棉花发根农杆菌转化体系的建立及生根优化  

Establishment and Rooting Optimization of Agrobacterium rhizogenes Transformation System in Cotton

作　　者：汪炜檬 魏云晓 唐云霓 刘苗苗 陈全家[1] 邓晓娟 张锐[2] WANG WeiMeng;WEI YunXiao;TANG YunNi;LIU MiaoMiao;CHEN QuanJia;DENG XiaoJuan;ZHANG Rui(College of Agronomy,Xinjiang Agricultural University/Engineering Research Center for Cotton,Ministry of Education,Urumqi 830052;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[1]新疆农业大学农学院/棉花教育部工程研究中心,乌鲁木齐830052 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科学》2025年第8期1479-1493,共15页Scientia Agricultura Sinica

基　　金：国家科技重大专项(2023ZD04040-01);国家重点研发计划(2022YFD1200304-3);中国农业科学院科技创新工程。

摘　　要：【背景】棉花是全球最主要的农作物之一,生物工程技术应用极大地提高了分子育种的效率,但棉花遗传转化目前存在受基因型限制、时间长和转化方法单一等问题。【目的】建立由发根农杆菌(Agrobacterium rhizogenes)介导的高效棉花遗传转化体系,丰富棉花遗传育种方法。【方法】以常用的棉花受体WC和R18为主要试验材料,利用mRUBY作为报告基因,通过诱导生根的激素组合和浓度的筛选优化、不同外植体和不同基因型棉花生根体系差异分析来对发根农杆菌介导的生根过程进行优化,在此基础上建立稳定的遗传转化体系,并将该体系应用于基因编辑。【结果】在生根培养基(RIM)中添加萘乙酸(naphthaleneacetic acid,NAA)和洛伐他汀(lovastatin)比单独添加NAA,或添加NAA+吲哚-3-丁酸(indole-3-butyric acid,IBA)、NAA+Lovastatin+IBA更易生根,进一步筛选出最适合诱导出毛状根的萘乙酸和洛伐他汀浓度均为2 mg·L^(-1)。子叶是最易诱导生根的外植体,WC子叶、子叶节和下胚轴单位材料的生根效率分别为398%、72%和39%,且子叶诱导生根时间最短(7 d),比子叶节至少短3 d,比下胚轴至少短8 d;R18最适合诱导生根的外植体也是子叶,但生根数量存在差异。基因型比较表明WC、R18、农大棉8号(NDM8)、新陆早61号(XLZ61)、Gb-1和Gb-2每片子叶在侵染后20 d的生根效率分别为398%、116%、199%、103%、57%和0,陆地棉的生根效率均在100%以上,海岛棉在100%以下,其中Gb-2在侵染后35 d才开始生根,陆地棉中常用的受体品种又表现出生根效率略高于生产品种的趋势。遗传转化的阳性率与生根率之间存在一定的差异,侵染后20 d NDM8、XLZ61、Gb-1和Gb-2的转化阳性率依次为59.8%、16.0%、38.5%和0。此外,以获得的阳性根作为后续试验的外植体,进行非胚性愈伤和胚性愈伤诱导,获得了转mRUBY植株,建立了完整的遗传转化体系,且发现植株颜色深浅和mRU【Background】Cotton is one of the most important crops globally.The application of bioengineering technology has greatly improved the efficiency of molecular breeding.However,current cotton genetic transformation faces challenges such as genotype dependency,lengthy timelines,and limited transformation methods.【Objective】This study aims to establish an efficient Agrobacterium rhizogenes-mediated genetic transformation system for cotton to expand genetic breeding methodologies.【Method】Using the common cotton receptor varieties WC and R18 as primary materials and m RUBY as a reporter gene,the root inducing process mediated by A.rhizogenes was optimized through screening hormone combinations (types and concentrations),analyzing differences in explant types and genotype-specific rooting systems.A stable genetic transformation system was subsequently developed and applied to gene editing.【Result】The addition of naphthaleneacetic acid (NAA) and lovastatin to the root inducing medium (RIM) promoted more efficient root formation compared to NAA alone or combinations of NAA+indole-3-butyric acid(IBA) or NAA+Lovastatin+IBA.The optimal concentrations for inducing hairy roots were both 2 mg·L^(-1) for NAA and lovastatin.Cotyledons were the most effective explants for root induction:WC cotyledons,cotyledon nodes,and hypocotyls exhibited rooting efficiencies of 398%,72%,and 39%,respectively.Cotyledons required the shortest induction time (7 d),3 d shorter than cotyledon nodes and 8 d shorter than hypocotyls.Cotyledons were also the optimal explants for R18,their rooting capacity differed.Genotype comparisons revealed that 20 days post-infection (dpi),the rooting efficiencies per cotyledon were 398%(WC),116%(R18),199%(NDM8),103%(XLZ61),57%(Gb-1),and 0 (Gb-2).Upland cotton varieties (WC,R18,NDM8,and XLZ61) exhibited rooting efficiencies above 100%,while sea island cotton varieties (Gb-1,Gb-2) were below 100%.Notably,Gb-2 began to root at 35 dpi.Receptor varieties of upland cotton generally showed slightly higher r

关 键 词：棉花 遗传转化 发根农杆菌 毛状根 基因编辑 

分 类 号：S562[农业科学—作物学]