棉花发根农杆菌转化体系的建立及生根优化

doi:10.3864/j.issn.0578-1752.2025.08.002

摘要/Abstract

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mRUBY表达量成正相关；同时获得了GhGI被编辑的棉花植株。【结论】优化了发根农杆菌介导的棉花生根过程，并建立了棉花遗传转化体系，同时成功将该体系应用于基因编辑，获得了转mRUBY和转GhGI的棉花植株。

关键词: 棉花, 遗传转化, 发根农杆菌, 毛状根, 基因编辑

Abstract:

【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 mRUBY 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 rooting efficiency than production varieties. There was a certain difference between the positive rate of genetic transformation and the rooting rate. The positive rates of NDM8, XLZ61, Gb-1 and Gb-2 at 20 dpi were 59.8%, 16.0%, 38.5% and 0, respectively. Using positive roots as explants, non-embryogenic and embryogenic callus induction yielded transgenic mRUBY-expressing plants, establishing a complete genetic transformation system. The intensity of plant coloration correlated positively with mRUBY expression levels. Additionally, cotton plants with edited GhGI genes were successfully obtained.【Conclusion】The study optimized the A. rhizogenes-mediated root induction process in cotton and established a robust genetic transformation system. This system was successfully applied to gene editing, generating transgenic cotton plants expressing mRUBY and edited GhGI genes.

Key words: cotton, genetic transformation, Agrobacterium rhizogenes, hairy root, gene editing

汪炜檬, 魏云晓, 唐云霓, 刘苗苗, 陈全家, 邓晓娟, 张锐. 棉花发根农杆菌转化体系的建立及生根优化[J]. 中国农业科学, 2025, 58(8): 1479-1493.

WANG WeiMeng, WEI YunXiao, TANG YunNi, LIU MiaoMiao, CHEN QuanJia, DENG XiaoJuan, ZHANG Rui. Establishment and Rooting Optimization of Agrobacterium rhizogenes Transformation System in Cotton[J]. Scientia Agricultura Sinica, 2025, 58(8): 1479-1493.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.08.002

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培养基Medium	培养基成分Medium component
液体1/2 MS Liquid 1/2 MS	2.22 g·L^-1 MS粉末+15 g·L^-1葡萄糖，pH 5.8 2.22 g·L^-1 MS powder+15 g·L^-1 glucose, pH 5.8
共培养基A1 Co-culture medium A1	MS+0.2 mg·L^-1萘乙酸+40 mg·L^-1乙酰丁香酮+30 g·L^-1葡萄糖+2.5 g·L^-1植物凝胶，pH 5.8 MS+0.2 mg·L^-1 NAA+40 mg·L^-1 acetosyringone (AS)+30 g·L^-1 glucose+2.5 g·L^-1 gelling agents, pH 5.8
生根培养基 Root inducing medium (RIM)	MS+250 mg·L^-1噻孢霉素+30 g·L^-1葡萄糖+2.5 g·L^-1植物凝胶，pH 5.8 MS+250 mg·L^-1 cefotaxime nasalt+30 g·L^-1 glucose+2.5 g·L^-1gelling agents, pH 5.8
愈伤诱导培养基 Callus inducing medium (CIM)	4.44 g·L^-1 MS粉末+0.1 mg·L^-1二氯苯氧乙酸+0.1 mg·L^-1激动素+250 mg·L^-1噻孢霉素+30 g·L^-1葡萄糖+2.5 g·L^-1植物凝胶，pH 5.8 4.44 g·L^-1MS powder+0.1 mg·L^-1 2,4-D+0.1 mg·L^-1 KT+250 mg·L^-1 cefotaxime nasalt+30 g·L^-1glucose+2.5 g·L^-1 gelling agents, pH 5.8
分化培养基 Shoot inducing medium (SIM)	4.44 g·L^-1MS+30 g·L^-1葡萄糖+2.5 g·L^-1植物凝胶，pH 6.5 4.44 g·L^-1 MS+30 g·L^-1 glucose+2.5 g·L^-1 gelling agents, pH 6.5
长苗培养基 Seedling medium (SM)	2.652 g·L^-1 MS+10 mL·L^-1 B5有机+1 g·L^-1谷氨酰胺+0.5 g·L^-1 L-天冬酰胺+30 g·L^-1葡萄糖+2.5 g·L^-1植物凝胶，pH 6.5 2.652 g·L^-1MS+10 mL·L^-1 B5 organic+1 g·L^-1 glutamine (Gln)+0.5 g·L^-1 L-asparagine (Asn)+30 g·L^-1glucose+ 2.5 g·L^-1gelling agents, pH 6.5

名称 Name	激素 Hormone	激素浓度 Hormone concentration (mg·L^-1)	名称 Name	激素 Hormone	激素浓度 Hormone concentration (mg·L^-1)
N	NAA	0.2	NL-4	NAA, Lovastatin	0.2, 3
NI	NAA, IBA	0.2, 0.1	NL-5	NAA, Lovastatin	0.2, 5
NL	NAA, Lovastatin	0.2, 2	NL-6	NAA, Lovastatin	0.1, 2
NLI	NAA, Lovastatin, IBA	0.2, 2, 0.1	NL-7	NAA, Lovastatin	1, 2
NL-1	NAA, Lovastatin	0.2, 0.1	NL-8	NAA, Lovastatin	2, 2
NL-2	NAA, Lovastatin	0.2, 1	NL-9	NAA, Lovastatin	3, 2
NL-3	NAA, Lovastatin	0.2, 2