Meiotic behavior during microsporogenesis of Alchornea triplinervia (Sprengel) Muller Argoviensis/Comportamento meiotico durante a microsporogenese de Alchornea triplinervia (Sprengel) Muller Argoviensis.
The Euphorbiaceae botanical family comprises around 290 genus and 7,500 species with great variations, ranging from woody to herbaceous plants. In Brazil there are approximately 70 genus and 1,000 species, native and exotic ones, representing one of the main families of the Brazilian flora and one of the most complex ones taxonomically (BARROSO, 1991; SOUZA & LORENZI, 2005). Several species belonging to this family have economic interest and among them are: the rubber tree (Hevea brasiliensis), cassava (Manihot esculenta) and beaver beans (Ricinus communis). There are also other species used in ornamentation (LORENZI, 2002; SOUZA & LORENZI, 2005).
Alchornea triplinervia is a specie of great environmental importance because it is indicated for reforestation of degraded areas. The aril that involves its seeds is used as food by birds and its wood can be used in the making of boxes, doors and crutches (LORENZI, 2002).
Cytologically, the Alchornea genus has been little studied, and the studies are limited to the counting of chromosome number. In the Alchornea genus has been cited x= 8, 9, 10 or 11 as basic chromosome numbers for most species (HANS, 1973). Cytogenetic data obtained from the observation of number and morphology of chromosomes as well as the behavior and meiotic division process, have been utilized to understand chromosome evolution, phylogeny and taxonomic relations (GUERRA & NOGUEIRA, 1990). In order to contribute to a better understanding of the species, this study reports the chromosome number and the abnormalities of the meiotic behavior through the analysis of the microsporogenesis A. triplinervia.
MATERIALS AND METHODS
Botanical material and inflorescences at the initial development stage of A. triplinervia were collected in the Municipal Park of the city of Paranavai, Parana--Brazil (23[degrees]05'S and 52[degrees]27'W), and at the Ecological Station of Caiua, located in the municipality of Diamante do Norte, Parana State - Brazil (22[degrees]41'S and 52[degrees]55'W).
Reproductive and vegetative parts of four plants were collected and herborized according to the usual techniques (FIDALGO & BONONI, 1989), and are stored at the Herbarium of Maringa State University (HUEM). The inflorescences at the initial development stage were collected for meiotic studies and, immediately after the collection, were fixed in a mixture of ethanol and acetic acid (3:1 v/v) for 24 hours at room temperature. After the fixation period, the material was washed in alcohol at 70% and stored in alcohol at 70% under refrigeration.
In the time of the study, the inflorescences were dissected with the help of a stereomicroscope for the withdrawal of anthers that were used to prepare semi-permanent slides by squashing technique, and staining with 1% acetic carmine. Microsporocytes were analyzed under binocular optical microscope Motic, B1 series, model 220A. Cells from the metaphase I to tetrad phases, besides microspores and pollen grains were observed. Photomicrographies were obtained with a digital camera, DCE- 2 Image Driving Soft Ware, and only contrast and brightness were altered.
Plants collected at the Municipal Park of Paranavai (MPP) will be named MPP I and MPP II and the plants collected at the Environmental Station of Caiua (ESC) will be named ESC I and ESC II.
RESULTS AND DISCUSSION
Chromosome counting during microsporogenesis (Figure 1A) showed that all analyzed plants presented 2n=8x=72 chromosomes. In diakinesis, the chromosomes were predominantly associated as bivalents, but univalents and tetravalent were also observed in a lower frequency (Figure 1A).
Considering x=9 as the basic number of chromosomes for the species of the genus, A. triplinervia is an octaploid (2n=8x=72). This statement is based on the described number of chromosome for the species of Alchornea genus, where n=9 are considered the haploid chromosome number of Alchornea tiliifolia Mull. Arg. (HANS, 1973) and Alchornea hirtella Benth. (SCHMELZER & GURIB-FAKIM, 2008), 2n=18 chromosomes as the diploid number of the Alchornea floribunda (HANS, 1973; SCHMELZER & GURIBFAKIM, 2008) and Alchornea laxiflora (Benth.) Pax & Hoffm species, and 2n=36 chromosomes for Alchornea cordifolia (Schumach. & Thonn.) Mull. arg. (SCHMELZER & GURIB-FAKIM, 2008).
No records were found in the literature on the meiotic behavior of the genus to which A. triplinervia specie belongs, and the few cytogenetic studies that were found are restrict to the counting of chromosome number for some species. In this study, the meiotic behavior of four A. triplinervia plants during microsporogenesis was analyzed. The number of analyzed cells and the percentage of irregularities in each meiosis phase can be observed on table 1.
In the chromosomes segregation process, the observed irregularities were: precocious chromosomes migration in metaphases (Figure 1C and I), laggard chromosomes in anaphases (Figure 1D and J), micronuclei in telophases (Figure 1F, K and L), and tetrads with microcytes (Figure 2D). These constituted the main abnormalities observed in the four studied A. triplinervia plants and according to SINGH (1993) this is a typical meiotic behavior of polyploids.
The greater frequency of precocious migration in metaphases I (Figure 1C) was observed in MPP I plant (13.04%) and the lowest frequency in ESC II plant (4.76%). The chromosomes in precocious migration in metaphases I may have originated from univalents in diakinesis or have been a result of precocious terminalization of chiasma and the late terminalization of these chiasmas makes the laggard chromosomes in anaphase I (SOUZA-KANESHIMA et al., 2010).
Laggard chromosomes (Figure 1D and J) were visualized in MPP I and MPP II plants, affecting 16.83% of meiocytes in anaphase I and 35.90% in anaphase II, respectively. The presence of irregular chromosome behavior in metaphases and anaphases in the Euphorbiaceae family was observed by NASSAR (2000) in a cytogenetic and evolutive study of Manihot esculenta Crantz, where there was precocious chromosome migration in metaphase I and II, and laggard chromosomes in anaphase I and II. RISSO-PASCOTTO et al. (2005), in an analysis of microsporogenesis in Brachiaria ruziziensis, it was reported the occurrence of precocious chromosome migration and laggard chromosomes.
[FIGURE 1 OMITTED]
Non-oriented bivalents (Figure 1 B) as well as chromosomes in precocious migration and laggards may lead to the formation of micronuclei in telophase I if they are not reintegrated to the telophasic nucleus (KONGPRAKHON et al., 2005). RISSO-PASCOTTO et al. (2003) suggest that non-oriented chromosomes result from a linking failure of spindle fibers to the kinetochore, making them disperse in the cytoplasm.
The frequency of chromosome stickiness (Figure 1E) ranged from absent in ESC II plant up to 23.88% in anaphases of MPP II plant. The stickiness is characterized as a chromosome grouping during some phases of the cellular cycle that according to PAGLIARINI (2000), may be caused by environmental causes as well as genetic control. GAULDEN (1987) suggest that chromosome stickiness its caused by a failure in the functioning of one of the two non-histone proteins involved in the process of chromosome separation and segregation. According to author, this failure could be caused by mutations in genes that codify these proteins, being the chromosome stickiness hereditary, or by mutagenic agents that act on these proteins which would lead to induced stickiness. In studies on corn, CAETANO-PEREIRA et al. (1995) report chromosome stickiness caused by aluminum saturation in the soil, showing an environmental factor as the cause of that abnormality.
Chromosome grouping during metaphase I and anaphase I led to the formation of chromosome stickiness bridges in telophase I (Figure 1G), observed only in MPP I. According to MENDES-BONATO et al. (2001), the occurrence of non-terminalization of chiasma or the presence of chromosome stickiness is a factor that competes for the formation of chromosome bridges which may persist until telophase or break down, forming micronuclei.
The presence of micronuclei in telophases I (Figure 1F) and II (Figure 1K and L) was observed in the four plants of this study, ranging from 1.64% to 20.60%. The formation of micronuclei was observed in Avena sativa by BAPTISTA-GIACOMELLI et al. (2000), in which the authors report the elimination of micronuclei in the tetrad phase, giving rise microspores with and amount of unbalanced genetic material. In A. triplinervia, despite the great number of micronuclei, few tetrads presented microcytes (Figure 2D). The most probable cause of micronucleus formation is the irregular segregation of chromosomes during the meiotic process (PAGLIARINI et al., 1993; MENDESBONATO et al., 2002).
[FIGURE 2 OMITTED]
Another observed abnormality, during the analysis of microsporogenesis of A. triplinervia, was the occurrence of irregular spindles. Three of the analyzed plants, MPP I, MPP II and ESC II, presented this irregularity. The presence of tripolar spindles during metaphase II (Figure 2A) and anaphase II ended with the fusion of nuclei in telophase II (Figure 2B). According to ENDOW (1999) tripolar spindle can also be called convergent spindle or V spindle and, this irregularity can result in the fusion of two close nuclei, originating, at the end of the meiosis, triads instead of tetrads. The irregular organization of spindles is due to a mutation in the gene that controls the meiotic division (SHAMINA et al., 2000).
PAGLIARINI et al. (1992), in a cytological observation of Ochna sp., report the phenomenon of the convergence of two spindles into a single cellular pole, thus resulting in the occurrence of telophase II with only three nuclei, one of them being 2n. In A. triplinervia, the formation of triads (Figure 2C) and dyads (Figure 2D) was observed; thus occurring the restitution of nuclei in two cell poles. These irregularities culminated in the formation of microspores 2n (Figure 2E), as well as the micronuclei produced microspores with reduced amount of genetic material, being called unbalanced microspores.
The presence of irregular spindles ending in the formation of restitution nuclei (2n) has been reported in different species of Brassica napus and Brassica campestris (SOUZA et al. 1999), Medicago sativa (MARIANI et al., 2000), Achillea species (SHEIDAI et al., 2009) and Passiflora genus plants (SOUZA et al., 2003; KIIHL et al., 2010).
Abnormal microspores give rise pollen grains with different sizes and amount of genetic material, pollen grains 2n and unbalanced (Figure 2G). All analyzed A. triplinervia plants presented pollen grains 2n (Figure 2F) and unbalanced (Figure 2H), ranging from 0.69% to 5.38% and 1.58% to 8.32%, respectively.
According to the analysis done on A. triplinervia species, it may be concluded that the species has chromosome number 2n=8x=72, a polyploid, and despite of the abnormalities occurred in the meiotic process, these did not interfere in the reproduction and development of plants because the frequency of unviable pollen grains is low.
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Sara Mataroli de Godoy (I) Andreia Rodrigues Alonso Pereira (I) Mariza Barion Romagnolo (II) Claudiceia Risso-Pascotto (I) *
(I) Biotecnologia Aplicada a Agricultura, Universidade Paranaense (UNIPAR), Av. Huberto Bruning, 360, 87706-560, Paranavai, PR, Brasil. E-mail: email@example.com. *Autor para correspondencia.
(II) Universidade Estadual de Maringa (UEM), Departamento de Biologia, Maringa, PR, Brasil.
Received 07.05.11 Approved 12.30.11 Returned by the author 04.20.12 CR-5627
Table 1--Frequencies of meiotic abnormalities and of microspores and pollen grains of four A. triplinervia plants. Number of analyzed cells (% of abnormal cells) Phase Abnormalities MPP I MPP II Metaphase I 230 (82.61%) 71 (78.87%) Precocious chromosome migration 31 (13.04%) 8 (11.27%) Non-oriented bivalent 10 (4.35%) 7 (9.86%) Anaphase I 208 (82.21%) 67 (76.12%) Laggard chromosome 35 (16.83%) -- Chromosome stickiness 2 (0.96%) 16 (23.88%) Telophase I 233 (75.97%) 163 (90.80%) Micronucleus 48 (20.60%) 15 (9.20%) Chromosome stickiness bridge 8 (3.43%) -- Prophase II 222 (99.10%) 135 (78.52%) Micronucleus 2 (0.90%) 29 (21.48%) Metaphase II 244 (79.51%) 94 (81.92%) Precocious chromosome migration 32 (13.11%) 10 (10.64%) Non-oriented univalent 12 (4.92%) 4 (4.25%) Tripolar spindle 6 (2.46%) 3 (3.19%) Anaphase II 242 (80.99%) 78 (61.54%) Laggard chromosome 41 (16.94%) 28 (35.90%) Tripolar spindle 5 (2.07%) 2 (2.56%) Telophase II 241 (91.29%) 246 (73.58%) Micronucleus 13 (5.39%) 50 (20.32%) Tripolar spindle 5 (2.08%) 8 (3.25%) Nuclear restitution 3 (1.24%) 7 (2.85%) Tetrad 223 (87.90%) 124 (83.87%) With microcytes 3 (1.34%) 5 (4.03%) Triad 21 (9.42%) 1 (0.81%) Dyad 3 (1.34%) 3 (2.42%) Dyad with microcytes -- 11 (8.87%) Microspores 319 (94.04%) 397 (96.98%) Unbalanced 5 (1.57%) 10 (2.52%) 2n microspore 14 (4.39%) 2 (0.50%) Pollen grain 316 (93.04%) 577 (90.99%) Unbalanced 5 (1.58%) 48 (8.32%) 2n Pollen grain 17 (5.38%) 4 (0.69%) Number of analyzed cells (% of abnormal cells) Phase Abnormalities ESC I ESC II Metaphase I 66 (83.33%) 63 (93.65%) Precocious chromosome migration 6 (9.09%) 3 (4.76%) Non-oriented bivalent 5 (7.58%) 1 (1.59%) Anaphase I 61 (98.36%) 58 (100%) Laggard chromosome -- -- Chromosome stickiness 1 (1.64%) -- Telophase I 60 (91.67%) 54 (94.44%) Micronucleus 5 (8.33%) 3 (5.56%) Chromosome stickiness bridge -- -- Prophase II 60 (98.33%) 65 (100%) Micronucleus 1 (1.67%) -- Metaphase II 57 (87.72%) 78 (75.64%) Precocious chromosome migration 6 (10.53%) 8 (10.26%) Non-oriented univalent 1 (1.75%) 7 (8.97%) Tripolar spindle -- 4 (5.13%) Anaphase II 53 (100%) 77 (96.10%) Laggard chromosome -- -- Tripolar spindle -- 3 (3.90%) Telophase II 61 (98.36%) 82 (92.68%) Micronucleus 1 (1.64%) 2 (2.44%) Tripolar spindle -- 3 (3.66%) Nuclear restitution -- 1 (1.22%) Tetrad 78 (96.16%) 69 (98.55%) With microcytes -- 1 (1.45%) Triad 2 (2.56%) -- Dyad 1 (1.28%) -- Dyad with microcytes -- -- Microspores 57(96.49%) 109 (91.74%) Unbalanced 2 (3.51%) 9 (8.26%) 2n microspore -- -- Pollen grain 123 (97.56%) 664 (91.57%) Unbalanced 2 (1.63%) 50 (7.53%) 2n Pollen grain 1 (0.81%) 6 (0.90%) * MPP: Collected plant in Municipal Park of Paranavai; ESC: Collected plant in Ecological Station of Caiua.