In previous study, we reported the screening and characterization of rice embryonic mutant from NMU- induced or callus culture-induced mutant libraries (Hong et al., 1995, Imamura et al., 2008). Among these, the club-shaped embryo1 (cle1) mutant was characterized by the “club-shaped” structure which extends along the apical-basal axis during the embryonic stage (see Fig. 1G-I compared with A-C). The cle1 embryo fails to develop most organs and tissues (i.e. SAM and RAM) except for the epithelium-like and the vascular-like
tissues. It should be noted that a rice homeobox gene OSH1, which is specifically expressed in the shoot apical region before the morphological development in WT is misexpressed in cle1 (Imamura et al., 2008). This expression pattern differs from that of other organless-mutants, (i.e. organless1) whose expression pattern is similar to that of the WT embryo (Kamiya et al., 2001). Here, we report a newly identified club-shaped embryo, cle3, which was also screened by the “club-shaped” phenotype of the embryo from the callus culture-induced
mutant libraries.
We utilized 7000 mutant lines from the callus culture-induced mutant library to screen for the club- shaped embryos, and identified four mutant lines, which were crossed with the cle1 for allelism test. From these, one line was found to be non-allelic to cle1 and thus, was named cle3 (Table 1). We studied the segregation of cle3 mutant, and the resulting frequency of segregation (17%) did not fit 3:1 (Table 2). This was also true for the segregation of the F2 progenies derived from crossing the cle3 mutant (japonica) pistil with Kasalath (indica) pollen. Interestingly, when the Kasalath pistil was crossed with the cle3 pollen, the frequency of segregation (24%) fitted 3:1 (Table 2). These results suggest that the inheritance of cle3 is not of Mendelian manner. A similar mode of inheritance was observed in cle1-1 (data not shown)
The histological analysis of the cle3 mutant during embryogenesis was studied. In the WT embryo, protrusion of the coleoptile as the first organ differentiation is observed at 4 days after pollination (DAP) (Ito et al., 2005 Fig. 1A). The development of the second leaf primordium from SAM, the radicle from RAM, as well as the vascular tisuue, and the epithelium tissue are completed at 7 DAP (Fig. 1B). The embryo then matures until 10 DAP (Fig. 1C). In contrast to the WT embryo, the protrusion of the coleoptile in the cle3 embryo was not observed at 4 DAP (Fig. 1D, compared with A). At 7 DAP, the cle3 embryo elongated along the apical- basal axis, and assumed a “club-shape” configuration. At this stage, the radicle was developed, however, SAM, vascular-like tissue, and epithelium-like tissue remained unformed (Fig. 1E). At 10 DAP, the cle3 embryo began to degenerate of tissues in allover the embryo including the radicle and the vascular-like tissue (Fig. 1F). In summary, the different phenotypes of cle3 compared with cle1 embryos are: (1) development of radicle was observed, (2) Establishment of epithelium-like tissue was not observed, and (3) the degeneration of tissues was observed 7 DAP (Fig. 1G-I, compared with D-F).
Towards the isolation of the CLE3 gene, we used the genomic DNAs of F2 heterozygous plants from a cross between the cle3 mutant and Kasalath in this study. We used 30 F2 heterozygous plants, which were identified by the segregation of the mutant phenotype in F3 embryos for the linkage analysis. The result demonstrated that CLE3 is located between TG105 and c16130 on chromosome 6 (Fig. 2). This presents a genetic distance of 9.0 cM (Fig. 2) as inferred from the RGP (Rice Genome Project) map (Harushima et al., 1998). We are currently narrowing down this region containing the CLE3 gene.
References
Hong S. K., T. Aoki, H. Kitano, H. Satoh and Y. Nagato, 1995. Phenotypic diversity of 188 rice embryo mutant. Dev. Genet. 16: 298-310.
Imamura K., H. Imai, Y. Nagato, M. Matsuoka, H. Kitano and T. Sazuka, 2008. Mapping and Analysis of the CLUB-SHAPED EMBRYO1, CLE1. Rice Genet. Newsl. 24: 22-24.
Itoh J., K. Nonomura, K. Ikeda, S. Yamaki, Y. Inukai, H. Yamagishi, H. Kitano and Y. Nagato, 2005. Rice plant development: From zygote to spikelet. Plant Cell Physiol. 46: 23-47.
Kamiya N., M. Ashikari, Y. Sato, Y. Nagato and H. Kitano and M. Matsuoka, 2001. Mapping of the ORGANLESS1 gene, ORL1. Rice Genet. Newsl. 18: 33.
Harushima Y., M. Yano, A. Shomura, M. Sato, T. Shimono, Y. Kuboki, T. Yamamoto, S. Y. Lin, B. A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G. S. Khush and T. Sasaki, 1998. A high-density rice genetic linkage map with 2275 makaers using a single F2 population. Genetics 148: 479-494.