KARYOTYPE ANALYSIS IN RICE METHOD FOR IDENTIFYING ALL CHROMOSOME NORI KURATA AND TAKESHI OMURA

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1 JAPAN. J. GENETICS Vol. 53, No. 4: (1978) KARYOTYPE ANALYSIS IN RICE I. ANEW METHOD FOR IDENTIFYING ALL CHROMOSOME PAIRS NORI KURATA AND TAKESHI OMURA Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, 46-1, Fukuoka 812 Received May 1, 1978 Several studies on the chromosome morphology in cultivated rice, Oryza sativa L. (2n=24), have been carried out (Ishii and Mitsukuri 1960; Shastry et al. 1960; Hu 1964; Chu 1967; Khan 1975), however, smallness and poor stainability of rice chromosomes have prevented a complete karyotype analysis. Ishii and Mitsukuri (1960) distinguished 8 chromosome pairs in the diploid 'Normn 8' and 10 chromosomes in its haploid. Hu (1958) also distinguished 10 chromosomes in the haploid `Taichung 65'. Although pachytene analysis was more critical in chromosome characterization than metaphase analysis (Shastry 1964), the identification of individual chromosomes remains obscure even in the pachytene analysis. In this paper we report a new method for demonstrating the clearly shaped rice chromosomes and a complete identification of all 12 pairs in a single metaphase cell. Our newly developed method which can characterize the rice chromosomes is derived from the modification of two conventional methods; the plant tissue macerating method (Gill and Kimber 1974) and the flame-drying technique commonly used in the animal chromosome preparation. MATERIALS AND METHODS A Japanese rice cultivar `Sekitori' was used as a material. Excised roots from the germinating seeds at C were pretreated with M 8-oxyquinoline for 2-3 h at C. Root tips (about 1 mm long of meristems) were immersed in M KC1 at room temperature for 20 min. Then the meristems were treated with the enzyme solution which contained 6% cellulase (Onozuka R-10) and 6% pectinase (Sigma) and was adjusted to ph 4.0 with HCl for about 60 min at 35 C in order to dissolve the cell wall. The macerated meristems were immersed in distilled water for 5-10 min at about 20 C to remove the enzyme solution. A piece of the meristem was put with a drop of fresh fixative (3:1, methanol : acetic acid) on a clean slide glass and thoroughly smashed with a needle. After a few drops of the fixative was again added, the slide glass was flame dried. When well flattened and spreaded cells were observed under a phase-contrast microscope, they were stained with Giemsa diluted 30-fold with 33 mm Sorensen's phosphate buffer (ph 6.8) for min, followed by rinsing with running water and air drying. Suitable cells for the chromosome identification were photo-

2 252 N. KURATA AND T. OMURA graphed and measured the length and the arm ratio (long arm/short arm) of individual chromosomes. RESULTS AND DISCUSSION The chromosomes at prometaphase and metaphase are shown in Fig. 1. The length of chromosomes at prometaphase ranged from 1.5 to 4.1 pm and the total length of haploid complement was 28.4 pm in Fig. 1-a. Meanwhile, the chromosomes were contracted at metaphase and ranged from 1.0 to 1.9pm and totaled 17.1 pm in Fig. 1-b. This stage was, therefore, unsuitable for the identification of individual chromosomes. Combining the staining pattern with the length and the arm ratio, all of the 12 pairs Fig. 1. A prometaphase nucleus (a) and a metaphase one (b). Scale: 5 pm. Fig pairs of chromosomes at prometaphase identified by their characteristics. They were arranged and designated in descending order of the length. Scale: 5 pm.

3 KARYOTYPE ANALYSIS IN RICE 253 of chromosomes could be distinguished clearly at prometaphase as shown in Fig. 2. The chromosomes were arranged in descending order of the length and designated by numbering from Kl to K12 to be distinguished from Nishimura's chromosome number of rice (Nishimura, 1961). Means of the relative length and the arm ratio calculated from 10 chromosomes (5 nuclei) in each pair are shown in Table 1. The relative length Table 1, Mean from values of the relative each 10 chromosomes length (% of the total length) and the arm ratio varied continuously from 15.2 to 5.1. It was found from the arm ratio that the diploid complement was composed of 5 metacentric pairs (K3, K6, K7, K8, Ku), 5 submetacentric pairs (Kl, K2, K5, K9, K12) and 2 sub-telocentric pairs (K4, K10) including a satellited pair. No other secondary constriction was recognized except for the nucleolar constriction of the satellited pair. Other than these features, the segmental differentiation of chromosomes in staining ability was observed. All chromosomes had deeply stained regions on both sides of the centromere. K8 and Kul chromosomes, however, had no differentially stained segment and were stained deeply in whole length at prometaphase. Deeply stained distal segments were observed on one side in 6 pairs (Kl, K2, K3, K5, K6, K9). Main characteristics of 12 pairs at prometaphase were as follows: Kl: Sub-metacentric pair. The telomeric segment was observed at the distal end of the short arm. The sizes of deeply stained regions adjacent to centromere on both arms were much the same. K2: Sub-metacentric pair. At the end of the short arm darkly stained region was visible. Deeply stained segments at the proximal regions were equivalent on both arms. K3: Metacentric pair. The distal end of the short arm was stained a little more darkly than the intercalary part. The proximal deeply stained regions were equivalent on both arms and that of the long arm was sometimes adjoined by scarcely stained part at early metaphase. This part like a secondary constriction was not constantly observed. K4: Sub-telocentric pair. The whole of the short arm was stained darkly. The deeply stained proximal segment on the long arm was as long as the short arm. K5: Sub-metacentric pair. This pair had deeply stained short arms. The deeply stained proximal region on the long arm was about half of the short arm in length. The distal end of the long arm was stained darkly in some degree.

4 254 N. KURATA AND T. OMURA K6: Metacentric pair. The whole short arm was stained deeply but in some cases they were divided into proximal and distal deeply stained segments by a bit of faintly stained part. Long arm had a deeply stained proximal segment, a semi-deeply stained distal segment and an intercalary faintly stained region. K7: Metacentric pair. The whole chromosome was stained deeply except for a bit of faintly stained part. At very early metaphase a small piece of faintly stained segment was inserted between the proximal and the distal deeply stained regions of the long arm. Meanwhile at a little more advanced metaphase, that position was recognized as a dim constriction. K8: Metacentric pair. The chromosome was stained deeply along its all length and did not differentiate morphologically except for the centromeric constriction. K9: Sub-metacentric pair. The short arm was in whole stained deeply. The long arm had a deeply stained proximal region as long as the short arm, a half-toned distal end and a small intercalary part stained faintly. K10: Sub-telocentric and satellited pair. Excluding the secondary constriction and the satellite, the chromosome length and the arm ratio were measured. This pair had deeply stained small short arms with stretched nucleolar constrictions and satellites. The long arm had a deeply stained segment at the proximal region by half of its length. In many cases, the constricted region and the satellite were amorphous because of their penetration into the nucleolus. This is a reason why the constricted region and the satellite were excluded in measuring the length. K11: Metacentric pair. The morphology was almost the same as the pair of K8, therefore, it is rather difficult to distinguish between them. The identification solely depended upon the difference in their length. K12: Sub-metacentric pair. This pair had deeply stained short arms. The long arm had a deeply stained proximal segment slightly longer than the short arm and a small piece of faintly stained distal region. This analytic system is reliable for the identification of all 12 pairs of chromosomes in only one metaphase nucleus. Almost all features of each chromosome are clear and constant in prometaphase but there are some indefinite characteristics, such as the secondary constriction-like slit in K3 chromosome, the amount of faintly stained segment in K7 chromosome and the intensity of stainability in the distal segments. However, these characteristics hardly obstructed the identification of chromosomes. The success in obtaining well shaped and differentially stained chromosomes is thought to be brought by the treatment of living cell with KC1 and enzyme solution containing HCI. Instantaneous fixation and thorough removal of cytoplasm by flame-drying may be also a factor for the clear characterization. The present technique will be an effective tool for characterizing plant chromosomes, especially for the small sized ones. We are now pursuring a more critical analysis of banded structure in the rice chromosomes. SUMMARY Mitotic chromosomes of rice, Oryza sativa L. (2n=24), were all identified by a newly developed technique. A new technique of cell suspending and flame-drying made

5 KARYOTYPE ANALYSIS IN RICE 255 chromosomes possible to be well flattened and spreaded. Tissue treatment with KCl and enzyme caused segmental differentiation in chromosome stainability with Giemsa. All chromosomes were able to be identified in a single prometaphase nucleus by individual characteristics. Twelve pairs of chromosomes were gradually different in length and composed of 5 metacentrics, 5 sub-metacentrics and 2 sub-telocentrics including a satellited pair. ACKNOWLEDGMENTS We wish to manuscript. thank Dr. Hiroshi Watanabe for his kind advice and reading the LITERATURE CITED Chu, Y. E., 1967 Pachytene analysis and observations of chromosome association in haploid rice. Cytologia 32: Gill, B., and G. Kimber, 1974 The Giemsa C-banded karyotype of rye. Proc. Natl. Acad. Sci. USA 71: Hu, C. H., 1958 Karyological studies of haploid rice plants. II. Analysis of karyotype and somatic pairing. Japan. J. Genetics. 33: (in Japanese with English summary). Hu, C. H., 1964 Further studies on the chromosome morphology of Oryza sativa L. In "Rice Genetics and Cytogenetics" pp Elsevier, Amsterdam. Ishii, K., and S. Mitsukuri, 1960 Somatic chromosomes of Oryza sativa L. Bull. Res. Coll. Agr. Vet. Med. Nihon University 11: Khan, S. H., 1975 A technique for staining rice chromosomes. Cytologia 40: Nishimura, Y., 1961 Studies on the reciprocal translocations in rice and barley. Bull. Natl. Inst. Agr. Sci. D9: (in Japanese with English summary). Shastry, S. V. S., D. R. Ranga Rao, and R. N. Misra, 1960 Pachytene analysis in Oryza. I. Chromosome morphology in Oryza sativa. Indian J. Genet. Pl. Breed. 20: Shastry, S. V. S., 1964 A new approach to the study of rice karyomorphology. In "Rice Genetics and Cytogenetics" pp Elsevier, Amsterdam.