CN105850737B - 一种高山杜鹃“红粉佳人”染色体加倍方法 - Google Patents
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Abstract
本发明属于生物育种领域，专用于高山杜鹃“红粉佳人”染色体倍性育种，特别是涉及一种高山杜鹃“红粉佳人”染色体加倍方法，利用高山杜鹃“红粉佳人”外植体诱导产生愈伤组织，用化学试剂秋水仙素处理愈伤组织使染色体加倍，再转接到分化培养基中进行培养，诱导不定芽产生，将不定芽转接到增殖培养基中进行增殖培养，再将壮苗接种到生根培养基中进行生根培养、炼苗移栽，在显微镜下观察染色体，筛选出加倍后的植株种苗。本发明首次提出观赏植物高山杜鹃“红粉佳人”染色体加倍的方法。本发明培育出的多倍体高山杜鹃“红粉佳人”，有效提高高山杜鹃“红粉佳人”的观赏价值。
Description
技术领域
本发明属于生物育种领域，专用于高山杜鹃“红粉佳人”染色体倍性育种，特别是涉及一种一种高山杜鹃“红粉佳人”染色体加倍方法。
背景技术
高山杜鹃“红粉佳人”(Rhododendron Delavayi“Cosmopolitan”)杜鹃科杜鹃属，原产于我国中西部地区，常绿灌木和小乔木，外粉红内白的复色品种，植株茂密，株型舒展，叶绿色光亮。多倍体育种是一种有效的育种手段，也是植物进化的重要途径之一。“巨大型”是多倍体最显著的外部形态特征，表现出茎干粗壮、叶片宽厚、叶面粗糙、叶色加深、花冠大而厚实、花瓣增多、颜色浓艳，此外，还具有延迟某些花卉种类的花期、抗逆性增强等优点，提高花卉的观赏价值和商品价值。随着市场对该品种需求越来越多，需要进一步对高山杜鹃植物种质资源进行创新及新品种培育。
发明内容
针对上述技术问题，本发明的目的提供一种高山杜鹃“红粉佳人”染色体加倍的技术方法，为实现发明的目的，本发明提供以下技术方案：
一种高山杜鹃“红粉佳人”染色体加倍方法，包括以下步骤：
(1)将高山杜鹃“红粉佳人”外植体消毒后转接到愈伤组织培养基中进行培养，诱导产生愈伤组织；
(2)使用化学试剂秋水仙素处理愈伤组织，实现染色体加倍；
(3)将经过化学试剂秋水仙素处理愈伤组织转接到不定芽培养基中进行培养；
(4)将分化出的不定芽转接到增殖培养基中进行增殖培养；
(5)经过增殖培养的健壮良好组培苗接种到生根培养基中进行生根培养；
(6)选择根系长到2-3cm的生根苗进行炼苗，用草炭土和珍珠岩配成的混合基质；
(7)炼苗移栽后在显微镜下进行染色体数目观察，筛选出染色体加倍后的植株苗。
步骤(1)中所述的外植体消毒的方法：用质量浓度75％的酒精处理外植体30S，用无菌水冲洗4-5遍，再用质量浓度0.1％的氯化汞处理1分钟；
所述的愈伤组织培养基，由以下方法制备所得：230ml的WPM基本培养基中添加蔗糖30g、琼脂粉6.5g、2-ip 6mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，暗培养，培养时间30天。
步骤(2)具体过程为：愈伤组织经过质量浓度0.05％的秋水仙素处理24h，用无菌水冲洗4次；
步骤(3)中所述的不定芽培养基，由以下方法制备所得：230ml的WPM基本培养基添加蔗糖30g、琼脂6.5g、TDZ 0.1mg、2-ip 1mg，定容水至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间45天。
步骤(4)中所述的增殖培养基，由以下方法制备所得：230ml的WPM基本培养基中添加蔗糖30g、琼脂粉6.5g、2-ip 3mg、NAA 0.5mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间60天。
步骤(5)生根培养基，由以下方法制备所得：230ml的WPM培养基中大量元素减一半，即其中WPM培养基中NH4NO3、(NH4)2SO4、MgSO4.7H2O、KH2PO4、KNO3、K2SO4量减少一半，添加蔗糖20g、琼脂粉6.5g、活性炭1g、IBA0.8mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间90天。
步骤(6)中所述的混合基质，包括重量比为5:1的草炭土和珍珠岩。
步骤(7)具体的过程为：选择植株的茎尖和根尖，做好切片，在显微镜下观察染色体加倍情况。
本发明提供的一种高山杜鹃“红粉佳人”染色体加倍方法，用化学物质秋水仙素处理高山杜鹃“红粉佳人”愈伤组织从而诱导多倍体，对高山杜鹃植物种质资源进行创新，用于进一步培养新品种，提高观赏价值。
具体实施方式
结合实施例说明本发明的技术方案，应用化学物质秋水仙素处理高山杜鹃“红粉佳人”愈伤组织从而诱导多倍体。
其中，1升WPM基本培养基成分为：
大量元素：NH4NO3 400mg；(NH4)2SO4 132mg；MgSO4.7H2O 370mg；KH2PO4170mg；KNO3400mg；K2SO4 900mg；
钙盐：CaCl2 96mg；Ca(NO3)2·4H2O 556mg；
微量元素：H3BO3 6.2mg；MnSO4.H2O 22.3mg；Na2MoO4·2H2O 0.25mg；CuSO4.5H2O0.025mg；ZnSO4·7H2O 8.6mg；
铁盐：Na2-EDTA 37.3mg；FeSO4.7H2O 27.8mg；
维生素：肌醇100mg；烟酸(维生素PP)0.5mg；盐酸吡多醇0.5mg；盐酸硫胺素0.5mg；甘氨酸2mg。
具体方法如下：
(1)采集外植体
在春天嫩梢生长积极，天气晴朗上午，选择生长势良好的高山杜鹃“红粉佳人”植株，剪取顶端的幼嫩组织和顶芽，取外植体30个，剥去顶芽部分包被的鳞片，准备消毒处理。
(2)消毒处理
首先每个外植体用牙刷蘸洗涤灵稀释液刷洗一遍，然后用自来水冲洗30分钟，在超净工作台上用75％酒精处理30S，用无菌水冲洗4-5次，0.1％的氯化汞处理1分钟，再用无菌水冲洗4-5遍。
(3)外植体诱导愈伤组织
将消毒后的外植体接种到愈伤组织培养基中，以230ml的WPM为基本培养基添加蔗糖30g，琼脂粉6.5g，2-ip 2mg，NAA 0.5mg，纯净水定容到1升，调整pH值至5.4，培养温度22±2℃，暗培养，培养时间30天。
(4)秋水仙素处理愈伤组织
在无菌环境下，将愈伤组织浸泡在0.05％的秋水仙素处理24h，用无菌水冲洗4次。
(5)愈伤组织经过再分化
选择质地呈颗粒状，颜色发红色的愈伤组织，转接到再分化培养基中。230ml的培养基为WPM基本培养基添加蔗糖30g，琼脂6.5g，TDZ 0.1mg，2-ip 1mg，定容水至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间45天。
(6)增殖培养
由愈伤组织经过再分化生长的芽，转接到增殖培养基，230ml的培养基以WPM为基本培养基添加蔗糖30g，琼脂6.5g，NAA 0.5mg，2-ip 3mg，定容水至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间60天。
(7)生根培养
经过增殖培养后，选择2-3cm长的芽苗转接到生根培养基中，230ml的培养基，其中WPM培养基中NH4NO3、(NH4)2SO4、MgSO4.7H2O、KH2PO4、KNO3、K2SO4量减少一半，添加蔗糖20g，琼脂6.5g，活性炭1g，IBA 0.8mg，IAA 0.3mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Iux，培养时间90天。
(8)炼苗移栽
在生根培养基中根长到2-3cm时，即可炼苗移栽。移栽前先将瓶苗置于温室中驯化7天左右，打开瓶盖放置3天，用镊子将苗轻轻取出，用温水洗净培养基后用1％的多菌灵浸泡4分钟，栽入育苗穴盘中，移栽组培苗基质比例为草炭土：珍珠岩为5:1，浇透水，用塑料薄膜遮阳3-5天。
(9)染色体检测
选取经过秋水仙素处理过高山杜鹃“红粉佳人”植株的茎尖和根尖，通过显微镜观察染色体数目，筛选加倍的植株。
Claims (3)
1.一种高山杜鹃“红粉佳人”染色体加倍方法，其特征在于，包括以下步骤：
（1）将高山杜鹃“红粉佳人”外植体消毒后转接到愈伤组织培养基中进行培养，诱导产生愈伤组织；
所述的外植体消毒的方法：用质量浓度75%的酒精处理外植体30S，用无菌水冲洗4-5遍，再用质量浓度0.1%的氯化汞处理1分钟；
所述的愈伤组织培养基，由以下方法制备所得：230ml的WPM基本培养基中添加蔗糖30g、琼脂粉6.5g、2-ip 6mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，暗培养，培养时间30天；
其中1升WPM基本培养基成分为：
大量元素：NH4NO3 400mg；(NH4)2SO4 132mg；MgSO4·7H2O 370mg；KH2PO4 170mg；KNO3400mg；K2SO4 900mg；
钙盐：CaCl2 96mg；Ca(NO3)2·4H2O 556mg；
微量元素：H3BO3 6.2mg；MnSO4·H2O 22.3mg；Na2MoO4·2H2O 0.25mg；CuSO4·5H2O0.025mg；ZnSO4·7H2O 8.6mg；
铁盐：Na2-EDTA 37.3mg；FeSO4·7H2O 27.8mg；
维生素：肌醇100mg；烟酸0.5mg；盐酸吡多醇0.5mg；盐酸硫胺素0.5mg；甘氨酸2mg；
（2）使用化学试剂秋水仙素处理愈伤组织，实现染色体加倍；
具体过程为：愈伤组织经过质量浓度0.05%的秋水仙素处理24h，用无菌水冲洗4次；
（3）选择质地呈颗粒状，颜色发红色的愈伤组织，转接到不定芽培养基中进行培养；
所述的不定芽培养基，由以下方法制备所得：230ml的WPM基本培养基添加蔗糖30g、琼脂6.5g、TDZ 0.1mg、2-ip 1mg，定容水至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Lux，培养时间45天；
（4）将分化出的不定芽转接到增殖培养基中进行增殖培养；
所述的增殖培养基，由以下方法制备所得：230ml的WPM基本培养基中添加蔗糖30g、琼脂粉6.5g、2-ip 3mg、NAA 0.5mg，用纯净水定容至1升，调整pH值5.4，培养温度22±2℃，光照强度3500Lux，培养时间60天；
（5）经过增殖培养的健壮良好组培苗接种到生根培养基中进行生根培养；
所述的生根培养基，由以下方法制备所得：230ml的WPM基本培养基中，添加蔗糖20g、琼脂粉6.5g、活性炭1g、IBA 0.8mg，用纯净水定容至1升，调整pH值5.4；其中WPM培养基中NH4NO3、(NH4)2SO4、MgSO4、7H2O、KH2PO4、KNO3、K2SO4量减少一半；培养温度22±2℃，光照强度3500Lux，培养时间90天；
（6）选择根系长到2-3cm的生根苗进行炼苗，用草炭土和珍珠岩配成的混合基质；
（7）炼苗移栽后在显微镜下进行染色体数目观察，筛选出染色体加倍后的植株苗。
2.根据权利要求1中所述的一种高山杜鹃“红粉佳人”染色体加倍方法，其特征在于，步骤（6）中所述的混合基质，包括重量比为5:1的草炭土和珍珠岩。
3.根据权利要求1中所述的一种高山杜鹃“红粉佳人”染色体加倍方法，其特征在于，步骤（7）具体的过程为：选择植株的茎尖和根尖，做好切片，在显微镜下观察染色体加倍情况。
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