Abstract
Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.
Similar content being viewed by others
References
Artelt P, Grannemann R, Stocking C, Friel J, Bartsch J and Hauser H (1991) The prokaryotic neomycin-resistance encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene 99: 249–254.
Christou P, Ford T and Kofron M (1991) Production of transgenic rice (Oryza sativa L.) from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9: 957–962.
Cluster PD, O'Dell M, Metzlaff M and Flavell RB (1994) Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol. Biol. 32: 1197–1203.
Datta SK, Peterhans A, Datta K and Potrykus I (1990) Genetically engineered fertile Indica rice recovered from protoplasts. Bio/Technology 8: 736–740.
De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Rao Movva N, Thompson C, Van Montagu M and Leemans J (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 6: 2513–2518.
Edwards K, Johnston C and Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analyses. Nucleic Acids Res. 98: 1349.
Fu X, Kohli A, Twyman RM and Christou P Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene single-copy transgenic locus in rice. Mol. Gen. Genet. (in press).
Jefferson RA, Kavanagh TA and Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.
Klein TM, Wolf ED, Wu R and Sandford JC (1987) High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73.
Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA and Christou P (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology-mediated recombination. Plant J. 17: 591–601.
Kohli A, Leech M, Vain P, Laurie DA and Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot-spots. Proc. Natl. Acad. Sci. USA 95: 7203–7208.
Kononov E, Bassuner B and Gelvin SB (1997) Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: Evidence for multiple complex patterns of integration. Plant J. 11: 945–957.
Linden RM, Winocour E, Berns KI (1996) The recombination signals for adeno-associated virus site-specific integration. Proc. Natl. Acad. Sci. USA 93: 7966–7972.
Matzk A, Mantell S and Schiemann J (1996) Localization of persisting Agrobacteria in transgenic tobacco plants. Mol. Plant-Microbe Interact. 19: 373–381.
Matzke MA, Matzke AJM and Eggleston WB (1996) Paramutation and transgene silencing: A common response to invasive DNA? Trends Plant Sci. 1: 382–388.
Muller AE, Kamisugi Y, Gruneberg R, Niedenhof I, Horold RJ and Meyer P (1999) Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum. J. Mol. Biol. 291: 29–46.
Nam J, Matthysse AG and Gelvin SB (1997) Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration. Plant Cell 9: 317–333.
Palmiter RD and Brinster RL (1986) Germline transformation of mice. Ann. Rev. Genet. 20: 465–491.
Pawlowski WP and Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc. Natl. Acad. Sci. USA 95: 12106–12110.
Ramanathan V and Veluthambi K (1996) Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTIA6 from the left terminus of T-L-DNA. Plant Mol. Biol. 28: 1149–1154.
Register JC, Peterson DJ, Bell PJ, Bullock PW, Evans IJ, Frame B, Greenland AJ, Higgs NS, Jepson I, Jiao S, Lewnau CJ, Sillick JM and Wilson HM (1994) Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol. Biol. 25: 951–961.
Rogers SO and Bendlich AL (1994) Extraction of total cellular DNA from plants, algae and fungi. In: Gelvin SB, Schilperoort AR (eds) Plant Molecular Biology Manual, 2nd edn. (pp. 1–8) KluwerAcademic Publishers, Dodrecht.
Salomon S and Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 17: 6086–6095.
Srivastava V, Vasil V and Vasil IK (1996) Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.) Theor. Appl. Genet. 92: 1031–1037.
Stoger E, Williams S, Keen D and Christou P (1998) Molecular characteristics of transgenic wheat and the effect on transgene expression. Transgenic Res. 7: 463–471.
Sudhakar D, Duc LT, Bong BB, Tinjuangjun P, Maqbool SB, Valdez M, Jefferson R and Christou P (1998) An efficient rice transformation system utilizing mature seed-derived explants and a portable, inexpensive particle bombardment device. Transgenic Res. 7: 289–294.
Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thomas S and Brettel R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11: 1369–1376.
Vain P, Worland B, Clarke MC, Richard G, Beavis M, Liu H, Kohli A, Leech M, Snape J, Christou P and Atkinson H (1998) Expression of an engineered cysteine protienase inhibitor (Oryzacystatin-IΔD86) for nematode resistance in transgenic rice plants. Theor. Appl. Genet. 96: 266–271.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Fu, X., Duc, L.T., Fontana, S. et al. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res 9, 11–19 (2000). https://doi.org/10.1023/A:1008993730505
Issue Date:
DOI: https://doi.org/10.1023/A:1008993730505