Caryologia

International Journal of Cytology, Cytosystematics and Cytogenetics

ISSN: 0008-7114 (Print) 2165-5391 (Online) Journal homepage: https://www.tandfonline.com/loi/tcar20

N-Band Staining in Plant Chromosomes with a

HCL-Giemsa Technique

G. D'Amato, G. Bianchi, R. Capineri & P. Marchi

To cite this article: G. D'Amato, G. Bianchi, R. Capineri & P. Marchi (1979) N-Band Staining
in Plant Chromosomes with a HCL-Giemsa Technique, Caryologia, 32:4, 455-459, DOI:
10.1080/00087114.1979.10796810

To link to this article: https://doi.org/10.1080/00087114.1979.10796810

Published online: 30 Jan 2014.

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 N-BAND STAINING IN PLANT CHROMOSOMES WITH
A HCI-GIEMSA TECHNIQUE *

G. D'AMATO, G. BIANCHI, R. CAPINERI and P. MARCHI

Istituto Botanico, Universita di Roma, Roma, Italy

Received: 71h June 1979

INTRODUCTION

TheN-banding technique was introduced by MATSUI and SASAKI (1973)

on mammalian and human chromosomes. The original method consisted in
incubating slides in a 3% trichloroacetic acid (TCA) solution, for 30 min.
at a temperature of 85-90°C and then hydrolizing them at 60°C for 30-45
min. in 0,1 N HCI. This method revealed bands located at secondary
constrictions of nucleolar chromosomes and was thus called N-bands.
An improved technique was introduced by FUNAKI et al. (1975) using
a phosphate buffer at 96°C instead of TCA and chloridric acid, on animal
and plant material, such as Vicia faba, Narcissus tazzetta, Secale cereale and
Zea mays. The results revealed that the localization of N-bands may occur
in various specific regions other than secondary constrictions (satellites,
centromeres and other heterochromatic segments).
GERLACH (1977) in Triticum obtained a N-band pattern similar, but
not identical with the C-hand pattern.
We have found that these methods are not always repeatable with
our material: the chromosomes are either faintly stained or «ghost like»
and no bands at all are often visible; moreover the use of high temperature
easily damages glyceroalbuminated slides that are routinely used in our
laboratory. To avoid these drawbacks we have attempted to combine a mild
HCl treatment together with the standard Giemsa technique for constitutive
heterochromatin.
In the present paper, the results obtained in three plant species,
where N-bands are mainly at the secondary constrictions, are described.

* Work supported by a grant from CNR (Rome).

[Caryologia, Vol. 32, n. 4, 1979

456 D'AMATO, BIANCHI, CAPINERI and MARCHI

MATERIALS AND METHODS

Root tips of Vicia faba L., Bellevalia dubia (Guss.) R. et S. and Ornithogalum
montanum Cyr. ex Ten. have been treated with 0,4% aqueous solution of
colchicine for 3-5 hours then fixed in a mixture of acetic acid, ethylic alcohol
(1/3 ), for 4-18 hours, squashed with glyceroalbuminated slides; these were
detached in absolute ethanol, left to dry for 2-3 days and then incubated in
0,2 N HCl at 60°C, for 20-40 min. . Subsequently, slides were briefly rinsed
in distilled water, soaked in a saturated Ba(OHh solution for 2-3 min., washed
in tap water and incubated in 2 X sse at 60°C for 60-90 min .. Staining was
carried out using 1% Giemsa (Gurr R 66) at pH 6,8 for 10 min ..

OBSERVATIONS AND DISCUSSION

Bellevalia dubia.
This species has a 2n=8 chromosome complement (CHIARUGI 1949)
with six secondary constrictions (GARBARI 1968 ), located on chromosomes
A, C and D, which can show a peculiar heteromorphism (MAGGINI 1972).
In this species there exists a positive correlation between the length of
secondary constrictions and the number of ribosomal RNA genes (MAGGINI
and DEDoMINICIS 1977). The karyotype of an individual after HCl-Giemsa
treatment, described here, is represented in Fig. 1. The figure shows dark
bands on chromosomes A, C, D which correspond to the nucleolar organizer
regions. It may be also observed the heteromorphism of couple D, in which
one chromosome shows a characteristic compound structure of eu- and
heterochromatic segments in the N.O. region. Furthermore a faint dot
shaped band may be observed on chromosome B, near the primary constriction
(arrows). Quinacrine or standard Giemsa techniques have never given
appreciable results in this plant so far.

Ornithogalum montanum.
In 0. montanum the number and distribution of Q-bands as well
as the size of secondary constrictions are highly variable (CAPINERI et al. 1975).
It seems also that Q-bands and C-bands are in agreement (CAPINERI et al.
unpublished). With our modified N-banding technique, dark heterochromatic
segments occur only at the secondary constrictions. The Figs. 2, 3, 4
represent the Quinacrine, Feulgen and N-banding patterns of an individual,
in which the size of s.c. is almost the same in the homologous chromosomes.
An individual with heteromorphic secondary constrictions is shown in Fig.
5 and dark N-bands of different size are represented in Fig. 6.
Fig. 1. - N-bands in the chromosome complement of Bellevalia dubia. 2600 x.
Fig. 2. - Part of Ornithogalum montanum complement after Q staining. Arrows show the
homomorphic NOR pair. 2600 x.
Fig. 3. - Ornithogalum montanum homomorphic NOR pair after Feulgen staining. Same
individual as in 2. 2000 x.
Fig. 4. - Ornithogalum montanum homomorphic NOR pair after method herein described
for N-bands. Note that no telomeric band is visible. Same individual as in 2. 2600 x.
Figs. 5-6. - Ornithogalum montanum heteromorphic NOR pair of chromosomes after Feulgen
and N-band staining. (Fig. 5: 2000 x; Fig. 6: 2600 x).
Fig. 7. - Vicia faba: N-band on chromosome M. 2600 x.
458 D'AMATO, BIANCHI, CAPINERI and MARCHI

Vicia faba.
In this plant, N-bands are located at secondary constrictions, on
M-chromosome (Fig. 7). Sometimes, we observed the persistence of e-bands
on the S-chromosomes, expecially when hydrolysis was performed for
shorter times. In fact, if incubation in Hel is only 15 min., the e-bands
on S-chromosomes are still visible, while the M-chromosomes appear
completey unhanded. Prolonged Hel treatment makes all the bands
disappear, so that good results seem to depend on a proper timing of
0,2 N Hel, Ba(OH)2 and sse exposure.
The use of mild hydrolysis in e-banding techniques is widely used,
expecially in plant chromosomes, to facilitate squash and to improve results.
This treatment is generally carried out on root tips but sometimes also on
slides after squash. VosA (1976) observed dark bands at the N.O. of
V. faba together with other positive and negative bands on M-chromosome
(bands 7 and 3 ), after a 45% acetic acid fixation followed by 10 min.
hydrolysis before SSe treatment. YEN and FILION ( 1977) used different
temperature of hydrolysis before e-banding, to distinguish between two
types of heterochromatin in Avena. They found that the 60oe 1 N Hel
hydrolysis produced centromeric bands, while room temperature gave
telomeric and intercalary bands. FISKESJO (1974, in Allium cepa) succeded
in obtaining good e-bands when using a 5 min. 0,1 N Hel hydrolysis,
before squash, and a successive 30 min. hydrolysis at room temperature,
between barium and SSe treatment. Our method may be an useful mean
to stain selectively N-bands in several plant species. Routinely it has given
good results in many other species, for example Vicia lutea, V. hybrida,
V. melanops, Ranunculus ficaria, Nigella damascena, but in the case of
Allium cepa the band pattern obtained was closely similar to e-banding.
However, such differences may account for the heterogeneous nature of
heterochromatin which in plants reacts differently according to the various
methods and species examined (VosA 1970, 1973, 1975; STACK et al. 1974;
YEN and FILION 1977).

Acknowledgement. - We thank Dr. C. G. VosA for the interest shown in our work
and for helpfull discussion.

REFERENCES

CAPINERI R., D'AMATO G. and MARCHI P., 1975. - Colorazione dilferenziale dei cromosomi
di Ornithogalum montanum Cyr. ed il suo impiego nell'analisi cariotipica di popola-
zioni calabre. Giorn. Bot. Ital., 109: 291 (Summary).
CHIARUGI A., 1949. - Saggio di una revisione cito-sistematica della flora italiana. I. Il tetra-
 N-BANDING IN PLANT CHROMOSOMES 459

ploidismo della Bellevalia webbiana Parl. e il suo diritto di cittadinanza nella flora
italiana. Caryologia, 1: 362-377.
FrsKESJO G., 1974. - Two types of constitutive heterochromatin made visible in Allium
by rapid C-banding. Hereditas, 78: 153-156.
FuNAKI K., MATSUI S. and SASAKI M., 1975. - Location of nucleolar organizers in animal
and plant chromosomes by means of an improved N-banding technique. Chromosoma,
49: 357-370.
GARBARI F., 1968. - Iconografia cromosomica di alcune Liliaceae. Atti Soc. Toscana Sc.
Nat., Mem., Ser. B, 75: 163-178.
GERLACH W. L., 1977. - N banded karyotypes of wheat species. Chromosoma, 62: 49-56.
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length of the nucleolar secondary constrictions in Bellevalia romana and B. dubia
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STACK S. M., CLARKE C. R., CARY W. E. and MUFFLY ]. T., 1974. - Different kinds of
heterochromatin in higher plant chromosomes. J. Cell Sci., 14: 499-504.
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- , 1973. - Heterochromatin recognition and analysis of chromosome variation in Scilla
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- , 1975. - The use of Giemsa and other staining techniques in karyotype analysis. Current
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SUMMARY

A method, similar to BSG method, which seems to stain selectively plant nucleolus
organizing regions is described. The critical step is a mild hydrolysis applied to Carnoy fixed
root apices after squashing. The method has been tested on Vicia faba and also on individuals
of Bellevalia dubia and Ornithogalum montanum (Liliaceae) selected because of heteromorphic
nucleolus organizing regions.