52. Characterization of shootless mutants in rice 

N. SATOH1, S.K. HONG2, M. MATSUOKA3, H. KITANO4 and Y. NAGATO1

1) Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113 Japan
2) Department of Agronomy, Kangweon National University, Chuncheon, 192-1 Korea
3) BioScience Center, Nagoya University, Nagoya, 464 Japan
4) Faculty of Agriculture, Nagoya University, Nagoya, 464 Japan
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The establishment of shoot apical meristem (SAM) is a key event for plant development, because plant body is constructed by the continuing activity of SAM. However, genetic basis of SAM differentiation have been poorly understood. In this report, we characterize nine recessive shootless mutations derived from four loci, SHL1 to SHL 4 (Hong et al. 1995). Out of nine, eight mutations were identified from M2 population of cv. Taichung 65 and one (shl2-5) from that of cv. Kinmaze, mutagenized with MNU.

The embryos of these shootless mutants do not differentiate SAM, leaves, coleoptile and epiblast but have normal radicles (Fig.1). Among them, shl1, shl2 and shl4 embryos are not morphologically distinguishable. The embryos of shl3 is unique in that the radicle is precociously germinated during seed maturation and that apical part of embryo is underdeveloped. This shl3 embryo is not viable in mature dry seed. Normal differentiation of scutellum in shl1, shl2 and shl4 was suggested by the presence of morphologically peculiar epidermal cells resembling scutellar epithelium. To confirm this, we performed in situ hybridization experiment on embryos at 2-4 days after imbibition probed with Ramy1A, a rice a-amylase gene, which is known to be specifically expressed in scutellar epithelium of wild-type embryos. In every mutant, hybridization signals were detected in epidermal cells facing endosperm. This indicates that scutellum is differentiated and functional in these mutants, suggesting that the differentiation of scutellum is regulated independently of SAM.

Fig. 1. Phenotypes of mature shootless embryos.
A: wild type, B: shl1-1, C: shl1-2, D: shl2-1, E: shl2-2, F: shl2-3, G: shl2-4, H: shl2-5, I: shl3, and J: shl4.
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Fig. 2. Calli of shootless embryos on regeneration medium.
A: wild type calli regenerating a large number of adventitious shoots, B: shl2-3 calli with no adventitious shoots, and C: adventitious leaves (arrows) produced on shl2-3 calli.

Fig. 3. In situ expression of OSHI in shootless embryos at 4-5DAP.
A: wild type embryo at 4DAP, B: shl1-1 at 5DAP, C: shl1-2 at 5DAP, D: shl2-l at 5DAP, E: shl2-2 at 5DAP. F: sh12-3 at 5DAP, G: shl2-4 at 5DAP, H: shl2-5 at 5DAP, I: shl3 at 5DAP, and J: shl4 at 5DAP. Hybridization signals were presented by black color.

To determine whether SHL genes are generally associated with adventitious shoot formation, we tried adventitious shoot regeneration from calli induced from shl1, shl2 and shl4 embryos. No adventitious shoots were regenerated from mutant calli, although many adventitious shoots were regenerated from wild-type calli (Fig. 2). It is concluded that SHL1, SHL2 and SHL4 are required not only for SAM differentiation during embryogenesis but also for general shoot formation.

It is noted that calli of shl1, shl2 and shl4 frequently generated small leaf-like organs (Fig. 2), which were also observed in wild-type calli. Plastic sections and scanning electron microscopy confirmed that they were adventitious leaves. That is, they had trichomes, stomata and vascular bundles. These adventitious leaves were not initiated from SAM but from epidermal cells of calli, and they soon became necrotic before elongation. These results suggest that these genes are not directly associated with the differentiation of leaves.

Finally, we examined the expression of a rice homeobox gene, OSH1, in embryos of shootless mutants by in situ hybridization (Fig. 3). In wild-type embryos at five days after pollination. OSH1 was expressed in the SAM, epiblast, peripheral region of radicle, and their intervening tissues, but not in coleoptile, scutellum, and a region where the first leaf primordium generally differentiates. OSH1 is considered to be required for the establishment and maintenance of SAM (Sato et al. 1996). In two alleles of shl1, OSH1 was expressed in a narrower region than in wild-type. In five shl2 embryos, the signals were not detected at all, while the expression of OSH1 in shl3 and shl4 was almost normal (Fig. 3). Since the OSH1 expression is affected by shl1 and shl2 mutations, SHL1 and SHL2 are estimated to function in the upstream of OSH1. On the other hand, SHL3 and SHL4, loss of function of whose do not modify the OSH1 expression, operate in the downstream or independently of OSH1.

The present results throw important light on embryo development; 1) the differentiation of coleoptile and epiblast depends on the presence of SAM, whereas scutellum and radicle are regulated independently of SAM, 2) adventitious leaves can be produced from tissues other than SAM, but only those produced from SAM would develop, and 3) SAM formation is a complex process involving a large number of genes differentially functioning in a cascade.
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References

Hong, S.-K., T. Aoki, H. Kitano, H. Satoh and Y. Nagato, 1995. Phenotypic diversily of 188 rice embryo mutants. Dev. Genet. 16: 298-310.

Sato, Y.,S.-K. Hong, A. Tagiri, H. Kitano, N. Yamamoto, Y. Nagato and M. Matsuoka, 1996. A rice homeobox gene, OSH1, is expressed prior to organ differentiation in a specific region during early embryogenesis. Proc. Natl. Acad. Sci. USA 93: 8117-8122.