Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation
- 2.2k Downloads
The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term “half-seed” to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5–7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T 1 generation by phenotypic assay using the herbicide Liberty® (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method.
KeywordsAgrobacterium tumefaciens Cotyledonary node Glufosinate Half-seed Soybean Transformation
The authors thank Joanna Harbaugh, Amanda Ehrler and Francois Torney for their technical assistance; Mike Webber, Bayer CropScience, USA for graciously giving us Liberty® herbicide; and Bronwyn Frame and John Pesek for critical review of the manuscript and valuable suggestions. This work was partially supported by the Iowa Soybean Promotion Board and the North Central Soybean Research Program.
- An G, Evert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoort RA (eds.) Plant molecular biology manual A3. Kluwer Academic Publishers, Dordrecht, pp 1–19Google Scholar
- Dan Y, Reichert NA (1998) Organogenic regeneration of soybean from hypocotyl explants. In Vitro Cell Dev Biol-Plant 34:14–21Google Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
- Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB (1989) Recovery of primary transformants of soybean. Plant Cell Rep 7:615–617Google Scholar
- Townsend JA, Thomas LA (1993) An improved method of Agrobacterium-mediated transformation of cultured soybean cells. US Patent WO 94/02620Google Scholar
- Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250CrossRefPubMedGoogle Scholar