尾巨桉DH32-28组培再生过程中激素浓度筛选与转基因愈伤组织获得  

Hormone Concentrations Transgenic Calli Production during Tissue Culture Regeneration of Eucalyptus urophylla×E.grandis DH32-28

作　　者：张吴滔 白卫国 苏国芬 费晓云 胡浩 付朝阳 韦利达 李蔷薇 李魁鹏 徐增富 ZHANG Wutao;BAI Weiguo;SU Guofen;FEI Xiaoyun;HU Hao;FU Chaoyang;WEI Lida;LI Qiangwei;LI Kuipeng;XU Zengfu(College of Forestry,Guangxi University/Key Laboratory of National Forestry and Grassland Administration on Cultivation of Fast-growing Timber in Central South China,Nanning 530004,Guangxi,China;Guangxi Zhuang Autonomous Region State-owned Dongmen Forest Farm,Fusui532108,Guangxi,China)

机构地区：[1]广西大学林学院/中南速生材繁育国家林业和草原局重点实验室,广西南宁530004 [2]广西壮族自治区国有东门林场,广西扶绥532108

出　　处：《桉树科技》2024年第2期1-10,共10页Eucalypt Science & Technology

基　　金：广西重点研发计划揭榜制科技项目(桂科JB22035001);广西基地和人才专项(桂科AD23026337)。

摘　　要：探索尾巨桉良种无性系DH32-28的组培再生体系,进而建立稳定的遗传转化系统,以期为进一步获得转基因桉树再生植株,以及开展桉树基因功能及分子育种研究提供参考。以DH32-28组培苗叶片和茎段为外植体,通过筛选植物生长调节剂噻苯隆(TDZ)、6-苄氨基嘌呤(6-BA)、1-萘乙酸(NAA)以及吲哚丁酸(IBA)的合适浓度,进行外植体愈伤组织诱导、不定芽分化和生根诱导;通过测定愈伤组织诱导阶段以及生根诱导阶段组培材料对卡那霉素的耐受力,确定卡那霉素的临界筛选浓度,用于农杆菌(Agrobacterium tumefaciens)介导的遗传转化实验。结果表明:以MS为基本培养基,0.025 mg·L^(-1) TDZ+0.15 mg·L^(-1) NAA、0.05 mg·L^(-1) TDZ+0.15 mg·L^(-1) NAA的激素配比分别最适于DH32-28叶片和茎段的愈伤诱导,愈伤诱导率均为98.3%。将诱导出的叶片和茎段愈伤组织转移到含有0.5 mg·L^(-1) 6-BA+0.1 mg·L^(-1) NAA的不定芽分化培养基中,不定芽分化率分别为44.4%和96.7%。以1/2MS为基本培养基,添加0.5 mg·L^(-1)IBA对DH32-28再生芽的不定根诱导率最高,不定根诱导率为72.8%。愈伤组织和不定芽诱导阶段的卡那霉素最适筛选浓度为5 mg·L^(-1),生根阶段最适浓度为20 mg·L^(-1)。本研究成功建立了尾巨桉无性系DH32-28高效的再生体系,并采用农杆菌介导的遗传转化方法将红色荧光蛋白报告基因DsRed2导入到DH32-28茎段受体材料,获得了表达DsRed2的转基因愈伤组织。A tissue culture regeneration system was explored to enable a stable genetic transformation system of Eucalyp-tus urophylla×E.grandis DH32-28 to be established with the objective of providing a benchmark reference for regener-ation of transgenic Eucalyptus and enable studies of gene function and molecular breeding in Eucalyptus.Leaves and stems were used in this study as explants,and the plant growth regulators thidiazuron(TDZ),6-benzylaminopurine(6-BA),1-naphthalene acetic acid(NAA),and indole-3-butyric acid(IBA)were used to induce callus,adventitious shoot differentiation,and rooting.The critical concentration of kanamycin for screening in Agrobacterium-mediated genetic transformation experiments was determined by assays of the resistance of the cultured materials to kanamycin at the stages of callus and root induction.The results showed that using MS as the basic culture medium,the hormone combina-tions of 0.025 mg·L^(‒1) TDZ+0.15 mg·L^(‒1) NAA and 0.05 mg·L^(‒1) TDZ+0.15 mg·L^(‒1) NAA were the most suitable for callus induction from leaves and stem segments of DH32-28,respectively,and provided a callus induction rate of 98.3%.The induced calli from leaves and stem segments were transferred to an adventitious shoot differentiation medium con-taining 0.5 mg·L^(‒1)6-BA+0.1 mg·L^(‒1) NAA,and the adventitious shoot differentiation rates were 44.4%and 96.7%,re-spectively.The highest induction rate of adventitious roots was 72.8%in DH32-28 regenerated shoots with 1/2 MS as the basic medium and 0.5 mg·L^(‒1) IBA.The optimal screening concentration of kanamycin was 5 mg·L^(‒1) at the stage of callus and adventitious bud induction,and 20 mg·L^(‒1) at the rooting stage.In this study,an efficient regeneration system for DH32-28 was successfully established.The Agrobacterium-mediated genetic transformation method was successfully used to introduce the red fluorescent protein reporter gene,DsRed2,into the recipient DH32-28 stem segments,and transgenic calli expressing DsRed2 were obtained.

关 键 词：尾巨桉DH32-28 再生 遗传转化 DsRed2 

分 类 号：S722.37[农业科学—林木遗传育种]