Russian Journal of Developmental Biology, Vol. 34, No. 1, 2003, pp. 1–13. Translated from Ontogenez, Vol.

34, No. 1, 2003, pp. 5–18.

Original Russian Text Copyright © 2003 by Zoshchuk, Badaeva, Zelenin.

REVIEWS

History of Modern Chromosomal Analysis.

Differential Staining of Plant Chromosomes
N. V. Zoshchuk, E. D. Badaeva, and A. V. Zelenin
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received June 1, 2001; in final form, April 4, 2002

Abstract—Differential staining methods found extensive use in karyotype studies of many plant and animal
species and provide for reliable identification of all chromosomes of the organism. Below we describe the most
widespread methods and history of their advent. In addition, we discuss specific structure of the chromosomes
and possible mechanisms responsible for differential segmentation.

Key words: differential staining, karyotype, mechanisms of differential segmentation.

Plants are the pivot of life on the Earth, which
explains permanent interest of the humankind to
their investigation. Plants were studied by various
methods at various organization levels including
chromosomes. Nevertheless, plant chromosomes are
much less explored then the animal ones. This is
largely due to complex isolation of the chromosome
samples related to the presence of rigid cell wall in
plants. Huge number of plant species and their con-
siderable variation by the number and dimensions of
the chromosomes even within single genus also mat-
ter. Scattering of chromosomal numbers within cer-
tain taxa of flowering plants can range from ten to
several hundreds.

Family Species 2n
Agavaceae Agave L. (agave) 50, 60, 90, 119, 120, 136, 140, 174, 184
Asteraceae(=Compositae) Chrysanthemum L. (chrysanthe- 18, 36, 54, 72, 80, 90, 198
mum)
Brassicaceae Dentaria L. (toothwort) 16, 32, 48, 80, 96, 128, 208
Cactaceae Mammillaria Haw (mammillaria) 22, 24, 44, 264
Cyperaceae Carex L. (sedge) 16, 18, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 55, 56, 58, 60, 62, 64, 66, 68, 70, 71, 72, 74, 76,
78, 80, 82, 84, 86, 90, 106, 108, 110, 112
Didiereaceae Alluaudia ~190–200, ~150
Malvaceae Hibiscus L. (hibiscus) 22–225
Rosaceae Alchemilla L. (ady’s mantle) 64, 96, 101–105, ~224
Violaceae Viola L. (violet) 10–128

The number of plant species living on the Earth also
remains unclear; the mentioned number of 250 thou-
sands does not include around 50 thousands not yet dis-
covered or described ones as well as vanishing spe-
cies—according to the data of World Conservation
Monitoring Centre the number of plant species
decreases by 5.4% every ten years (Global Biodiver-
sity…, 1992).
Plant genome has been studied for over a hundred
years (or all of 150 years considering Gregor Mendel’s
works). Now chromosomal numbers have been deter-
mined for ~25% species; however, these values are
incomplete or incorrect in many cases since they were
calculated for a single specimen or population (Ben-
nett, 1998). The bottom known chromosomal number
2n = 4 has been observed in four flowering plants
(dicotyledons Haplopapus gracilis and Brachychome
dichromosomatica, Asteraceae family, and monocoty-
ledons Zingeria biebersteiniana and Colpodium versi-
cola). The least possible animal chromosomal number
2n = 2 has been revealed in Australian ant Myrmecia
pilosa. Although no plant with such chromosomal num-

1062-3604/03/3401-0001$25.00 © 2003 MAIK “Nauka /Interperiodica”

2 ZOSHCHUK et al.
ber has been revealed so far, it is possible (and even
probable) to find it among 200 000 unexplored species.
Any plant organism with single chromosome will
become an amazing subject for genetic studies. At the
moment a small crop Z. biebersteiniana (2n = 4) is best
explored among plant species with low chromosomal
number (Bennett, 1998). Nevertheless, Arabidopsis
(2n = 2x = 10) and rice (2n = 2x = 20) remain the stan-
dard subject for genetic inquiries due to small size of
their genome.
In 1920–1930s active progress of chromosomal
analysis was chiefly related to plant objects—plant
comparative karyology went far ahead of the animal
one. Russian cytogeneticist S.G. Navashin was among
the first to considerably contribute to the studies of
plant chromosomes. His report at the Imperial Acad-
emy of Sciences in 1912 “On Nuclei Dimorphism in
Somatic Cells of Galtonia candicans (Liliaceae Fam-
ily)” became famous later. Navashin has discovered
permanent minute processes attached to the chromo-
somes by “fibrils” and called them “satellites.” He has
demonstrated that the satellites are permanent compo-
nents of two out of four chromosomes of the lily since
they “segregated together with the rest of the chromo-
somal body” during division of the nucleus (Navashin,
1912).
Navashin was the first to demonstrate possible dis-
crimination of the chromosomes by their specific struc-
ture and his review on external properties of internal
structure of the chromosomes was published in 1916 in
the anniversary issue dedicated to K.A. Timiryazev.
Liliaceous chromosomes (Fritillaria and Galtonia gen-
era) were the subjects of this inquiry (Navashin, 1916).
Works of another Russian classical cytogeneticist
G.A. Levitskii also played important role in plant kary-
otaxonomy. In his work “Morphology of Chromo-
somes” published in 1931 he proposed a new technique
for fixing and staining the samples revealing specific
chromosome structure of cultured plants (rye, barley,
pea, wheat, and white beet) as well as classical plants
for cytology—nais Najas and grape hyacinth Muscari.
Scope of the scientist’s interests included the problems
of taxonomy and evolution (Levitskii and Kuz’mina,
1927; Levitskii, 1931a, 1931b). Levitskii is one of the
founders of plant radiation genetics (Levitskii and
Araratyan, 1931); due to his activity the Russian School
of chromosome studies became internationally known
and was called classical.
It is generally accepted that Levitskii introduced the
term karyotype and developed the theory of karyotype
(Levitskii, 1931b; Dubinin, 1976). At present karyo-
type is considered as a set of characters that can be used
to identify specific chromosome set: the number and
size of chromosomes, their shape (primarily defined by
location of the centromeres), the presence of secondary
constrictions and satellites, alternation of euchromatic
and heterochromatic regions, their pattern, etc. “Kary-
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
otype is the passport of species” (Inge-Vechtomov,
1989).
Hence, by mid 1930s it was established that the
chromosomes can be described by the length, cen-
tromere position, and presence or absence of secondary
constrictions and satellites, which was used to describe
and classify them, reveal differences between karyo-
types, etc.
However, many plant species have similar chromo-
somes indistinguishable by monochrome staining (ace-
tocarmine, acetic orsein, and Feulgen staining)
(Zurabishvili et al., 1974), which limits application of
karyotype analysis. This naturally substantiated the
search for additional tests for differentiation of chromo-
somes. Heitz (1930, 1933a, 1933b) proposed terms
“euchromatin” and “heterochromatin” to define differ-
ing individual chromosomes or their regions. On the
basis of cytological observations Heitz proposed that
eu- and heterochromatic regions of the chromosomes
have different contribution to the cell metabolism: the
compact heterochromatic ones should have lower phys-
iological activity or should be passive by contrast to the
active euchromatic regions (Heitz, 1929, 1932). He
proposed that the majority of active genes lies within
euchromatic regions of the chromosome. These conclu-
sions agreed with the genetic data obtained by Müller
and Painter (1932) on drosophila where the revealed
inert regions of the chromosomes coincided with the
heterochromatic regions described by Heitz. Com-
monly, two types of heterochromatin—constitutive and
facultative—are recognized. Prokofieva-Bel’govskaya
(1986) stated that “facultative heterochromatin is quite
an equivocal term,” and it is not so basically different
from the constitutive (structural) one as initially pro-
posed (Schwarzacher, 1976).
The first techniques for differential staining reveal-
ing linear heterogeneity along the chromosomes
appeared in 1930–1940s. For instance, staining with
basic dyes revealed cooled heterochromatin (H-seg-
ments) in certain plant (and amphibian) species as
weakly stained regions on the metaphase chromo-
somes. They appeared after cooling the sample to
0…+3°C for 3–5 days (Darlington and La Cour, 1940;
Callan, 1942). Alternatively, an unusual differentiation
of heterochromatic segments of the chromosomes, par-
ticularly, in the vicinity of the centromere was observed
after X-irradiation combined with incubation in the
cold in wake-robin (Trillium), spiderwort (Trades-
cantia), and horse bean (Vicia) (Darlington and La
Cour, 1945; Kurabayashi, 1953).
During the following 30 years these particularly
interesting approaches were not widely distributed in
practical research despite single publications. As a
result, identification of individual chromosomes was
not practicable for many species including plants
before 1970s.
Development of the technique for differential stain-
ing of chromosomes (banding) in early 1970s provided
Vol. 34 No. 1 2003
 HISTORY OF MODERN CHROMOSOMAL ANALYSIS
cytogeneticists with a powerful tool for identification
both individual chromosomes as a whole and their
regions, thus, facilitating analysis of the genome. This
equally applies to plants and animals including
humans.
Fluorescence differential staining of plant chromo-
somes (Vicia faba) was first demonstrated by Caspers-
son et al. (1968). The new method was called Q-band-
ing (after quinacrine). Caspersson et al. used two acri-
dine compounds: quinacrine and its alkylating
derivative quinacrine mustard that provided for diffuse
thin fluorescence of the chromosome all along. Brightly
fluorescing centromeric and satellite regions contrasted
against this background. Selective staining has also
been revealed in heterochromatic regions of horse bean
and Chinese hamster chromosomes. We discuss this
discovery elsewhere (Zelenin and Zoshchuk, 2000).
Caspersson et al. presented the first publication on
differential staining of plant chromosomes. Later they
demonstrated pronounced fluorescence bands typical
for each pair of horse bean chromosomes using various
fluorescence agents (Caspersson et al., 1969a, 1969b)
and colocalization of the fluorochrome-binding regions
and heterochromatin proteins revealed after incubation
in the cold.
In addition to horse bean Caspersson et al. (1969a,
1969b) studied chromosomes of many other plant spe-
cies: wake-robin Trillium erectum, Siberian squill
Scilla sibirica, Madonna lily Lilium candidum, Turk’s
cap lily L. martagon, hyacinth Hyacinthus spp., pea
Pisum sativum, onion Allium cepa, and smooth hawk’s-
beard Crepis capillaris. The pattern of differential fluo-
rescence proved to be chromosome-specific and was
clearly reproduced in various cells and, as was demon-
strated later, was identical in various tissues of the
organism (Caspersson et al., 1972). No difference has
been revealed between the chromosomes isolated from
the root tip and endosperm cells of horse bean.
These works primed investigation of differentiation
of plant chromosomes. In 1970 Vosa reproduced
Caspersson’s results on Vicia faba, Tulbaghia, Allium
cepa, and A. carinatum (Vosa, 1970). Investigation of
other plant species using different dyes and sample
treatment techniques were initiated. Of particular inter-
est was the search for GC- and AT-specific fluoro-
chromes for revealing heterochromatic regions of the
chromosomes. Specificity of Q-bands for AT-rich DNA
regions has been confirmed in the experiments with
quinacrine, quinacrine mustard, and acridine orange
(Weisblum and de Haseth, 1972; Ellison and Barr,
1972).
After a while fluorochromes of mithramycin group
specifically binding GC-base pairs were demonstrated
to provide for the staining pattern inverse to that of
Q-banding. For instance, treatment of chromosomes of
three plant species (Vicia faba, Scilla sibirica, and
Ornithogalum caudatum) with chromomycin Ä3 and
related mithramycin (AT-specificity) provided for
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
3
strong fluorescence of heterochromatic regions (C-
bands) and nucleolus organizer regions, while DAPI-
staining (GC-specificity) provided for dim fluorescence
of these regions (Schweizer, 1976). In particular, large
proteins of intercalary and telomeric heterochromatin
of Scilla siberica chromosomes demonstrated active
fluorescence after treatment with chromomycin Ä3 and
mithramycin but remained DAPI-negative. Bright chro-
momycin bands similar to those in S. siberica were
absent in V. faba and O. caudatum and large regions of
high-colored heterochromatin were revealed in O. cau-
datum after DAPI-staining. This allowed Schweizer to
argue that “intercalary banding” is due to variable
nucleotide composition of DNA along the chromo-
some.
The most fundamental consequence of the Caspers-
son’s group activity was development of the method
providing for a clear staining pattern specific for indi-
vidual human chromosomes and complete identifica-
tion of all individual chromosomes (Paris Conference,
1971). Starting from this moment development of
human and animal chromosomal analysis kept ahead of
that for plants.
Considerable progress in revealing structural differ-
entiation along mitotic chromosomes is related to
application of Giemsa staining (Azure-Eosin and Meth-
ylene Blue are active components of the dye)—C- and
G-banding. The method of C-banding was accidentally
discovered by Pardue and Gall (1970) experimenting
with in situ hybridization of mouse chromosomes with
tritium-labeled satellite DNA. After autoradiography
the samples were stained with Giemsa. The centro-
meric regions of the chromosomes proved to be stained
more actively as compared to the other regions. Soon
this technique was modified to produce C-staining
(centromeric) (Arrighi and Hsu, 1971, 1974). C-blocks
proved to correspond to the regions of constitutive het-
erochromatin (Prokofieva-Bel’govskaya, 1986). A set
of experiments demonstrated the presence of particular
classes of satellite DNA in the constitutive heterochro-
matin psychologically revealed as C-bands (Langridge
et al., 1970; Zakharov et al., 1982). In 1960–1970s a
number of hypotheses were proposed on the functional
role of satellite DNA: it was related to maintaining
structural integrity of the chromosomes (housekeeping
functions) as well as to “evolutionary” role (Britten and
Kohne, 1968, 1970). Since phenotypic and evolution-
ary functions of satellite DNA are not completely clear
it was called “selfish” and “parasitic” DNA. Britten and
Kohne believed that satellite DNA has no specific func-
tion per se but can increase the organism capacity to
form new genetic information via multiple mutations,
translocations, and recombinations with other
sequences. Some researchers believe that large
stretches of tandemly repeated satellite DNA sequences
can be involved in evolution (Bostok and Samner,
1981). Appearance of satellite DNA changed the mor-
phology of chromosomes. Hatch et al. (1976) supposed
that the satellite DNA supplemented the genome with-
Vol. 34 No. 1 2003
4 ZOSHCHUK et al.
out losing non-satellite DNA. They also noted that the
presence of satellite DNA increases the probability of
chromosomal rearrangements and favors their fixation.
Thus, such changes induced appearance of new linkage
groups and affect evolutionary process.
Size increase in chromosome organs through accu-
mulation of satellite DNA in the genome could induce
genetic isolation of two populations. Thus, according to
the hypothesis of Hatch et al. (1976) satellite DNA
mediated rapid change of the karyotype during evolu-
tion.
Imai (1991) noted “weird” behavior of constitutive
heterochromatin at cytological level: C-bands are
ample and variable in the chromosomes of amphibians,
grasshoppers, and plants but are relatively poor in
mammals. Quantitative analysis of the changes in chro-
mosomal constitutive heterochromatin during evolu-
tion allowed dividing higher eukaryotes into two
groups according to their C-banding pattern (Imai,
1991). The first type includes mammals, fish, and ants,
while amphibians, grasshoppers, and plants constitute
the second one. C-bands are relatively poor in the chro-
mosomes of the first type and are located in their peri-
centromeric regions, while interstitial or terminal
C-bands prevail in the second type chromosomes.
From the very beginning of its development
C-banding technique was widely used for investigation
of animal and human chromosomes; and even superfi-
cial list of these works is hardly possible. By contrast,
only single publications on plant chromosome investi-
gation by this approach appeared for a long time. Pri-
marily, they were devoted to improving C-banding
technique for the analysis of plant chromosomes.
Wake-robin Trillium grandiflorum, three fritillary spe-
cies (Fritillaria meleagris, F. recurva, and F. lan-
ceolata), Siberian squill Scilla sibirica, horse bean
Vicia faba smooth hawk’s-beard Crepis capillaris (Sch-
weizer, 1973), and rye Secale cereale (Sharma and Na-
tarajan, 1973) were selected for the investigation.
After C-staining of the Trillium grandiflorum sam-
ples Schweizer observed clear pronounced bands on the
pro- and metaphase chromosomes, which allowed him
to precisely describe the plant karyotype. The centro-
meric region was actively stained in all chromosomes
visible as dark spots on each chromatid in the region of
primary constriction or as small proximal bands on the
both arms of the chromosome at the background of
unstained centromere.
Schweizer managed to reveal the bands only in two
(Californian F. recurva and F. lanceolata) out of three
fritillary species. The nucleus of F. meleagris looked
quite homogeneous and the chromosomes had no
bands. The absence of actively stained regions was not
explained by limitations of the technique, since a tiny
terminal or subterminal band was revealed on the short
arm of certain subtelocentric chromosomes. In all three
fritillary species the region of primary constriction
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
remained unstained and no C-bands was revealed in the
regions adjacent to the centromere.
Soon comparison of the data obtained by Schweizer
with the already available fluorescence banding of the
plant chromosomes became possible (Vosa and Marchi,
1972). The segments on Tulbaghia leucantha and
Allium carinatum chromosomes revealed by Giemsa-
staining corresponded to the chromosome regions
stained by quinacrine. Vosa and Marchi noted consider-
able difference in the methods: Giemsa-staining cannot
distinguish the regions of weak and active fluorescence
and they are evenly stained. Hence, they recommended
joint application of fluorescence banding and C-differ-
ential Giemsa-staining for the studies of chromosome
polymorphism in the plant populations. Several analo-
gous works on liliaceous plants chromosomes (Lili-
aceae)—Scilla siberica, S. bifolia, S. drunensis, and
S. vindobonensis—have been published (Greilhuber
and Speta, 1977, 1978).
In parallel Schweizer used the method of fluores-
cence differential staining of Fritillaria lanceolata and
F. meleagris by quinacrine and quinacrine mustard as
well as C-banding. The patterns obtained by different
methods coincided.
The C-banding technique was also successful on the
classical cytogenetic species Vicia faba: the revealed
bands located at M chromosome arms adjacent to the
centromere (Schweizer, 1973). In addition, two more
proximal bands were usually revealed on the long arm
of M chromosome. Several proximal dark regions were
also noted on S chromosomes as well. Increased dura-
tion of the staining exposed additional bands at the sub-
median region of the long arm of certain S chromo-
somes; however, the rest of the chromosome appeared
overstained in this case.
The dark regions were revealed in all chromo-
somes without exception in smooth hawk’s-beard
(Crepis capillaris) but the bands were less pronounced
as compared to other studied plant species. As with
horse bean a certain variation in the depth and pattern
of Giemsa staining was observed between the samples.
In the case of longer chromosomes the bands were
always observed at the distal end of the short arm, while
short chromosomes had a tiny submedian band in the
long arm and dark distal region in the short one. The
obtained data pointed to applicability of the C-tech-
nique for revealing heterochromatic regions on the
plant chromosomes as well as for studying the origin of
species and chromosomal polymorphism (Schweizer,
1973; Greilhuber and Speta, 1977, 1978).
In this context there is an interesting work on three
tulip varieties—Queen of Night (2n = 24), Spring Song
(2n = 24), and Turkestanica (2n = 48) (Filion, 1974).
Pronounced bands were obtained on the chromosomes
of all three cultures. The top and bottom numbers of
bands were revealed in the chromosomes of Queen of
Night and Turkestanica, respectively. On the basis of
the depth and constancy of the bands all revealed
Vol. 34 No. 1 2003
 HISTORY OF MODERN CHROMOSOMAL ANALYSIS
Giemsa-positive regions were divided into two
groups—the main and secondary bands. The main
bands were observed in all cells without exception and
looked solid and dark. The secondary ones were subdi-
vided into weakly stained bands of variable depth pre-
sented in all cells and faint and often missing bands.
All bands revealed in the three tulip varieties had
either terminal or interstitial localization, no centro-
meric bands have been observed, since the main and
secondary bands in the tulips represent telomeric and
intercalary constitutive heterochromatin, respectively.
Hence, C-banding of plant chromosomes has been
realized and constant pattern of the bands was demon-
strated by mid 1970s; however, the number of
described species remained limited.
Application of C-banding for studying cereal chro-
mosomes was an important step in development of
plant karyology (Zurabishvili et al., 1974; Gill and
Kimber, 1974a, 1974b; Shchapova, 1974; Iordansky
et al., 1978; Badaev et al., 1983, 1985; Bol’sheva et al.,
1984; Zelenin et al., 1987; Badaeva et al., 1994). Chro-
mosome identification based on linear length and pro-
portion of the arms is hardly applicable to wheat, rye,
and barley. In many cases only satellite chromosomes
can be identified due to the secondary constriction
(Okamoto, 1962). After C-banding technique became
available it was used for identification and analysis of
related genomes of the cultivated cereals and their wild-
lings.
First clearly discernible C-bands have been obtained
on the chromosomes of rye Secale cereale (Gill and
Kimber, 1974a; Shchapova, 1974; Sharma and Natara-
jan, 1973). The terminal bands on the short and long
arms of the chromosomes were mostly large and dark;
the centromeric bands had middle depth; while the
interstitial ones appeared small and dim. The experi-
ments demonstrated species- and chromosome-specific
pattern of the C-heterochromatic regions. This is the
basis for using differential staining methods in evolu-
tionary studies (determination of relationship between
species and origin of polyploid genomes), description
of varieties and geographical populations, and identifi-
cation of alien chromosomes in the hybrids. The first
cereal genus with all species studied by differential
staining was barley Hordeum (Linde-Laursen, 1985,
1986a, 1986b, 1989).
Despite considerable number of publications on
cereals, only morphology of monochromatically
stained chromosomes has been described in most Triti-
cum (2n = 14, 28, 42) and Aegilops species (Kihara,
1919, 1924, 1940, 1947, 1954; Sorokina, 1928; Senya-
ninova-Korchagina, 1932; Vavilov, 1935; Flyaksberger,
1935; Lilienfeld, 1951; Chennaveeraiah, 1960). Long
after introduction of the methods for differential stain-
ing of chromosomes only soft wheat and certain
Aegilops species were studied (Gill and Kimber, 1974b;
Iordansky et al., 1978; Jewel, 1979; Endo and Gill,
1984; Badaeva et al., 1996; Friebe et al., 1999). These
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
5
investigations have been completed just recently. For
instance, an author of this review (Badaeva, 2001) has
karyotyped 26 Aegilops and 16 wheat species. The pat-
terns of heterochromatic regions on the chromosomes
of A- and B- genomes in tetra- and hexaploid species
are basically similar; i.e., formation of the hexaploid
species was not accompanied by considerable changes
in the parent genomes. Virtually all heterochromatic
regions present in the hexaploid wheats can be found in
the tetraploid ones, although, depth of their staining
considerably varies. At the same time, certain bands
present in the tetraploid wheat species were lost by the
hexaploid ones. Changed system of polymorphism
through elimination of certain heterochromatic seg-
ments and amplification of the other ones was proposed
to mediate wheat hexaploidization. Triticum spelta
holding “intermediate” position between tetra- and
hexaploid forms was proposed as the primary hexap-
loid form from which other forms diverged. Unfortu-
nately, the ancestor tetraploid wheat of the hexaploid
species has not been established yet.
Maize (Zea mays L.)—one of the most important
cultivated cereals on Earth—became another subject of
active inquiry. Its chromosomal number 2n = 24 was
established slightly less than a century ago (Kuwada,
1910) followed by morphological investigation of the
meiotic chromosomes using knobs as psychological
markers for chromosome identification (McClintock,
1929). Identification of maize mitotic chromosomes
remained a painstaking job due to small chromosomes
and failure to expose the knobs by monochromatic
staining of the mitotic chromosomes. Later maize
mitotic chromosomes were described by relative
lengths of the chromosomes, arm index, and the pres-
ence of satellite (Chen, 1969; Filion and Walden,
1973). Appearance of the techniques for differential
staining of the chromosomes made them applicable to
studying maize chromosomes. In particular, the tech-
nique modification by Vosa and Marchi (1972) allowed
identification of Zea mays mitotic chromosomes (Had-
laczky and Kalman, 1975; Savchenko et al., 1986).
There were attempts to apply C-banding for the
investigation of chromosomes in gymnosperms. For
instance, investigation of five pine species (2n = 24)—
Strobus koraiensis, S. strobus, Pinus restinosa, P. nigra,
and P. banksianna—demonstrated localization of all
intercalary C-bands in the region of secondary constric-
tions (MacPherson and Filion, 1981). Unfortunately,
these data has not been confirmed until recent publica-
tions on fluorescent banding in gymnosperms
(Doudrick et al., 1995; Dagher-Kharrat et al., 2001).
Blocks of constitutive heterochromatin were also
described in the chromosomes of liverworts and mosses
(Bryophita), gymnosperms (Gymnospermae), and
angiosperms (Angiospermae).
Usually the chromosomes of higher plants carry rel-
atively large C-segments located both in the pericentro-
meric regions and close to telomeres in the interstitial
Vol. 34 No. 1 2003
6 ZOSHCHUK et al.
regions of the chromosomes. Small size of C-blocks
which are hard to reveal by the classical C-staining is
specific for paleoherbs and Magnoliidae (Rodionov
et al., 2001).
In parallel with discovery of C-technique in 1971
application of other pretreatment methods allowed
Sumner et al. (1971) to describe the technique of
G-staining (Giemsa-staining) first on human chromo-
somes and after a year on plant species (Sumner, 1972).
They demonstrated coincidence of the G-positive seg-
ments and brightly fluorescing Q-segments with a few
exceptions in humans.
At the same time, the presence of G-bands in plants
remained debatable for a long time. This discussion
was primed by Greilhuber (1977) who stated that all
cases of G-band description in plants were wrong due
to miscomprehension of G-banding nature. He believed
that the error is caused by misuse of the term. Accord-
ing to Greilhuber, it is not possible to obtain G-banding
in plants due to high degree of their chromosomes com-
paction; G-staining would be also impossible in human
chromosomes (due to insufficient resolution of light
microscopy) compacted to the size of been or wheat
chromosomes. Hence, appearance of G-bands in plant
chromosomes is only possible after specific treatment
so that their size exceeds that of human chromosomes.
Impossibility of obtaining G-banding in plants was also
explained by considerably higher quantity of DNA in
metaphase plant chromosomes as compared to verte-
brate chromosomes of the corresponding length. Greil-
huber introduced the index of compaction—the propor-
tion between content of nuclear DNA (pg) and the
length of metaphase chromosome (µm)—to compare
plant and human chromosomes. In plants this index
varied from 0.07 in Antirrhinum majus to 0.33 in Orni-
thogalum virens, while it was 0.02 pg/µm in humans.
This discussion continued later as well. On the basis
of length and volume measurements of the chromo-
somes in certain plant and animal species Anderson
et al. (1982) proposed that there is no considerable dif-
ference in the level of animal and human chromosomes
compaction; while the hypothesis of Greilhuber was
based on initially erroneous assumption. Later Wang
and Kao (1988) explained inability to reveal G-bands
on plant chromosomes by specific pretreatment of the
samples (i.e., acid or enzymatic hydrolysis), which
changed the structure of chromosomes and made them
resistant to G-staining procedures. Thus, omitting the
stage of acid or enzymatic hydrolysis allowed them to
obtain Vicia hajastana chromosome samples with
G-like banding.
Starting from 1980s more and more data on G- or at
least G-like staining of plants are published (Drewry,
1982). The subjects of the inquiry include the chromo-
somes of pine Pinus resinosa (2n = 24) with the size
ranging from 15 to 20 µm; the G-bands proved to be
very narrow and their pattern was invariable. The com-
paction index introduced by Greilhuber was also men-
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
tioned; it equaled 0.138 pg DNA per µm metaphase
chromosomes, i.e., the pine metaphase chromosomes is
seven times more compact than the human one (the
human index is 0.02 pg/µm).
This applies to large chromosomes, while investiga-
tion of species with small chromosomes (chamomile
and flax) requires decondensation of the mitotic chro-
mosomes and improvement of the techniques increas-
ing the resolution. Introduction of intercalating
agents—ethidium bromide or 9-aminoacridine—
proved to elongate the chromosomes (Hsu et al., 1973;
Muravenko et al., 1998b), thus, facilitating identifica-
tion of the bands revealed after the subsequent differen-
tial staining. For instance, pretreatment of wild chamo-
mile (Matricaria chamomilla L.) seedlings with the
ethidium solution (10 µg/ml for 12 h at 26°C) increases
the metaphase chromosomes from 3–6 to 5–10 µm
(Muravenko et al., 1998a); the pericentromeric regions
were particularly extended. The experiments on the
ethidium treatment indicate that it inhibits the effects of
colcemid and colchicine at the stage of spindle forma-
tion in addition to inhibiting compaction of the chromo-
somes; hence, one should double colcemid dose when
using it as an intercalating agent. 9-Aminoacridine is
more and more commonly used in the experiments
instead of the ethidium (Muravenko et al., 1998a).
In parallel with development of the main techniques
for differential staining (Q-, C-, and G-banding) other
methods based on different approaches appeared. Their
number is over twenty and detailed description of these
methods goes beyond the scope of this paper. The com-
plex of methods successfully used for plant chromo-
somal analysis can be divided into several groups:
(1) the proper differential staining techniques including
N-, R-, T-, and restriction banding in addition to Q-, G-,
and C-banding; (2) in situ hybridization techniques
aimed at localization of genes or cloned DNA
sequences of various nature on the chromosomes; and
(3) functional methods indicating functioning of indi-
vidual genes or their families at specific chromosomal
loci. These include replication banding, revealing sister
chromatid exchange (SCE or harlequin staining), and
ribosomal genes activity assay on the chromosomes
(Zakharov et al., 1982).
The most simple and fast way to reveal active nucle-
olar organizer genes is silver staining technique
described for animal (Goodpasture and Bloom, 1975;
Howell et al., 1975) and later plant chromosomes
(Schubert et al., 1979; Hizume et al., 1980; Sato et al.,
1980; Friebe, 1983; Cermeno et al., 1984; Lacadena
et al., 1984). Transcriptionally active nucleolar orga-
nizer regions (NOR) look as black bands on olive chro-
mosomes after staining. The molecular mechanisms of
silver staining are not yet completely clear. One of the
best-founded hypothesis is silver-staining of phosphop-
roteins (C23-nucleolin 110 kDa/pl 5.1) (Ochs and
Busch, 1984). However, the absence of correlation
between the presence or absence of the nucleolin and
Vol. 34 No. 1 2003
 HISTORY OF MODERN CHROMOSOMAL ANALYSIS
formation of Ag-NOR-bands has also been reported
(Biggiogera et al., 1989, 1991). Specificity of the
method for mammalian chromosomes is quite high. Sil-
ver staining is not NOR-specific for certain animal spe-
cies from other orders. Silver-staining of active genes
on Rhynchosciara polythene chromosomes (Stocker
et al., 1978) and Triturus cristatys carnifex lampbrush
chromosomes (Varley and Morgan, 1978) were
reported.
Ag-NOR-banding is successfully used to analyze
nucleolar activity and the processes of genomes inter-
action within polyploid plants—Aegilops, Allium, and
Hordeum species (Orellana et al., 1984; Cermeno et al.,
1984; Friebe and Fuleen, 1989; Schubert and Kunzel,
1990; Linde-Laursen et al., 1992). This technique is
also widely used to analyze interspecific hybrids and
chromosome addition lines (Santos et al., 1984; Lac-
adena et al., 1984; Lacadena and Cermeno, 1985; Cer-
meno et al., 1987; Friebe et al., 1987).
The technique of N-differential staining of chromo-
somes was initially developed to study NOR in the
plant and animal chromosomes (Matsui and Sasaki,
1973; Matsui, 1974; Funaki et al., 1975). NOR local-
ization was carried out on 27 sample types including
chromosomes of mammals, birds, amphibians, fish,
insects, and plants (Funaki et al., 1975). In most cases
N-bands were clearly revealed at specific chromosomal
loci—the regions of secondary constriction, satellite,
centro-, and telomeres. Based on the available cytolog-
ical and biochemical data Funaki et al. proposed that
N-bands represent (describe) certain structural nonhis-
tone proteins specifically associated with nucleolar
organizers of eukaryotic chromosomes.
Later N-differential staining technique proved to
reveal also the regions of constitutive heterochromatin
in cereals and, hence, can be used for identification of
chromosomes (Gerlach, 1977; Jewell, 1979, 1981;
Islam et al., 1981; Singh and Tsuchia, 1982; Endo and
Gill, 1983, 1984; Schlegel and Gill, 1984; Kakeda
et al., 1991).
For instance, clear bands have been revealed in nine
(4A, 7A, and all chromosomes of the B-genome) out of
21 pairs of chromosomes in hexaploid wheat Triticum
aestivum var. Chinese Spring (genomes AABBDD).
The bands were either dim or absent on the other chro-
mosomes (Endo and Gill, 1984). Chromosome staining
of tetraploid wheat T. dicoccoides (AABB) was similar
to that of the hexaploid wheat. The number of bands in
diploid species T. monococcum, T. boeoticum,
T. urartu, and Aegilops squarrosa was small (or no
bands were revealed) as could be expected from the
donors of A- and D-genomes. Chromosomes of A. spel-
toides carried a lot of N-bands (as could be expected
from the probable donor of B-genome). Since N-bands
were not observed on certain chromosomes with NOR,
Gerlach (1977) postulated the absence of correlation
between the presence of NOR and N-banding.
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
7
The regions for N-banding are relatively inexpen-
sive and the technique is simple. However, karyotype
comparison for the chromosomes stained by the tradi-
tional C-technique and N-technique in wheat variety
Chinese Spring (Gill and Kimber, 1974b; Gerlach,
1977; Gill et al., 1991a, 1991b) demonstrated consider-
able number of regions where either of the staining
types (C- or N-) is revealed despite their coincidence in
some cases. C-banding reveals all bands on the chro-
mosomes by contrast to N-differential staining of chro-
mosomes unsuitable for studying certain plant species
(since it does not reveal all types of heterochromatin
stained by C-banding) (Schlegel and Gill, 1984; Gill
et al., 1991a, 1991b; Kakeda et al., 1991). However,
C-banding is not very useful for the subsequent molec-
ular-genetic manipulations (in situ hybridization and
microdissection). At the same time, a modification of
N-banding compatible with in situ hybridization has
been recently developed (Jiang and Gill, 1993, 1994a–
1994c), which increases resolution of the hybridization.
All this applies to the species with large chromo-
somes. The possibilities of C-banding are limited for
plants with small chromosomes. It follows that neither
of the differential staining methods produces com-
pletely specific pattern of heterochromatic segments.
The most complete information on the functional struc-
ture of chromosomes can be obtained by using several
types of differential staining at the same time. Some-
times high number of the chromosomes and amazing
similarity between the small chromosomes reduce reli-
ability of identification and classification of chromo-
some pairs. The restriction banding and SCE detection
can complement the standard banding techniques (C
and G) to study the chromosomes. In addition to the
above-mentioned techniques there is a lot of differen-
tial staining types yielding additional information for
chromosomes identification.
Different methods of differential staining of chro-
mosomes have great value for practical cytogenetics;
they stimulate investigation of the structure and chemi-
cal nature of the chromosome components subjected to
differential staining. Clearly, differential staining tech-
niques will be used for chromosomes identification
later, thus, improving their resolution: there is a search
for new dyes for chromosomes differentiation and
sometimes the techniques for a more accurate karyo-
type identification.
Development of in situ hybridization techniques
made it possible to combine them with differential
staining of chromosomes (Szabo and Ward, 1982; Jiang
and Gill, 1994c); however, combination of these tech-
niques found wide application after the appearance of
fluorescence in situ hybridization (FISH) technique
(chiefly developed for mapping human chromosomes)
(Pinkel et al., 1986; Lichter et al., 1990; Wiegant et al.,
1991). Various combinations of individual binding
techniques (G, Q, and replication banding) with in situ
hybridization are commonly used to analyze human
Vol. 34 No. 1 2003
8 ZOSHCHUK et al.
chromosomes (McNeil et al., 1991). Simultaneous
observation of the hybridization signal and identifica-
tion of G-bands became possible with the help of 5-bro-
modeoxyuridine (Bhatt et al., 1988; Lawrence et al.,
1990). This technique is also very efficient for studying
human chromosomes. Other variants are more com-
monly used with plant chromosomes: sequential band-
ing (sequential staining of the sample by different
methods—C, G, or N) combined with in situ hybridiza-
tion (Hutchinson and Seal, 1983; Jiang and Gill, 1993)
as well as genomic in situ hybridization (GISH)—a
very important modification of in situ hybridization
(Langer-Safer, 1982; Le et al., 1989; Schwarzacher
et al., 1989; Heslop-Harrison and Schwarzacher, 1996;
Mukai, 1996). Genomic DNA of one of parents is used
as a probe and excessive fragmented genomic DNA of
another parent or the hybrid is added to the hybridiza-
tion mixture (to inhibit cross-hybridization). After
GISH the chromosomes (or their regions) inherited
from different species have different staining pattern.
GISH technique is used to analyze closely related
genomes: it allows us to analyze the origin of hybrids
or polyploid forms directly on the chromosome sam-
ples and is a unique approach for localization of the
breakpoints after intergenomic translocations.
Successful application of chromosomal technolo-
gies in plant genomics requires their considerable
development. Detailed description of chromosomes,
development of techniques for differential staining of
chromosomes suitable for subsequent DNA cloning
and generation of chromosome-, locus-, and band-spe-
cific libraries, improvement of in situ hybridization
methods (FISH), as well as resolving the problem of
repeated sequences are essential for plants targeted for
sequencing or investigation by comparative genomics.
Using the main approaches for generating chromo-
some- and locus-specific libraries of humans and some
other animals is complicated for plants—there is no
real success in isolating pure fractions of individual
chromosomes by flow sorting and there are serious lim-
itations for using microdissection technique.
This approach is based on cutting out individual
chromosomes or its region with the help of microman-
ipulator under the microscope or burning out unwanted
part of the sample by a laser beam (Jung et al., 1992,
Fukui et al., 1992; Schondelmaier et al., 1993; Rodova
et al., 1995; Ponelies et al., 1997; Zhou et al., 1999). A
fragment of the chromosome is amplified and cloned
for subsequent generation of chromosome- or locus-
specific markers as well as for “fishing up” specific
clones from the genomic library of a given organism.
However, application of this approach for plant chro-
mosomes meets considerable difficulties. Many plants
have very small chromosomes that can hardly or cannot
be identified even using the modern techniques of dif-
ferential staining.
The currently used methods for plant chromosome
banding, particularly, C-staining, make chromosomal
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
DNA unusable for cloning and subsequent investiga-
tion by the methods of molecular biology. Application
of G-like staining for identification of individual chro-
mosomes or their loci (Muravenko et al., 1998a, 1998b)
offers the opportunity for considerable expansion of the
application range for microdissection techniques in
plant genomics.
Chromosomal technologies already have and will
have great significance in the near future for compara-
tive and evolutionary genomics of plants (Zelenin et al.,
2001). Investigation of plant karyotype using classical
cytogenetic approaches gaining more and more from
rapidly developing methods of molecular biology and
computer technologies will remain the key approach
for describing plant genome for many years (Jiang and
Gill, 1994c; Gill and Friebe, 1998). The systems for
computer image processing are now applied for karyo-
typing. Note that Caspersson et al. (1971) were the first
to apply photometric methods for chromosomal stud-
ies. The system for computer image processing proved
to be particularly promising for investigating plant
karyotypes with small chromosomes with similar mor-
phology: rice, sugar cane, white beet, cotton, chamo-
mile, sunflower, and carrot (Fukui and Iijima, 1991;
Iijima et al., 1991; Muravenko et al., 1998b).
Application of the above-mentioned approaches is
particularly important for investigation of genomic sta-
bility and variability of the karyotype not only at the
level of individual organisms but also at the levels of
population, variety, and species. Finally, it is hard to
imagine how can the number and spectrum of chromo-
somal rearrangements (aberrations and bridges) be
evaluated without differential staining techniques; and
this criterion seems quite promising for environmental
monitoring by the status of plant genome.
In conclusion, we want to outline that the modern
chromosomal analysis of plants has a variety of meth-
ods for the most complete description of chromosomes
in the studied species. The genomic age has already
come for humans, while plant genetics only enters it
and the methods for chromosomal analysis will have
more and more important role.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research (project nos. 00-04-49036, 01-04-
48716, and 02-04-81022) and Russian State Scientific
Schools Program (project no. 00-15-97833).
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