CARYOLOGIA Vol. 61, no. 2

CARYOLOGIA
Vol. 61, no. 2: 154-159, 2008
Morphological aspects of Passiflora edulis f. flavicarpa chromosomes using acridine orange banding and rDNA-FISH tools
Praça Milene Miranda, Carlos Roberto Carvalho*, Francismar Correa Marcelino
and Maria Andréia Correa MendonçA
Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Universidade Federal de Viçosa,
36570-000, Viçosa–MG, Brazil.
Abstract — Passiflora edulis f. flavicarpa is considered the most important species of the genus Passiflora, mainly
because of its botanical and commercial importance, as well as for crop breeding and genomic programs. However, its chromosome characterization has been conflicting on what concerns the number and localization of secondary constrictions (SC) and nucleolus organizer regions (NORs). In this context, conventional and molecular
cytogenetic methodologies have been adapted to resolve these morphology problems. Cellular dissociation, airdrying techniques and Giemsa staining, acridine orange fluorochrome and fluorescent in situ hybridization (FISH)
methodologies were used. We identified four secondary constrictions, in the subterminal portion of the long arm
of chromosomes 1 and 8 and in the subterminal portion of short arm of chromosomes 2 and 7. Regions emitting
yellowish fluorescence were observed on chromosomes 7 and 8 after acridine orange staining. The same pairs were
evidenced with the rDNA 18S probe used in the FISH technique, indicating that only two of the four secondary
constrictions are related with NORs.
Key words: Acridine orange, NOR, Passiflora edulis f. flavicarpa, passion fruit, rDNA-FISH, secondary constriction.
INTRODUCTION
Besides its agroeconomical and ecological
importance, the genus Passiflora is also of great
interest for cytogenetics, considering the amount
of published works. However, the karyotype of
Passiflora edulis Sims f. flavicarpa Deg. (2n=18)
still presents conflicts concerning the morphology
data of its chromosomes. Due to their importance
for systematics, crop breeding and genomic programs of this plant, it becomes relevant that these
conflicts be eliminated.
The number and localization of secondary constrictions (SC), namely the morphological aspect
of this chromosome region identified without any
special banding, have been described differently.
OLIVEIRA (1996) found one SC in chromosome 8,
while SOARES-SCOTT (1998) showed two SC, one
in chromosome 4 and another in chromosome 7.
The latter author later related the presence of at
least three SC, as cited by SOUZA et al. (2003). In
contrast, CUCO et al. (2003) observed the presence
of SC in chromosomes 8 and 9.
* Corresponding author: phone: +55 31 38992568/
38991295; fax: +55 31 38992549; e- mail: [email protected]
Contradictory data have also been described
in relation to the identification and chromosomic
allocation of nucleolus organizer regions (NORs),
meaning the region with active rDNA genes identified by positive silver nitrate staining. Using this
staining, MAYEDA (1997) detected NORs associated with the SC of chromosomes 8 and 9, although
not specifying in which arm.
With the advent of molecular cytogenetics, fluorescent in situ hybridization (FISH) has been the
technique of choice for the specific localization of
active or inactive rDNA genes (LÓPEZ-LEÓN et al.
1999; SUMNER 2003). Using FISH in P. edulis f. flavicarpa chromosomes, MELO and GUERRA (2003)
described differing morphological data regarding
SC and NOR locations, in relation to previous authors. They observed sites of 45S rDNA on the
long arm of chromosomes 7 and 9, and 5S rDNA
localized on the long arm of chromosome 5.
In addition to these methods, another procedure has been used to localize active or inactive
NOR of plant chromosomes, based on indirect
evidence yielded by the flanking heterochromatin
associated with rDNA genes (ALMEIDA and CARVALHO 2004). These authors used a hot denaturation pre-treatment and acridine orange fluorochrome to stain maize and pepper chromosomes
155
cytogenetic banding tools in passiflora
in a similar usage to the reverse banding (RFA)
procedure described by VERMA and LUBS (1976)
for human cytogenetics.
Another improved cytogenetic tool has also
been successfully applied for obtaining information from high quality plant chromosomes. As
evidenced by CARVALHO and SARAIVA (1993; 1997)
and ALMEIDA and CARVALHO (2004), the cellular
dissociation and air-drying methods have demonstrated to be appropriate plant cytogenetic tools
for excellent chromosome preparation for showing well-resolved morphological aspects.
We applied the cellular dissociation and airdrying procedures, associated with acridine orange
fluorochrome staining and the FISH techniques,
for revising the NORs and SC number/position in
the chromosomes of P. edulis f. flavicarpa.
MATERIAL AND METHODS
Plant material - P. edulis f. flavicarpa seeds (Passion fruit round yellow) were obtained from ISLA
PAK Sementes Ltda., Porto Alegre, RS, Brazil
(www.isla.com.br). The analyses were carried out
at the Plant Cytogenetics and Cytometry Laboratory of the Federal University of Viçosa-UFV
(Brazil). Probe labeling for the FISH technique
was carried out at the Molecular Laboratory/
BIOAGRO-UFV.
Pre-treatment and fixation - Seeds were germinated on distilled water in a Petri dish at 30 ºC. Seeds
showing 1 cm roots were treated with a 3 µM amiprophos-methyl (APM, Nihon Bayer Agrochem
K. K®) solution (DOLEZEL et al. 1999) for 16 h
and 25 min at 4 ºC, washed with distilled water
for 15 min, and then fixed in fresh cold methanol:acetic acid solution (3:1). The fixative was
changed three times and the seeds were stored at
-20 ºC.
Slide preparations - Root-tips were washed three
times with distilled water and placed in 1 mL of
freshly prepared enzymatic solution consisting of
10 µL Flaxzyme (Novo FermentTM), which contains pectinolytic, cellulolytic and hemicellulolytic
activities (CAIXETA and CARVALHO 2001), and 300
µL distilled water, for 1 h 30 min at 34 ºC. Roots
were washed for 20 min with distilled water and
fixed again at -20 ºC. Slides were prepared by
meristematic cellular dissociation, air-dried and
placed on a hot-plate (50 ºC) for 20 min (CARVALHO and SARAIVA 1993; 1997). Some slides were
immediately stained with a 5% Giemsa (Merck®)
solution in phosphate buffer (pH 6.8) for 4 min,
washed twice in distilled water and air-dried.
Acridine Orange (AO) staining - Slides were aged
for 15 – 20 days, then incubated in phosphate
buffer (pH 4.7 at 85 ºC for 18 min) and stained
with 0.01% (w/v) acridine orange for 15 min (ALMEIDA and CARVALHO 2004).
Fluorescent in situ hybridization - FISH was based
on the methods of OSUJI et al. (1998). rDNA
probes containing the ribosomal 18S sequence
(1100 pb) of Zea mays were cloned in pGEM®
-T Vector (Promega®). The probes were labeled
with reaction buffer by mixing 8 µL of fluor-12dUTP with 92 µL of 5x nucleotide buffer, by
random priming, according to the recommendations of the Prime It™ kit (Stratagene®) manual.
Slides with well-spread chromosomes of P. edulis
f. flavicarpa were incubated with RNase (100 mg/
ml in 2xSSC) for 1 h in a moist chamber at 37 ºC,
and then treated three times with 2x SSC (0.03
M sodium citrate and 0.3 M sodium chloride) in
baths of 3 min. The material was dehydrated by 3
min incubation in each component of an alcohol
series (50%, 75% and 100%). Samples of 15 mL
of the probe were added to each hybridization area and the slide was covered with a coverslip and
incubated for 3 min at 80 ºC, and overnight at
37 ºC. After hybridization, the slides were washed
twice for 2 min in 2x SSC containing 50% (v/v)
formamide, at 45 ºC; four times for 2 min in 2x
SSC at room temperature; then once in PBS for 2
min. The preparation was counterstained with 25
mL propidium iodide (Sigma®) solution (1mg/
mL), washed in phosphate-buffered saline (PBS)
for 1 min, and mounted with 15 mL Vectashield
(Vector®).
Image analysis - Images of chromosomes were
captured with a CoolSNAP-Pro cf (Roper ScientificTM) video camera of 12 bits on an OlympusTM
BX-60 fluorescence microscope, with a 100x objective lens and a WB filter for analysis of acridine
orange, and WG filters for FISH analysis. The
frame was digitized using an Image Pro®-Plus 4.5
software (Media CyberneticsTM). Image analysis
was performed on a Power Macintosh G4 computer, using the freely available (http://reg.ssci.liv.
ac.uk) Image SXM software (BARRETT 2002). This
is a spin-off of the public domain image analysis
application NIH Image, which was developed by
RASBAND (1998).
RESULTS
Metaphase images were obtained showing a
complete set of 18 chromosomes, well-spread and
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without overlapping on the same focal plane on
the slide. The chromosome morphology showing
well-defined primary and SC facilitated the characterization of the homologous pairs and, consequently, the karyogram assembly (Fig. 1 a).
The analyses of pro- and metaphasic karyotypes stained with Giemsa revealed six pairs of
metacentric (2-7) and three pairs of submetacentric (1, 8 and 9) chromosomes (morphometric
data not shown). The SC were observed in the
subterminal long arm of chromosomes 1 and 8
and subterminal short arm of chromosomes 2 and
7 (Figs. 1 b, c).
The chromosomes stained with acridine orange showed four bright yellowish fluorescence
regions, one localized on the terminal short arm
of chromosome 7 and one on the terminal long
arm of chromosome 8 (Fig. 2 a).
Using 18S rDNA probes in FISH, the target sequence was identified in interphasic nuclei showing four fluorescent spots (Fig. 2 b) and in two
pairs of chromosomes showing four fluorescent
signals. These signals were located on the distal
short arm of chromosome 7 and on the distal long
arm of chromosome 8 (Fig. 2 c).
The chromosomes 7 and 8 were selected in order to highlight the morphological comparisons
between the Giemsa staining, acridine orange fluorescence and FISH procedures (Fig. 2 d).
praça, carvalho, correa marcelino and correa mendonça
DISCUSSION
Root-tips treatment with APM, a phosphoric
amide herbicide with significantly higher affinity
to plant tubulins (DOLEZEL et al. 1999), enzymatic
maceration, cellular dissociation and air-drying
were considered important steps for providing
slides showing high quality cytogenetic preparations. The APM treatment was considered adequate for accumulation of cells with a fine range
of pro- and metaphasic condensation, for screening minor differences between chromosomes of
the same class with apparent similar lengths (Figs.
1a – c). The cellular fixation, maceration, dissociation and air-drying steps resulted in well-spread
and morphologically preserved chromosomes.
These methodologies, associated to the digital
image analysis, contributed to discriminate subtle measurements between chromosomes. Similar
results were obtained in Zea mays (CARVALHO and
SARAIVA 1993; 1997), Capsicum annuum (ALMEIDA
and CARVALHO 2004) and Aniba rosaeodora (CONTIM et al. 2005).
Four SC were found at the present work, localized in the subterminal long arm of chromosomes 1 and 8 and in the subterminal short arm
of chromosomes 2 and 7 (Figs. 1 b, c). In contrast, using the conventional squashing technique,
various authors differently described either the
Fig. 1 — Chromosomes of P. edulis f. flavicarpa (2n = 18 chromosomes) treated with APM 3 µM for 16 h and 25 min
at 4 ºC and stained with Giemsa 5%. (a) Well-spread metaphase and (b) karyogram derived from prometaphasic
and (c) metaphasic chromosomes. Notice the defined primary constrictions and the SC present in the distal long
arm of chromosomes 1 and 8 and in the distal short arm of chromosomes 2 and 7 in both karyograms. Bar = 5 µm.
cytogenetic banding tools in passiflora
157
Fig. 2 — Chromosomes obtained from meristematic cells of P. edulis f. flavicarpa, treated with APM 3 µM during
16 h and 25 min at 4 ºC, and interphasic nuclei. (a) Karyogram stained with 0.01% acridine orange. Notice the yellowish fluorescence signals on the distal short arm of chromosome 7 and on the terminal long arm of chromosome
8. FISH using the rDNA 18S probe in (b) interphasic nuclei and (c) metaphasic chromosomes. Notice the presence
of four yellowish fluorescent signals both in (b) nuclei and on (c) the distal arm of chromosomes 7 (short arm) and
8 (long arm). (d) Collection of chromosomes 7 and 8 stained with (G) Giemsa, (AO) acridine orange and using (F)
FISH with 18S rDNA labeled probe. Notice the correspondence between the SC with fluorescent signals in the
short arm of chromosome 7 and long arm of chromosome 8. Bar = 5 µm.
number or the localization of SC in P. edulis f.
flavicarpa. OLIVEIRA (1996) found only one SC in
chromosome 8, while MAYEDA (1997) related the
presence of two pairs of chromosomes with satellites. SOARES-SCOTT (1998) showed a karyotype
with two SC, one in chromosome 4 and another
in chromosome 7, both in the long arm. In later
works, SOARES-SCOTT related the presence of at
least three SC, but did not specify their position
(cited by SOUZA et al. 2003). CUCO et al. (2003)
observed the presence of SC in chromosomes 8
and 9. According to MAYEDA (1997) differences
on chromosome characterization may occur as a
result of either the methods used or the quality of
the sample preparation. In addition, the morphological identification of a particular SC in a chromosome without any special banding techniques
depends on the level of chromatin condensation.
The acridine orange staining, in accordance
with the protocol used by ALMEIDA and CARVALHO
(2004), showed a differential fluorescence pattern similar to that observed by these authors on
chromosomes of pepper and maize. However, in
these species, yellowish fluorescence bands were
observed flanking the SC regions of the chromosomes 11 (pepper) and 6 (maize), while in passion
fruit the fluorescent signals were observed on the
terminal SC chromosomes 7 and 8, although not
discriminating the flanking bands (Fig. 2 a). The
differential fluorescence pattern in the SC region
was obtained by treatment with buffer solution
at pH 4.7 and 85 ºC for 18 min. Shifts for lower
or higher values than these, in order to search
for better resolution of the flanking regions, did
not present satisfactory results. If the yellowish
fluorescence of the SC of chromosomes 7 and 8 of
passion fruit stained in a process similar to that of
ALMEIDA and CARVALHO (2004), these regions also
correspond to NOR-associated heterochromatin.
Using FISH with 18S rDNA probe two fluorescent signals were observed, one on the short arm
of chromosome 7 and another on the long arm
of chromosome 8 (Fig. 2 c). Similar results were
obtained by MELO and GUERRA (2003), who iden-
158
tified two sites of 45S rDNA on the long arm of
chromosomes 7 and 9 and one site of 5S rDNA on
the long arm of chromosome 5 in the karyotype of
P. edulis f. flavicarpa. However, these data diverge
mainly on what concerns the position of the two
45S rDNA fluorescent signals corresponding to
the SC, in short, whether one signal is present on
the short or long arm of the chromosome 7, and
whether the other signal is on chromosome 8 or 9.
Such differences can be explained by the authors’
criteria, such as whether chromosome 7 had been
oriented up or downwards, and if the positions of
chromosomes 8 and 9 had also been exchanged in
the karyogram.
Although the origin of these punctual morphologic chromosome differences can be justified
by interpretational problems related to the small
size of the chromosomes, condensation degree or
the cytogenetic methodology used, the differences between the 18S (present paper) and the 45S
rDNA (MELO and GUERRA 2003) probes used did
not prevent the identification of SC that contain
the genes. As shown by SUMNER (2003), the 45S
unit includes the 18S sequence, since the NORs
correspond to the sequences of repeated DNA
encoding for 18S, 5.8S and 26S rRNAs (BESENDORFER et al. 2002; SCHROEDER-REITER et al. 2006)
and, cytologically, these regions are localized in
SC in metaphasic chromosomes (BRASILEIRO-VIDAL et al. 2003).
According to BATTISTIN et al. (1999), all the
NORs are localized in SC, but not all SC are NOR
sites. With assistance of the techniques used in the
present work, it was possible to identify four SC
and two NOR sites.
In conclusion, considering that the SC containing rDNA sites could be identified by different staining methods, the Ag-NOR technique
used by other authors could only identify the SC
holding rDNA genes that were active in nucleolus
during preceding interphase. In this sense, some
identification differences can be found between
genotypes, populations and ecotypes. However,
the acridine orange, which can be used for localization of heterochromatin associated with rDNA
genes, and rDNA-FISH methods allow the location of all rDNA genes, active or non-active during interphase.
Thus, if cases of intraspecific, genotypic or ecotypic differences are not present in these cytogenetic studies, our results suggest four SC (chromosomes 1, 2, 7 and 8) and two rDNA sites, at the
chromosomes 7 (short arm) and 8 (long arm) in
P. edulis f. flavicarpa.
praça, carvalho, correa marcelino and correa mendonça
Acknowledgements — We thank the Fundação
de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG, Brazil) and the Conselho Nacional de
Pesquisa (CNPq, Brazil) for the financial support.
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Received April 13th 2007; accepted January 2th 2008