BGN 11: An improved Giemsa N-banding technique for the identification of barley chromosomes  
BARLEY GENETICS NEWSLETTER, VOL. 11, III. GENETIC AND CYTOLOGICAL TECHNIQUES
Singh, pp. 84-85

III. 2. An improved Giemsa N-banding technique for the identification of barley chromosomes. (1)

R. J. Singh, Department of Agronomy, Colorado State University, Fort Collins, Colorado 80523 U.S.A.

(1) Supported partly by USDA/SEA Competitive Research Grant 5901-0410-9-0334-0 to T. Tsuchiya.

The Giemsa N-banding technique was developed from the modification of N-banding (Funaki et al. 1975; Garlach, 1977; Islam, 1980) and C-banding techniques (Singh and Röbbelen, 1975; Linde-Laursen, 1975).

1. Collection of root tips: Seeds of barley lines were germinated in petri dishes on moist filter paper. For rapid and uniform germination, petri dishes with seeds were kept in a refrigerator (5°C) for 4 to 7 days. Petri dishes were left at room temperature (ca. 23°C) for germination after cold treatment. Root tips were collected when they were about 1-2 cm long. It is important to make sure that roots should be touching the blotting paper during their growing period.

2. Pretreatment: Vials with roots in tap water were kept in a container which had cold water covering the top of the vials. Vials were further surrounded by and covered with a thick layer of ice (Tsuchiya, 1971). Pretreatment period was 15 h only. Longer pretreatment makes chromosomes shorter and inadequate for banding technique. The ice cold water pretreatment was preferred because large numbers of cells were arrested at early and mid-metaphase which is a prerequisite in obtaining cells with optimal N-banded chromosomes.

3. Fixation: Root tips were fixed in freshly prepared ethanol-acetic acid (3:1) solution. Root tips were kept in the fixative at least overnight. The longer fixing time resulted in poor quality of the bands.

4. Preparation of Acetocarmine-Giemsa stained chromosomes: Root tips were transferred in acetocarmine (0.3%) for about 2 to 3 hours, and slides were prepared by squash method. Photographs of well spread cells were taken by phase contrast. The basic principle of this technique was established by Nakata et al. (1977) to identify rye chromosomes.

After removal of the cover slip by the dry ice method, slides were placed in 96% ethanol for 2 to 4 hours. Air dried slides were kept in a desiccator over silica gel for at least 2 weeks (Linde-Laursen, 1975). The slides which were kept longer in the desiccator showed improved banding pattern but required more time for staining.

Slides were incubated in 1M NaH2P04 H20 solution, pH 4.15 for 5 minutes at 94°C (Funaki et al., 1975; Garlach, 1977). Slides were washed in several changes of distilled water and air dried. The slides were stained for 20 minutes to 24 hours in 1% Giemsa Sigma No. G04507 in 1/15 M Sörensen's phosphate buffer (pH = 6.8). After optimal staining of the cells which were previously photographed in acetocarmine preparation, the slides were rinsed in distilled water, air dried, then kept in xylene overnight. Slides were air dried and mounted in Permount.

The results of the karyotype analysis by this technique were partly reported by Singh and Tsuchiya (1981).

References:
Funaki, K., S. Matsui and M. Sasaki. 1975. Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosoma (Berl.) 49:357-370.

Garlach, W. L. 1977. N-banded karyotypes of wheat species. Chromosoma (Berl.) 62:49-56.

Islam, A.K.M.R. 1980. Identification of wheat-barley addition lines with N-banding of chromosomes. Chromosoma (Berl.) 76:365-373.

Linde-Laursen, Ib. 1975. Giemsa C-banding of the chromosomes of 'Emir' barley. Hereditas 81:285-289.

Nakata, N., Y. Yasumuro and M. Sasaki. 1977. An acetocarmine - Giemsa staining of rye chromosomes. Japan. J. Genet. 52:315-318.

Singh, R. J. and G. Röbbelen. 1975. Comparison of somatic Giemsa banding pattern in several species of rye. Z. Pflanzenzuchtg 74:270-285.

Singh, R. J. and T. Tsuchiya. 1981. Preliminary studies on identification of telocentric chromosomes in telotrisomic barleys by Giemsa N-banding technique. Barley Genet. Newsletter 11:71-74.

Tsuchiya, T. 1971. An improved acetocarmine squash method, with special reference to the modified Rattenbury's method of making a preparation permanent. Barley Genet. Newsletter 1:71-72.

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