Establishment of Alfalfa Tissue Culture System and Research on Agrobacterium-mediated Transformation System in Alfalfa

Abstract

Abstract: In order to establish high-efficiency alfalfa genetic transformation system，the influence factors of Agrobacterium-mediated transformation system were studied. The callus and adventitious bud of alfalfa were induced by culturing leaves as explants in 8 different kind of medium，named M1-M8, respectively. The results showed that M7 medium（MS+1 mg/L 2，4-D + 2 mg/L CH + 0.25 mg/L KT + 2 mg/L agar） was the best one which can induced the much more ammount of callus and adventitious bud of alfalfa than others. The results showed that the most suitable conditions for kanamycin was 75 mg/L; OD600 of agrobactium concerntration value for 0.45-0.6.

Key words: Alfalfa, Tissue culture, High frequency regeneration, Genetic transformation, Agrobacterium, Kanamycin OD₆₀₀

Zhou Xiaofu, Lü Jie, Miao Lu, Gao Feng, Xu Hongwei . Establishment of Alfalfa Tissue Culture System and Research on Agrobacterium-mediated Transformation System in Alfalfa[J]. Biotechnology Bulletin, 2013, 0(4): 63-68.

References

［1］ Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP. Signaling in plant-microbe interactions[J]. Science, 1997, 276 ：726-733.
［2］王世玉, 郑用涟, 刘亚, 等. 玉米成熟胚胚性愈伤组织的诱导、高频再生及转化的研究[J]. 作物学报, 2008, 34（3）：423- 428.
［3］ Deak M, Kiss GB, Korkz C, et al. Transformation of Medicago by Agrobacterium mediated gene transfer[J]. Plant Cell Reports, 1986, 5（2）：97-100.
［4］ Shahin EA, Spielmann A, Sukhapinda K, et al. Transformation of cultivated alfalfa using disarmed Agrobacterium tumefaciens[J].
Crop Science, 1986, 26 ：1235-1239.
［5］ Mariotti D, Davey MR, Draper J, et al. Crown gall tumorigenesis in the forage legume Medicago sativa L[J]. Plant Cell Physiology, 1984, 25 ：473-482.
［6］ Chabaud M, Passiatore JE, Cannon F, Buchanan-wollaston V. Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation[J]. Plant Cell Reports, 1988, 7 ：512-516.
［7］ Du S, Erickson L, Bowley S. Effect of plant genotype on the transformation of cultivated alfalfa（Medicago sativa） by Agrobacterium tumefaciens[J]. Plant Cell Reports, 1994, 13 ：330-334.
［8］ Desgagnes R, Laberge S, Allard G, et al. Genetic transformation of commercial breeding lines of alfalfa（Medicago sativa ）[J]. Plant Cell, Tissue and Organ Culture, 1995, 42 ：129-140.
［9］ Chabaud M, Decarvalhoniebel F, Barker DG. Efficient transformation of Medicago truncatula cv. Jema-long using hypervirulent Agrobacterium tumefaciens strain AGL1[J]. Plant Cell Reports, 2003, 22 ：46-51.
［10］ Aoki T, Kamizawa A, Ayabe S. Efficient Agrobacterium-mediated transformation of Lotus japonicus with reliable antibiotic selection [J]. Plant Cell Reports, 2002, 21 ：238-243.
［11］ Slavica N, Jovanka MD, Branka V, Mirjana N. Improved transformation of alfalfa somatic embryos using a superbinary vector[J].
Acta Biologica Cracoviensia Series Botanica, 2004, 46 ：139-143.
［12］吴丽芳, 丁伟. 不同激素配比对紫花苜蓿不同外植体愈伤组织诱导的影响[J]. 安徽农业科学, 2010, 38（2）：620-621, 628.
［13］张立军, 赵成昊, 葛超. 玉米再生体系建立及其影响因素的研究[J]. 玉米科学, 2008, 16（2）：77-79, 81.
［14］王关林, 方宏筠. 植物基因工程［M］. 第2 版. 北京：科学出版社, 2002 ：527-528.
［15］傅荣昭. 植物遗传转化技术手册［M］. 北京：中国科学技术出版社, 1994.
［16］王紫萱，易自力. 卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价[J]. 中国生物工程杂志，2003，23（6）: 9-12.

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