CN110268978A - 一种利用二甲戊乐灵在田间诱导枣树同质多倍体的方法 - Google Patents
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Abstract
一种利用二甲戊乐灵在田间诱导枣树同质多倍体的方法，即以安全性高、成本低的二甲戊乐灵为诱变剂，在田间活体条件下，通过愈伤组织途径诱导枣树同质多倍体。对枣树健壮枝条进行截枝处理，迅速用生长调节剂处理形成层，并保湿24 h；约4~6 d后，当截面形成层有透明微弱愈伤出现时，用二甲戊乐灵（含有0.1%二甲基亚砜）处理愈伤组织24~48 h并保湿；当愈伤组织上的再生芽长至2~3 cm后，去除所有覆盖物，对再生芽进行染色体倍性检测。与对照相比（以秋水仙素做诱变剂），利用二甲戊乐灵做诱变剂，诱变率提高了30.3%，成本降低5000倍，而且安全性更高。
Description
技术领域
本发明涉及一种利用二甲戊乐灵在田间诱导枣树同质多倍体的方法。
背景技术
枣树是原产于我国的特色果树，是我国古代著名的“五果”之一，同时也是我国现今第一大干果和第七大果树树种。枣树耐干旱、瘠薄、盐碱，枣果营养成分高，经济效益高,目前枣产业已成为我国山、沙、碱、旱地区重要的产业支柱。但现有的枣树品种都是古老的地方品种，分化严重，抗性下降 ,在生产上迫切需要创新品种去更新换代。
在枣树的种质资源中，已经发现自然三倍体品种赞皇大枣、苹果枣，表现出果个大、适应性强等优点；枣树的近缘种毛叶枣（Ziziphus mauritiana Lam.）的栽培品种多为多倍体，也都表现为果个大、适应性强的优点。从21世纪开始，本课题组先后开展了多种途径的枣树多倍体诱变，其中通过田间愈伤组织途径诱导枣树同质多倍体已经成为了枣树多倍体诱变的重要途径，课题组发明的“一种在田间快速获得枣树同质多倍体的方法”（ZL201010033318.1）、“一种同步获得两种以上同质多倍体的枣树多倍体种质创新方法”（ZL201210530993.4）先后获得了国家发明专利。
但是，上述方法均是利用秋水仙素作为诱变剂，由于秋水仙素存在毒性大、价格昂贵的缺点，所以科学工作者们开始探究新型的化学诱变剂。前人曾以氨磺乐灵为诱变剂成功获得了马铃薯、玫瑰、湖北紫荆、百合的多倍体和猕猴桃六倍体，以氟乐灵为诱变剂成功诱导了辣椒、甜瓜、桑树、西瓜、萝卜多倍体，以二甲戊乐灵为诱变剂成功获得了金线莲、大蒜、万年青、西瓜、矮牵牛等多倍体，二甲戊乐灵的诱变效果要好于秋水仙素，但是二甲戊乐灵在木本果树多倍体诱变中的研究较少，而且多是在组培条件下进行的。
本课题组（路芳等）曾在离体条件下以二甲戊乐灵为诱变剂获得了酸枣八倍体。在田间活体条件下，二甲戊乐灵用于愈伤组织途径枣树同质多倍体诱变还未见报道。
发明内容
在田间活体条件下，以二甲戊乐灵为化学诱变剂，通过愈伤组识途径进行枣树同质多倍体诱变，安全性高、成本低廉、效果显著。
本发明解决其技术问题所采用的技术方案是：首先对枣树健壮枝条进行截枝处理，利用生长调节剂进行愈伤组织诱导，其次利用适宜浓度的二甲戊乐灵对愈伤组织细胞进行染色体加倍，最后使染色体加倍的愈伤组织细胞进行芽再生，从而获得同质多倍体。
本发明的具体内容，即利用二甲戊乐灵在田间诱导枣树同质多倍体的方法如下：
（1）愈伤组织诱导 于5~7月份，对直径1.5~3 cm的枣树健壮枝条进行截枝处理，尽量保持截面光滑、无毛边，可使枝皮开裂，增加与药液接触的形成层的面积；迅速用脱脂棉覆盖截面形成层，将含有4.0~5.0 mg/L TDZ+2.0 mg/L AgNO3的药液1~2 ml滴加在脱脂棉上，以浸湿脱脂棉为度，覆上黑色塑料袋，避光处理；24 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹截面，用塑料袋全部套住并绑紧袋口，便于保湿；
（2）愈伤组织细胞的染色体加倍和芽再生 约4~6 d后，当截面形成层有透明微弱愈伤出现时，去除覆盖物；将0.01%~0.05%二甲戊乐灵（含有0.1%二甲基亚砜）1~2 ml滴加到脱脂棉上，用脱脂棉覆盖愈伤组织，迅速用黑色塑料袋套住保湿；24~48 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹愈伤组织，用塑料袋全部套住并绑紧袋口；当愈伤组织上的再生芽长至2~3 cm后，去除所有覆盖物，15~20 d后对再生芽进行染色体倍性检测。
获得本发明专利的有益效果是，以安全性高、成本低的二甲戊乐灵为诱变剂，在田间活体条件下，通过愈伤组织途径诱导枣树同质多倍体，而且诱变效率高。
具体实施方式：
于5月中旬，以10年生二倍体‘月光’枣为试材，对直径在3cm左右的枝条进行截枝处理，用脱脂棉覆盖截面形成层，并用注射器将5.0 mg/L TDZ + 2.0 mg/L AgNO3的混合液1.5ml注入脱脂棉，用黑色塑料袋扎紧，进行避光处理；24 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹截面，用塑料袋全部套住并绑紧袋口，便于保湿；约4~6 d后，当截面形成层有透明微弱愈伤出现时，去除覆盖物；将0.025%二甲戊乐灵（含有0.1%二甲基亚砜）1.5ml滴加到脱脂棉上，用脱脂棉覆盖愈伤组织，迅速覆盖上黑色塑料袋保湿；24~48 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹愈伤组织，用塑料袋全部套住并绑紧袋口；当愈伤组织上的再生芽长至2~3 cm后，去除所有覆盖物，15 d后对再生芽进行染色体倍性检测。与对照相比（以秋水仙素做诱变剂），利用二甲戊乐灵做诱变剂，诱变率提高了30.3%，成本降低5000倍，而且安全性更高。
Claims (1)
1.一种利用二甲戊乐灵在田间诱导枣树同质多倍体的方法，其特征在于以安全性高、成本低的二甲戊乐灵为诱变剂，在田间活体条件下，通过愈伤组织途径诱导枣树同质多倍体，而且诱变效率高，包括以下2个步骤：
（1）愈伤组织诱导 于5~7月份，对直径1.5~3 cm的枣树健壮枝条进行截枝处理，尽量保持截面光滑、无毛边，可使枝皮开裂，增加与药液接触的形成层的面积；迅速用脱脂棉覆盖截面形成层，将含有4.0~5.0 mg/L TDZ+2.0 mg/L AgNO3的药液1~2 ml滴加在脱脂棉上，以浸湿脱脂棉为度，覆上黑色塑料袋，避光处理；24 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹截面，用塑料袋全部套住并绑紧袋口，便于保湿；
（2）愈伤组织细胞的染色体加倍和芽再生 约4~6 d后，当截面形成层有透明微弱愈伤出现时，去除覆盖物；将0.01%~0.05%二甲戊乐灵（含有0.1%二甲基亚砜）1~2 ml滴加到脱脂棉上，用脱脂棉覆盖愈伤组织，迅速用黑色塑料袋套住保湿；24~48 h后去除脱脂棉和塑料袋，使用略微湿润的脱脂棉包裹愈伤组织，用塑料袋全部套住并绑紧袋口；当愈伤组织上的再生芽长至2~3 cm后，去除所有覆盖物，15~20 d后对再生芽进行染色体倍性检测。
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