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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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.
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