Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T1 progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T1 progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency.
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Altpeter F, Baisakh N, Beachy R, Bocket R et al (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327. doi:10.1007/s11032-004-8001-y
Bhatnagar M, Prasad K, Pooja Bhatnagar-Mathur P, Narasu ML, Waliyar F, Sharma KK (2010) An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.). Plant Cell Rep 29:495–502. doi:10.1007/s00299-010-0838-4
Bingham ET (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research. Crop Sci 31:1098
Brown DCW, Atanassov A (1985) Role of genetic background in somatic embryogenesis in Medicago. Plant Cell Tissue Organ Cult 4:111–122
De Block M, Debrouwer D (1991) Two T-DNA’s co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82:257–263. doi:10.1007/BF02190610
De Buck S, Jacobs A, Montagu MV, Depicker A (1998) Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplast. Mol Plant Microbe Interact 11:449–457
de Vetten N, Wolters AM, Raemakers K, van der Meer I, ter 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. doi:10.1007/s11627-007-9089-7
Depicker A, Herman L, Jacobs A, Schdl J, Van Montagu M (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet 201:477–484
Doshi KM, Eudes E, Laroche A, Gaudet D (2007) Anthocyanin expression in marker free transgenic wheat and triticale embryos. In Vitro Cell Dev Biol Plant 43:429–435. doi:10.1007/s11627-007-9089-7
Dutt M, Li ZT, Dhekney SA, Gray DJ (2008) A co-transformation system to produce transgenic grapevines free of marker genes. Plant Sci 175:423–430. doi:10.1016/j.plantsci.2008.06.014
Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada K, Komamine A (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20:383. doi:10.1007/s002990100344
Gough KC, Hawes WS, Kilpatrick J, Whitelam GC (2001) Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation. Plant Cell Rep 20:296–300. doi:10.1007/s002990100337
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994. doi:10.1007/BF00014672
Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5(10):446–451
Jia H, Liao M, Verbelen J-P, Vissenberg K (2007) Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs. Plant Cell Rep 26:1961–1965. doi:10.1007/s00299-007-0403-y
Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302
Miller M, Tagliani L, Wang N, Berka B, Bidney D, Zhao ZY (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11:381–396. doi:10.1023/A:1016390621482
Park J, Lee YK, Kang BK, Chung WI (2004) Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants. Theor Appl Genet 109:1562–1567. doi:10.1007/s00122-004-1790-x
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
RamanaRao MV, Veluthambi K (2010) Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection. Plant Cell Rep 29:473–483. doi:10.1007/s00299-010-0836-6
Ramessar K, Peremarti A, Gomez-Galera S, Naqvi S, Moralejo M, Munoz P, Capell T, Christou P (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgenic Res 16:261–280. doi:10.1007/s11248-007-9083-1
Rosellini D, Capomaccio S, Ferradini N, Savo Sardaro ML, Nicolia A, Veronesi F (2007) Non-antibiotic, efficient selection for alfalfa genetic engineering. Plant Cell Rep 26:1035–1044. doi:10.1007/s00299-007-0321-z
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
Samac DA (1995) Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 43:271–277
Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour
Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298. doi:10.1007/BF00021308
Weeks JT, Ye J, Rommens CM (2008) Development of an in planta method for transformation of alfalfa (Medicago sativa). Transgenic Res 17:587–597. doi:10.1007/s11248-007-9132-9
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415. doi:10.1093/nar/gkg595
Funding was provided by the Italian Ministry of University and Scientific research, Project: ‘Impact of genetic engineering on the alfalfa genome and strategies to reduce it’ (PI Daniele Rosellini). Nicoletta Ferradini acknowledges the European Union (Programme POR FSE 2007–2013) for financial support. Patrizia Lepri provided valuable help with tissue culture work.
Communicated by P. Ozias-Akins.
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Ferradini, N., Nicolia, A., Capomaccio, S. et al. Assessment of simple marker-free genetic transformation techniques in alfalfa. Plant Cell Rep 30, 1991–2000 (2011). https://doi.org/10.1007/s00299-011-1107-x
- Genetic engineering
- Marker-less transformation
- Medicago sativa
- Selectable markers