Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

Plant Cell Rep. 2003 Feb;21(6):599-604. doi: 10.1007/s00299-002-0560-y. Epub 2002 Dec 13.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Agrobacterium tumefaciens / genetics*
  • Blotting, Western
  • Brassica napus / drug effects
  • Brassica napus / genetics*
  • Brassica napus / physiology
  • Culture Techniques
  • Green Fluorescent Proteins
  • Hypocotyl / drug effects
  • Hypocotyl / genetics
  • Hypocotyl / physiology*
  • Indoles / pharmacology
  • Kanamycin / pharmacology
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Plant Growth Regulators / pharmacology
  • Plant Roots / drug effects
  • Plant Roots / genetics
  • Plant Roots / physiology*
  • Plant Shoots / drug effects
  • Plant Shoots / genetics
  • Plant Shoots / physiology
  • Plants, Genetically Modified / drug effects
  • Plants, Genetically Modified / genetics*
  • Plants, Genetically Modified / physiology
  • Regeneration
  • Transformation, Genetic*

Substances

  • Indoles
  • Luminescent Proteins
  • Plant Growth Regulators
  • indolebutyric acid
  • Green Fluorescent Proteins
  • Kanamycin