Abstract
Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing “cytokinin” vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.
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References
Agrawal PK, Kohli A, Twyman RM, Christou P (2005) Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels. Mol Breeding 16:247–260
Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ, Fauquet C, Huang N, Kohli A, Mooibroek H, Nicholson L, Nguyen TT, Nugent G, Raemakers K, Romano A, Somers DA, Stoger E, Taylor N, Visser R (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327
Assad-García N, Ochoa-Alejo N, García-Hernández E, Herrera-Estrella L (1992) Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants. Plant Cell Rep 11:558–562
Ballas N, Citovsky V (1997) Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc Natl Acad Sci USA 94:10723–10728
Ballester A, Cervera M, Peña L (2007) Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination. Plant Cell Rep 26:39–45
Barry GF, Kishore GM, Krohn BM (1998) Isoamylase gene, compositions containing it, and methods of using isoamylases. US Patent 5750876
Bent AF (2006) Arabidopsis thaliana floral dip transformation method. Methods Mol Biol 343:87–103
Bukovinszki A, Diveki Z, Csanyi M, Palkovics L, Balazs E (2007) Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a ‘shooter mutant’ A. tumefaciens. Plant Cell Rep 26:459–465
Cardoza V, Stewart CN (2003) Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep 21:599–604
Chichkova NV, Kim SH, Titova ES, Kalkum M, Morozov VS, Rubtsov YP, Kalinina NO, Taliansky ME, Vartapetian AB (2004) A plant caspase-like protease activated during the hypersensitive response. Plant Cell 16:157–171
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 Breeding 7:25–33
de Vetten N, Wolters AM, Raemakers K, van der Meer I, der Stege R, Heeres E, Heeres P, Visser R (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21:439–442
Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94:2117–2121
Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system. Plant J 30:115–122
Garbarino JE, Belknap WR (1993) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol Biol 24:119–127
Garbarino JE, Oosumi T, Belknap WR (1995) Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol 109:1371–1378
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37
Hanson B, Engler D, Moy Y, Newman B, Ralston E, Gutterson N (1999) A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J 19:727–734
Kepinski S (2006) Integrating hormone signaling and patterning mechanisms in plant development. Curr Opin Plant Biol 9:28–34
Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773
Kobori S, Masuda Y, Horii M, Marubashi W (2007) High levels of the cytokinin BAP suppress programmed cell death in hybrid tobacco cells (Nicotiana suaveolens × N. tabacum) expressing hybrid lethality. Plant Biotechnol 24:375–381
Kondrak M, van der Meer IM, Banfalvi Z (2006) Generation of marker- and backbone-free transgenic potatoes by site-specific recombination and a bi-functional marker gene in a non-regular one-border Agrobacterium transformation vector. Transgenic Res 15:729–737
Kononov ME, Bassuner B, 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
Li Y, Hagen G, Guilfoyle TJ (1992) Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs. Dev Biol 153:386–395
Molinier J, Thomas C, Brignou M, Hahne G (2002) Transient expression of ipt gene enhances regeneration and transformation rates of sunflower shoot apices (Helianthus annuus L.). Plant Cell Rep 21:251–256
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497
Myskja B (2006) The moral difference between intragenic and transgenic modification of plants. J Agric Env Ethics 19:225–238
Nielsen KM (2003) Transgenic organisms––time for conceptual diversification? Nat Biotechnol 21:227–228
Popelka JC, Xu J, Altpeter F (2003) Generation of rye (Secale cereale L.) plants with low transgene copy number after biolistic gene transfer and production of instantly marker-free transgenic rye. Transgenic Res 12:587–596
Puchta H (2003) Marker-free transgenic plants. Plant Cell Tissue Organ Cult 74:123–134
Que Q, Jorgensen RA (1998) Homology-based control of gene expression patterns in transgenic petunia flowers. Dev Genet 22:100–109
Rios G, Lossow A, Hertel B, Breuer F, Schaefer S, Broich M, Kleinow T, Jasik J, Winter J, Ferrando A, Farras R, Panicot M, Henriques R, Mariaux JB, Oberschall A, Molnar G, Berendzen K, Shukla V, Lafos M, Koncz Z, Redei GP, Schell J, Koncz C (2002) Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes. Plant J 32:243–253
Romano A, Raemakers K, Bernardi J, Visser R, Mooibroek H (2003) Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes. Transgenic Res 12:461–473
Rommens CM (2004) All-native DNA transformation: a new approach to plant genetic engineering. Trends Plant Sci 9:457–464
Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang L, Perry R, Swords K (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135:421–431
Rommens CM, Bougri O, Yan H, Humara JM, Owen J, Swords K, Ye J (2005) Plant-derived transfer DNAs. Plant Physiol 139:1338–1349
Rommens CM, Haring MA, Swords K, Davies HV, Belknap WR (2007) The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci 12:397–403
Sallaud C, Meynard D, van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PB, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408
Simpson J, Montes-Hernandez S, Gutierrez-Campos R, Assad-Garcia N, Herrera-Estrella L (1995) Genetic transformation in Physalis species (Tomatillo). In: Bajaj YPS (ed) Plant protoplasts and genetic engineering VI. Biotechnology in agriculture and forestry, vol. 34. Springer-Verlag, Berlin Heidelberg, pp 228–239
Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899
Yan H, Rommens CM (2007) Transposition-based plant transformation. Plant Physiol 143:570–578
Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in plants. Plant Physiol 141:1508–1518
Acknowledgements
We thank Scott Simplot, Bill Whitacre, and Dr. Kathy Swords for fruitful discussions and continued support. Dr. Jingsong Ye, Dr. Oleg Bougri, Kristi Fessenden, and Jeffery Hein are acknowledged for excellent technical contributions and Alexey Kromin for a critical review of the manuscript.
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Richael, C.M., Kalyaeva, M., Chretien, R.C. et al. Cytokinin vectors mediate marker-free and backbone-free plant transformation. Transgenic Res 17, 905–917 (2008). https://doi.org/10.1007/s11248-008-9175-6
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DOI: https://doi.org/10.1007/s11248-008-9175-6