Tissue culture and Agrobacterium-mediated transformation of carrot (Daucus carota L.)

Transgenic carrot (Daucus carota L.) plants were obtained using disarmed Agrobacterium tumefaciens strains EHA 105 (leucinopine) and MOG 101 (nopaline).Each of the two strains harboured a binary plasmid containing either an acidic chitinase gene from petunia (pMOG196), or a basic chitinase gene from either tobacco (pMOG198) or bean (pGA492-CHN) driven by the constitutive 35-S promoter from Cauliflower Mosaic Virus (CaMV).In addition, the neomycin phosphotransferase (NPT II) gene from Tn5 encoding kanamycin resistance was present.The influence of the Agrobacterium strain, plasmid, carrot cultivar, age of explant, and co-cultivation time were evaluated.The highest frequency of transformation (1 2.1 %) based on production of somatic embryos on Murashige and Skoog medium (MS) with 4.5 pM 2,4dichlorophenoxyacetic acid (2,4-D) and 100 mg L-1 of kanamycin, was obtained with epicotyl segments of the cultivar Nanco cocultivated for 2 or 3 days with the supervirulent A. tumefaciens strain EHA 105.Suspension cultures were also initiated from the embryogenic calli in liquid MS medium with 0.5 pM 2,4-D and 50 mg L-I of kanamycin.Plantlet development occurred within 2-4 months after plating either calli or cell suspensions onto MS medium without growth regulators or kanamycin; excised shoots were rooted on MS medium containing kanamycin (50 mg L-1) before transferring to soil.Transformation was confirmed by PCR amplification of the NPT II coding region and by Southern hybridization analysis using an 800 bp Digoxigenin-UTP labelled probe specific for the NPT II gene.A single hybridizing band was seen indicating the integration of one T-DNA copy in the genome of the transgenic plants.iii Dedication To my parents and my husband Chris for their understanding, patience, and encouragement.