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

Abstract

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

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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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

Establishment and Rooting Optimization of Agrobacterium rhizogenes Transformation System in Cotton

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