K. FUKUI1, K. KAKEDA2, J. HASHIMOTO1,3 and S. MATSUOKA1
1) National Institute of Aerobiological Resources, Tsukuba, lbaraki 305, Japan
2) Faculty of Agriculture, Kyoto University, Kyoto, 606 Japan
3) Present address: National Institute of Animal Health, Tsukubu, Ibaraki, 305 Japan
Identification of mitotic rice chromosomes has been very difficult because of their small size. They are 1-2μm in length and almost all of them are of metacentric type at the mid-metaphase (Fukui 1987). Therefore, techniques which can be used to identify each chromosome other than ordinary karyotype analysis, have been long awaited. The in situ hybridization technique is one of the most promising methods for identifying rice chromosomes. However, reports of successful in situ hybridization to rice chromosomes are few (Wu et al. 1986; N. Kurata, personal communication).
The rRNA fraction was collected from total extracted rice RNA by centri- fugation in sucrose density gradients. rRNA was fractionated into 28S and 18S rRNA by agarose gel electrophoresis. Each rRNA was labelled with 125I (Prensky et al. 1973) to a specific activity of 5 X 106cpm/μg rRNA. 28S rRNA probe was used for in situ hybridization to rice chromosomes.
Rice chromosome samples were prepared by a modified enzymatic maceration and flame drying method (Karata and Omura 1978). In situ hybridization was carried out on the glass slides for 20 hrs at 40°C in solution (3xSSC, 50% formamide) containing the radioactive probe after denaturation. After washing with 2 X SSC solution several times, the preparation was dipped into Sakura NR-M2 emulsion and exposed for one to six weeks (Fukui 1984).
Autoradiograms were lightly and uniformly labelled with silver grains in general. However, clusters of silver grains were evident for two chromosomes (Fig. 1). In most cases other chromosomes were labelled with only one or two independent silver grains. Although the shapes of the labelled chromosomes were somewhat ambiguous, it was obvious that the sizes of the chromosomes were relatively small, and that the hybridization region was located on one end of both chromosomes. These results were consistent with the size of chromosome K10 and the position of the nucleolar organizing region of rice.
Therefore, it could be concluded that in situ hybridization is an effective method to identify specific chromosomes. In the future, it will be necessary to develop specific probes that can hybridize to each of the arms of the twelve rice chromosomes, in order to investigate chromosomal aberrations occurring in regenerated plants after tissue culture, or progenies of mutagenic treatment.
Fig. 1. In situ hybridization of 28S rRNA to chromosome K10. Arrows show the nucleolus-organizing regions.
Fukui, K. 1984. In situ hybridization method. In: Experimental methods for plant genetic engineering, by H. Uchimiya, K. Tanaka and A. Sugiura (eds.). Kodansha Scientific, Tokyo, p. 165-168. (in Japanese)
Fukui, K. 1987. Comparison of the Giemsa and orcein staining methods in rice chromosomes. La Kromosomo II-43 (in press)
Kurata, N. and T. Omura, 1978. Karyotype analysis in rice. I. A New method for identifying all chromosome pairs. Jpn. J. Genet. 53: 251-255.
Prensky,W., D. M. Steffensen and W. L. Hughes 1973. The use of iodinated RNA for gene localization. Proc. Nat. Acad. Sci. USA 70: 1860-1864.
Wu, H. K.,M. C. Chung, Y. Xie and R. Wu 1986. Chromosomal localization of rice rbcS gene. RGN 3: 117-119.