Abstract
High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.
Similar content being viewed by others
References
An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoot RA (eds) Plant Mol Biology Manual. Martinus Nijhoff, Dordrecht, The Netherlands, pp A3:1–19
Armstrong CL (1999) The first decade of maize transformation: a review and future perspective. Maydica 44:101–109
Armstrong CL, Green CE (1985) Establishment and maintenance of friable, embryogenic maize callus and the involvement of l-proline. Planta 164:207–214
Armstrong CL, Romero-Severson J, Hodges TK (1992) Improved tissue culture response of an elite maize inbred through backcross breeding, and identification of chromosomal regions important for regeneration by RFLP analysis. Theor Appl Genet 84:755–762
Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980
Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672–3676
Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Establishment of an high-frequency medium for anther culture of rice, through comparative experiments on the nitrogen sources. Sci Sin 18:659–668
Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version 2. Plant Mol Biol Rep 1:19–22
Der G, Everitt B (2001) A handbook of statistical analyses using SAS, 2nd edn. CRC, Boca Raton, p 376
Dong Q, Lawrence CJ, Schlueter SD, Wilkerson MD, Kurtz S, Lushbough C, Brendel V (2005) Comparative plant genomics resources at Plant GDB. Plant Physiol 139:610–618
Frame B, Shou H, Chikwamba R, Zhang Z, Xiang C, Fonger T, Pegg SE, Li B, Nettleton D, Pei D, Wang K (2002) Agrobacterium-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22
Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ, Paz MM, Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024–1034
Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321–325
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180
Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301
Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, Fry J (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010–1019
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol 14:745–750
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Ke XY, McCormac AC, Harvey A, Lonsdale D, Chen DF, Elliot MC (2002) Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat. Euphytica 126:333–343
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) High-frequency soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735
Olhoft P, Lin K, Galbraith J, Nielsen N, Somers D (2001) The role of thiol compounds in increasing Agrobacterium-mediated transformation of soybean cotyledonary-node cells. Plant Cell Rep 20:731–737
Olhoft P, Somers D (2001) l-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep 20:706–711
Perl A, Lotan O, Abu-Abied A, Holland D (1996) Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape–Agrobacterium interactions. Nat Biotechnol 14:624–628
Paz MM, Shou H, Z Guo, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179
Shou H, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789
Yu W, Lamb JC, Han F, Birchler JA (2006) Telomere-mediated chromosomal truncation in maize. Proc Natl Acad Sci USA 103:17331–17336
Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 10:1157–1168
Zhang Z, Xing A, Staswick PE, Clemente TE (1999) The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tiss Organ Cult 56:37–46
Zhao ZY, Gu W, Cai T, Pierce D (1999) Methods for Agrobacterium-mediated transformation. United States Patent No 5981840
Zhao ZY, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2002) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333
Zeng P, Vadnais D, Zhang Z, Polacco J (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.]. Plant Cell Rep 22:478–482
Acknowledgments
We thank Regina Wamsley (from Zhanyuan J. Zhang’s laboratory) for her excellent technical assistance; CAMBIA (Australia) for providing pCAMBIA3301; Aventis CropScience (Research Triangle Park, NC, USA) for herbicide Liberty®, and James A. Birchler and Seth D. Findley (University of Missouri-Columbia) for a critical review of this manuscript. University of Missouri-Columbia Life Science Mission Enhancement program supported Angela R. Kennon and, in part, Xinlu Chen (from Zhanyuan J. Zhang’s lab). All transformation experiments were conducted in the Plant Transformation Core Facility at the University of Missouri-Columbia.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by P. Ozias-Akins.
Rights and permissions
About this article
Cite this article
Vega, J.M., Yu, W., Kennon, A.R. et al. Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27, 297–305 (2008). https://doi.org/10.1007/s00299-007-0463-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-007-0463-z