CN105409768B - 花叶良姜愈伤再生体系建立的方法 - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了花叶良姜愈伤组织再生体系建立的方法,以种子为外植体,经外植体灭菌、无菌萌发、愈伤组织的诱导、不定芽的分化和生根培养等步骤,建立其高频高效的再生体系,污染率控制在5%以下,萌发率达79.59%,诱导率达88.46%,增殖率达10.07倍,生根率达98.85%。本发明可为规模化、周年生产种苗提供一种新、高效的方法,同时为育种和遗传转化等基因工程研究奠定基础。
Description
技术领域
本发明属于园林科学中观赏植物组培快繁技术,具体是花叶良姜愈伤组织再生体系建立的方法。
背景技术
花叶良姜(学名:Alpinia zerumbet‘Variegata’)又称花叶艳山姜、彩叶姜、斑纹月桃等,是姜科山姜属多年生草本植物。花叶良姜叶色艳丽,花姿优美,花香清纯,是非常有观赏价值的观叶观花植物,是园林绿化工程不可或缺的素材。随着“美丽中国”的建设,花叶良姜在园林中的应用日益广泛、工程用量日益增多。传统分株繁殖的方法存在增殖系数低的问题,不是高效的繁殖方法,不能满足市场的需求。所以,需要研究开发更加高效的繁殖方法。
目前,花叶良姜的组织培养主要利用根状茎和茎尖诱导丛生芽、以芽繁芽的方法进行繁殖,仍存在增殖系数较低的问题。如潘梅.花叶艳山姜茎尖离体快繁技术花叶艳山姜茎尖离体快繁技术[J].热带生物学报,2012(3):267-270,以茎尖为外植体诱导丛生芽,增殖倍数为4.0,生根率为93%;秦丽凤,文清岚,宋西娟.花叶良姜组培生产与栽培管理技术[J].南方园艺2013,24(5),以花叶良姜块茎上开始萌发的芽为外植体,经过诱导培养基培养得到丛生芽,再依次经过继代增殖、诱导生根培养得到花叶良姜组培苗,增殖倍数为3.8倍,生根率为100%;陈玉梅.花叶良姜组培快繁体系建立的探讨[J].中国科技信息,2014(12):51-52,以花叶良姜根状茎为组培外植体,经过初代培养后得到无菌芽,把芽再进行继代增殖,得到的继代再经过生根诱导培养,最终得到花叶良姜组培苗,此方法的增殖系数最高为7.03,有效苗率(苗生根率)为62.13%。但是,未见关于花叶良姜通过诱导愈伤组织和分化不定芽的途径进行植株再生的研究报道。
发明内容
本发明的目的是提供花叶良姜愈伤组织再生体系建立的方法,克服现有组培育苗技术增殖系数低的问题,提高组培育苗增殖系数,降低育苗成本,满足市场需要。
本发明解决现有技术问题的技术方案如下:
花叶良姜愈伤组织再生体系建立的方法,包括以下步骤:
1)外植体选择及预处理:选择成熟、饱满的当年种子作为外植体,用800倍高锰酸钾浸种20min后清水冲洗10min,用浓硫酸进行预处理5min后清水冲洗30min;
2)无菌萌发:将预处理的种子用75%乙醇处理1min后无菌水冲洗3次,0.1%升汞处理10min后无菌水冲洗5次,5%次氯酸钠处理3min后无菌水冲洗5次,接种于萌芽培养基M0,30天后统计污染率和萌发率;
3)愈伤组织诱导:当植株高1~2cm将根切除,接种于愈伤组织培养基M1进行愈伤组织诱导,30天后统计诱导率;
4)不定芽分化:将愈伤组织接种于不定芽分化培养基M2进行不定芽诱导,30天后统计增殖率;
5)生根培养:当不定芽高3~4cm时,接种与生根培养基M3进行生根培养,30天后统计生根率。
所述的萌芽培养基M0,其成分是:K2SO4800mg/L,MgSO4·7H2O 350mg/L,KH2PO4180mg/L,NH4NO3600mg/ml,MnSO4·4H2O 22.5mg/L,Ca(NO3)2·4H2O 556mg/L,FeSO4·7H2O 27.8mg/L,Na2EDTA 37.3mg/L,ZnSO4·7H2O 8.6mg/L,H3BO36.2mg/L,CuSO4·5H2O0.25mg/L,Na2MoO4·2H2O 0.25mg/L,肌醇100mg/L,甘氨酸2.0mg/ml,维生素B11.0mg/L,维生素B60.5mg/L,烟酸0.5mg/L。
所述的愈伤组织诱导培养基M1,其成分是:M0+1~2mg/L苯噻隆+0.5~1mg/L 2,4-二氯苯氧乙酸。
所述的不定芽分化培养基M2,其成分是:M0+1~2mg/L苯噻隆+0.05~0.1mg/L 2,4-二氯苯氧乙酸。
所述的生根培养基M3,其成分是:1/2M0+0.5~1mg/L ABT1号生根粉,所述的ABT1号生根粉是ABT生根粉系列之一,是中国林业科学研究院研制成功的一种新型广谱高效植物生长调节剂,市面上有售。ABT1号生根粉,主要用于难生根植物及珍贵植物的扦插育苗,如红松、泡桐、银杏、金花茶、苹果、柑桔、龙眼、荔枝、玉兰等。
与现有的技术相比,本发明具有如下优点:
1.以茎尖和根状茎诱导丛芽的增殖率最高达到7倍,而通过愈伤组织诱导不定芽的增殖率高达10倍,提高增殖率,提高效率,降低成本。
2.外植体选取容易、灭菌效果好。
3.发明了萌发培养基、愈伤组织诱导培养基、不定芽分化培养基和生根培养基,提供一种新的植株再生的方法。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
2014年9月15日,选择成熟、饱满的种子作为外植体,用清水漂洗,除去漂浮于水面的种子,挑选100粒种子备用。用800倍高锰酸钾浸种20min后清水冲洗10min,用浓硫酸进行预处理5min后清水冲洗30min。将预处理的种子用75%乙醇处理1min后无菌水冲洗3次,0.1%升汞处理10min后无菌水冲洗5次,5%次氯酸钠处理3min后无菌水冲洗5次,接种于萌芽培养基M0。30天后,外植体污染5粒,污染率为5%,萌芽72粒,萌发率达75.79%。植株高1-2cm时将根切除,接种于含M0+1mg/L苯噻隆+0.5mg/L 2,4-二氯苯氧乙酸的培养基进行愈伤组织诱导。30天后,62株产生愈伤组织,诱导率达86.11%。将愈伤组织接种于含M0+1mg/L苯噻隆+0.05mg/L 2,4-二氯苯氧乙酸的培养基进行不定芽诱导。30天后,愈伤组织产生不定芽共496个,平均为8个,增殖率达8倍。当不定芽高3-4cm时,接种于含1/2M0+0.5mg/L ABT1号的培养基进行生根培养,30天后,472株生根,长出3条根,根长2cm,生根率达95.16%。
实施例2
2014年9月30日,选择成熟、饱满的种子作为外植体,用清水漂洗,除去漂浮于水面的种子,挑选100粒种子备用。用800倍高锰酸钾浸种20min后清水冲洗10min,用浓硫酸进行预处理5min后清水冲洗30min。将预处理的种子用75%乙醇处理1min后无菌水冲洗3次,0.1%升汞处理10min后无菌水冲洗5次,5%次氯酸钠处理3min后无菌水冲洗5次,接种于萌芽培养基M0。30天后,外植体污染3粒,污染率为3%,萌芽75粒,萌发率达77.32%。植株高1-2cm时将根切除,接种于含M0+2mg/L苯噻隆+0.8mg/L 2,4-二氯苯氧乙酸的培养基进行愈伤组织诱导。30天后,64株产生愈伤组织,诱导率达85.33%。将愈伤组织接种于含M0+2mg/L苯噻隆+0.07mg/L 2,4-二氯苯氧乙酸的培养基进行不定芽诱导。30天后,愈伤组织产生不定芽共556个,平均为8.7个,增殖率达8.7倍。当不定芽高3-4cm时,接种于含1/2M0+0.8mg/LABT1号的培养基进行生根培养。30天后,541株生根,长出4条根,根长2cm,生根率达97.30%。
实施例3
2014年10月15日,选择成熟、饱满的种子作为外植体,用清水漂洗,除去漂浮于水面的种子,挑选100粒种子备用。用800倍高锰酸钾浸种20min后清水冲洗10min,用浓硫酸进行预处理5min后清水冲洗30min。将预处理的种子用75%乙醇处理1min后无菌水冲洗3次,0.1%升汞处理10min后无菌水冲洗5次,5%次氯酸钠处理3min后无菌水冲洗5次,接种于萌芽培养基M0。30天后,外植体污染2粒,污染率为2%,萌芽78粒,萌发率达79.59%。植株高1-2cm时将根切除,接种于含M0+1.5mg/L苯噻隆+1mg/L 2,4-二氯苯氧乙酸的培养基进行愈伤组织诱导。30天后,69株产生愈伤组织,诱导率达88.46%。将愈伤组织接种于含M0+1.5mg/L苯噻隆+0.1mg/L 2,4-二氯苯氧乙酸的培养基进行不定芽诱导。30天后,愈伤组织产生不定芽共695个,平均为10.07个,增殖率达10.07倍。当不定芽高3-4cm时,接种于含1/2M0+1mg/LABT1号的培养基进行生根培养。30天后,687株生根,长出4条根,根长3cm,生根率达98.85%。
Claims (1)
1.花叶良姜愈伤组织再生体系建立的方法,其特征在于包括以下步骤:
1)外植体选择及预处理:选取健壮、无病虫害、成熟植株上饱满的种子作为外植体,用800倍高锰酸钾浸种20min后清水冲洗10min,用浓硫酸进行预处理5min后清水冲洗30min;
2)无菌萌发:将预处理的种子用75%乙醇处理1min后无菌水冲洗3次,0.1%升汞处理10min后无菌水冲洗5次,5%次氯酸钠处理3min后无菌水冲洗5次,接种于萌芽培养基M0;所述萌芽培养基M0的成分:K2SO4 800mg/L,MgSO4·7H2O 350mg/L,KH2PO4 180mg/L,NH4NO3600mg/L,MnSO4·4H2O 22.5mg/L,Ca(NO3)2·4H2O 556mg/L,FeSO4·7H2O 27.8mg/L,Na2EDTA37.3mg/L,ZnSO4·7H2O 8.6mg/L,H3BO3 6.2mg/L,CuSO4·5H2O 0.25mg/L,Na2MoO4·2H2O0.25mg/L,肌醇100mg/L,甘氨酸2.0mg/L,维生素B1 1.0mg/L,维生素B6 0.5mg/L,烟酸0.5mg/L;
3)愈伤组织诱导:当植株高1~2cm时将根切除,接种于愈伤组织培养基M1进行愈伤组织诱导;所述愈伤组织培养基M1的成分:M0+1~2mg/L苯噻隆+0.5~1mg/L 2,4-二氯苯氧乙酸;
4)不定芽分化:将愈伤组织接种于不定芽分化培养基M2进行不定芽诱导;所述不定芽分化培养基M2的成分:M0+1~2mg/L苯噻隆+0.05~0.1mg/L 2,4-二氯苯氧乙酸;
5)生根培养:当不定芽高3~4cm时,接种于生根培养基M3进行生根培养,所述生根培养基M3的成分:1/2M0+0.5~1mg/L ABT1号生根粉。
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