va-Meiotic behavior of several Brazilian soybean varieties

Despite the importance of soybeans little cytogenetic work has traditionally been done, due to the small size and apparent similarity of the chromosomes. Fifteen soybean [ Glycine max (L.) Merrill] varieties adapted for cultivation in two distinct regions of Brazil were analyzed cytogenetically. A low frequency of meiotic abnormalities was noted in all varieties, although they were not equally affected. Irregular chromosome segregation, chromosome stickiness, cytoplasmic connections between cells, cytomixis and irregular spindles were the main abnormalities observed, none of which had been described previously in soybeans. All of these abnormalities can affect pollen fertility. Pollen fertility was high in most varieties and was correlated with meiotic abnormalities. Although soybean is not a model system for cytological studies, we found that it is possible to conduct cytogenetic studies on this species, though some modifications in the standard methods for meiotic studies were necessary to obtain satisfactory results.


INTRODUCTION
The genus Glycine, which includes the cultivated soybean, comprises predominantly diploid (2n = 2x = 40) and tetraploid (2n = 4x = 80) species. Soybean contains 2n = 40 small (1.42-2.82 µm), morphologically similar somatic chromosomes (Sen and Vidyabhusan, 1960) that do not show sufficiently different banding patterns to allow chromosome identification (Ladizinsky et al., 1979). Palmer (1976) has pointed out the usefulness of cytogenetic methods for improvement of soybeans. However, information on the cytogenetics of cultivated soybean is minimal when compared with other important crops. The causes of this lack of information include: i) the small but numerous chromosomes which are indistinguishable from each other and ii) the fact that the techniques usually used for cytological studies in other plant species are inadequate for soybean. In recent years, several important sterile male soybean mutants have been described (see Graybosch and Palmer, 1988;Palmer et al., 1992) and a cytogenetic map of the 20 soybean chromosomes has been constructed for the relatively uncondensed pachytene chromosomes (Singh and Hymowitz, 1988). More recently, in situ hybridization has been used to characterize individual soybean metaphase chromosomes (Griffor et al., 1991).
Despite its considerable economic importance for Brazil, there have been no detailed cytogenetic studies of soybeans in this country. Paraná State, which is home to the National Center for Soybean Research, is responsible for a large part of Brazilian soybean production. Current research at this center involves the analysis of spontaneous meiotic mutants that cause male sterility which may be useful in hybridization programs. To further our under-standing of soybean cytology and to improve the technique for meiotic studies we have examined the meiotic behavior and pollen fertility of 15 varieties of soybean adapted for cultivation in two different regions of Brazil.  (Maringá, PR), where the soil was prepared for soybean cultivation.

MATERIAL AND METHODS
Flower buds were collected from five plants of each variety for meiotic analysis and were fixed in FAA (ethanol:formaldehyde:acetic acid, 2:1:1 v/v) for 24 h, after which they were transferred to 70% alcohol and stored at 4 o C. Pollen mother cells (PMCs) were prepared by the squash technique and stained with 1% acetic carmine. At least 250 PMCs in different phases of meiosis were evaluated for each plant and any abnormalities seen were recorded. The same procedures and stain used for meiotic analysis were employed with open flowers to test pollen sterility. One thousand pollen grains/plant were examined.
The data were analyzed statistically by analysis of variance in a completely randomized design. Initially, the va-rieties were compared in the same group, and then the two groups were compared. The mean percentage of normal PMCs/variety in each group was compared using the Duncan test.

RESULTS
The 15 soybean varieties had a low frequency of meiotic abnormalities ( Table I). Analysis of variance revealed significant differences (P < 0.05) in meiotic behavior among the varieties in group I cultivated in Paraná State. In this group, the variety EMBRAPA 48 was the most affected by meiotic abnormalities. The varieties in group II, adapted for cultivation in central Brazil, had a more normal meiotic behavior than those in group I, though analysis of variance showed differences among them. There was also a significant difference between the meiotic behavior of the two groups (P < 0.05) as determined by analysis of variance.
The meiotic abnormalities observed among the varieties included chromosome segregation, chromosome stickiness, cytoplasmic connections among cells, cytomixis and irregular spindle. The meiotic phases generally most affected by these abnormalities were prophase I and metaphase I.
Precocious migration of univalents to the poles (Figure 1a) was observed in all varieties, with the exception of EMGOPA 314, in which all of the cells had normal meiosis. Another frequent segregational abnormality observed in metaphase I of two varieties was non-oriented bivalents at the equatorial plate ( Figure 1b). This abnormality occurred in the varieties OCEPAR 14 and EMBRAPA 48, with a significantly higher frequency in the latter. Laggard chromosomes in anaphases I and II were observed at a low fre-quency in some varieties. As a consequence of precocious migration of univalents, non-oriented bivalents and laggard chromosomes, some micronuclei (Figura 1c-e) were observed in telophase I and meiosis II. These micronuclei gave rise to microcytes in the tetrads (Figure 1f).
Chromosome stickiness was observed only in the MT/BR-45, EMBRAPA 48 and EMBRAPA 62 varieties, and affected all meiotic phases (Figure 2a-d). The phenomenon ranged from slight stickiness to an indistinct compact chromatin mass involving the entire complement (Figure 2a,b), which impaired chromosome segregation. Bridges were observed in telophase I ( Figure 2c) and pycnosis also occurred in some cells (Figure 2d).
The most common abnormality in all varieties was cytoplasmic connections involving two or more microsporocytes ( Figure 3a). The mean percentage of cytoplasmic connections ranged from 3.2 to 24.5 (Table II). Analysis of variance showed significant differences (P < 0.05) in this characteristic among the varieties. Although cytoplasmic connections were frequent, only one case of true chromosome transfer among cells (cytomixis) was observed (Figure 3b), although evidence of chromosome transfer was found in some cells with extra chromosomes (Figure 3c,d).
An irregular spindle was observed in only a few cells. In meiosis I, tripolar rather than bipolar spindles were present (Figure 4a,b), whereas in meiosis II the spindles were convergent (Figure 4c).
The test for pollen fertility showed a low percentage of sterile pollen grains ( Figure 4d). In most of the varieties, pollen fertility was significantly correlated with meiotic abnormalities, although in a few cases there was no relationship (Table I), as in the case of variety EMGOPA 314, which had the highest meiotic stability but the lowest pollen fertility.

DISCUSSION
Spontaneous chromosomal aberrations are relatively rare in Glycine compared with other important genera (Singh and Hymowitz, 1991a) and generally involve polyploidization and aneuploidy. Spontaneous meiotic mutations that cause male sterility have also been reported in soybeans (see Graybosch and Palmer, 1988;Palmer et al., 1992). We found meiosis to be relatively normal in 15 varieties of cultivated soybeans with few abnormalities when compared with other crops (Moraes-Fernandes, 1982;Souza et al. 1997;Baptista-Giacomelli, 1999). The abnormalities involved chromosome segregation, chromosome stickiness, irregular spindle formation and connections among cells, and had not been described in soybeans.
The observed precocious chromosome migration to the poles may have resulted from univalent chromosomes at the end of prophase I or precocious chiasma terminalization in diakinesis or metaphase I. Univalents may originate from an absence of crossing-over in pachytene or from synaptic mutants. However, prophase I stages were not analyzed because of the poor quality of the squash preparations. Chiasmata are responsible for the maintenance of bivalents which permit normal chromosome segregation. This process ensures pollen fertility. While precocious migration of univalents to the poles is a very common abnormality among plants (Pagliarini, 1990;Pagliarini and Pereira, 1992;Defani-Scoarize et al., 1995a,b;, the other segregational abnormality (non-oriented bivalents) observed in the varieties OCEPAR 14 and EMBRAPA 48 is rare, but is known to occur in Chlorophytum comosum (Pagliarini et al., 1993). The behavior of these and of the laggard chromosomes is characteristic in that they generally lead to micronucleus formation (Koduru and Rao,  1981). In soybean, the percentage of cells with meiotic abnormalities was higher in metaphase I and decreased until telophase II, indicating that some chromosomes were included in the main nucleus. This seems to be normal behavior for many species (Koduru and Rao, 1981). Sticky chromosomes were first reported in maize (Beadle, 1932) and are seen as intense chromatin clustering in the pachytene stage. The phenotypic manifestation of stickiness may vary from mild, when only a few chromosomes of the genome are involved, to intense, with the formation of pycnotic nuclei that may involve the entire genome, culminating in chromatin degeneration (for a review, see . In the soybean varieties, the stickiness was of both types. Some cells showed mild stickiness, in which case it was possible to identify the meiotic stage. In other cells, the intense phenotypic manifestation led to the formation of pycnotic nuclei. Chromosome stickiness may be caused by genetic or environmental factors. Genetically controlled stickiness has been described in other cultivated plants such as maize (Beadle, 1932;Golubovskaya, 1989;Caetano-Pereira et al., 1995), pearl millet (Rao et al., 1990) and wheat (Zanella et al., 1991). Several agents have been reported to cause chromosome stickiness, including X-rays (Steffensen, 1956), gamma rays (Rao and Rao, 1977;Al Achkar et al., 1989), temperature (Erikisson, 1968), herbicides (Badr and Ibrahim, 1987) and some chemicals present in soil (Levan, 1945;Steffensen, 1955;Caetano-Pereira et al., 1995). However, the primary cause and biochemical basis of chromosome stickiness are still unknown. Gaulden (1987) postulated that sticky chromosomes may result from the defective functioning of one or two types of specific nonhistone proteins involved in chromosome organization, which are needed for chromatid separation and segregation. The altered functioning of these proteins leading to stickiness is caused by mutations in the structural genes coding for them (hereditary stickiness) or by the action of mutagens on the proteins (induced stickiness).
Cytoplasmic connections, the most common abnormality observed in soybeans, is a phenomenon widely described in angiosperms (see Heslop-Harrison, 1966;Risueño et al., 1969;Whelan, 1974). The first description was made by Gates (1908), who observed delicate threads of cytoplasm connecting adjacent pollen mother cells in Oenothera. Gates (1911) subsequently suggested that these connections must form an important avenue of exchange between PMCs, and described the transfer of nuclear material through them from one meiocyte to another, calling the process "cytomixis". According to Heslop-Harrison (1966) and Risueño et al. (1969), the role of cytoplasmic channels is related to the transport of nutrients between meiocytes. Investigations in angiosperms have provided evidence that massive protoplasmic connections are formed among microsporocytes. Our study showed that the frequency of cytoplasmic connections among varieties varied from 3.2 to 24.5%. Although cytoplasmic connections are very common in angiosperms, the movement of nuclear material through them is rare. In the soybean varieties studied here, only one case of chromosome transfer (cytomixis) among microsporocytes was observed. In general, cytomixis has been detected at a higher frequency in genetically imbalanced species such as hybrids, as well as in apomictic, haploid and polyploid species (see Yen et al., 1993). Among the factors proposed to cause cytomixis are the influence of genes, fixation effects, pathological conditions, herbicides and temperature (see Caetano-Pereira and Pagliarini, 1997). Cytomixis may have serious genetic consequences by causing deviations in chromosome number and may represent an additional mechanism for the origin of aneuploidy and polyploidy (Sarvella, 1958).
The abnormal spindles observed in a few cells have also been reported for other genera (see Harlan and De Wet, 1975;Veilleux, 1985). The spindle apparatus is normally bipolar and acts as a single unit, playing a crucial role in the alignment of metaphase chromosomes and their poleward movement during anaphase. Distortion in meiotic spindles may be responsible for unreduced gamete formation. While the tripolar spindles seen in metaphase I of some cells may cause genome fractionation, convergent spindles in metaphase II rejoin the homologues segregated in meiosis I, leading to the formation of unreduced gametes. Although the formation of unreduced gametes has been investigated in studies of evolution (Harlan and De Wet, 1975) and in breeding programs (Veilleux, 1985), the frequency of convergent spindles in metaphase II in soybean was very low (0.3 to 1.4%) and not enough to be useful in breeding programs.
In normal soybean genotypes meiotic abnormalities are rare whereas they are common in meiotic mutants that cause male sterility. Chromatin bridges and micronuclei were described for the first time in interspecific hybrids of Glycine max x Glycine soja by Ahmad et al. (1977), who found that the extent of abnormalities was influenced by environmental conditions. The same abnormalities were reported by Ahmad et al. (1984), who concluded that chromosome behavior and fertility depended on the parentage of the hybrids and on environmental temperature. Their results, obtained in greenhouse and controlled environmental studies, suggest that at least three factors (genotype, temperature and genotype x temperature interaction) influence chromosome behavior and fertility.
All of the meiotic abnormalities found in the soybean varieties analyzed here have been reported to be responsible for pollen sterility. Fertility depends on the efficiency of the meiotic process. Studies on different plant species have shown that the decline in seed production is correlated with meiotic irregularities (La Fleur and Jalal, 1972;Dewald and Jalal, 1974;Moraes-Fernandes, 1982;Smith and Murphy, 1986;Pagliarini and Pereira, 1992;Pagliarini et al., 1993;Khazanehdari and Jones, 1997). In most of the soybean varieties, pollen fertility showed a close relationship with meiotic abnormalities. Most of the varieties had few meiotic abnormalities and, as a consequence, a high pollen fertility. Soybean is an autogamous, diploid and genetically stable species that produces a low number (300 to 800) of pollen grains per anther (Palmer et al., 1978). For this reason high meiotic stability is required in order to guarantee seed production. From our study, we suggest that the differential seed production observed among varieties is due to genetic control and not only to meiotic abnormality.
Soybean has not been considered a model system for cytological studies. According to Singh and Hymowitz (1991b), this may explain why soybean cytogenetics has lagged behind genetic studies of maize, barley and tomato. Our experience with soybean cytogenetics confirms this conception. Squash preparations of PMCs routinely employed for other species did not give good results. Some small modifications in the smear and stain in relation to the standard methods were necessary to obtain satisfactory results. The fact that the plants were cultivated in fields probably affected the analysis since, according to Palmer and Kilen (1987), greenhouse-grown plants yield a higher percentage of acceptable preparations, whereas plants grown under hot and dry conditions give very poor results. Despite the difficulties, we conclude that it is possible to conduct cytogenetic studies on soybean.