农杆菌介导的玉米叶片遗传转化体系的建立

doi:10.16035/j.issn.1001-7283.2026.03.019

摘要/Abstract

摘要：

由于幼胚转化受幼胚质量影响较大，需要建立一种替代或补充玉米幼胚的遗传转化体系。通过在萌发培养基中添加10 mg/L噻苯唑（thiabendazole）来显著抑制种子的真菌污染，将萌发12 d左右的幼苗基部叶片切碎，经6 min热激预处理，辅以含有BBM基因的质粒进行农杆菌介导的遗传转化，从而建立一种以玉米叶片为外植体的转化方法。利用该体系已成功转化6个玉米自交系，转化植株正常结实。测试的6个自交系均获得转基因阳性植株，证明该方法具有普遍适用性。但不同自交系间转化效率存在较大差异，其中M013转化效率最高，达11.0%，PH4CV最低，为1.1%。与幼胚转化方法相比，该体系具有材料获取简便、操作简单、不受季节和空间限制等优势，可为玉米基因功能研究、分子育种和遗传改良提供理论依据。

关键词: 玉米, 叶片, 农杆菌介导, 遗传转化, 噻苯唑

Abstract:

Since immature embryo transformation is greatly affected by explant quality, it is necessary to establish a genetic transformation system that can replace or supplement the transformation of maize immature embryos. Adding 10 mg/L thiabendazole to the germination medium significantly inhibited fungal contamination of seeds. The basal leaves of approximately 12-day-old seedlings were cut into pieces, and used for Agrobacterium- mediated genetic transformation with a plasmid containing the BBM gene after a six-minute heat shock pretreatment. Thus, a genetic transformation method using maize leaves as explants was established. Using this system, six maize inbred lines were successfully transformed, and the transformed plants produced seeds normally. Transgenic positive plants were obtained from all six tested inbred lines, demonstrating the broad applicability of this method. However, there were significant differences in transformation efficiency among different inbred lines, among which M013 had the highest transformation efficiency of 11.0%, and PH4CV had the lowest efficiency of 1.1%. Compared with the immature embryo transformation method, this system has advantages such as easy material acquisition, simple operation, and freedom from seasonal or spatial limitations, which can provide a theoretical basis for maize gene function research, molecular breeding, and genetic improvement.

Key words: Maize, Leaf, Agrobacterium-mediated, Genetic transformation, Thiabendazole

盛奇明, 徐永盛, 荆风雪, 苏纯洁, 解光宁, 孙晓莎. 农杆菌介导的玉米叶片遗传转化体系的建立[J]. 作物杂志, 2026 (3): 141–146

Sheng Qiming, Xu Yongsheng, Jing Fengxue, Su Chunjie, Xie Guangning, Sun Xiaosha. Establishment of Agrobacterium-Mediated Genetic Transformation System for Maize Leaves[J]. Crops, 2026(3): 141–146

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培养基Medium	培养基组分Medium composition
种子萌发培养基 Seed germination medium (G)	MS+20 g/L蔗糖+5 g/L琼脂+10 mg/L噻苯唑，pH 5.6
侵染液培养基 Infection medium (I)	1.204 g/L MgSO₄+5 g/L麦芽糖+20 mg/L乙酰丁香酮+0.5 mg/L羟敌草腈+0.01% (v/v) BREAK-THRU S233，pH 5.6
共培培养基 Co-cultivation medium (C)	MS+20 g/L麦芽糖+10 g/L葡萄糖+0.8 mg/L CuSO₄+0.7 g/L脯氨酸+0.5 g/L 2-吗啉乙磺酸+2 mg/L 2,4-二氯苯氧乙酸+10 mg/L维生素C+20 mg/L乙酰丁香酮+3.4 mg/L硝酸银+300 mg/L半胱氨酸+8 g/L琼脂，pH 5.6
恢复培养基 Resting medium (Rs)	M5605 (Phytotech)+20 g/L蔗糖+0.6 g/L葡萄糖+0.3 g/L酸水解酪蛋白+0.8 mg/L酸水解酪蛋白+1.2 mg/L麦草畏+3.4 mg/L硝酸银+200 mg/L特美汀+0.025 mg/L植物磺肽素+6 g/L 琼脂，pH 5.6
愈伤组织诱导培养1 Calli inducing medium 1 (CIM1)	N492 (Phytotech)+20 g/L蔗糖+2.88 g/L脯氨酸+650 mg/L MgCl₂+300 mg/L谷氨酰胺+0.1 g/L酸水解酪蛋白+ 2.2 mg/L 2,4-二氯苯氧乙酸+10 mg/L硝酸银+200 mg/L特美汀+0.025 mg/L植物磺肽素+8 g/L琼脂，pH 5.8
愈伤组织诱导培养2 Calli inducing medium 2 (CIM2)	N492+20 g/L蔗糖+2.88 g/L脯氨酸+650 mg/L MgCl₂+0.1 g/L酸水解酪蛋白+2.2 mg/L 2,4-二氯苯氧乙酸+ 10 mg/L硝酸银+200 mg/L特美汀+0.025 mg/L植物磺肽素+8 g/L琼脂，pH 5.8
分化培养基 Maturation medium (M)	MS+30 g/L蔗糖+0.5 g/L 2-吗啉乙磺酸+1.3 mg/L CuSO₄+0.1 mg/L噻苯隆+0.5 mg/L玉米素+1 mg/L吲哚乙酸+ 0.1 μmol/L脱落酸+200 mg/L特美汀+3.5 g/L植物凝胶，pH 5.8
生根培养基Rooting medium (R)	MS+30 g/L蔗糖+0.5 g/L 2-吗啉乙磺酸+1.3 mg/L CuSO₄+200 mg/L特美汀+3.5 g/L植物凝胶，pH 5.8

编号 Number	自交系 Inbred line	杂种优势群 Heterotic group	起始种子数 Number of starting seeds	碎叶片数 Number of chopped leaves	阳性苗数 Number of positive seedlings	转化效率 Transformation rate (%)
1	M013	Lancaster	11	1925	212	11.0
2	M018	Lancaster	10	1900	86	4.5
3	DH351	Reid	6	1092	36	3.3
4	F003	Reid	2	372	12	3.2
5	Qi319	PB	16	2752	52	1.9
6	PH4CV	Lancaster	3	540	6	1.1