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《Acta Genetica Sinica》 1982-02
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Wall Degradation Hypotonic Method of Preparing Chromosome Samples in Plant and Its Significance in the Cytogenetics

Chen Ruiyang Song Wenqin Li Xiulan (Department of Biology, Nankai University, Tianjin)  
In this paper the wall degradation hypotonic method of preparing mitotic chromosome sample in plant has been studied. Prom the chromosome count, karyotype analysis, Giemsa banding and SEM observation of chromosome obtained by testing 105 materials of 37 branches, it is shown that this method is of great signifieane.Some of the main factors and processes of this method are narrated as follows:1. Material culture: Roots from germineting seeds growing at 25℃.2. Pretreatment: Treating the attached root with 0.01%-0.2% colchicine or 0.002 M 8-hydroxyquinoline for 3-4 hours prior to the peak time of mitotic activity.3. Pre-hypotonie treatment: Excising the good root tips and immersing them in 0.075 M KC1 solution for 30 minutes at 20- 25℃.4. Wall degradation: Treating with 2.5% enzyme mixture for 2-4 hours at 25-26℃.5. Post-hypotonic treatment: Rinsing the material 2-3 times in 25-30℃ redistilled water and then immersing it in the distilled water for 5-10 minutes.Preparation of samples:Ⅰ. Suspension method:Ⅰ 6. Preparation of cell suspension: Pour out the water. Tear the material with a pair of forceps to make well a cell suspension.Ⅰ 7. Fixation: Add 2-3 ml of fresh prepared methanlo: acetic acid (3:1) to the suspension.Ⅰ 8. Removing of precipitate: Keep the solution for a moment to precipitate the residues. Use the upper suspension.Ⅰ9. Removing of supernatant: Keep the suspension for 20-30 minutes. Remove the supernatant with a pipette and take 1 ml of concentrated suspension for chromosome preparation.Ⅰ 10. Preparation of chromosomes: Drop 2-3 drops of the suspension on a chilled slide kept in distilled water. Lift one end of the slide and blow gently to spread the cells. Flame the slide gently above an alcohol burner to have it dry.Ⅰ 11. Staining: Stain the dried slides in a 1 -. 20 or 1: 40 Giemsa solution for 4-5 minutes, then rinse them in distilled water, and have them air-dried and mounted in damar balsam.Ⅱ. Smear method:Ⅱ 6. Fixation: Pour out the distilled water. A dd fresh prepared methanol: acetic acid (3:1) to the material.Ⅱ 7. Smear: Put on 3-4 root tipe on the slide which was kept in the freezed distilled water. Add drop fixation solution to them. Tear the material with a pair of forceps and Remove the supernatant.Ⅱ 8. Flame-drying: Add 2 drops fixation solution on the slide, then bake it on the alcohol burner.Ⅱ 9. Staining: see 111.
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