15. Increased frequency of chimeric plants in progenies of dwarf chimeric rice plant treated with 5-azacytidine

M. SHIKARI, A. YOSIMURA and N. IWATA

Faculty of Agriculture. Kyushu University, Fukuoka, 812-81 Japan

A dwarf chimeric rice plant was identified by its production of normal tillers and varying number of dwarf tillers which produce short but wide dark green leaves, compact panicles and small round grains. These dwarf or tall tillers of a chimera may develop at different stages of the plant growth. Some are produced at the vegetative stage while others appeared even at the heading stage.

Small round seeds from dwarf tillers grew mostly as dwarf plants and all chimeric tillers, and again produced chimeric plants. On the other hand, normal seeds from the normal tillers generally give rise to normal as well as chimeric plants. In extremely rare instances, however, small round seed may give rise to a normal plant while a normal seed may develop into a chimeric plant. Hence, selfed progenies of a chimera may be classified into three plant types: dwarf, chimeric and normal plants. Selfed progenies of these plants perpetuate again into these plant types but with varying frequencies that do not follow Mendelian inheritance.

The development of normal and chimeric tillers in a dwarf chimeric rice may be compared to epigenetic phenomena. Epigenetic phenomena may be defined as the meiotically and mitotically heritable changes in gene expression that cannot be accounted for by changes in the DNA sequence (Bender and Fink 1995). Many epigenetic phenomena have been associated with DNA methylation in eukaryotes. In fact, evidence emphasizes its role in gene expression and some epigenetic phenomena has been accumulating in recent years. In this report, we present the results of our experiment on the effect of 5-azacytidine treatment that inhibits DNA methylation on the frequency of occurrence of chimeric plants in the progenies of dwarf and normal plants.

Fig. 1.   Frequency of chimeric plants in 5-azacytidine treated seedlings from small round seeds of
the dwarf chimeric plants A, B and C.

Selfed progenies of a rice chimera were classified into three plant types: dwarf, chimeric and normal plants. In this generation, dwarf and normal plants were harvested. In the next progenies, small round seeds generated from the dwarf plants (dwarf plot) and normal seeds from the normal plants (normal plot) were used for 5-azacytidine treatment. The treatment was carried out by immersing three-days old seedlings in 0.3mM 5-azacytidine water for three days (Sano et al. 1990). There were three replications in each plot and about 400 seedlings/replication were treated. The seedlings were grown in the glass-house for one month and then transplanted to rice field for observation of chimeric expression.

On the plants grown from small round seeds, about 7-15% increase in the frequency of chimeric plants with normal parts was observed in 5-azacytidine treated seedlings of 3 dwarf plants (A, B and C) as compared with the control (Fig. 1). These results show that inhibiting DNA methylation increases chimeric expression in progenies of dwarf chimera since the original dwarf plants were highly methylated. On the contrary, plants from normal seeds showed a reduction in frequency of chimeric plants (1-5%) when seedlings were subjected to 5-azacytidine treatment. It also appears that the progenies of dwarf plants (small round seeds) were more methylated compared with the progenies (normal seeds) of normal plants. We are now trying to isolate the gene controlling chimeric expression in rice so that we can study more deeply the mechanisms involved in the alternate expression of the gene in dwarf chimeric rice.

References

Bender, J. and G.R. Fink, 1995. Epigenetic control of an endogeneous gene family is revealed by a novel blue fluorcscent mutant. Cell 83: 725-734.

Sano, H., I. Kamada and S. Youssefian. 1996. A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undcrmethylation of genomic DNA. Mol. Gen. Genet. 220: 441-447.