磷酸二氢钾170、七水硫酸亚铁27.8、乙二胺四乙酸二钠37.3;
微量元素:四水硫酸锰22.3、七水硫酸锌8.6、硼酸3.1、二水钼酸钠0.25、碘化钾0.83、五水硫酸铜0.025、六水氯化钴0.025;
有机物:肌醇100.0、甘氨酸2.0、盐酸吡哆醇(VB6)0.5、烟酸0.5、盐酸硫胺素(VB1)0.1;
其它:琼脂8000、蔗糖40000、pH 6.0;
若要继续扩增,应在培养20-25天时继代,此时苗高3cm左右,根系较短,通过分段切割重新转入MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP3330.1mg/L培养基即可;若要生根移栽,则应继续培养至35天,此时苗高3-5cm,植株健壮、根系良好,移入炼苗棚炼苗10天后即可移栽。
步骤5)所述的移栽,洗净基部培养基,将瓶苗均匀地分为2-3芽/丛的小丛芽,组培苗基部在多菌灵600倍液中浸泡5min后移栽入备用的基质中,在泥炭土和珍珠岩的混合基质(泥炭土和珍珠岩体积比为3:1)中成活率可达100%,移栽入黄心土中成活率也可达90%以上;所用基质和容器移栽前均用0.5%高锰酸钾溶液消毒备用,移栽后加强管理,待苗高30cm时即可供大田栽培或山坡造林。
与现有技术相比,本发明具有如下优点:
1.本方法在同一种培养基上进行扁桃斑鸠菊的继代培养和生根培养,实现了继代培养和生根培养同时进行的一步成苗,大大简化了生产流程,节约了物料成本和人工成本,为扁桃斑鸠菊的规模化组培快繁提供了工艺方法。
2.本方法中设计的MS改良培养基MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP3330.1mg/L完全适合扁桃斑鸠菊的试管苗繁殖,不仅保证了较高的增殖系数和生根率,而且有效解决了扁桃斑鸠菊在组培过程中出现的玻璃化、徒长等问题。
3.目前扁桃斑鸠菊的组织培养快繁技术尚无相关报道,本方法与改进前的扁桃斑鸠菊常规组培快繁方法相比,其主要技术参数和效果对比:
具体实施方式
下面以实施例对本发明作进一步说明,但本发明并不局限于这些实施例。实施例:
扁桃斑鸠菊的组培快繁育苗方法,包括以下步骤:
1)外植体采集:选取生长健壮、无病虫害的优良植株,剪取木质化程度较轻的当年生嫩枝为外植体;
2)外植体消毒:将采回的茎段截成长1.2-1.5cm的带节小段,剪去叶片,只保留叶柄基部(长约0.5cm),流水冲洗20min备用,在超净工作台上,用75%酒精浸泡10s,0.1%升汞振荡消毒8min,再用无菌水冲洗5次洗去残留药液,用无菌滤纸吸干水分后,切去茎段两端和叶柄上部少许(保留长度约0.2cm),备用;
3)无菌芽诱导:在超净工作台上将完成消毒的外植体茎段接种于诱导培养基上,诱导培养基为MS基本培养基附加6-苄氨基腺嘌呤(6-BA)0.5mg/L和萘乙酸(NAA)0.05mg/L,即MS+6-BA0.5mg/L+NAA0.05mg/L,蔗糖和琼脂粉添加量分别为每L培养基30g、6g,灭菌前培养基pH调整为6.0,培养室温度为(23±2)℃,光照强度1500-2500lx,光照时间为12h/d;
在诱导培养基中,侧芽易于诱导且生长迅速,诱导率可达70%,以春夏季消毒效果更佳,以茎段消毒诱导的无菌芽多为腋芽,即“以芽繁芽”的方式获得无菌芽,虽有愈伤组织产生,但愈伤组织很少成苗,降低了变异率,保证了种苗的品质;
4)继代-生根培养:待无菌芽生长至2cm时,将其分段切割,转入附加有特定植物生长调节剂的MS-z培养基进行培养,培养周期为35天,所用MS-z培养基即MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP3330.1mg/L,培养室光照时间为16h/d,光照强度2000-3500lx,培养室温度控制在(21±2)℃以减少玻璃化、促使组培苗健壮;若要继续扩增,应在培养20-25天时继代,此时苗高3cm左右,根系较短,通过分段切割重新转入MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP3330.1mg/L培养基即可;若要生根移栽,则应继续培养至35天,此时苗高3-5cm,植株健壮、根系良好,移入炼苗棚炼苗10天后即可移栽;
5)移栽:洗干净基部培养基,将瓶苗均匀地分为2-3芽/丛的小丛芽,组培苗基部在多菌灵600倍液中浸泡5min后移栽入备用的基质中,在泥炭土和珍珠岩的混合基质(泥炭土和珍珠岩体积比为3:1)中成活率可达100%,移栽入黄心土中成活率也可达90%以上,所用基质和容器移栽前均用0.5%高锰酸钾溶液消毒备用,移栽后加强管理,待苗高30cm时即可供大田栽培或山坡造林。
对比试验:
表1 MS-z培养基成分(每升中所含的组分及浓度)
注:MS的蔗糖是30000mg、pH5.8,MS-z培养基主要针对扁桃斑鸠菊在组培过程中易出现的玻璃化现象而设计,增加了蔗糖和升高pH是为了减缓玻璃化,在此改良的基础上再添加特定种类和浓度的植物生长调节剂,即MS-z+6-BA0.3mg/L+IBA0.1mg/L+PP3330.1mg/L,可以有效控制试管苗玻璃化和徒长现象,得到健壮继代苗和生根苗。
表2 不同培养基配方对扁桃斑鸠菊继代苗生长的影响
Claims (2)
1.一种扁桃斑鸠菊的简化组培快繁方法,其特征在于,包括以下步骤:
1)外植体采集:选取生长健壮、无病虫害的优良植株,剪取木质化程度较轻的当年生嫩枝为外植体;
2)外植体消毒:对步骤1)得到的外植体进行浸泡消毒,并用无菌水冲洗;
3)无菌芽诱导:将步骤2)完成消毒的外植体茎段接种于诱导培养基上进行培养;
4)继代-生根培养:将步骤3)培养得到的无菌芽分段切割,转入继代-生根培养基进行培养;
5)移栽:待根系发育完成,洗净基部培养基,移栽到备用的基质中;
步骤3)所述的诱导培养基为MS+6-BA0.5mg/L+NAA0.05mg/L;
步骤4)所述的继代-生根培养基为MS-z+6-BA0.3mg/L+IBA0.15mg/L+PP333 0.1mg/L,所述的MS-z培养基每升中所含的组分及浓度mg/L如下:
大量元素:硝酸钾1900、硝酸铵412.5、二水氯化钙880、七水硫酸镁370、磷酸二氢钾170、七水硫酸亚铁27.8、乙二胺四乙酸二钠37.3;
微量元素:四水硫酸锰22.3、七水硫酸锌8.6、硼酸3.1、二水钼酸钠0.25、碘化钾0.83、五水硫酸铜0.025、六水氯化钴0.025;
有机物:肌醇100.0、甘氨酸2.0、盐酸吡哆醇0.5、烟酸0.5、盐酸硫胺素0.1;
其它:琼脂8000、蔗糖40000;pH 6.0。
2.根据权利要求1所述的一种扁桃斑鸠菊的简化组培快繁方法,其特征在于:步骤2)所述的外植体消毒,是指将步骤1)得到的外植体截成长1.2-1.5cm的带节小段,剪去叶片,只保留叶柄基部,流水冲洗20min备用;在超净工作台上,用75%酒精浸泡10s,0.1%升汞溶液振荡消毒8min,再用无菌水冲洗5次洗去残留药液,用无菌滤纸吸干水分后,切去茎段两端和叶柄上部少许,备用。
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