Many genes are involved in embryogenesis in plants. We have isolated 12 recessive mutants, derived from four independent loci; gle1 at GLE1 (GROBULAR EMBRYO 1), two alleles (gle2-1, gle2-2) at GLE2, gle3 at GLE3 and two alleles (gle4-1, gle4-2) at GLE4 (Hong et al. 1995), and seven mutants of Organ Differentiation Mutant (odm) category, that cease embryogenesis at early globular stages, from M2 population of cv. Taicung65 mutagenized with MNU. In this report, we classified these mutants into three classes from histological analysis.

These mutants examined do not differentiate any embryonic organs, namely radicles, epiblasts, coleoptiles and leaves. Thus, even at the fully matured stage, these embryos keep globular morphology. Our histological analysis revealed that these mutants could be classified into three types (Fig. 1, 2). Type1 mutant embryos were almost same with wild-type embryos in size at the globular stage, but further development does not occur, and remain globular even at the full-mature stage. Type1 is the most frequently isolated type and includes gle1, gle2-1, gle2-2, gle3, odm42 and odm57. The type2 embryo grew obviously larger than

embryo of wild type until the globular stage, and the outermost layer of embryos of these mutants seem to differentiated into epithelium. Differentiation of epithelium was confirmed on one of type2 mutants, gle4-1 by in situ hybridization analysis with probe for Ramy1A gene that has been used as a marker of epithelium (Sugimoto et al. 1997). This category includes gle4-1 and gle4-2. In Type3, the size of mutant embryo at full-mature stage is clearly smaller than other types. The activity of cell division in the embryogenesis of Type3 mutants was low and a number of cells of mutant embryo were very few. The features that senescence begins from early developmental stage and the cells arrangement in the outer layer of matured embryo is irregular also characterized Type3 mutants. This category included odm63, odm78, odm142 and odm157.

To characterize these mutant classes in detail, we performed in situ hybridization analysis with OSH1 probe on mutant embryos at about five days after pollination. OSH1 is one of rice homeobox genes, and in the wild-type rice embryos is considered to be required for the establishment and maintenance of shoot apical meristem (SAM) (Sato et al. 1996). In every 12 mutants we examined, hybridization signals in the SAM region were detected as did in the wild-type embryo (Fig. 2). This result indicated that SAM is regionalized also in these mutants. However, in type2 and type3, in situ signals for OSH1 expression disappeared at later stages, suggesting that these mutants lack some machinery for maintenance of SAM region. It is also plausible that senescence occurred at later stages of the embryogenesis, in particular, in the case of type3 embryos in where vacuolation was severely occurred (Fig. 1).

Our results suggest the existence of genes that maintain activity of cell division in embryo before organ determination (Type1, 2, 3). Detailed analyse of these mutants will provide a lot of new information of regulatory mechanisms of organ determination in rice embryo.

References

Hong, S.K., Aoki, T., Kitano, H., Satoh, H. and Nagato, Y. (1995). Phenotypic diversity of 188 rice embryo mutants. Developmental Genetics, 16: 293-310.

Sato, Y., Honk, S.K., Tagiri, A., Kitano, H., Yamamoto, N., Nagato, Y. and Matsuoka, M. (1996). A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Natl Acad. Sci. USA, 93: 8117-8112.

Sugimoto, N., Takeda, G., Nagato, Y. and Yamaguchi, J. (1997). Temporal and spatial expression pattern of rice aamylase gene Ramy1A during seed development. RGN, 14: 148-150.