CN107242139B - 一种高山杜鹃‘粉金蝶’体细胞胚的诱导方法 - Google Patents
一种高山杜鹃‘粉金蝶’体细胞胚的诱导方法 Download PDFInfo
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Abstract
本发明属于观赏植物生物技术领域，涉及一种高山杜鹃‘粉金蝶’体细胞胚的发生方法。本发明利用高山杜鹃‘粉金蝶’的叶片诱导产生体细胞胚的主要步骤包括：将幼嫩叶片经消毒处理后接种到含wpm+0.2mg/LTDZ+2mg/L2ip+0.25mg/LNAA、附加蔗糖、琼脂的培养基中，暗培养后，转光照培养。本发明提供的观赏植物高山杜鹃‘粉金蝶’体细胞胚的诱导方法，为高山杜鹃遗传转化体系的建立、原生质体培养、优良无性系的繁殖、基因工程和/或突变体筛选等研究奠定基础。
Description
技术领域
本发明属于观赏植物生物技术领域，具体公开了一种利用高山杜鹃‘粉金蝶’的叶片诱导体细胞胚的方法。
背景技术
高山杜鹃(Rhododendron hybrides)的品种‘粉金蝶’(Cosmopolitan)为杜鹃科杜鹃属的杂交栽培品种，常绿灌木和小乔木，花粉红色，边缘褪为粉白色，带有一个褐色的大红斑，植株茂密，株型舒展，叶绿色光亮，耐寒-23℃不落叶，抗病力强，具极高的观赏价值。目前高山杜鹃‘粉金蝶’的研究主要集中在栽培技术和生理生化方面的研究，开展分子生物学方法得研究甚少，原因是缺乏其遗传转化体系的建立，在遗传转化体系的建立过程中，通过体细胞胚诱导达到植株再生是研究顺利开展的瓶颈。
本发明首次提出观赏植物高山杜鹃‘粉金蝶’体细胞胚的诱导方法，为高山杜鹃建立遗传转化体系、原生质体培养、优良无性系的繁殖、基因工程和突变体筛选等研究奠定基础。
发明内容
本发明目的包括：
提供一种高山杜鹃‘粉金蝶’体细胞胚的诱导方法；
提供一种高山杜鹃‘粉金蝶’体细胞胚的诱导方法的应用；
本发明公开了诱导获得高山杜鹃‘粉金蝶’体细胞胚的方法，包括以下步骤：
用消毒试剂对高山杜鹃‘粉金蝶’外植体灭菌后，将将高山杜鹃‘粉金蝶’外植体转接到合适培养基中进行培养，诱导产生体细胞胚。消毒处理前，可先采集高山杜鹃‘粉金蝶’外植体，所述外植体如叶片等，优选幼嫩叶片。
其中，消毒处理可选择性的包括前处理步骤，如使用洗涤灵刷洗外植体，用水冲洗干净。
本发明公开的高山杜鹃‘粉金蝶’的体细胞胚诱导方法的一种实施方式是，包括以下步骤的方法：
1)取高山杜鹃‘粉金蝶’植株的叶片，消毒处理；
2)将步骤1)处理后的叶片在培养基中避光培养后，再光照培养。
优选的，步骤1)所述叶片为幼嫩叶片；
步骤1)所述消毒处理方法为：以体积浓度50-99％的乙醇水溶液浸泡10-100秒后，无菌水冲洗，再用HgCl2溶液灭菌1-5分钟，无菌水冲洗；
步骤1)所述的消毒处理方法为：体积浓度75％的乙醇水溶液浸泡30秒后，无菌水冲洗，再用质量百分浓度0.1％的HgCl2溶液灭菌2-2.5分钟，无菌水冲洗。所述HgCl2溶液，例如可以是氯化汞的水溶液。
优选的，步骤2)所述的培养基含有：wpm、0.1-0.5mg/L的TDZ、0.1-0.5mg/L的2ip、0.1-0.5mg/L的NAA，以及10-50mg/L的蔗糖和1-10mg/L的琼脂，所述培养基的PH值为5-7；
进一步的，步骤2)所述的培养基含有：wpm、0.2mg/L的TDZ、2mg/L的2ip、0.25mg/LNAA、25mg/L的蔗糖和6.5mg/L的琼脂，以无菌水定容；所述培养基的PH值为5.4-5.6。
步骤2)所述培养，优选的，叶片在20-25℃的温度条件下，遮光培养5-10d后，在3000-4000Iux的强度下光照培养50-80d；
进一步的，步骤2)所述叶片在22±2℃温度条件下，遮光培养7d，再转入光照培养60d，光照强度为3500Iux。
本发明所述的高山杜鹃粉金蝶的体细胞胚诱导方法的一种实施方式是，包括以下步骤的方法：
1)将高山杜鹃‘粉金蝶’的幼嫩叶片以体积浓度75％的乙醇水溶液浸泡30秒后，无菌水冲洗3次，再用质量浓度0.1％的HgCl2溶液灭菌2-2.5分钟，无菌水冲洗3次；
2)将步骤1)处理后的叶片转接至培养基，在22±2℃温度条件下，遮光培养7d，再转入光照培养60d，光照强度为3500Iux；
所述培养基含wpm、0.2mg/L的TDZ、2mg/L的2ip、0.25mg/LNAA、25mg/L的蔗糖和6.5mg/L的琼脂，以无菌水定容；其中培养基的PH值为5.4-5.6。
此外，本发明还公开了以上任一所述的诱导方法在高山杜鹃‘粉金蝶’遗传转化体系建立、原生质体培养、优良无性系繁殖、基因工程和突变体筛选中的用途。
以上所述wpm，指母本植物培养基(Woody Plant medium)；
所述TDZ指thidiazuron，中文名为噻苯隆；
2ip指Riboside，中文名为异戊烯基腺嘌呤核苷；
NAA指α-naphthy-acetic-acid，中文名为α-萘乙酸。
有益效果
植物体细胞胚的培养成功与否是开展基因工程技术的前提。本发明首次提出观赏植物高山杜鹃‘粉金蝶’体细胞胚的诱导方法，为高山杜鹃遗传转化体系的建立、原生质体培养、优良无性系的繁殖、基因工程和突变体筛选等研究奠定基础。
附图说明
图1a、图1b、图1c为胚性愈伤组织诱导观察结果。
图2a、图2b、图2c、图2d为体细胞胚的形态学观察。
经显微镜观察体细胞胚的发育过程，表明经历了球形期，心形期，鱼雷期和子叶期。
具体实施方式
实施例一、借助组织培养的方法诱导高山杜鹃‘粉金蝶’叶片经过脱分化产生体细胞胚
步骤1：采集外植体
选择生长势良好的植株，剪取顶端的幼嫩叶片，准备消毒处理。
步骤2：消毒处理首先用牙刷蘸洗涤灵稀释液刷洗，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HgCl2处理2-3分钟，再用无菌水冲洗4-5次。
步骤3：培养基制备：
1升WPM基本培养基中含大量元素：NH4NO3 400mg；(NH4)2SO4 132mg；MgSO4.7H2O370mg；KH2PO4 170mg；KNO3 400mg；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；CuSO4.5H2O 0.025mg；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，添加蔗糖30g，琼脂6.5g，TDZ 0.2mg，2ip 2mg，NAA 0.25mg，完全溶解后，调整pH值5.4-5.6。
步骤4：体细胞胚培养
在培养温度22±2℃的条件下，暗培养7d后转入光照培养12h，光照强度3500Iux，培养时间60d。
图1a-图1b为胚性愈伤组织诱导观察结果；图2a-图2b为体细胞胚的形态学观察结果。体细胞胚的形态学观察结果表明，培养物经历了球形期，心形期，鱼雷期和子叶期，得到了高山杜鹃‘粉金蝶’的体细胞胚。
Claims (1)
1.一种高山杜鹃‘粉金蝶’的体细胞胚诱导方法，包括以下步骤：
1)将高山杜鹃‘粉金蝶’的幼嫩叶片以体积浓度75％的乙醇水溶液浸泡30秒后，无菌水冲洗3次，再用质量浓度0.1％的HgCl2溶液灭菌2-2.5分钟，无菌水冲洗3次；
2)将步骤1)处理后的叶片转接至培养基，在22±2℃温度条件下，避光培养7d，再转入光照培养60d，光照强度3500Lux；所述培养基为WPM+0.2mg/L TDZ+2mg/L2ip+0.25mg/LNAA+30g/L蔗糖+6.5g/L琼脂，以无菌水定容；其中培养基的pH值为5.4-5.6。
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