刺槐离体叶片高效再生体系的优化  

Optimization of Efficient Regeneration System of Robinia pseudoacacia Leaves in vitro

作　　者：韩娟 李亚鹏 田彦挺 郭琪 李云[2] 孙宇涵[2] 邓永平 牛东升 苏立琢 李秀宇 彭祚登[1] Han Juan;Li Yapeng;Tian Yanting;Guo Qi;Li Yun;Sun Yuhan;Deng Yongping;Niu Dongsheng;Su Lizhuo;Li Xiuyu;Peng Zuodeng(Engineering Technology Research Center of Black Locust of National Forestry and Grassland Administration,College of Forestry,Beijing Forestry University,Beijing 100083;National Engineering Laboratory for Tree Breeding Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083;Natural Resources Bureau of Qingshui County,Gansu Province,Qingshui 741400;Seed Garden of Robinia pseudoacacia in Jixian County,Shanxi Province,Jixian 042200;Linghai Ecological Construction and Development Center,Liaoning Province,Linghai 121228)

机构地区：[1]国家林业和草原局刺槐工程技术研究中心、北京林业大学林学院,北京100083 [2]国家林业和草原局刺槐工程技术研究中心、林木育种国家工程实验室、林木、花卉遗传育种教育部重点实验室、北京林业大学生物科学与技术学院,北京100083 [3]甘肃省清水县自然资源局,清水741400 [4]山西省吉县刺槐种子园,吉县042200 [5]辽宁省凌海市生态建设发展中心,凌海121228

出　　处：《林业科学》2023年第4期68-78,共11页Scientia Silvae Sinicae

基　　金：北京林业大学青年教师科学研究中长期项目(2015ZCQ-SW-03);国家自然科学基金项目(31971675);国家重点研发课题(2017YFD0600503)。

摘　　要：【目的】通过优化再生过程中的重要影响因素,建立刺槐离体叶片高效的再生体系,这将为刺槐苗木繁殖、诱变育种、遗传转化以及进一步开展刺槐分子育种等奠定良好的基础。【方法】以刺槐组培苗离体的叶片为外植体,观察叶片再生不定芽的整个过程,探究基因型、取材时期、黑暗处理时长以及植物生长调节剂对刺槐叶片再生不定芽的影响,同时筛选适合不定芽生根的IBA浓度。【结果】1)二倍体基因型普18-22再生能力最佳;2)组培苗继代45天为最佳外植体取材时期;3)黑暗处理14天为最佳黑暗处理时长;4)最佳的叶片再生不定芽的培养基为:MS+6-BA3.0 mg·L^(-1)+KT 1.0 mg·L^(-1)+2,4-D 0.1 mg·L^(-1)+蔗糖30 g·L^(-1)+琼脂6.5 g·L^(-1);普18-22不定芽诱导率达96.67%,平均每个叶片再生7.4个不定芽;5)不定芽生根最佳培养基为:1/2MS+蔗糖30 g·L^(-1)+琼脂6.5 g·L^(-1)+IBA 0.3 mg·L^(-1);生根率达100%。【结论】以继代培养45天的二倍体刺槐组培苗普18-22叶片为外植体,接种至MS+6-BA 3.0 mg·L^(-1)+KT 1.0 mg·L^(-1)+2,4-D 0.1 mg·L^(-1)+蔗糖30 g·L^(-1)+琼脂6.5 g·L^(-1)的再生培养基中,黑暗处理14天后转移至正常光照培养,45天后将再生的不定芽转移至生根培养基1/2MS+蔗糖30 g·L^(-1)+琼脂6.5 g·L^(-1)+IBA 0.3 mg·L^(-1)中,待不定芽生根后进行炼苗移栽,得到完整的再生植株。【Objective】Leaves are rich in sources,easy to sample,and have little impact on the plant after collection,such they are ideal materials for plant asexual reproduction.However,the regeneration efficiency of Robinia pseudoacacia leaves is not high enough to meet the requirements of genetic transformation.In this study,an efficient regeneration system of R.pseudoacacia leaves in vitro was established by optimizing the important factors in the regeneration process,which would lay a good foundation for R.pseudoacacia seedling reproduction,mutagenesis breeding,genetic transformation and further development of molecular breeding of R.pseudoacacia.【Method】The isolated leaves of R.pseudoacacia tissue culture seedlings were used as explants,the whole process of regeneration of adventitious buds from the isolated leaves was observed to investigate the effects of genotype,sampling period,dark treatment duration as well as plant growth regulators on the regeneration of adventitious buds from R.pseudoacacia leaves.Meanwhile,the IBA concentration suitable for rooting of adventitious buds was screened.【Result】1)Diploid genotype Pu-18-22 had the best regeneration ability;2)Subculture of tissue culture seedlings for 45 days was the best time for selecting explants;3)Darkness treatment for 14 days was the best darkness treatment time;4)The optimal medium for adventitious bud regeneration was MS(culture medium)+N-(Phenylmethyl)-9H-purin-6-amine 3.0 mg·L^(-1)+Kinetin 1.0 mg·L^(-1)+2,4-Dichlorophenoxyacetic acid 0.1 mg·L^(-1)+Sucrose 30 g·L^(-1)+Agar 6.5 g·L^(-1);The adventitious bud induction rate of Pu-18-22 was 96.67%,and 7.4 adventitious buds were regenerated per leaf on average;5)The best rooting medium for adventitious buds was 1/2MS+Sucrose 30 g·L^(-1)+Agar 6.5 g·L^(-1)+3-Indolebutyric acid 0.3 mg·L^(-1),and the rooting rate was 100%.【Conclusion】The leaves of diploid R.pseudoacacia Pu-18-22 tissue culture plantlets are used as explants that are inoculated into MS culture medium+N-(Phenylmethyl)-9H-purin-

关 键 词：刺槐 叶片 黑暗处理 植物生长调节剂 再生体系 

分 类 号：S722.37[农业科学—林木遗传育种]