CARYOLOGIA
Vol. 52, n. 1-2: 97-103, 1999
Longitudinal differentiation in chromosomes of some Sesbania
Scop, species (Fabaceae)
ELIANA REGINA FORNI-MARTINS'• and MARCELO GUERRA2
1
Departamento de Botanica, Institute de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, Campinas, SP, 13083-970,
Brasil; 2 Departamento de Botanica, CCB, Universidade Federal de Pernambuco, Recife, PE, 50670-420, Brasil.
Abstract — Three different techniques were applied to analyze the patterns of longitudinal
chromosome differentiation in five Sesbania species (S. exasperata, S. punicea, S. sesban, S.
tetraptera, and two different geographical populations of S. virgata). For all the species investigated, the prophase chromosome condensation was always proximal. After staining with the
fluorochromes chromomycin A, and DAPI, each karyotype revealed two or four CM AY
DAPI~ heterochromatic blocks, apparently corresponding to the nucleolus organizing regions.
However, in S. virgata two or four CMA+ bands were observed, depending on the population
studied. No DAPI+ bands were observed. C-banding showed the most diversified patterns,
being a more useful technique to distinguish karyorypes of Sesbania species. The largest
amount of heterochromatin was observed in S. virgata (subgenus Daubentonia) whereas the
smallest was found in S. sesban (subgenus Sesbania).
Key words: chromosomes, karyotypes, chromosome banding, prophase condensation, Giemsa
C-banding, fluorochromes, Sesbania spp.
INTRODUCTION
Differences in chromosome morphology and
in the arrangement and amount of heterochromatin have been found in several plant
groups. They have been largely applied to the
understanding of karyotype evolution and systematics, as in the case of the genus Scilla
(GREILHUBER et al. 1981) or the family Arecaceae (RosER 1994). The longitudinal differentiation of metaphase chromosomes has become
more precise in the last 30 years, owing to the
application of techniques as C-banding and the
use of fluorochromes. These staining techniques
can be useful to identify individual chromosomes,
distinguish karyotypic structural variations and
reveal phylogenetic relationships between species
(see, among others, DEUMLING and GREILHUBER
1982; MOSCONE et al. 1996). The chromosome
condensation pattern in mi-totic prophase is
another karyotypic feature as specific as the
banding pattern or the conven-
* Corresponding author: fax +55-19-2893124; e-mail:
[email protected]
tional metaphase chromosome morphology. The
comparison
of
chromatin
condensation
characteristics in prophase and interphase nuclei
has contributed to the taxonomic characterization below and above the species level
(OKADA 1975; WATANABE and SMITH-WHITE 1987).
The genus Sesbania belongs to the family Fabaceae, tribe Robinieae. It comprises 32 species
in Africa, 10 in Australia, some 10-11 in tropical
Asia and 8 in America (MONTEIRO 1984). Chromosome counts are available for nearly half of
the species, although details of chromosome
morphology are known from only a few of them
(LuBis et al. 1981; JOSHUA and BHATIA 1989;
SALIMUDDIN and RAMESH 1993; HEERING and HANSON
1993; FORNI-MARTINS et al. 1994; Vi-JAYAKUMAR and
KURIACHAN 1995; ABOU-EL-ENAIN et al. 1998). These
studies suggested that the karyotypes were stable,
with little difference in the form and size of
chromosomes.
The present study aims to investigate the
pattern of prophase chromosome condensation,
C-bands and fluorescent CMA/DAPI bands in
the chromosomes of some Sesbania species.
98
FORNI-MARTINS and GUERRA
MATERIAL AND METHODS
The seeds were obtained from the "Institute de
Zootecnia" (IZ, municipality of Nova Odessa, State
of Sao Paulo, southeastern Brazil) (Table 1). The
vouchers are deposited in the herbarium IZ at that
institution. The IZ seeds used in this study belong to
the same introductions analyzed byFORNI-MARTINS et al.
(1994). Seeds of S. virgata (Cav.) Pers. were also
collected from plants growing alongside a highway
(BR-101), in the municipality of Recife, state of Pernambuco, northeastern Brazil, which vouchers are
deposited in the Herbarium of the Campinas State
University (DEC 35.739).
Root tips of recently germinated seeds were pretreated in a solution of 8-hydroxyquinoline for 1
hour at room temperature followed by 23 hours at
ca. 6°C. The root tips were fixed inCarnoy (ethanolglacial acetic acid 3:1 v/v) for 24 hours, transferred
to 70% ethanol and stored in a freezer. This initial
procedure was identical for the three techniques
used. For conventional staining root tips were hydrolyzed with 5N HC1 for 20 minutes at room temperature, squashed in 45% acetic acid and stained with
2% Giemsa for about 20 minutes(GUERRA 1983). The
chromosome condensation pattern wasanalysed with
this technique on all species exceptS. punicea, from
which only a very few root tips were obtained.
All five species were double stained withchromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI). The fixed root tips were washed, and
macerated in enzymatic solution of 2%pectinase —
20% cellulase during 1 to 2 hours(SCHWARZACHER et al.
1980). They were then squashed directly between
slide and coverslip in a 45% acetic acid drop. The
coverslip was removed with liquid nitrogen. Theslides
were stained with CMA for 1 hour and withDAPI
for 30 minutes, in agreement with DEUMLING and
GREILHUBER(1982).
For C-banding, the procedure ofSCHWARZACHER et al.
(1980) was followed with minor modifications.The
slides were hydrolyzed in 45% acetic acid at 60°C
for 10 minutes, treated with 5% barium hyd roxide at
room temperature for 10 minutes,incu-
bated in 2xSSC at 60°C for 80 minutes, and stained
with 2% Giemsa in a pH 6.8 Sorensen phosphate
buffer for about 20 to 30 minutes.
The idiograms indicating the C-banding patterns
are based on the chromosome morphology described
by FORNI-MARTINS et al. (1994). The photo micrographs
were taken with an Agfa Copex Pan film ASA 25 for
light microscopy and Kodak Tri-X Pan ASA 400 for
fluorescence microscopy.
RESULTS
All species presented the same chromosome
number (2n=l2). The nuclear interphase structure varied from reticulate to chromomeric
types, with some conspicuous chromocentres.
The pattern of prophase chromosome condensation was of the proximal type, with some terminal segments uncondensed until prometaphase (Fig. 1A,B)- The condensed segments
displayed a particular pattern for each chromosome pair. The proportion of condensed segments in prophase-prometaphase varied among
different chromosomes of the same cell and
among arms of the same chromosome. Such
patterns were identical for homologous chromosomes, but varied among homeologues of
different species.
The C-banding revealed that the species of
Sesbania have a very diversified heterochromatin distribution pattern. Heterochromatic regions
were observed in centromeric, intercalate and
terminal positions. The bands varied innumber
and size, according to the species. They may
appear as relatively large heterochromatic bands
or as dots in some chromosomes.
The karyotype of S. tetraptera had a simple
banding pattern, with centromeric bands in each
chromosome plus a terminal, heavy band in two
chromosome pairs (Fig. 1C). All the
LONGITUDINAL DIFFERENTIATION IN CHROMOSOMES OF SESBANIA
99
Fig. 1. — Pattern of prophase chromosome condensation and C-bands in Sesbania species. A - B: conventionally stained prometaphase (A), metaphase and interphase (B) of S. tetmptera. Chromosome pairs are identified to show the differential condensation of each
homologue pair during prometaphase. C-E: C-banded metaphases of £ tetraptera (C), S. virgata from Miranda (D) and S. sesban (E). F,
Idiograms of S. sesban (above) and S. virgata from Miranda (below). Bars in A (valid for all photographs) and in F (only for idiograms)
represent 5µm
chromosomes of S. sesban exhibited centromeric bands, plus one intercalate band in pairs 1
and 2, one terminal band in pair 2, and two
terminal bands in pair 3 (Fig. 1D). Chromosomes of both samples of S. virgata displayed
many heterochromatic bands in terminal, centromeric and intercalate positions (Fig. 1E).
Both the number of heterochromatic areas and
their size were smaller in chromosomes of S. ses-
ban than in those of S. virgata. Figure 1F
presents two idiograms showing the pattern of
heterochromatic bands of S. virgata from Miranda and S. sesban. The number and size of
heterochromatic bands of S. exasperata were
similar to those of S. sesban.
After fluorochrome staining, one or two
chromosome pairs showing CMA+/DAPI bands
were evidenced in all species studied.
100
The CMA+ bands were often adjacent to the
secondary constrictions and two to four CMA+
blocks were also observed in the nucleolus during
the interphase. In chromosomes of S. exasperata, the heterochromatic CMA+ bands occurred in a medium-sized pair and in the smallest
one (Fig. 2A). S. sesban presented a CMA+
terminal band in the two smallest chromosome
pairs, one of them sometimes weakly stained
(Fig. 2B). In both samples of S. virgata the
FORNI-MARTINS and GUERRA
CMA+ bands appeared in one of the smallest
chromosome (Fig. 2D), whereas in the Miranda
sample, CMA+ bands were also observed in a
medium to large size pair. S. punicea had a telomeric CMA+ band in a single chromosome pair
of medium to small size, similar to S. virgata
from Recife. S. tetraptera exhibited a CMA+
band in the large arms of a large pair and a small
sized one (Fig. 2F). No DAPI+ band was clearly
found in none of the species (Fig. 2C,E,G).
Fig. 2. — CMA/DAPI staining of metaphase cells of Sesbania exaspemta (A), S. sesban (B, C), S. virgata from Recife (D, E) and S.
tetraptera (F, G). Figures A, B, D, E, CMA fluorescence; C, E, G, DAPI fluorescence. Arrows points to CMA + blocks. Figures F, G
show two clumped metaphases (left) with CMA + blocks observed as dot pairs. Bar in G corresponds to 5 µ,m.
LONGITUDINAL DIFFERENTIATION IN CHROMOSOMES OFSESBANIA
DISCUSSION
Almost all species of Sesbania so far studied
have 2n=12, except S. pachycarpa with 2n=14,
and S. formosa, S. grandiflora and S. sericea with
2n=24 (PAWAR and KULKARNI 1955; FRAHMLELIVELD 1953,1957; LUBIS et al. 1981; SALLMUDDIN and RAMESH 1993; VIJAYAKUMAR and
KURIACHAN 1995; ABOU-EL-ENAIN et al. 1998). S.
sesban presented diploid and tetraploid cyto-types
(JOSHUA and BHATIA 1989; SALIMUDDIN and RAMESH
1993). The chromosomes shape and size
exhibited little variation among the species
analyzed (LUBIS et al. 1981; JOSHUA and BHATIA
1989; SALIMUDDIN and RAMESH 1993; HEERING and
HANSON 1993; FORNI-MARTINS et al. 1994;
VIJAYAKUMAR and KURIACHAN 1995; ABOU-EL-ENAIN
et al. 1998). Nevertheless, small karyotypic
differences do occur, and they were consistently
reported at the subgenus level (FORNI-MARTINS et al.
1994).
The species of Sesbania studied here displayed a great similarity in the structure of the
interphase nuclei, which was always reticulate
to chromomeric (GUERRA 1987; LUCENO et al.
1998). The interphase nuclear strucuture is generally similar in species of the same genus although it may vary within some genera, like
Habenaria (FELIX and GUERRA 1998).
The pattern of prophase chromosome condensation was proximal for all the species of
Sesbania studied here. However, the proportion
of condensed/uncondensed areas varied among
species. A detailed study of these areas is
needed to show their relationship with the heterochromatin and the meaning of their variation.
According to WATANABE et al. (1975), three
chromatin types can be recognized in prophase:
1) The heterochromatin sensu HEITZ (1928),
which is condensed during the whole cellular
cycle and heteropycnotic in interphase; 2) The
early-condensing euchromatin, heteropycnotic
in interphase but indistinguishable of the
heterochromatin in prophase; 3) The latecondensing euchromatin, weakly stained during
the prophase but indistinguishable from the
early-condensing euchromatin in metaphase and
anaphase. In Sesbania the condensed and the
uncondensed areas on prophase and prophaseprometaphase
chromosomes
seemed
to
correspond to the early-and late-condensing
chromatin, respectively. However, both areas
101
may contain heterochromatin as well as euchromatin, as observed by MORAWETZ (1981), on
uncondensed chromosomal regions oiLiriodendron, and by GUERRA (1988), on condensed regions
of prophase chromosomes ofCostus.
The analysis through CMA/DAPI did not
reveal DAPI+ bands, but two to four CMA+ terminal bands, which were generally associated
with the secondary constrictions and adjacent
heterochromatin. In interphase, the nucleolus
often showed two to four bright regions, indicating that CMA+ bands were associated with
NORs, as reported by many authors (e.g.
DEUMLING and GREILHUBER 1982; MOSCONE et al.
1996). The variation in number of CMA+ bands
(two to four) was not significant to discriminate
groups of species, since the same variation was
observed within a single species —S. virgata.
The number of terminal CMA+ bands rarely
coincided with the number of secondary constrictions described by FORNI-MARTINS et al.
(1994). S. tetraptera was the only species whose
number and position of CMA+ bands, two pairs in
this case, coincided with the secondary constrictions. S. sesban, S. exasperata and S. virgata
from Miranda showed two pairs of CMA+ bands,
but only one pair of chromosomes with
secondary constriction. Actually, the number of
secondary constrictions observed depends on its
activation on previous interphase and may be
underestimated, but not the number of CMA+
blocks. The chromosome size showed little variation, hindering the exact determination of the
pair carrying the secondary constrictions or
CMA+ bands. JOSHUA and BHATIA (1989), FORNIMARTINS et al. (1994) and HEERING and HANSON
(1994) recorded the presence of a single
chromosome pair (#3 or 4) with a secondary
constriction in S. sesban. SALIMUDDIN and RAMESH
(1993) and VIJAYAKUMAR and KURIACHAN (1995)
observed satellites in the largest chromosome
pair of the same species. LUBIS et al. (1981)
observed satellites in the fourth and fifth pairs or
in the fifth and sixth pairs, depending on the
origin of the S. sesban plants.
As the C-banding technique showed a larger
number of bands, it seems more adequated for
karyotypic differentiation at the subgenus level.
In a general way, the C-banding method is more
efficient to show the total amount of hetero-
102
chromatin (e.g. BERG and GREILHUBER 1993;
MOSCONE et al. 1996). In S. virgata, large and
numerous heterochromatic bands were observed,
whereas in S. tetraptera, S. sesban and S.
exasperata the amount of hetero chromatin was
smaller. These results corroborate the karyotypic
differentiation at the subgenus level observed by
FORNI-MARTINS et al. (1994). These authors
observed the largest chromosomes in the
subgenus Pterosesbania (S. tetraptera), the
smallest chromosomes in the subgenus Daubentonia (S. punicea and S. virgata), and chromosomes of intermediate size in the subgenus Sesbania (S. sesban and S. exasperata). The latter
one is considered the most primitive, as for
morphology and geographical distribution
(MONTEIRO 1984). The small amount of heterochromatin observed in their species may represent the primitive condition, as reported in some
other taxa (GREILHUBER et al. 1981; MO-SCONE et
al. 1996). The large difference in het-erochromatin
amount between S. virgata (Dau-entonia) and S.
tetraptera (Pterosesbania) seems to represent
different evolutionary trends in the genus (see also,
FORNI-MARTINS et al. 1994).
The polymorphic number of CMA+ bands
observed in S. virgata may indicate the occurrence of cytogeographic races. MONTEIRO (1984)
did not mention the occurrence of S. virgata in
northeastern Brazil. Our observations indicated
that this species should be considered ruderal in
Recife and other localities in Pernam-buco,
where it occurs in disturbed areas. The
occurrence of S. virgata in Pernambuco enlarges its known geographical distribution. Intraspecific chromosome polymorphisms are often
associated to the species geographical distribution.
WATANABE et al. (1975), for example, used the
polymorphism of nucleolar organizing regions to
distinguish
four
cytodemes
within
a
chromosome races ot Brachycome lineariloba.
Some conclusions can be drawn from the
three patterns of chromosome longitudinal differentiation in Sesbania. 1) C-banding pattern
cannot be deduced from the pattern of prophase
condensation, since the numerous terminal Cbands, mainly in S. virgata, cannot be predicted
by the prophase condensation pattern. 2) The
number of CMA+ bands varies within the genus
and may also vary within a single species. 3) The
C-banding pattern is very di-
FORNI-MARTINS and GUERRA
versified within the genus and seems to be the
most useful method to analyze the karyotype
evolution in the group.
Acknowledgments — To the Institute of Zootecnia, Nova
Odessa, SP, Brazil, for supplying the Sesbania seeds used in
this work; to Ana Emilia Barros and Silva, for her kind collaboration to the photographic documentation; and to FAEP/
UNICAMP for the financial support.
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Received 17 July 1999; accepted 17 Agust 1999