Introduction and expression of chitinase encoding genes in carrot followingAgrobacterium-mediated transformation

Abstract

Transgenic carrot (Daucus carota L.) plants were obtained following transformation with disarmedAgrobacterium tumefaciens strains MOG 101 (octopine type) and EHA 105 (leucinopine type). The strains harbored a binary plasmid that contained either an acidic chitinase gene from petunia (pMOG196), or a basic chitinase gene from tobacco (pMOG198) or bean (pGA492-CHN), transcriptionally fused to the constitutive 35S promoter from cauliflower mosaic virus. Each construct also contained the neomycin phosphotransferase gene (npt II) conferring kanamycin resistance. The influence of theAgrobacterium strains, plasmids, carrot cultivars, ages of explant, and cocultivation times were evaluated. The frequency of transformation (i.e., development of somatic embryos on Murashige and Skoog medium with 4.5 μM 2,4-D and 100 mg/liter of kanamycin) was highest (12.1%) with 2-5-wk-old epicotyl segments of the cultivar Nanco cocultivated for 2–3 d with the supervirulentA. tumefaciens strain EHA 105. Transformation efficiency was not influenced by explant age or binary plasmid, but was significantly influenced by cocultivation period,Agrobacterium strain, and carrot cultivar. Transformed embryogenic calluses from five independent transformation events (out of about 15) were multiplied in suspension cultures (liquid MS medium with 0.5 μM 2,4-D and 50 mg/liter of kanamycin). Within 4–6 wk following plating of cell suspensions onto MS medium without growth regulators or kanamycin, plantlets developed. Excised shoots were rooted on MS medium with kanamycin (50 mg/liter) before transferring to soil. Transformation, was confirmed in the five independent lines by polymerase chain reaction (PCR)-amplification of thenpt II coding region and Southerm hybridization analysis using an 800 bp Digoxigenin-UTP labeled probe specific for thenpt II gene. One to four hybridizing bands were seen in the transgenic plants, indicating the integration of one to four T-DNA copies in the carrot genome. Transgenic plants of cultivars Golden State, Danvers Half Long, and Nanco, which contained either the acidic or basic chitinase genes were obtained. Expression of the introduced basic chitinase genes was confirmed by protein dot-blot analysis and immunostaining with anti-bean and anti-tobacco antibodies.