Universidade Federal de Viçosa

DNA content of maize metaphasic A and B chromosomes determined by image cytometry

— Rosado, TB; Carvalho, CR; Saraiva, LS

Maize (Zea mays) was the first species in which B chromosomes were detected. Since then they have been subjected to many cytogenetical and molecular studies to elucidate their origin, evolution, structure and behavior. Maize plants of Black Mexican inbred line with B chromosomes, gently supplied by the Maize Stock Center (University of Illinois, USA), were analyzed by image cytometry and cytogenetics to measure the DNA content of the A and B chromosomes.

Chromosome slide preparations suitable for optical density analysis by image cytometry was made in root tips from Black Mexican diploid inbred line seeds without B (P1/0B), with 2 B’s (P3/2B) and 4 B’s (P4/4B). DNA content from interphasic nucleus (G1) with 2C values were 5.43, 5.81 and 6.24 picograms (pg), respectively. These values were previously determined in leaves by flow cytometry. Seeds from these plants were germinated in Petri dishes with distilled water at 28–30°C. When the root tips were 0.5 to 1.0 cm long they were treated with Oryzalin 3 μM for 20h 30min at 4°C, and fixed with methanol-acetic acid solution 3:1 (v/v) e finally macerated in enzymatic solution (0.1% cellulase, 0.5% hemicellulase and 0.3% pectinase) for 1h 45min at 37°C. The slides were prepared by cellular dissociation and air drying technique and afterwards treated by Feulgen reaction, with HCl 5N hydrolysis for 18min at 20°C.

Metaphase images were captured with a 100× objective (microscope: Olympus BX60), a neutral density filter (ND6) and another of interference green color (IF550), by means of a CCD 12 bits monochromatic camera attached to a computer. To make a Feulgen calibration curve was used the tools of software Pro Plus 4.5 and a set of filters with standard optical density values known (DO = 0.04; 0.22; 0.42; 0.60; 0.79; 0.99; 1.18; 1.36; 1.55; 1.75; 1.95 and 2.13). With the aid of the digital tools the values of integrated optical density (DOI) were transformed in DNA picogram values, using the plant P1/0B (2C = 5.43 pg or 4C = 10.86 pg) as standard.

This methodology has been used in our lab to make good slide preparations with the chromosomes in the same plane of the focus without overlaps, adequated to caryogram assemblage (Figures 1, 2 and 3) and also to quantitative analysis. The determination of DNA content value of the metaphasic A chromosome set and the B chromosomes were included in Table 1. The A chromosome complement of the plants P3/2B and P4/4B did not differ in DNA content more than 0.03 pg in comparison with the standard plant, while B chromosomes varied from 0.36 to 0.44 DNA pg in these metaphases. From these data associated to cytogenetic analysis it was estimated that each B metaphasic chromosome increased, on average, 0.40 DNA pg (3.75%) to A genome of the plants analyzed. In our conclusion, the Image Cytometry technique was sensible in determination of small DNA amount variations on metaphases with or without maize B chromosomes. This methodology can be useful to determinations of the DNA content in samples with reduced amount of cellular material, as a single root tip as demonstrated in the present analysis. This work was supportated by a grant from the FAPEMIG, CNPq, Brazil.

Figure 1. Maize karyogram of the plant P1/0B, used as standard, with 2n = 20 chromosomes after Feulgen reaction. Bar = 5 μm

Figure 2. Maize karyogram of the plant P3/2B with 2n=20A+2B chromosomes after Feulgen reaction. Bar = 5 μm

Figure 3. Maize karyogram of the plant P4/4B with 2n = 20A+4B chromosomes after Feulgen reaction. Bar = 5 μm

Table 1. DNA content of maize root tips metaphasic A and B chromosomes treated with Feulgen reaction and analysed by image cytometry

C Value Chromosomes Plants (DNA/pg)
  P1/0B P3/2B P4/4B
4 20A 10.86 10.88 10.83
2 B   0.36 0.40
2 B   0.38 0.38
2 B     0.44
2 B     0.43
Sub-total 20A 10.86 10.88 10.83
Sub-total B   0.74 1.65
Total A+B 10.86 11.62 12.48

pg = picogram

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