Staining Procedure for Watermelon Somatic Chromosomes

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Cucurbit Genetics Cooperative Report 13:47-48 (article 19) 1990

H. T. Skorupska and N. G. Allgood
Dept. of Agronomy & Soils and Biological Sciences, Clemson University, Clemson, SC 29634

The application of polyploidy in watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) breeding, and the development of aneuploid stocks for future genome mapping and chromosome engineering, requires the characterization of the watermelon chromosome karyotype. This report presents methodology which can be used routinely for chromosome screening. The procedure allows for the arresting of chromosome divisions at metaphase, which is very suitable for chromosome counting. P-dichlorobenzene (PDB) is used as a pretreatment factor. PDB causes straightening of the chromosome arms, prevents excessive clumping, and simulates contraction. It has been employed successfully as a pretreatment reagent in karyotype studies on a number of plants, particularly legumes. A root-tip squash technique which applied paradichlorobenzene was used for soybean. Forty, uniformly shaped, small chromosomes were described (2). Skorupska and Palmer (1) using PDB treatment were able to identify soybean monosomics (2n139 chromosomes).

The seed of three cultivars, ‘Sugar Baby’, ‘Charleston Gray’, and ‘Southern Bell Hybrid’, were germinated and positive chromosome staining was obtained. Chromosomes were stained dark purple and the cytoplasm was clear enough to provide a good contrast for more detailed analyses of chromosome morphology.

Root-tip Squash Preparation

  1. Germinate scarified seed for 72 to 96 hours at 27 to 28C on 10×15 regular weight germination paper.
  2. Harvest root-tips at 8:20 a.m., excise 1 cm and partly slit tips with a razor blade for better chemical penetration.
  3. Prefix root-tips in covered vials in saturated PDB at 12.5C for 2½ hours.
  4. Transplant seedlings into peat-pots.
  5. Wash root-tips in distilled water, transfer to 3:1 fixative (95% ethanol:glacial acetic acid) for 24 to 48 hours in covered vials at 28 to 30C.
  6. Wash root tips in distilled water hydrolyze in 1N HCl for 8 to 10 minutes, at 60C.
  7. Place root tips in Feulgen’s stain, in covered vials 45 to 60 minutes in the dark, at room temperature.
  8. Place root-tips in ice cold water and allow to set 20 minutes.
  9. Place root-tips in a porcelain spot plate in pectinase. Cover plate with parafilm and incubate for 1¼ hours at 30C.
  10. Transfer root-tips to 70% ethanol and store in refrigerator.
  11. Put treated root-tip on a slide and with a razor blade remove and discard the unstained root cap. Place ½ to 1 mm of the root-tip in a drop of 0.5% aceto-carmine stain. Tap gently but thoroughly with a glass rod. Apply a cover slip and press firmly under filter paper, or a pellet press.

PDB Preparation

750 mg PDB, 50 ml distilled water, in stoppered 125 ml Erlenmeyer flask, corked, incubate overnight at 60C. Cool, then shake vigorously before using.

Leuco-basic Fuchsin (Feulgen Stain) Preparation*

  1. Put 1 g basic fuchsin in a 500 ml Erlenmeyer flask.
  2. Pour 200 ml boiling water into flask, shake well.
  3. Cool to 50C check temperature frequently with a thermometer (further cooling may cause precipitation).
  4. In a Buchner funnel, vacuum filter through 2 layers #1 filter paper.
  5. Add: 30 ml 1N HCl

3 g potassium metabisulfite (K2S2O5)

  1. Shake well, pour into a well-stoppered bottle, store in the dark at room temperature for 24 to 48 hours.
  2. Add 1 g powdered declorizing charcoal, shake well. Vacuum filter in a Buchner funnel through 2 layers of #1 filter paper. If solution is still slightly colored, you may add another 1 g charcoal, and refilter.
  3. Store in a well-stoppered amber bottle, or bottle wrapped in foil, in the refrigerator. Allow stain to “mature” 1 to 2 days before using.

*Adapted from “The Handling of Chromosomes.” 1970. C. D. Darlington and L. F. LaCour. Hafner Publishing Company, Inc., Darien, Conn.

Literature Cited

  1. Skorupska, H. and R. G. Palmer. Monosomics from synaptic KS mutant. Soybean Genet. News1. 1987:174-178.
  2. Palmer, R. G. and H. Heer. 1973. A root-tip squash technique for soybean chromosomes. Crop Sci. 389-391.