Recalibration of “c-tester” line as a standard to estimate nuclear DNA content/pg by Feulgen microdensitometry

--C. Tito, L. Poggio and C. A. Naranjo

When nuclear DNA content is measured by microdensitometry using Feulgen stain, the results are obtained in arbitrary units (A.U.). It is assumed that there is a proportionality between stain density and DNA amount, and through a standard species the arbitrary units can be converted into absolute units in picograms (pg).

Bennett and Smith (Proc. Roy. Soc. London B 274:227-274, 1976) advised using as a standard a species, cultivar or seed of defined population with DNA content similar to one of the species being studied. Moreover, they should be easy to culture under laboratory conditions, and root-tips should be readily available for use as standards throughout the year. This procedure tends to minimize some technical errors inherent in Feulgen microdensitometry. These authors pointed out that the accuracy of absolute DNA amounts calculated by this method depends “first, on the accuracy with which each species is measured relative to the standard species used for calibration, and secondly, on the accuracy of the assumed DNA amount of the standard species”. The majority of plant DNA amounts measured by Feulgen microdensitometry have been calibrated using Allium cepa as a standard (2C=33.55 pg). Bennett and Smith did not find evidence of detectable intravarietal variation in DNA amount in A. cepa var. Ailsa Craig, which was considered suitable for use as a standard for calibrating other species. Moreover, this species was used to recalibrate several species as other standards.

The data available for species of Zea in the literature vary between 2C=4.96 pg for Gaspe Flint and 2C=11.36 pg for Z. perennis (Laurie and Bennett, Heredity 55:307-313, 1985; Rayburn et al., J. Exp. Bot. 40:11-1183, 1989). The standard used by these authors is not easily available to us and the available ones (e.g., A. cepa var. Ailsa Craig) have remarkably high 2C value to be used when the genus Zea is being studied.

The c-tester inbred line has all the characteristics suggested by Bennett and Smith to justify its use as standard. Therefore, three experiments were done on different days to recalibrate carefully the 2C value of c-tester line, against A. cepa var. Ailsa Craig.

DNA content was measured in telophase nuclei (2C) of the root apex of germinating seeds. Seeds were placed in Petri dishes on wet filter paper. Roots of 0.5-1 cm length were fixed in 3:1 (absolute ethanol:acetic acid) during 1-4 days. After fixation the roots were rinsed for 30 minutes in distilled water. Hydrolysis was carried out with 5N HCl at 20 C. Different times of hydrolysis were investigated and the optimum period was found to be 30 minutes. They were then given three washes in distilled water for 10 or 15 minutes and stained for 120 min in Schiff's reagent at pH 2.2. The material was then rinsed three times in SO2 water for 10 minutes each rinse, kept in distilled water (5 to 15 minutes) and squashed in 45% acetic acid. The coverslip was removed after freezing with CO2 and the slide dehydrated in absolute alcohol and mounted in Euparal. The amount of Feulgen staining per nucleus, expressed in arbitrary units, was measured at a wavelength of 570 nm using the scanning method in a Zeiss Universal Microspectrophotometer (UMSP 30). The DNA content per basic genome expressed in pg was calculated using A. cepa var. Ailsa Craig as a standard. The differences in DNA content were tested through an analysis of variance and comparisons between means using Scheffe's method.

The results obtained are shown in the table.
      DNA content (2C) pg  
Experiment Individual No. nuclei measured X S.E.
1 1 24 5.9289 0.1103
  2 36 5.5411 0.1055
2 3 26 5.8013 0.1085
  4 36 5.7059 0.1033
3 5 34 5.7817 0.0673
  6 33 5.8196 0.1044
  7 28 6.0128 0.0762

An Anova test was made, and it was determined that the results do not show significant differences among themselves (F=2.43; p<0.01). The results obtained point out a DNA average amount for “c-tester” of 2C=5.78±0.0965 pg.

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