title: Assessment of simple marker-free genetic transformation techniques in alfalfa.
abstract: 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 T(1) progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T(1) progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency.
authors.affiliation: Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo XX giugno 74, 06121, Perugia, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Ferradini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Nicolia
authors.forename: Stefano
authors.initials: S
authors.lastname: Capomaccio
authors.forename: Fabio
authors.initials: F
authors.lastname: Veronesi
authors.forename: Daniele
authors.initials: D
authors.lastname: Rosellini
mesh_terms: D015139:Blotting, Southern; D020090:Chromosome Segregation / Q000235:genetics; D003433:Crosses, Genetic; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D018628:Gene Dosage / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D005819:Genetic Markers; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000378:metabolism; D000455:Medicago sativa / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; D005819:Genetic Markers; C036483:T-DNA; D005966:Glucuronidase