Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS

Abstract

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. This disease is hard to be completely controlled by chemical sprays, and the selection of citrus genotypes resistant to the pathogen becomes one of the most important ways to control the disease. The limited genotypes that have been found up to now tolerant to Xcc are not commercially cultivated. Sweet orange [Citrus sinensis (L.) Osbeck] is one of the most important citrus species widely cultivated in world and all the orange cultivars are susceptible to canker disease. Therefore, this study aims at the establishment of an efficient protocol for in vitro ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange somaclones tolerant to citrus canker disease. Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 h (the lethal concentration). A crude extract produced from the pathogen solution was used as the selection agent. Sweet orange leaves inoculated by Xcc-crude extract showed the symptoms similar to those inoculated by Xcc bacterial inoculum. The young shoots of susceptive genotypes cultured in 10 % of Xcc-crude extract solution become brown and died in 1 week, while the resistant citron C-05 grew normally under the same conditions. After two steps (cell suspension and plants) of selection by Xcc-crude extract solution, the survival plants were tested by in vitro and in vivo inoculation with Xcc. One somaclone, named DG-2, was identified to be resistant to the canker disease. The results suggested that the established protocol for somaclones variation by treating sweet orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the pathogen crude extract was effective. The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar selection procedures.