title: Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.
abstract: A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.
authors.affiliation: Department of Plant Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli-24, 620 024, Tamil Nadu, India. vinogenes@gmail.com
authors.forename: S
authors.initials: S
authors.lastname: Vinoth
authors.forename: P
authors.initials: P
authors.lastname: Gurusaravanan
authors.forename: N
authors.initials: N
authors.lastname: Jayabalan
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D018551:Solanum lycopersicum / Q000382:microbiology*; D018519:Meristem / Q000235:genetics / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000382:microbiology*; D014170:Transformation, Genetic / Q000235:genetics