Abstract
Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance (bar) or antibiotic resistance (aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes (gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15–20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T0 plants or T1 progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aldemita R, Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199:612–617
Bec S, Chen L, Ferriere NM, Legave T, Fauquet C, Guideroni E (1998) Comparative histology of microprojectile-mediated gene transfer to embryonic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability. Plant Sci 138:177–190
Cheng M, Fry JE, Pang S, Zhou H, Hironica CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980
De Buck S, De Wilde C, Van Montagu M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468
Dong J, Teng W, Buchholz G, Hall TC (1996) Agrobacterium-mediated transformation of Javanica rice. Mol Breed 2:267– 276
Dong J, Kharb P, Teng W, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7:187–194
Garfinkel M, Nester EW (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:732–743
Hellens R, Mullineaux P (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451
Hellens RP, Edwards A, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832
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 T-DNA. Plant J 6:271–282
Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205–218
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
James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:533–561
Jefferson RA, Kavanagh TA, Bevan MW (1987) β-glucuronidase as a sensitive and versatile fusion marker in higher plants.EMBO J 6:3901–3907
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
Martineau B, Voelker TA, Sanders RA (1994) On defining T-DNA. Plant Cell 6:1032–1033
McCormac AC, Fowler MR, Chen D, Elliot MC (2001) Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgen Res 10:143–155
Sivamani E, Shen P, Opalka N, Beachy RN, Fauquet CM (1996) Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile plants using the biolistic method. Plant Cell Rep 15:322–327
Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376
Upadhyaya NM, Surin B, Ramm K, Gaudron J, Schunmann PHD, Taylor W, Waterhouse PM, Wang MB (2000) Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amarroo using modified promoters and selectable markers. Aust J Plant Physiol 27:201–210
Vain P, Finer K, Engler D, Pratt R, Finer JJ (1996) Intron-mediated gene expression enhancement in maize (Zea mays L.) and bluegrass (Poa pratensis L.). Plant Cell Rep 15:489–494
Vain P, Worland B, Clarke MC, Richard G, Beavis M, Liu H, Kohli A, Leech M, Snape JW, Christou P, Atkinson H (1998) Expression of an engineered proteinase inhibitor (Oryzacystatin-I Δd86) for nematode resistance in transgenic rice plants. Theor Appl Genet 96:266–271
Vain P, Worland B, Kohli A, Snape JW, Christou P, Allen GC, Thompson WF (1999) Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J 18:233–242
Vain P, James VA, Worland B, Snape JW (2002) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105:878–889
Wang MB, Upadhyaya NB, Brettell RIS, Waterhouse PM (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Genet Breed 51:325–334
Wang MB, Li ZY, Matthews PR, Upadhyaya NM, Waterhouse PM (1998) Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hortic 461:401–407
Yin Z, Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470
Acknowledgements
We gratefully acknowledge The Rockefeller Foundation for its support. We thank Drs E. Guiderdoni, C. Sallaud and J.C. Breitler (CIRAD, France) for advice and insightful discussions. We thank V.A. James (University of Florida, USA) for proofing the manuscript. This document is an output from projects (Plant Sciences Research Programme R8031) funded by the UK Department for International Development (DFID) and administered by the Centre for Arid Zone Studies (CAZS) for the benefit of developing countries. The views expressed are not necessarily those of DFID.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by C. Möllers
Rights and permissions
About this article
Cite this article
Vain, P., Afolabi, A.S., Worland, B. et al. Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107, 210–217 (2003). https://doi.org/10.1007/s00122-003-1255-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00122-003-1255-7