Factors affecting the transformation of 'Marshall McIntosh' apple by Agrobacterium tumefaciens

被引：39
作者：
Bolar J.P. [1 ]
Brown S.K. [1 ]
Norelli J.L. [2 ]
Aldwinckle H.S. [2 ]
机构：
[1] Department of Horticultural Sciences, Cornell University, New York State Agric. Exp. Station, Geneva
[2] Department of Plant Pathology, Cornell University, New York State Agric. Exp. Station, Geneva
来源：
Plant Cell, Tissue and Organ Culture | 1998年 / 55卷 / 1期
关键词：
Agar/Gelrite combination; Aminoglycosides; Malus x domestica Borkh; Marshall McIntosh; Neomycin phosphotransferase; β-glucuronidase;
D O I：
10.1023/A:1026414031217
中图分类号：
学科分类号：
摘要：
The goal of this research was to develop an efficient transformation system for 'Marshall McIntosh' apple. To determine the optimum combination of agar and Gelrite gelling agents in the media to maximize regeneration and minimize hyperhydicity (vitrification), the following combinations of agar (A)+Gelrite (G) in g l-1 were tested: 7.0 A+0 G; 5.2 A+0.6 G; 3.5 A+2.5 G; 1.7 A+1.8 G; and 0 A+2.5 G. Both 5.2 A+0.6 G and 3.5 A+1.2 G provided greatest regeneration of healthy non-hyperhydric shoots. To determine the optimal concentration of aminoglycoside for the selection and regeneration of transgenic 'Marshall McIntosh' on agar-Gelrite-based media, kanamycin was tested at 0, 10, 25, 50, 75 and 100 mg l-1, and paromomycin was tested at 0, 50, 100, 150, 200 and 250 mg l-1. Kanamycin was more effective than paromomycin in the initial selection of transgenics. For selection of transformants of 'Marshall McIntosh', the use of kanamycin at 25 mg l-1 on 5.2 A+0.6 G solidified medium is suggested. By optimizing the medium and selection conditions, a protocol was developed that resulted in four transgenic lines as confirmed by a GUS assay, NPT II ELISA, PCR, and Southern analysis. In repeated experiments with this protocol, transformation efficiencies of 3.1 and 2.6% were obtained.
引用
收藏
页码：31 / 38
页数：7
相关论文
共 22 条
  • [1] Atkinson R.G., Gardner R.C., Agrobacterium-mediaied transformation of Pepino and regeneration of transgenic plants, Plant Cell Rep., 10, pp. 208-212, (1991)
  • [2] Chen C.R., Wang L., Winans S., Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBO542, Mol. Gen. Genet., 230, pp. 302-309, (1991)
  • [3] Cheung W.Y., Hubert N., Landry B.S., A simple and rapid DNA microextraction method for plant, animal, and insect suitable for RAPD and other PCR analyses, PCR Methods Appl., 3, pp. 69-70, (1993)
  • [4] De Bondt A., Eggermont K., Penninckx I., Goderis I., Broekaert W.F., Agrobacterium-mediated transformation of apple (Malus X domestica Borkh.): An assessment of factors affecting regeneration of transgenic plants, Plant Cell Rep., 15, pp. 549-554, (1996)
  • [5] Fasolo F., Zimmerman R.H., Fordham I., Adventitious shoot formation on excised leaves of in vitro grown shoots of apple cultivars, Plant Cell Tiss. Org. Cult., 16, pp. 75-87, (1989)
  • [6] Fulton T.M., Chunwongse J., Tanksley S.D., Microprep protocol for extraction of DNA from tomato and other herbaceous plants, Plant Mol. Bio. Rep., 13, pp. 207-209, (1995)
  • [7] Hoekema A., Hirsch P.R., Hooykaas P.J.J., Schilperoort R.A.A., Binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid (Plant Genetics), Nature, 303, pp. 179-180, (1983)
  • [8] James D.J., Agrobacterium-mediated transformation of apple (Malus pumila Mill.), Woody Plant Biotechnology, pp. 213-226, (1991)
  • [9] James D.J., Uratsu S., Cheng J., Negri P., Viss P., Dandekar A.M., Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple, Plant Cell Rep., 12, pp. 559-563, (1993)
  • [10] Jefferson R.A., Kavanagh T.A., Bevan M.W., GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J., 6, pp. 3901-3907, (1987)
123