Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants.
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.
Cardoza
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Department of Plant Sciences and Landscape Systems, University of Tennessee, Rm 252 Ellington Plant Sciences, 2431 Center Drive, Knoxville, TN 37996-4561, USA.
Stewart
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CN
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
D007211:Indoles; D008164:Luminescent Proteins; D010937:Plant Growth Regulators; C014612:indolebutyric acid; D049452:Green Fluorescent Proteins; D007612:Kanamycin