CN110810251B - 一种薄壳山核桃的微体嫁接方法 - Google Patents
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Abstract
本发明涉及一种薄壳山核桃的微体嫁接方法，包括接穗的准备、砧木的培育以及微体嫁接步骤，首先选取三年生嫁接苗带腋芽的茎段作为外植体，清洗消毒后切成茎段，接入腋芽诱导培养基培养，将产生的腋芽转入进行继代培养，选取无褐化现象、茎长2～4cm、带有叶片的、生长良好的无根试管苗作为接穗；取成熟薄壳山核桃种子清洗消毒后剥取种仁，切取胚轴，诱导培养后作为砧木苗，炼苗5‑10d后，采用劈接的方法进行嫁接，嫁接后将嫁接苗移至嫁接苗培养基上培养25d，然后解绑，开盖练苗一周后移栽到栽培基质中。与现有技术中薄壳山核桃的嫁接方法相比，本发明的微体嫁接方法具有嫁接成活率高、成本低、占地面积小，可有效人为操控等优点。
Description
技术领域
本发明涉及一种薄壳山核桃的微体嫁接方法，属于林木育种技术领域。
背景技术
薄壳山核桃[Caryaillinoinensis(Wangenh.)K.Koch]又名美国山核桃或长山核桃，是胡桃科山核桃属植物。它原产美国和墨西哥北部，是世界上著名的干果树种之一。其坚果个大、壳薄，出仁率高，取仁容易，产量高。其果仁色美味香、无涩味、营养丰富，是理想的保健食品或面包、糖果等食品的添加材料。薄壳山核桃亦是重要的木本油料植物，种仁油脂含量高达70％以上，其中不饱和脂肪酸含量高达97％，是上等的烹调用油和色拉油。薄壳山核桃还是优良的材用和庭园绿化树种。薄壳山核桃不仅能为市场提供富含营养的干果、优质的木材等林产品，而且能绿化荒山、荒滩、荒地，美化环境，集经济、生态、社会三大效益于一身，其多样的产品已经成为改善人民群众饮食结构和国民经济建设的重要物质基础。
我国引种薄壳山核桃已有100多年的历史，但仍未形成大规模商品化生产，薄壳山核桃传统的繁殖方法以播种繁殖为主，但是播种实生苗10-15年后才能正常结果，由播种至结果苗龄所需的时间漫长，给栽培者造成很大压力，从而影响了薄壳山核桃种植者的积极性。而且实生繁殖不易保持其品种的优良特性，后代容易发生性状分离。因此薄壳山核桃的繁殖方法必须依靠无性繁殖来解决，目前薄壳山核桃的无性繁殖手段主要是嫁接，而嫁接技术不成熟、嫁接成活率低是限制薄壳山核桃产业化发展的主要因素之一(彭方仁，2012)。近年来，通过我国科技工作者的不懈努力，薄壳山核桃的嫁接技术已有所突破，采用改良的生长季方块芽接技术，可使薄壳山核桃的嫁接成活率达60％以上(Zhang et al,2015)，但仍然普遍存在枝接成活率不高、不同品种嫁接成活率差异明显等问题(翁春余等,2012)。
发明内容
本发明的目的在于解决现有技术中薄壳山核桃的嫁接成活率低的问题，提供一种薄壳山核桃的微体嫁接方法。
技术方案
一种薄壳山核桃的微体嫁接方法，包括如下步骤：
(1)接穗的准备
在薄壳山核桃优良品种中，选取三年生嫁接苗带腋芽的茎段作为外植体，清洗后用70-75v％的乙醇溶液浸泡20-30s，无菌水冲洗后用0.05-0.1wt％的HgCl2溶液消毒处理10-15min，然后用无菌水冲洗，切成1cm长的茎段，接入腋芽诱导培养基进行培养，培养15-20d后，将产生的腋芽转入继代培养基进行培养，得到无根试管苗，选取无污染、无褐化现象，茎长2～4cm、带有叶片的、生长状况良好的无根试管苗作为接穗；
(2)砧木的培育
取成熟薄壳山核桃种子，用自来水冲洗后浸泡3d，然后在无菌室内用70-75v％的乙醇溶液浸泡20-30s，无菌水冲洗后用0.05-0.1wt％的HgCl2溶液消毒处理10-15min，用无菌水冲洗后置于超净工作台上剥去果壳，剥取种仁放入0.1％HgCl2溶液中进行消毒，从消毒过的种仁中切取种胚，将其竖直插入种胚诱导培养基，在21-25℃、空气相对湿度保持为70-80％、全黑暗的条件下进行培养，培养25-30d后，得到组培苗，作为砧木苗；
(3)微体嫁接
将砧木苗放于日光灯下炼苗5-10d，待浅红色嫩茎变成青绿色、顶部叶片展开时进行嫁接，先将砧木苗取出，切除胚芽的上部，保留胚轴以上2cm的部分作为砧木，在横切面的中央垂直下切，劈开砧木，切口深0.5-1cm，将接穗下端两侧对称切削，成一楔形，切口长0.5-1cm，然后将接穗插入砧木切口中，用保鲜膜包扎，即完成嫁接；嫁接后，将嫁接苗移至嫁接苗培养基上培养，待嫁接苗培养25d后解绑，开盖练苗一周后移栽到栽培基质中。
步骤(1)中，所述腋芽诱导培养基配方为：基本培养基+6-BA2.0-3.0mg/L+IBA0.01-0.02mg/L+蔗糖30g/L+琼脂5.8g/L，PH值为5.8-6.0，基本培养基选自WPM、MS或DKW中任意一种。
步骤(1)中，所述继代培养基配方为：改良DKW+1.0mg/L 6-BA+0.01mg/LIBA+5.8g/L琼脂+30g/L蔗糖+0.1-1.0g/L抗褐化剂，pH5.8-6.0。所述改良DKW的配方为(mg/L)：硝酸铵1416.0，硼酸4.8，无水氯化钙112.5，硝酸钙1367.0，五水硫酸铜0.25，Na2-EDTA 45.4，七水硫酸铁33.8，硫酸镁361.49，一水硫酸锰33.5，钼酸钠0.39，六水硫酸镍0.005，磷酸二氢钾265.0，硫酸钾1559.0，六水硝酸锌17.0，盐酸硫胺素5.22，肌醇100，烟酸1.0。
进一步，步骤(1)中，优选采用0.1wt％的HgCl2溶液消毒处理10min，此时污染率最低，萌芽率最高，可达77％。
进一步，步骤(1)中，腋芽诱导培养基的配方优选为：MS+6-BA 2.0mg/L+IBA0.02mg/L+蔗糖30g/L+琼脂5.8g/L，pH值为5.8-6.0。萌芽率最高。
进一步，步骤(1)中，培养条件为：温度25℃±2℃，光照强度2400～2600lx，日光灯作为光源，光照时间14h/d，空气相对湿度为70-80％。
进一步，步骤(1)的继代培养基配方中，抗褐化剂选自活性炭、聚乙烯吡咯烷酮或维生素C中任意一种。更优选为聚乙烯吡咯烷酮，褐化率最低。
进一步，步骤(2)中，种胚诱导培养基配方：改良DKW+KT 2mg/L+6-BA1mg/L+IBA0.01mg/L，pH值为5.70-5.85。此时，种胚萌发率最高，种胚污染率最低。所述改良DKW的配方为(mg/L)：硝酸铵1416.0，硼酸4.8，无水氯化钙112.5，硝酸钙1367.0，五水硫酸铜0.25，Na2-EDTA 45.4，七水硫酸铁33.8，硫酸镁361.49，一水硫酸锰33.5，钼酸钠0.39，六水硫酸镍0.005，磷酸二氢钾265.0，硫酸钾1559.0，六水硝酸锌17.0，盐酸硫胺素5.22，肌醇100，烟酸1.0。
进一步，步骤(3)中，所述嫁接苗培养基配方为：MS+6-BA 1mg/L+IAA3.0mg/L，pH值为5.70-5.85。
进一步，步骤(3)中，所述栽培基质为河沙或重量比为1:1:1的珍珠岩、蛭石和腐殖土的混合物。
本发明的有益效果：首先，本发明完成了品种苗带腋芽茎段的外植体消毒和无菌培养过程，这是一个薄壳山核桃组织培养过程中的突破点，以往的研究中皆以实生苗的茎段为外植体材料，未见有品种苗茎段组培成功的先例，因为作为无性繁殖的材料，必须来源于良种化的无性系才有价值；其次，本发明完成了试管内的微体嫁接技术，并嫁接成活，且移栽成功。与现有技术中薄壳山核桃的嫁接方法相比，本发明的薄壳山核桃的微体嫁接方法具有嫁接成活率高、成本低、占地面积小，可有效人为操控等优点。
具体实施方式
下面结合具体实施例对本发明的技术方案作详细说明。下述实施例中，改良DKW的配方为(mg/L)：硝酸铵1416.0，硼酸4.8，无水氯化钙112.5，硝酸钙1367.0，五水硫酸铜0.25，Na2-EDTA 45.4，七水硫酸铁33.8，硫酸镁361.49，一水硫酸锰33.5，钼酸钠0.39，六水硫酸镍0.005，磷酸二氢钾265.0，硫酸钾1559.0，六水硝酸锌17.0，盐酸硫胺素5.22，肌醇100，烟酸1.0。
实施例1
一种薄壳山核桃的微体嫁接方法，包括如下步骤：
(1)接穗的准备
以薄壳山核桃品种‘波尼’(‘Pawnee’)三年生嫁接苗幼嫩带腋芽的茎段为外植体，洗洁精浸泡20min后，自来水冲洗2h，在无菌操作台用70-75v％酒精浸泡30s，无菌水冲洗4次，用0.1wt％的HgCl2溶液消毒处理10min，然后用无菌水冲洗6次，切成1cm长的茎段，接入腋芽诱导培养基(MS+6-BA 2.0mg/L+IBA0.02mg/L+蔗糖30g/L+琼脂5.8g/L，pH值为5.8～6.0)进行培养，培养15-20d后，统计腋芽诱导情况(腋芽萌发率为81.9％)，将产生的腋芽转入继代培养基(改良DKW+1.0mg/L 6-BA+0.01mg/L IBA+5.8g/L琼脂+30g/L蔗糖+0.1g/L聚乙烯吡咯烷酮，pH5.8～6.0)进行培养(培养条件：温度25℃±2℃，光照强度2400～2600lx，日光灯作为光源，光照时间14h/d，空气相对湿度为70-80％)，得到无根试管苗(褐化率为25％)，选取无污染、无褐化现象，茎长2～4cm、带有叶片的、生长状况良好的无根试管苗作为接穗；
(2)砧木的培育
取成熟薄壳山核桃‘波尼’的种子，用自来水冲洗后浸泡3d，然后在无菌室内用70-75v％的乙醇溶液浸泡30s，无菌水冲洗后用0.1wt％的HgCl2溶液消毒处理10min，用无菌水冲洗后置于超净工作台上剥去果壳，剥取种仁放入0.1％HgCl2溶液中消毒5min，从消毒过的种仁中切取种胚，将其竖直插入种胚诱导培养基(种胚诱导培养基配方：改良DKW+KT2mg/L+6-BA 1mg/L+IBA0.01mg/L)，没入培养基三分之一即可，在21-25℃、空气相对湿度保持为70-80％、全黑暗的条件下进行培养，培养30d后，得到组培苗，作为砧木；
(3)微体嫁接
将作为砧木的组培苗放于日光灯下炼苗10d，待浅红色嫩茎变成青绿色、顶部叶片展开时进行嫁接，先将砧木苗取出，切除胚芽的上部，保留胚轴以上2cm的部分作为砧木，在横切面的中央垂直下切，劈开砧木，切口深0.5-1cm，将接穗下端两侧对称切削，成一楔形，切口长0.5-1cm，然后将接穗插入砧木切口中，用保鲜膜包扎，即完成嫁接；嫁接后，将嫁接苗移至嫁接苗培养基(嫁接苗培养基配方为：MS+6-BA 1mg/L+IAA 3.0mg/L)上培养，待嫁接苗培养25d后解绑，开盖练苗一周后移栽到栽培基质(重量比为1:1:1的珍珠岩、蛭石和腐殖土的混合物)中。
本实施例中，嫁接成活率为86.7％，移栽后成活率为92％。
实施例2
步骤(1)中，腋芽诱导培养基为：WPM+6-BA 2.0mg/L+IBA 0.02mg/L+蔗糖30g/L+琼脂5.8g/L，PH值为5.8～6.0。统计得到：腋芽萌发率为80％。
其余与实施例1相同。
实施例3
步骤(1)中，继代培养基为：改良DKW+1.0mg/L 6-BA+0.01mg/L IBA+5.8g/L琼脂+30g/L蔗糖+0.1g/L维生素C，pH5.8～6.0)。褐化率为30％。
其余与实施例1相同。
实施例4
步骤(1)中，继代培养基为：改良DKW+1.0mg/L 6-BA+0.01mg/L IBA+5.8g/L琼脂+30g/L蔗糖+1.0g/L活性炭，pH5.8～6.0)。褐化率为35％。
其余与实施例1相同。
实施例5
步骤(3)中炼苗7d，其余与实施例1相同。嫁接成活率为80％，移栽成活率85.6％。
实施例6
步骤(3)中，栽培基质为河沙，其余与实施例1相同。嫁接成活率为83.3％，移栽成活率86.7％。
Claims (4)
1.一种薄壳山核桃的微体嫁接方法，其特征在于，包括如下步骤：
（1）接穗的准备
在薄壳山核桃优良品种中，选取三年生嫁接苗带腋芽的茎段作为外植体，清洗后用70-75v%的乙醇溶液浸泡20-30s，无菌水冲洗后用0.05-0.1wt%的 HgCl2溶液消毒处理10-15min，然后用无菌水冲洗，切成1cm长的茎段，接入腋芽诱导培养基进行培养，培养15-20d后，将产生的腋芽转入继代培养基进行培养，得到无根试管苗，选取无污染、无褐化现象，茎长2~4cm、带有叶片的、生长状况良好的无根试管苗作为接穗；
（2）砧木的培育
取成熟薄壳山核桃种子，用自来水冲洗后浸泡3 d，然后在无菌室内用70-75v%的乙醇溶液浸泡20-30s，无菌水冲洗后用0.05-0.1wt%的 HgCl2溶液消毒处理10-15min，用无菌水冲洗后置于超净工作台上剥去果壳，剥取种仁放入0.1%HgCl2溶液中进行消毒，从消毒过的种仁中切取种胚，将其竖直插入种胚诱导培养基，在21-25℃、空气相对湿度保持为70-80%、全黑暗的条件下进行培养，培养25-30d后，得到组培苗，作为砧木苗；
（3）微体嫁接
将砧木苗放于日光灯下炼苗5-10d，待浅红色嫩茎变成青绿色、顶部叶片展开时进行嫁接，先将砧木苗取出，切除胚芽的上部，保留胚轴以上2cm的部分作为砧木，在横切面的中央垂直下切，劈开砧木，切口深0.5-1cm，将接穗下端两侧对称切削，成一楔形，切口长0.5-1cm，然后将接穗插入砧木切口中，用保鲜膜包扎，即完成嫁接；嫁接后，将嫁接苗移至嫁接苗培养基上培养，待嫁接苗培养25d后解绑，开盖炼苗一周后移栽到栽培基质中；
步骤（1）中，腋芽诱导培养基的配方为：MS+2.0mg/L 6-BA +0.02mg/L IBA +30g/L 蔗糖+5.8g/L琼脂，pH值为5.8-6.0；
步骤（1）中，所述继代培养基配方为：改良DKW+1.0mg/L 6-BA+0.01mg/L IBA+5.8g/L琼脂+30 g/L蔗糖+0.1-1.0g/L抗褐化剂，pH5.8-6.0，抗褐化剂选自活性炭、聚乙烯吡咯烷酮或维生素C中任意一种；
步骤（2）中，种胚诱导培养基配方：改良DKW+2mg/L KT +1mg/L 6-BA +0.01mg/L IBA ，pH值为5.70-5.85；
所述改良DKW的配方为（mg/L)：硝酸铵1416.0，硼酸4.8，无水氯化钙112.5，硝酸钙1367.0，五水硫酸铜0.25，Na2-EDTA 45.4，七水硫酸铁 33.8，硫酸镁 361.49，一水硫酸锰33.5，钼酸钠 0.39，六水硫酸镍 0.005，磷酸二氢钾 265.0，硫酸钾 1559.0，六水硝酸锌17.0，盐酸硫胺素 5.22，肌醇100，烟酸1.0；
步骤（3）中，所述嫁接苗培养基配方为：MS+6-BA 1mg/L+IAA 3.0mg/L，pH值为5.70-5.85。
2.如权利要求1所述薄壳山核桃的微体嫁接方法，其特征在于，步骤（1）中，采用0.1wt%的HgCl2溶液消毒处理10min。
3.如权利要求1所述薄壳山核桃的微体嫁接方法，其特征在于，步骤（1）中，培养条件为：温度25℃±2℃，光照强度2400~2600lx，日光灯作为光源，光照时间14h/d，空气相对湿度为70-80%。
4.如权利要求1至3任一项所述薄壳山核桃的微体嫁接方法，其特征在于，步骤（3）中，所述栽培基质为河沙、重量比为1:1的珍珠岩与蛭石的混合物或重量比为1:1:1的珍珠岩、蛭石和腐殖土的混合物中的任意一种。
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