Abnormal meiotic behavior in three species of Crotalaria

The objective of this work was to compare the meiotic behavior and pollen grain viability of three species of Crotalaria. Slides for meiotic analysis were prepared by the air-drying technique. Pollen grain viability was measured by three staining procedures (Alexander’s solution, tetrazolium chloride and fl uorescein diacetate) and in vitro germination in a sucrose solution. Eight bivalents were observed, confi rming previous reports on populations from other regions of Brazil, as well as from other countries. All species showed abnormal meiotic behavior as follows: in Crotalaria micans, cytomixis and abnormal chromosome pairing in diakinesis; in C. spectabilis, abnormal chromosome pairing in diplotene; in C. zanzibarica, shrunk nuclei in leptotene and zygotene. Pollen grains of all three species show low viability, which may be associated with the irregularities of the meiotic behavior.


Introduction
Crotalaria L. belongs to the tribe Crotalarieae and is the third largest genus of the subfamily Faboideae (Fabaceae), comprising around 600 herbaceous and shrub species distributed in the tropics and subtropics (Polhill, 1982).In Brazil, there are 31 native and 11 introduced species of Crotalaria (Flores et al., 2006).Some are used in agriculture (nitrogen fi xation in crop rotating systems and biological control of nematodes); in the paper and fi ber industry, in landscaping (Polhill, 1982;Mendonça et al., 1999) and in phytoremediation (Pereira et al., 2002).
However, little is known on the meiotic behavior of Crotalaria species.Verma & Raina (1980) evaluated twenty species and observed chromosome stability based on predominance of normal bivalents, but also related the occurrence of univalents, multivalents, and bridges with or without fragments in some species.These irregularities were considered as evidence for structural changes during the evolution of the genus.Almada et al. (2006) observed regular meiosis in diploid and polyploid taxa of Crotalaria, with ring bivalents as the main confi guration in diakinesis.Interestingly, even though some laggard chromosomes Pesq. agropec. bras., Brasília, v.44, n.12, p.1641-1646, dez. 2009 and bridges without fragments were found in all taxa, meiosis was more regular in polyploids than in diploids.The authors considered that polyploid species are probably allopolyploids.
Increasing information on the meiotic behavior of Crotalaria species may give important insights on the numerical and structural chromosome changes involved in the evolution of the genus, as shown by Verma & Raina (1980) and Almada et al. (2006).
The objective of this work was to compare the meiotic behavior and pollen grain viability of three species of Crotalaria.Slides for meiotic analysis were prepared from cell suspensions, according to Viccini et al. (2005), with modifi cations in the enzymatic digestion of anthers.This step was adjusted for Crotalaria using Pectinex Ultra SP-L (Novozymes), at 34°C, in a water bath, for four to six hours.Slides were air-dried, stained with 10% Giemsa solution and evaluated under light microscope (Leica DMLS).All meiotic phases were analyzed and representative fi gures were digitalized using a digital microcamera (Nikon Digital Sight DS-Fi1).The meiotic index: MI (%) = 100(number of normal tetrads/total meiocytes) was calculated from 5,000 meiocytes per species.

Materials and Methods
Pollen grain viability was evaluated using three staining methods: Alexander's solution, for 24 hours with fi xed and fresh material, at 4ºC; 2,3,5-triphenyltetrazolium chloride in 5 (TTC 5%) and in 50% (TTC 50%) sucrose solution, for 2 hours with fresh material, at room temperature in the dark; and fl uorescein diacetate (FDA) 6.25 μg mL -1 in 25% sucrose solution, for 30 min with fresh material, at room temperature in the dark.Slides were prepared by squashing the anthers (fresh or fi xed) in a drop of staining solution.In each method, percentage of viable pollen grains was obtained from 1,000 mature microsporocytes observed in fi ve slides per species.Slides for the Alexander and TTC methods were evaluated under a light microscope (Leica DMLS), while those for FDA were evaluated under epifl uorescence using an Olympus BX60 microscope (460-490 nm excitation and 515-550 nm emission fi lter).Fresh pollen grains were cultured in liquid media containing sucrose at 15, 20 and 50% for C. spectabilis, C. micans and C. zanzibarica, respectively, for 24 hours, in a humid chamber, at 28°C.All pollen grains from fi ve slides per species were analyzed under a light microscope (Leica DMLS) to estimate germination percentage.
Ring and rod bivalents were detected in 0.33% diakineses for C. zanzibarica (Figure 1 D) and in 0.73% diakineses for C. spectabilis (Figure 1 F).In the latter species, ring pairing was more frequent as also observed by Almada et al. (2006).In C. micans, neither rod nor ring bivalents were observed (Figure 1 E), while Almada et al. (2006) described their occurrence.
All three species had irregular meiotic behavior with different kinds of abnormalities.In C. zanzibarica, approximately 14% of the meiocytes were irregular.Of these, 89.19% were in prophase I, presenting strongly condensed nuclei, different from the normal leptotene and zygotene pattern (Table 1 and Figure 2  In C. micans, ca.8% of the meiocytes were irregular, presenting: sticky chromosomes (Figure 3 A); laggard or lost chromosomes in metaphase I (Figure 3 B); irregular meiotic spindle in metaphase II and asynchronic telophase II (Figure 3 C and D).Laggard or lost chromosomes and bridges in anaphases I and II were reported by Almada et al. (2006).
This species also showed migration of genetic material between cells, evidencing the occurrence of cytomixis (Figure 3 E to I), which corresponded to 16% of the total irregularities.Cytomixis was observed only in prophase I, corresponding to 11.08% of the irregularities in leptotene/zygotene and 4.92% of those in pachytene.Ocurrence of cytomixis during prophase I, mainly in pachytene, has been reported in species of Fabaceae (Wang et al., 2002;Belluci et al., 2003;Haroun et al., 2004;Sidorchuk et al., 2004).
Partial or complete chromatin migration involving cytomitic cells was observed (Figure 3 G), with some acting as donor and others as receptor meiocytes (Figure 3 H and I).Some authors suggested that chromatin and chromosome migration is not random but directional.For example, Falistocco et al. (1995) observed that, in Dactylis, cytomixis always occurred from a donor to a receptor cell and that, in several cases, complete or almost total genetic material migration occurred not only between two cells, but also amongst several cells at the same time.Pesq. agropec. bras., Brasília, v.44, n.12, p.1641-1646, dez. 2009 Cytomixis in meiosis can have important implications in evolution, as it can lead to the formation of unbalanced gametes, including non-reduced ones.If these gametes are viable, they will originate aneuploid or polyploid plants.This was suggested by Belluci et al. (2003) working with Medicago sativa.High pollen grain viability (87.1 to 95.1%) of Dactylis plants showing cytomixis was reported by Falistocco et al. (1995), evidencing that cytomixis can be potentially important in the production of non-reduced pollen grains (2n).
In C. spectabilis, approximately 12% of the meiocytes were irregular.There was a high percentage of irregular diplotene (64.28%) showing multivalents (Figure 4 A).There were also: sticky chromosomes (Figure 4  Each species showed a predominant type of irregularity.Most of the abnormal meiocytes in C. zanzibarica had strongly condensed nuclei in prophase I. Crotalaria spectabilis and C. micans presented multivalents and, in the latter, cytomixis was also observed (Table 1).However, meiotic index values were high: C. zanzibarica, 92.19%; C. micans, 91.32%; and C. spectabilis, 88.18%.
Low pollen grain viability could be either associated to the observed meiotic irregularities, since they have potential for chromosome number alteration, or to post-meiotic events.Still, considering that these species produce large amounts of seeds, both in natural populations and under cultivation, such low viability must be compensated by the large number of pollen grains.  (1Alexander's solution in fi xed (A1) and fresh (A2) pollen grains. (2)5% 2,3,5-triphenyltetrazolium chloride in 5% sucrose solution.
A). Sticky chromosomes (Figure 2 B), laggard or lost chromosomes in metaphase I and prophase II (Figure 2 C and D), irregular meiotic spindle (Figure 2 E), and triads with micronuclei (Figure 2 F) were also observed.
B); early chromosome segregation in metaphase I (Figure 4 C); laggard chromosomes in prophase II (Figure 4 D); irregular meiotic spindle in metaphase II (Figure 4 E) and telophase II, as well as chromosome asynchronism and micronuclei (Figure 4 F).In populations of C. spectabilis from Argentina, Almada et al. (2006) found laggard or lost chromosomes and bridges, but all tetrads were normal.

Figure 3 .
Figure 3. Meiotic abnormalities in Crotalaria micans (n = 8).(A) Sticky chromosomes; (B) metaphase I with laggard chromosomes; (C) metaphase II and (D) asynchronic telophase II with irregular spindles; (E) cytomixis between meiocytes at the beginning of pachytene; (F) meiocytes in zygotene with simple chromatin bridge; (G) total chromatin migration from one meiocyte to another in prophase I; (H-I) chromatin migration among three or more cells in prophase I, with micronuclei.Bar: 5 μm.

Table 1 .
Percentage of irregularities observed per meiotic phase of Crotalaria species.
Crotalaria zanzibarica, C. micans and C. spectabilis have irregular meiotic behavior, with predominance of shrunk nuclei in leptotene and zygotene in C. zanzibarica, cytomixis and abnormal chromosome pairing in C. micans and abnormal chromosome pairing in C. spectabilis.2. Abnormal meiotic behavior does not affect the tetrad production.3. Viability of pollen grains of C. zanzibarica, C. micans and C. spectabilis is low. 1.

Table 2 .
Pollen grain viability percentage determined by staining tests and in vitro germination percentage (G) of Crotalaria species.