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. MAGGINI
F., 1972. - The chromosome complement of Betlevalia dubia (Guss.) R. et S. and
the problem of Bellevalia webbiana Parl. Ann. Bot. (Roma), 31: 115-123.
MAGGINI F. and DE DoMINICIS R. 1., 1977. - The ribosomal RNA gene numbers and the length of
the nucleolar secondary constrictions in Bellevalia romana and B. dubia (Liliaceae): a possible
correlation. Caryologia, 30: 97-103.
MATSUI S. and SASAKI M., 1973. - Differential staining of nucleolus organisers in mammalian
chromosomes. Nature, 246: 148-150.

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.
VosA C. G., 1970. - Heterochromatin recognition with fluorochromes. Chromosoma, 30:
366-372.
- , 1973. -Heterochromatin recognition and analysis of chromosome variation in Scilla sibirica.
Chromosoma, 43: 269-278.
- , 1975. - The use of Giemsa and other staining techniques in karyotype analysis. Current Adv. in Plant
Sci., 14: 495-510.
- , 1976. -Heterochromatin classification in Vicia faba and Scilla sibirica. Chromosomes
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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.