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Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection

  • Genetic Transformation and Hybridization
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Abstract

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study—selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50–80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of the aroA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

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References

  • Aldemita RR, Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199:612–617

    CAS  Google Scholar 

  • Altpeter F, Vasil V, Srivastava V, Stöger E, Vasil IK (1996) Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep 16:12–17

    CAS  Google Scholar 

  • Amoah BK, Wu H, Sparks C, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Bot 52:1135–1142

    Article  CAS  PubMed  Google Scholar 

  • Barro F, Cannell ME, Lazzeri PA, Barcelo P (1998) The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants. Theor Appl Genet 97:684–695

    CAS  Google Scholar 

  • Barry G, Kishore G, Padgette S, Taylor M, Kolacz K, Weldon M, Re D, Eichholtz D, Fincher D, Hallas L (1992) Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. In: Sinch BK, Flores HE, Shannon JC (eds) Biosynthesis and molecular regulation of amino acids in plants. American Society of Plant Physiologists, Rockville, Md., pp 139–145

  • Becker D, Brettschneider R, Lörz H (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 5:299–307

    CAS  PubMed  Google Scholar 

  • Brown SM, Santino CG (1994) Enhanced expression in plants. U.S. Patent no. 5,424,412

  • Chan MT, Lee TM, Chang HH (1992) Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Plant Cell Physiol 33:577–583

    CAS  Google Scholar 

  • Chan MT, Chang HH, Ho SL, Tong WF, Yu SM (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/ß-glucuronidase gene. Plant Mol Biol 22:491–506

    CAS  PubMed  Google Scholar 

  • Chen L, Zhang S, Bachy RN, Fauquet CM (1998) A protocol for consistent, large-scale production of fertile transgenic rice plants. Plant Cell Rep 18:25–31

    Google Scholar 

  • Cheng M, Fry JE (2000) An improved efficient Agrobacterium-mediated plant transformation method. Int Patent Publ WO 00/34491

  • Cheng M, Fry JE, Pang S, Zhou H, Hironaka C, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    CAS  Google Scholar 

  • Cheng M, Hu T, Layton J, Liu CN, Fry JE (2001) Desiccation of Agrobacterium-inoculated precultured plant tissues significantly enhances T-DNA delivery, and subsequently increases stable transformation in wheat (abstract). In Vitro 37:24A

    Google Scholar 

  • Conner TW, Santino CG (2000) Plant expression vectors. Int Patent Publ WO0011200 A2

  • Dai S, Zhang P, Marmey P, Zhang S, Tian W Chen S, Beachy R, Fauquet C (2001) Comparative analysis of transgenic plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33

    Article  CAS  Google Scholar 

  • Dong J, Tang W, Buchholz GB, Hall TC (1996) Agrobacterium-mediated transformation of Japanica rice. Mol Breed 2:267–276

    CAS  Google Scholar 

  • Flavell RB (1994) Inactivation of gene expression in plants as consequence of specific sequence duplication. Proc Natl Acad Sci USA 91:3490–3496

    Google Scholar 

  • Finnegan J, McElroy D (1994) Transgenic inactivation: plants fight back. Biotechnology 12:883–888

    Google Scholar 

  • Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick SJ, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JE, Galluppi GR, Goldberg SB, Hoffmann NL, Woo SC (1983) Expression of bacterial genes in plants. Proc Natl Acad Sci USA 80:4803–4807

    CAS  PubMed  Google Scholar 

  • Galun E, Breiman A (1997) Transgenic plants. Imperial College Press, London

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    CAS  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann N, Eicholz D, Rogers SG, Fraley RT (1983) A simple and general method for transferring genes into plants. Science 227:1229-1231

    Google Scholar 

  • Iser M, Fettig S, Schring F, Viertel K, Hess D (1999) Genotype-dependent stable genetic transformation in German spring wheat varieties selected for high regeneration potential. J Plant Physiol 154:509–561

    CAS  Google Scholar 

  • 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. Nat Biotechnol 14:745–750

    Google Scholar 

  • Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 230:1299–1302

    Google Scholar 

  • Kishore GM, Padgette SR, Fraley RT (1992) History of herbicide-tolerant crops, methods of development and current state of the art-emphasis on glyphosate tolerance. Weed Technol 6:626–634

    CAS  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174

    CAS  PubMed  Google Scholar 

  • McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171

    CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    CAS  Google Scholar 

  • Nehra N, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha K (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellum tissues following microprojectile bombardment with two distinct gene constructs. Plant J 5:285–297

    CAS  Google Scholar 

  • Padgette SR, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1461

    CAS  Google Scholar 

  • Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76

    CAS  Google Scholar 

  • Russell DA, DeBoer DL, Stark DM, Preiss J, Fromm ME (1993) Plastid targeting of E. coli β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells. Plant Cell Rep 13:24–27

    Google Scholar 

  • Russell JA, Roy MK, Sanford JC (1992) Major improvements in biolistic transformation of suspension-cultured tobacco cells. In Vitro Cell Dev Biol Plant 28P:97–105

    CAS  Google Scholar 

  • Sanford JC (1988) The biolistic process. Trends Biotechnol 6:299–302

    CAS  Google Scholar 

  • Sawahel WA, Hassan AH (2002) Generation of transgenic wheat plants producing high levels of osmoprotectant proline. Biotechnol Lett 9:721–725

    Article  Google Scholar 

  • Shah D, Horsch RB, Klee HJ, Kishore GM, Winter J, Turner N, Hironaka CM, Sanders PR, Gasser CS, Aykent S, Siegel N, Rogers SG, Fraley RT (1986) Engineering herbicide tolerance in plants. Science 233:478–481

    CAS  Google Scholar 

  • Smith RH, Hood EH (1995) Agrobacterium tumefaciens transformation of monocotyledons. Crop Sci 35:301–309

    Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium-mediated barley transformation. Plant J 11:1369–1376

    CAS  Google Scholar 

  • Tzafrir I, Torbert KA, Lockhart BEL, Somers DA, Olszewski NE (1998) The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots. Plant Mol Biol 38:347–356

    Article  CAS  PubMed  Google Scholar 

  • Vasil V, Catillo A, Fromm M, Vasil I (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology 10:667–673

    CAS  Google Scholar 

  • Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured embryos. Biotechnology 11:1553–1558

    Google Scholar 

  • Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. Methods Enzymol 153:3–11

    PubMed  Google Scholar 

  • Weeks JT, Anderson OD, Blechl A (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.). Plant Physiol 102:1077-1084

    Google Scholar 

  • Zhang J, Xu R, Elliott MC, Chen DF (1997) Agrobacterium-mediated transformation of elite indica and japonica rice cultivars. Mol Biotechnol 8:223–231

    CAS  PubMed  Google Scholar 

  • Zhang L, Rybczynski JJ, Langenberg WG, Mitra A, French R (2000) An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration. Plant Cell Rep 19:241–250

    CAS  Google Scholar 

  • Zhou H, Arrowsmith JW, Fromm ME, Hironaka CM, Taylor ML, Rodiguez D, Pajeau ME, Brown SM, Santino CG, Fry JE (1995) Glyphosate tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Rep 15:159–163

    Google Scholar 

  • Zhou H, Berg JD, Biest NA, Blank SE, Buehler RE, Chay CA, Chen G, Eskelsen SR, Fry JE, Gigax DR, Hoi S, Horak MJ, Hu T, Isakson PJ, Lawton MB, Metz SG, Rempel CB, Ryerson DK, Sansone AP, Schneider RW, Shook AL, Starke RJ, Taylor LW, Tichota JM, Valenti SA, Frohberg RC, Quick JS, Rudd JC and Zemetra R (2003) Development of Roundup Ready wheat through transgenic approach. Crop Sci (in press)

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Acknowledgements

The authors are grateful to Drs. C. Armstrong, J. Huang and G. Ye for their critical review of the manuscript. Special thanks are extended to L. Taylor, C. Langbecker, J.D. Berg, S. Hoi, A. Sansone and our many colleagues and collaborators at Monsanto for their significant contributions to this project.

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Author notes

  1. M. Cheng

    Present address: Monsanto Mystic, 62 Maritime Dr, Mystic, CT 03655, USA

Authors and Affiliations

  1. Monsanto Company, 700 Chesterfield Parkway West, St. Louis, MO 63017, USA

    T. Hu, S. Metz, C. Chay, H. P. Zhou, N. Biest, G. Chen, M. Cheng, X. Feng, M. Radionenko, F. Lu & J. Fry

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  1. T. Hu
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  2. S. Metz
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Correspondence to T. Hu.

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Communicated by G.C. Phillips

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Hu, T., Metz, S., Chay, C. et al. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21, 1010–1019 (2003). https://doi.org/10.1007/s00299-003-0617-6

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  • DOI: https://doi.org/10.1007/s00299-003-0617-6

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