| Xanthomonas citri subsp. citri is the causal agent of citrus canker. Citrus canker disease has been one of the serious diseases in the south of China, and caused serious damages. Sweet orange [Citrus. sinensis (L.) Osbeck] is the most important citrus species widely cultivated in the world, but all the sweet orange cultivars are highly susceptible to the disease. The essential and effective solution is to breed a new commercial citrus cultivars with good quality, high yield and high resistant to canker disease.Therefore, in this paper, the epicotyl internodal stem segments of ’Dahong’ sweet orange seedlings were treated with ethyl methylanesulphonate (EMS) solution and screened by the Xcc-crude extracts produced by Xanthomonas citri subsp. citri, via a multi-step screening method. The regenerated shoots were inoculated Xcc by in vitro and in vivo, in order to provide an important materials and theoretical basis for study the mechanism of citrus resistance of canker disease. Meanwhile, we analyzed the expression of LYP2 and CERK1 genes in the mutants after inoculation with Xcc and peptidoglycan which from Xcc’s cell wall, and analyzed the expresstion of CsLob1 gene that a kind of disease suscepbility gene in the mutants and ’Bingtang’ sweet oranges after inoculation with Xcc. The main results are as follow:1. The epicotyl internodal stem segments of ’Dahong’ sweet orange seedlings were treated with different concertration of EMS solution, while treated under the concertration of EMS is 1.8%(v/v) for 2h which resulted in the effect of the "semi lethal dose", established a systym of ethylmethane sulphonate (EMS) mutagenesis techniques of sweet orange.2. The Xcc-crude extracts has been verified on the harmful effects of ’Bingtang’ sweet orange shoots. The epicotyl internodal stem segments of ’Dahong’ sweet orange seedlings were treated with different concertration of Xcc-crude extracts, and the appropriate selection pressure was that the stem segments were cultured in medium supplemented with 4%(v/v) of Xcc-crude extracts.3. There were 10 sweet orange mutants resistant to Xcc-crude extracts were selected after mutation and twice discontinuous screening, and which weres successed grafted on the root stock of Poncirus trifoliata (L.) Raf. After in vivo inoculation with Xcc, the mutant strains 149,278,376 were performed steady capability of resistant to canker disease.4.’Bingtang’ sweet orange and the mutants were inoculated with Xcc and the peptidoglycan (PGN(Xcc)) which were extracted from Xanthomonas citri subsp. citri by in vivo. The results showed that, the expression levels of CERK1 and LYP2 decreased after inoculation with Xcc and PGN(Xc) in ’Bingtang’sweet orange. The expression level of CERKl increased after inoculation with Xcc and PGN(Xcc) in the mutant strains 149,278 and 376, the differential expression of 376 was the most significant; the expression level of LYP2 increased in 376, but the expression level of LYP2 have no significant difference in 149 and 278 after inoculation with Xcc or PGN(Xcc). Therefore, CERK1 and LYP2 were considered as the key genes in the process of initial resistance canker disease in the mutants. And in citrus, it may exists a recognition system that were constituted by CERK1 and LYP2 to recognize the peptidoglycan.5. The expression levels of CsLOBl increased significant in ’Bingtang’ sweet orange, and the mutant strains 149 and 278, but the expression levels of CsLOB1 in’ Bingtang’ sweet orange are much higher than in the mutants, while have on significant differential in the mutant strain 376. So, according to the expression levels of susceptibility gene CsLOB1, the mutant strain 376 was considered to be the most resistant to canker disease, and the mutant strain 149 and 278 were considered moderate their resistance canker disease. |
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