| C-banding technique for wheat chromosomes Kazuhiko NODA Kihara Institute for Biological Research Mutsukawa 3-122-21, Minami-ku, Yokohama, Japan C-banding techniques applicable to plant chromosomes have been reported in several crops; wheat (GILL & KIMBER 1974a), maize (HADLACZKY & KALMAN 1975), rye (SARMA & NATARAJAN 1973, DARVEY & GUSTAFSON 1975), barley (LINDE-LAURSEN 1975) and rice (KURATA & OMURA 1978). These techniques are mainly based on the Sumner's BSG (Barium/Saline/Giemsa) method (SUMNER 1972). However, the BSG technique has the technical difficulties in making well-separated chromosome preparations. GILL & KIMBER (1974a) added a emzyme maceration step to BSG method. NODA & KASHA (1978), using barley chromosomes, applied hot HCl treatment, which was the maceration step of the Feulgen stain procedure, and 0.07 N NaOH instead of Ba(OH)2 solution. The present experiment was attempted to examine if the method of NODA & KASHA (1978) is applicable to wheat chromosomes. Materials and Methods Seeds of Triticum monococcum var. vulgare and Aegilops speltoides var. typica (KU-2), which were kindly provided by Dr. M. Tanaka, Plant Germ Plasm Institute, Kyoto University, were germinated and about 2 cm long root-tips were placed in ice-cold water for 20 hr to accumulate cells at metaphase stage. They were fixed in freshly prepared acetic-alcohol (1:3) and stored in a refrigerator until used. Maceration: The root-tips were placed in 1N HCl at 60C for 10 min. This step was important to differentiate C-bands. The root-tips were treated with 100x diluted pectinase solution (Pectinase Solution, 10000 units/104 ml, Sigma Co.) at 35C for 15 min when further maceration was required. Slide preparation: The meristematic part of root-tips was removed by a razor blade and placed on a clean slide in a drop of 45% acetic acid. The root-tip was squashed under a cover slip with no adhesive applied to slide or cover slip. The slides were examined under a phase contrast microscope to determine if cells with well-separated chromosomes were present. Cover slips were removed by dry ice method. The slides were dried at room temperature overnight. C-banding: The slides were placed in 1N HCl solution at 60C. The treatment time of hot 1N HCl was different depending on species used. It was five minutes for T. monococcum chromosomes and nine minutes for Ae. speltoides chromosomes. After the hot HCl treatment, the slides were rinsed in tap water and placed in 0.07N NaOH solution for 35 seconds. Chromosomes were stained in 60x diluted Giemsa solution (BDH Giemsa Staining Solution) or 5x diluted Leishman solution (BDH Leishman Staining Solution) in 1/15 M phosphate buffer (pH 6.8). The slides were rinsed in tap water, air-dried and mounted in Canada balsam. Results and Discussion In the present experiment, ten minutes treatment of the first hot HCl was required for chromosomes of T. monococcum and Ae. speltodies to differentiate C-bands. NODA and KASHA (1978), using barley chromosomes, reported that seven minutes of treatment of hot HCl in the maceration step was appropriate to produce C-bands. Also there is a difference in the time of the second hot HCl treatment between T. monococcum and Ae. speltoides chromosomes. It was five minutes for T. monococcum chromosomes and nine minutes for Ae. speltoides chromosomes. These differencies in the treatment time of hot HCl among different species may reflect chemical and/or structural variation in the constitutive heterochromatin. In the present experiment, stain ability of Giemsa stain solution was compared with that of Leishman stain solution. Leishman solution gave better differential staining of heterochromatin and euchromatin than Giemsa solution as DARVEY and GUSTAFSON (1975) reported. |
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