Assessment of simple marker-free genetic transformation techniques in alfalfa

• Published in: Plant cell reports 2011-11, Vol.30 (11), p.1991-2000
• Main Authors: Ferradini, Nicoletta, Nicolia, Alessandro, Capomaccio, Stefano, Veronesi, Fabio, Rosellini, Daniele
• Format: Article
• Language: English
• Subjects: Agrobacterium; Alfalfa; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Blotting, Southern; Cell Biology; Cell culture; Chromosome Segregation - genetics; copy number; Crops; Crosses, Genetic; DNA, Bacterial - genetics; DNA, Plant - genetics; DNA, Plant - isolation & purification; Embryos; Fundamental and applied biological sciences. Psychology; Gene Dosage - genetics; Genes, Plant - genetics; Genetic crosses; Genetic Markers; Genetic Techniques; Genetic Vectors - genetics; Glucuronidase - metabolism; Integration; Kanamycin; Life Sciences; Medicago sativa - genetics; NPTII gene; Original Paper; Plant Biochemistry; Plant Sciences; Plants, Genetically Modified; Pollinators; Polymerase Chain Reaction; Somatic embryos; T-DNA; Transformation; Transformation, Genetic; Transgenes; Transgenes - genetics; Transgenic plants
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-011-1107-x

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 npt II 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 hem L SMG and one with the unselected npt II 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.