农杆菌介导的甜椒遗传转化体系的建立
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摘要
以低温渗透系甜椒CL122为材料,采用其子叶节为外植体材料,研究了农杆菌介导的甜椒遗传转化体系的主要影响因子,如卡那霉素选择压、农杆菌菌液浓度、乙酰丁香酮浓度、侵染时间、共培养时间、激素浓度等,初步建立了一个高效的甜椒遗传转化体系。结果表明:最适的卡那霉素选择压为50 mg/L,乙酰丁香酮浓度在100μmol/L时可以明显提高外植体的抗性芽数,最适合的预培养时间和共培养时间为2 d,最佳侵染时间5 min,外植体在MS+0.2 mg/L IAA(吲哚乙酸)+400 mg/L特美汀培养基上生根率最高。
The low-temperature infiltrated Capsicum annuum CL122 was used as the material, and the cotyledonary node was used as the explant material to study the main factors affecting the Agrobacterium-mediated sweet pepper genetic transformation system, such as kanamycin(Kan) selection pressure, Agrobacterium liquid concentration, acetyl syringone(AS) concentration, infection time, co-culture time, different hormone concentrations. A highly-efficient sweet pepper genetic transformation system was initially established. The results indicated that the optimal Kan selection pressure was 50 mg/L, and the AS could significantly increase the number of resistant buds from explants at 100 μmol/L. The most suitable pre-culture and co-culture time was 2 d. The best time for infection was 5 min. The rooting rate was the highest on MS+0.2 mg/L IAA(indole acetic acid)+400 mg/L Timentin medium.
The low-temperature infiltrated Capsicum annuum CL122 was used as the material, and the cotyledonary node was used as the explant material to study the main factors affecting the Agrobacterium-mediated sweet pepper genetic transformation system, such as kanamycin(Kan) selection pressure, Agrobacterium liquid concentration, acetyl syringone(AS) concentration, infection time, co-culture time, different hormone concentrations. A highly-efficient sweet pepper genetic transformation system was initially established. The results indicated that the optimal Kan selection pressure was 50 mg/L, and the AS could significantly increase the number of resistant buds from explants at 100 μmol/L. The most suitable pre-culture and co-culture time was 2 d. The best time for infection was 5 min. The rooting rate was the highest on MS+0.2 mg/L IAA(indole acetic acid)+400 mg/L Timentin medium.
引文
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[19] 黄好,张金谌,戴雄泽,等.影响辣椒T-DNA标签突变体库构建的转化因素探讨[J].湖南农业科学,2008(2):36-38.
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[21] 敬国兴,李伏钦,陈永双,等.保加利亚尖椒遗传转化体系的建立[J].江苏农业科学,2009(1):31-34.
[2] Hoekema A,Hirsch P R,Hooykaas P J J,et al.A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens,Ti-plasmid[J].Nature,1983,303(5913):179-180.
[3] 陈丽梅,潘俊松,何欢乐,等.农杆菌介导的基因转化研究进展[J].甘肃科学学报,2005,17(2):61-63.
[4] Márton L,Wulle G J ,Molendijk L ,et al.In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens[J].Nature,1979,277(5692):129-131.
[5] 张玉,蒋欣梅,于锡宏.ICE1基因表达载体的构建及对番茄的转化[J].中国蔬菜,2010,1(18):27-33.
[6] 张丽,宋玉霞,巩檑,等.农杆菌介导的马铃薯转化研究进展[J].分子植物育种,2015,13(7):1660-1667.
[7] 杜利娟,薛杨,张旺,等.根癌农杆菌介导VirE2-N-EGFP在2种茄科植物中的表达分析[J].安徽农业科学,2015,43(33):254-256.
[8] Dale P J,Irwin J A,Scheffler J A.The experimental and commercial release of transgenic crop Plants[J].Plant Breeding,2010,111(1):1-22.
[9] 袁静,吴涛,刘中来.影响农杆菌介导的辣椒基因转化因素的研究[J].华中师范大学学报:自然科学版,2003,37(2):227-230.
[10] 罗齐军.农杆菌介导的基因转化辣椒条件的优化[J].江西农业学报,2008,20(5):14-16.
[11] 黎定军.辣椒高效离体再生与遗传转化体系建立及抗病转基因研究[D].长沙:湖南农业大学,2000.
[12] 张正国,张丽莉,卢翠华,等.农杆菌介导BADH耐盐基因转化马铃薯的研究[J].作物杂志,2012(3):28-31.
[13] 王丽,张俊莲,王蒂,等.抗生素对根癌农杆菌的抑菌效果及对烟草叶片分化的影响[J].中国烟草学报,2006,12(1):32-37.
[14] 董喜才,杜建中,王安乐,等.乙酰丁香酮在植物转基因研究中的作用[J].中国农学通报,2011,27(5):292-299.
[15] Aarrouf J ,Castro-Quezada P ,Mallard S ,et al.Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum):a new and efficient tool for functional analysis of genes[J].Plant Cell Reports,2012,31(2):391-401.
[16] 武小霞,李静,王志坤,等.乙酰丁香酮浓度和共培养pH对大豆再生频率的影响[J].东北农业大学学报,2010,41(5):1-4.
[17] Kim S ,Kim S R ,An C S ,et al.Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.)[J].Molecules & Cells,2001,12(2):221.
[18] 杨国顺,谢丙炎,杨宇红,等.应用绿色荧光蛋白报告基因优化辣椒的遗传转化体系[J].园艺学报,2004,31(6):737-742.
[19] 黄好,张金谌,戴雄泽,等.影响辣椒T-DNA标签突变体库构建的转化因素探讨[J].湖南农业科学,2008(2):36-38.
[20] 张国刚.农杆菌介导茄子遗传转化体系研究[D].上海:上海交通大学,2015.
[21] 敬国兴,李伏钦,陈永双,等.保加利亚尖椒遗传转化体系的建立[J].江苏农业科学,2009(1):31-34.