Andréa Beatriz Mendes-Bonato

Chromosome numbers and microsporogenesis in Brachiaria brizantha (Gramineae)

The genus Brachiaria, native to the African tropical savannas, has achieved significance as a pasture grass in many tropical and subtropical countries, including Brazil. Many species and accessions are polyploid and apomictic, which complicates the improvement of breeding stocks through hybridization. In support of breeding programs, cytogenetic characterization, including chromosome counts and evaluation of the meiotic behavior in the accessions of the Brachiaria has been undertaken at the Embrapa Beef Cattle Center. In this study, 22 accessions of B. brizantha were analyzed of which one was found to be diploid (2n = 2x = 18), 18 were tetraploid (2n = 4x = 36) and three were hexaploid (2n = 6x = 54). The meiotic chromosome behavior was slightly irregular in the diploid and in some tetraploid accessions, and highly irregular in most tetra- and hexaploid accessions. Meiotic abnormalities were those common to polyploidy, i.e., multivalent chromosome association at diakinesis and irregular chromosome segregation leading to micronuclei formation in the tetrad stage. Low frequencies of multivalent chromosome associations among polyploids suggest that they may be segmental allopolyploids.

UNUSUAL CYTOLOGICAL PATTERNS OF MICROSPOROGENESIS IN BRACHIARIA DECUMBENS: ABNORMALITIES IN SPINDLE AND DEFECTIVE CYTOKINESIS CAUSING PRECOCIOUS CELLULARIZATION

Cytogenetic studies carried out in the tetraploid accession BRA001068 of Brachiaria decumbens, also known as cv. Basilisk, revealed an unusual pattern of microsporogenesis. The spindle in metaphase I and anaphase I became heavily stained with propionic carmine. In telophase I, the interzonal microtubules continued to be intensely stained, and during the phragmoplast formation the fibers were pushed to the cell wall, persisting until prophase II, even after cytokinesis. Due to its tetraploid condition, the accession presented many cells with precocious chromosome migration to the poles in metaphase I and laggards in anaphase I that gave rise to micronuclei in telophase I. While in other polyploid accessions of Brachiaria micronuclei remained in this condition until the second cytokinesis, the micronuclei in this accession organized their own spindle in the second division. In several microsporocytes, the micronuclei with their minispindle were divided further into microcytes by additional cytokinesis. Some curious planes of cytokinesis were found in some cells, with partitioning of cytoplasm into cells of irregular shape. The result consisted of a high frequency of abnormal products of meiosis. Quadrivalents were observed in diakinesis at low frequency, which suggests a segmental allotetraploid and the inability of both genomes to co‐ordinate their activities, leading to multiple spindle and precocious cellularization. In spite of abnormal meiotic products reducing pollen fertility, seed production was normal. Enough normal pollen was available to fertilize the central‐cell nucleus of the embryo sac and produce normal endosperm in this pseudogamous aposporous apomictic accession.

Chromosome number and meiotic behaviour in Brachiaria jubata (Gramineae)

The production of new genetic variability through hybridization has been undertaken in Brachiaria, an important forage grass genus of African origin widely used in pastures in the Brazilian tropics. Although the genus comprises about 100 species, only a few of them present favourable agronomic attributes and are explored. In the genus Brachiaria, the majority of species and accessions are polyploid and apomictic, which makes breeding through hybridization more complex. Sexuality has been found in diploids and eventually among tetraploids with normal meiosis. Brachiaria jubata presents good forage value and adaptation to riverbanks and lake margins, and is therefore relevant to pasture systems i n the tropics. Furthermore, the most interesting form of resistance to spittlebugs—antibiosis—was identified in one a ccession of B. jubata. Thus its interest in the Brazilian breeding programme lies in the possibility of using hybridization to transfer high resistance to this insect to susceptible ac cessions. This paper reports chromosome numbers and meiotic behaviour in 21 accessions of this species as a tool in selecting promising ones for crossing. Only one accession was found to be diploid; the others were tetraploid. Microsporogenesis was normal in the diploid accession, while among the tetraploid ones, irregular chromosome segregation leading to unbalanced gamete formation was common but variable. Pollen fertility among tetraploid accessions was hig h. Cytogenetic studies were carried out on accessions of Brachiaria jubatafrom the Brachiaria germplasm collection at Embrapa Beef Cattle kept in the field in Campo Grande (state of Mato Grosso do Sul, Brazil), which comprises 475 accessions of 15 species collected in Africa in the mid-1980s by CIAT (Colombia). Inflorescences were collected and

Normal microspore production after cell fusion in Brachiaria jubata (Gramineae)

Cytogenetic studies were carried out on 22 accessions of Brachiaria jubata from the Embrapa Beef Cattle Brachiaria collection. One accession was diploid (2n = 2x = 18) and the remaining 21 were tetraploid (2n = 4x = 36). Among five tetraploid accessions, a specific and constant pattern of cell fusion involving only two microsporocytes was recorded. Meiosis proceeded normally from prophase I to the end, giving rise to an octad with normal microspores that developed into fertile pollen grains. Regular octad formation was possible because each cellular chromosome set was maintained in its proper domain, spindles were correctly positioned, and cytokinesis planes were formed in the correct places. Such behavior of meiosis in syncytes has never been reported in any other plant species.

Cytogenetic evidence for genome elimination during microsporogenesis in interspecific hybrid between Brachiaria ruziziensis and B. brizantha (Poaceae)

Microsporogenesis was analyzed in an interspecific hybrid between an artificially tetraploidized sexual accession of Brachiaria ruziziensis (R genome) and a natural apomictic tetraploid accession of B. brizantha (B genome). Chromosomes associated predominantly as bivalents. From this phase to the end of meiosis, chromosomes presented irregular segregation and abnormal arrangement in the metaphase plate. During metaphase I, in 27.8% of meiocytes, bivalents were distributed in two metaphase plates. In anaphase I, two distinct and typical bipolar spindles were formed. In 29.7% of pollen mother cells, one genome did not divide synchronically, with chromosomes lagging behind or not segregating at all. The second division was very irregular, resulting in polyads. Based on previous results from analysis of a triploid hybrid between these species, where the R genome was eliminated by asynchrony during meiosis, it is suggested that the laggard genome in this hybrid also belongs to B. ruziziensis.

Abnormal spindle orientation during microsporogenesis in an interspecific Brachiaria (Gramineae) hybrid

This paper reports a case of abnormal spindle orientation during microsporogenesis in an interspecific hybrid of the tropical grass Brachiaria. In the affected plant, prophase I was normal. In metaphase I, bivalents were regularly co-oriented but distantly positioned and spread over the equatorial plate. In anaphase I, chromosomes failed to converge into focused poles due to parallel spindle fibers. As a consequence, in telophase I, an elongated nucleus or several micronuclei were observed in each pole. In the second division, the behavior was the same, leading to polyads with several micronuclei. A total of 40% of meiotic products were affected. The use of this hybrid in production systems needing good-quality seeds is discussed.

Abnormal pollen mitoses (PM I and PM II) in an interspecific hybrid ofBrachiaria ruziziensis andBrachiaria decumbens (Gramineae)

Microgametogenesis was analysed in an interspecific hybrid resulting from a cross between an artificially tetraploidized sexual accession of Brachiaria ruziziensis (2n = 4x = 36) and the apomictic cultivar (cv. Basilisk) of B. decumbens (2n = 4x = 36). Although each microspore initiated its differentiation by pollen mitosis, polarization of the nucleus was not observed in 28.85% of the microspores, and the typical hemispherical cell plate was not detected as well. The usual asymmetry was not seen at first pollen mitosis (PM I), and microspores therefore lacked differentiation between the vegetative and the generative cell. After telophase, each cell entered PM II, but this was not always followed by cytokinesis. Tripolar spindle and restitutional nuclei were observed among microspores that lacked the first cytokinesis. Pollen from such abnormal divisions were sterile and larger than those resulting from the normal ones. Male gametogenesis in flowering plants depends on a determinative asymmetry in the cell division at pollen mitosis I (PM I), which gives rise to a larger vegetative cell and a smaller generative cell. To achieve this asymmetry, the microspore undergoes several unique cellular events, including the establishment of cell polarity through nuclear migration, development of an asymmetric mitotic spindle, and an expected process of cytokinesis to form a hemispherical cell plate. Control of gametophytic cytokinesis is, therefore, a critical process in pollen cell fate determination which results in the asymmetric distribution of cellular components that presumably include cell fate determinants (Twell et al. 1998; Park and Twell 2001). During cell division in somatic cells, a pre-prophase band of microtubules marks the future division plane and the exact site of cytokinesis, and the cell plate arises from the phragmoplast and grows toward the cell wall (Sylvester 2000). On the other hand, in gametophytic cytokinesis at PM I the pre-prophase band is absent and a unique hemispherical cell plate is formed and surrounds the generative nucleus (Terasaka and Niitsu 1990). Asymmetric cytoplasm cleavage is ensured by a curved profile of phragmoplast microtubules that appear to guide the centrifugal growth of the cell plate from its margin (Brown and Lemmon 1991). Mutations altering cell division asymmetry during pollen mitosis have been reported in Arabidopsis thaliana (Park et al. 1998) and B. decumbens (Junqueira Filho et al. 2003). This paper reports a new occurrence of symmetry in pollen mitoses (PM I and PM II) in a hybrid involving B. decumbens as the male genitor. Absence of cytokinesis following PM I and PM II was also observed.

Abnormal meiosis in tetraploid genotypes of Brachiaria brizantha (Poaceae) induced by colchicine: its implications for breeding.

Meiotic behavior was analyzed in 6 progenies from 3 artificially induced tetraploid (2n = 4x = 36) sexual genotypes (C31, C41, and C48) of the normally apomicticBrachiaria brizantha (Hochst. ex A. Rich.) Stapf., syn.Urochloa brizantha (Hochst. ex A. Rich.) R. Webster. These are key plants to allow intraspecific hybridization of this important forage species, widely used for pastures in the tropics. The percentage of abnormal cells among the plants ranged from 39.8% to 63.2%. In the single plant derived from C48, only the common meiotic abnormalities typical of polyploids were observed, while in plants derived from C31 and C41, a distinct behavior was found. In the majority of cells of those plants, the chromosomes remained scattered in the cytoplasm in the first division, without forming a metaphase plate. This abnormality blocked chromosome movements at anaphase I. Several micronuclei of various sizes were formed and, after the occurrence of an irregular first cytokinesis, the meiocytes progressed normally to the second division, generating polyads with unbalanced microspores. Pollen viability was not correlated with meiotic abnormalities. The importance of these findings to theBrachiaria breeding program is discussed. The sexual progeny of C48 seems most suitable as female parents to be used in intra-and interspecific hybridization.

Desynapsis and precocious cytokinesis in Brachiaria humidicola (Poaceae) compromise meiotic division.

The forage grass species Brachiaria humidicola is native to African savannas. Owing to its good adaptation to poorly drained and infertile acid soils, it has achieved wide utilization for pastures in Brazilian farms. Among the 55 accessions of B. humidicola analysed from the Embrapa Beef Cattle collection, one (H022), presented desynapsis and an abnormal pattern of cytokinesis in the first meiotic division. Among 28 inflorescences analysed in this accession, 12 were affected by the anomaly. In affected meiocytes, the first cytokinesis occurred in metaphase I and was generally perpendicular to a wide-metaphase plate, dividing the genome into two parts with an equal or unequal number of chromosomes. The normal cytokinesis after telophase I did not occur, and the meiocytes entered metaphase II, progressing to the end of meiosis with the occurrence of the second cytokinesis. As the first cytokinesis occurred precociously, whereas the second was normal, tetrads were formed but with unbalanced chromosome numbers in microspores. Abnormal cytokinesis occurred only in those meiocytes that underwent desynapsis after diakinesis. The implications of this abnormality in the Brachiaria breeding programme are discussed.

Chromosome numbers and meiotic analysis in the pre-breeding of Brachiaria decumbens (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.