CN107410032B - 一种温郁金组织培养再生植株的方法 - Google Patents
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Abstract
本发明公开了一种温郁金组织培养再生植株的方法,所述方法为:将消毒后的外植体接种至丛生芽培养基中,在20~25℃,光照1000~1200lux条件下培养,获得丛生芽;将丛生芽切割为带顶芽的个体后,接种至生根培养基,在20~25℃,光照1000~1200lux条件下培养,形成带根的完整植株;本发明方法外植体培养过程中无任何污染、克服了以前报道的温郁金培养容易污染的严重问题。形成丛生芽和再生完整植株的过程快速高效。外植体培养25‑30天即可得到大量的芽生丛,丛生芽切割后在生根培养基上15‑20天,即可到达含粗壮根系的完整植株。
Description
(一)技术领域
本发明涉及一种温郁金组织培养再生植株的方法。
(二)背景技术
温郁金(Curcuma wenyujin Y.H.Chen et C.Ling),别名温莪术、黑郁金,主要产于浙江温州瑞安一带,为姜科植物,其块根是著名的“浙八味”药材之一。温郁金作为浙江的道地药材,其性味辛、苦、温,归肝、脾经,具行气破血、消积止痛功效。现代药理研究表明,温郁金具有抗肿瘤、抗血栓、抗菌、抗病毒作用,对呼吸系统、消化系统、泌尿生殖系统、循环系统和皮肤溃疡都有较好的疗效。由于其药用功能非常广泛,在中医临床治疗中广泛应用。
温郁金主要以根茎繁殖,易感染病毒,且受到多种病虫害的危害。由于其长期的无性繁殖,导致种群退化,病毒化严重,严重影响产量和品质,难以得到安全、有效、稳定、可控的温郁金药材。为了提纯复壮温郁金资源,快速繁殖获得良种,因此,通过植物组织培养技术建立温郁金的快速高效的繁殖体系非常必要。目前虽然有个别报道,利用组织培养获得了温郁金的再生植株,但均存在培养过程中外植体污染严重,且再生植株频率低等问题。
(三)发明内容
本发明目的是提供一种无污染的、高效的快速繁殖温郁金的方法,克服了目前温郁金组织培养污染严重、再生频率低、再生过程时间长等问题,在生产实践中有利于温郁金的品种改良。
本发明采用的技术方案是:
本发明提供一种温郁金组织培养再生植株的方法,所述方法为:(1)外植体的获取及诱导培养:选取长势良好、无病虫害的温郁金外植体消毒,获得消毒后的外植体;将消毒后的外植体接种至丛生芽培养基中,在20~25℃,光照1000~1200lux条件下培养,获得丛生芽;所述丛生芽培养基为含6-BA(6-苄基腺嘌呤)0.25~2.5mg/L+IAA(吲哚乙酸)0.25~2.5mg/L+ZT(玉米素)0.25~2.5mg/L+Cef(头孢霉素)100-1000mg/L的MS基本培养基;(2)生根培养:将步骤(1)获得的丛生芽切割为带顶芽的个体后,接种至生根培养基,在20~25℃,光照1000~1200lux条件下培养15-20天,形成带根的完整植株;所述生根培养基为含6-BA0.25~2.5mg/L+0.25~2.5mg/L IAA的MS基本培养基。
进一步,步骤(1)所述外植体为下列之一:温郁金块根、温郁金块根发芽的芽尖段、温郁金实生苗的茎段或顶端的幼叶。
进一步,步骤(1)所述外植体为温郁金实生苗的芽尖段。
进一步,所述芽尖段按如下方法获取:取健壮的块根,埋入用体积比4:1的泥炭和蛭石混合而成的培养土中,保持培养土湿润,20-25℃条件下30-40天,待块根长出1-1.5cm长的芽尖,从离块根0.1-0.2cm处剪取芽尖,即为温郁金实生苗的芽尖段。
进一步,步骤(1)所述消毒方法为:先将外植体用含体积浓度0.1%洗洁精的自来水清洗液浸泡10分钟,再用自来水漂洗3次,最后在消毒液中浸泡5-50min,消毒后的外植体用无菌的双蒸馏水冲洗3次;所述消毒液为质量浓度0.1%升汞水溶液、质量浓度3%双氧水或质量浓度6%次氯酸钠水溶液(即稀释5倍的安替福民)。
进一步,步骤(1)所述丛生芽培养基为含6-BA (6-苄基腺嘌呤)1~2.5mg/L+IAA(吲哚乙酸)0.5~1.5mg/L+ZT(玉米素)0.5~1.5mg/L+Cef(头孢霉素)400-600mg/L的MS基本培养基;更优选所述丛生芽培养基为含6-BA 2mg/L+ZT 1mg/L+IAA 1mg/L+Cef 500mg/L的MS基本培养基。
进一步,步骤(2)所述生根培养基为含6-BA 0.3~1.0mg/L+0.3~1.0mg/L IAA的MS基本培养基,更优选所述生根培养基为含0.5mg/L IAA+0.5mg/L 6-BA的MS基本培养基。
进一步,所述MS基本培养基终浓度组成为:NH4NO3 1.65g/L、KNO3 1.9g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O 0.37g/L、KH2PO4 0.17g/L、KI 0.83mg/L、H3BO35.2mg/L、MgSO4·7H2O 22.3mg/L、ZnSO4·7H2O 3.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2EDTA 37.3mg/L、FeSO4·7H2O27.8mg/L、肌醇100mg/L、甘氨酸2mg/L、盐酸硫胺素0.1mg/L、盐酸吡哆醇0.5mg/L、烟酸0.5mg/L、蔗糖30g/L、琼脂粉10g/L,溶剂为水,pH为5.6~6.0。
与现有技术相比,本发明有益效果主要体现在:(1)外植体培养过程中无任何污染、克服了以前报道的温郁金培养容易污染的严重问题。(2)形成丛生芽和再生完整植株的过程快速高效。外植体培养25-30天即可得到大量的芽生丛,丛生芽切割后在生根培养基上15-20天,即可到达含粗壮根系的完整植株。
(四)附图说明
图1外植体芽尖段接种于MS+6-BA 2mg/L+ZT 1mg/L+IAA 1mg/L+Cef 500mg/L培养基中分化出的小芽。
图2外植体芽尖段在MS+6-BA 2mg/L+ZT 1mg/L+IAA 1mg/L+Cef 500mg/L培养基中培养25天后形成的丛生芽。
图3移栽在含培养土的盆钵中成活的再生植株(移栽后10天)。
图4移栽在含培养土的盆钵中成活的再生植株(移栽后30天)。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
本发明所述MS基本培养基终浓度组成为:NH4NO3 1.65g/L、KNO3 1.9g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O 0.37g/L、KH2PO4 0.17g/L、KI 0.83mg/L、H3BO35.2mg/L、MgSO4·7H2O 22.3mg/L、ZnSO4·7H2O 3.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2EDTA 37.3mg/L、FeSO4·7H2O27.8mg/L、肌醇100mg/L、甘氨酸2mg/L、盐酸硫胺素0.1mg/L、盐酸吡哆醇0.5mg/L、烟酸0.5mg/L、蔗糖30g/L、琼脂粉10g/L,溶剂为水,pH为5.6~6.0。
实施例1:
1、外植体的选择:
利用健壮的温郁金块根,埋入用泥炭:蛭石=4:1(v/v)配比配制的培养土中,保持培养土湿润,20-25℃条件下30-40天,待块根长出1-1.5cm长的芽尖,从离块根0.1-0.2cm处剪取芽尖作为芽尖段为外植体。
再取温郁金块根及田间生长的温郁金实生苗的茎段、顶端的幼叶为外植体。
2、外植体的消毒
每种外植体取下来后,先用含少量洗洁精的自来水清洗液(洗洁精/自来水(v/v)=1/1000)浸泡10分钟,再用自来水漂洗3次,最后转入不同的消毒液中消毒(表1,稀释5倍安替福民即为质量浓度6%次氯酸钠水溶液)。消毒后的外植体用无菌的双蒸馏水冲洗3次,转入MS+6-BA 2mg/L+ZT 1mg/L+IAA1mg/L的培养基中,每种消毒方法外植体接种20个,20-25℃,光照1000~1200lux,12小时光照/天,观察外植体污染情况,结果见表1。
表1不同消毒方法外植体污染情况
3、外植体的消毒方法对丛生芽的影响
将步骤2中各个方法消毒后无污染的外植体接种在MS+6-BA 2mg/L+ZT 1mg/L+IAA1mg/L的培养基中,20-25℃,光照1000~1200lux,12小时光照/天。20天后,统计培养情况见表2。
表2不同消毒方法以及不同外植体再分化情况
根据表1和表2的结果可知,只有0.1%升汞10min和15min消毒的芽尖段才能分化出丛生芽。其中,0.1%升汞10min灭菌,分化丛生芽频率最高。为此,选择在0.1%升汞10min的灭菌方法,利用芽尖段作为外植体,做进一步优化实验。
4、芽尖段不同部位以及在添加头孢霉素培养基上的污染和再分化情况
利用健壮的块根,埋入用泥炭:蛭石=4:1(v/v)配比配制的培养土中,保持培养土湿润,20-25℃条件下30-40天,待块根长出1-1.5cm长的芽尖,从离块根0.1-0.2cm处剪取芽尖。先用含少量洗洁精的自来水清洗液(洗洁精:自来水=1:1000(v/v))浸泡10分钟,再用自来水漂洗3次。芽尖的顶端向下放入2mL的平底冻存管中,用加样器加入1.8mL 0.1%的升汞,盖严瓶盖,期间每间隔2分钟,颠倒冻存管数次。10min后,用加样器吸除消毒液0.1%的升汞。随后每次加入1.8mL ddH2O漂洗5次,再用剪子将芽尖剪成长均等的2段。接种于MS+6-BA 2mg/L+ZT 1mg/L+IAA 1mg/L+Cef(头孢霉素)500mg/L的丛生芽培养基中,在20-25℃,光照1000~1200lux,12小时光照/天下培养。结果显示,在接种的100个外植体中,全部无污染;而且茎尖段靠基部的区段,3-5天即可见芽段有萌动(图1),在15-20天即可见分化出小芽丛,且所有接种的外植体均能全部再分化出芽丛;但茎尖的顶端只生长,不能分化出芽生丛。外植体培养25-30天后,每个外植体均可分化出大量的丛生芽(图2)。将再生的丛生芽切割后,转入MS+6-BA 0.5mg/L+IAA 0.5mg/L的生根培养基中,在20-25℃,光照1000~1200lux,12小时光照/天下培养。丛生芽在生长过程中无任何污染出现,且生长旺盛。再经过15-20天,即可到达含粗壮根系的完整植株。洗除植株根的基部培养基,移栽用泥炭:蛭石=4:1(v/v)配比配制的培养土中,18-28℃、散射光下10天后(图3),转入室外在自然条件下生长,即获得健壮的实生苗(图4)。
利用本发明的方法,能够实现温郁金外植体培养过程中无任何污染出现,在50天内即可获得完整的再生植株,在60天内即可获得成活的实生苗。
Claims (5)
1.一种温郁金组织培养再生植株的方法,其特征在于所述方法为:(1)外植体的获取及诱导培养:选取长势良好、无病虫害的温郁金外植体消毒,获得消毒后的外植体;将消毒后的外植体接种至丛生芽培养基中,在20~25℃,光照1000~1200lux条件下培养,获得丛生芽;所述丛生芽培养基为含6-BA 0.25~2.5mg/L+IAA 0.25~2.5mg/L+ZT 0.25~2.5mg/L+Cef 100-1000mg/L的MS基本培养基;所述外植体为温郁金块根发芽的芽尖段,所述芽尖段按如下方法获取:取健壮的块根,埋入用体积比4:1的泥炭和蛭石混合而成的培养土中,保持培养土湿润,20-25℃条件下30-40天,待块根长出1-1.5cm长的芽尖,从离块根0.1-0.2cm处剪取芽尖,剪成均等的两段,取茎尖段靠基部的区段即为温郁金块根发芽的芽尖段;(2)生根培养:将步骤(1)获得的丛生芽切割为带顶芽的个体后,接种至生根培养基,在20~25℃,光照1000~1200lux条件下培养,形成带根的完整植株;所述生根培养基为含6-BA 0.25~2.5mg/L+0.25~2.5mg/L IAA的MS基本培养基。
2.如权利要求1所述温郁金组织培养再生植株的方法,其特征在于步骤(1)所述消毒方法为:先将外植体用含体积浓度0.1%洗洁精的自来水清洗液浸泡10分钟,再用自来水漂洗3次,最后在消毒液中浸泡5-50min,消毒后的外植体用无菌的双蒸馏水冲洗3次;所述消毒液为质量浓度0.1%升汞水溶液、质量浓度3%双氧水溶液或质量浓度6%次氯酸钠水溶液。
3.如权利要求1所述温郁金组织培养再生植株的方法,其特征在于步骤(1)所述丛生芽培养基为含6-BA 2mg/L+ZT 1mg/L+IAA 1mg/L+Cef 500mg/L的MS基本培养基。
4.如权利要求1所述温郁金组织培养再生植株的方法,其特征在于步骤(2)所述生根培养基为含0.5mg/L IAA+0.5mg/L 6-BA的MS基本培养基。
5.如权利要求1所述温郁金组织培养再生植株的方法,其特征在于所述MS基本培养基终浓度组成为:NH4NO3 1.65g/L、KNO3 1.9g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O 0.37g/L、KH2PO4 0.17g/L、KI 0.83mg/L、H3BO3 5.2mg/L、MgSO4·7H2O 22.3mg/L、ZnSO4·7H2O3.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L、肌醇100mg/L、甘氨酸2mg/L、盐酸硫胺素0.1mg/L、盐酸吡哆醇0.5mg/L、烟酸0.5mg/L、蔗糖30g/L、琼脂粉10g/L,溶剂为水,pH为5.6~6.0。
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