| The technique of giemsa staining of cereal chromosomes
G. KIMBER, B. S. GILL J.M. RUBENSTEIN and G.L. BARNHILL Department of Agronomy, Univ. of Missouri, columbia, Mo., U.S.A. Recently developed staining techniques that result in differential banding of somatic metaphase chromosomes permit the identification of individual chromosomes and have considerably enhanced cytogenetic studies in mammals. With these methods all of the chromosomes have been identified in man, mouse, and many other animal genera ; further, in mouse almost all the linkage groups have been correlated with specific chromosomes and chromosome arms. Unfortunately, the application of these techniques to plant chromosomes has not been particularly successful. One of the differential staining techniques, Giemsa C-banding (C =constitutive heterochromatin), which was first applied to animal chromosomes, involves denaturation-reassociation of DNA, with the highly repetitive DNA reassociating faster and appearing as dark bands. Attempts have been made to identify individual plant chromosomes with conventional staining methods, but the interpretation of the results is difficult. In this communication we report a Giemsa staining procedure on grass chromosomes that can be routinely used and by which the individual chromosomes can be easily identified. Technique: 1. Germination of seeds: A. Place seeds on damp filter paper in Petri dish for 24 hours at 25C, 24 hours at 2C, and then 24 hours at 25C. 2. Root tip treatment: A. Collect root tips when 0.5 to 1.5 centimeters long. B. Place the detached root tips in a freshly prepared saturated solution of mono-bromonapthalene in tap water. This solution can be made by placing one or two milliliters of monobromonapthalene in 250 ml. of water and violently agitating. The time of treatment at room temperature (72oF) varies from three hours for diploid species to three and a half for polyploid species. Too long a treatment will cause over-contraction and obscure faint bands. C. Fix in glacial acetic acid overnight (twelve hours minimum, three days maximum) in refrigerator (2C). 3. Enzyme softening: A. Wash roots in distilled water and transfer to tubes filled with enzyme solution. B. Leave root tips in enzyme for one to one-and-a-half hours at room temperature. 4. Root squashing (put 100cc of 2 x SSC and dish in oven to begin warming to 60C) : A. Transfer root tip to distilled water for washing. B. Place two or three tips on slide and cut off meristematic portion of the tip. Wipe off excess water and the elongated portion of the root. C. Add small drop of 45% acetic acid and macerate completely. D. Place cover slip on slide and tap vigorously. E. Heat slide till warm to touch and press under filter paper. F. Remove cover slip: Freeze the macerated material under the coverslip by either placing the slide in contact with solid carbon dioxide or spraying it from below with liquid CO2, Pry off the cover slip before the frozen material melts. 5. Dehydration: A. Place slide in fresh 95% ethyl alcohol (see discussion). B. Remove slide and air dry. 6. Barium hydroxide denaturation: A. Place slide in barium hydroxide solution for five minutes. (Keep dish covered to prevent carbonation.) B. Rinse slide in distilled water for ten minutes, changing the water three times. C. Air dry. (Start filtering Giemsa stock solution) 7. 2x SSC renaturation: A. If slide has a film of barium hydroxide on it, place in cold SSC for four minutes. B. Place in hot (60C) 2x SSC for one hour. C. Wash in distilled water for ten minutes and change water three times. D. Air dry. (During the drying prepare the staining solution and remove the scum from the surface.) 8. Giemsa staining: A. Stain in Giemsa for appropriate time. B. Wash in distilled water. If it is not stained enough, restain. If it is overstained, decolorize in 95% ethyl alcohol. C. Air dry. Place in xylene overnight. Mount with Canada balsam. |
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