title: Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants.
abstract: An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.
authors.affiliation: Department of Plant Sciences and Landscape Systems, University of Tennessee, Rm 252 Ellington Plant Sciences, 2431 Center Drive, Knoxville, TN 37996-4561, USA.
authors.forename: V
authors.initials: V
authors.lastname: Cardoza
authors.forename: C N
authors.initials: CN
authors.lastname: Stewart
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015153:Blotting, Western; D029688:Brassica napus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D046508:Culture Techniques; D049452:Green Fluorescent Proteins; D018546:Hypocotyl / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D018520:Plant Shoots / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D012038:Regeneration; D014170:Transformation, Genetic
chemical_list: D007211:Indoles; D008164:Luminescent Proteins; D010937:Plant Growth Regulators; C014612:indolebutyric acid; D049452:Green Fluorescent Proteins; D007612:Kanamycin