Microsporogenesis in sexual Brachiaria hybrids (Poaceae)

Three sexual interspecific hybrids of Brachiaria (HBGC076, HBGC009, and HBGC014) resulting from crosses between B. ruziziensis (female genitor) and B. decumbens and B. brizantha (male genitors) produced by Embrapa Beef Cattle in the 1980s were cytologically analyzed by conventional methods for meiotic studies. The cytogenetic analysis showed the occurrence of common meiotic abnormalities among them. The most frequent abnormalities were those related to irregular chromosome segregation due to polyploidy. Other abnormalities, such as chromosome stickiness, absence of cytokinesis, irregular cytokinesis, abnormal spindle orientation, and abnormal nucleolus disintegration, were found in the three hybrids, while, chromosome disintegration was detected only in HBGC014. All the abnormalities, except for abnormal nucleolus disintegration, can cause unbalanced gamete formation, leading to pollen sterility. Multivalent chromosome association at diakinesis revealed genome affinity between the two parental species in the hybrids, suggesting some possibility for gene introgression. Presently, the Brachiaria breeding program has the objective of releasing, primarily, apomictic hybrids as new cultivars since they do not segregate but preserve the genetic makeup indefinitely. Besides, they result in homogeneous pastures which are easier to manage. The sexual hybrids, however, are paramount in the breeding program: they work as 'bridges' to introgress traits of interest into the apomictic genotypes. The cytogenetic analyses of these three hybrids substantiate their maintenance in the breeding program due to low frequency of meiotic abnormalities, complemented by interesting agronomic traits. They may be used in crosses to generate new cultivars in the future.     

Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae)

Microsporogenesis was evaluated in the Brachiaria humidicola collection of the Embrapa Beef Cattle Center, represented by 60 accessions. One accession (H121) presented an abnormal pattern of cytokinesis that had never been reported in this genus. Among 900 meiocytes analyzed in the first division, 10.7% underwent precocious and multiple cytokinesis in metaphase I, fractionating the genome and the cytoplasm into two or more parts. The expected cytokinesis after telophase I did not occur. The abnormal meiocytes from the first division entered the second division but the second cytokinesis after telophase II was also abnormal. Among the 857 meiocytes analyzed in the second division, 10.9% presented abnormal, incomplete or total absence of cytokinesis. Dyads and binucleated microspores were recorded among the meiotic products. The use of this accession in the Embrapa breeding program is compromised.     

Microsporogenesis in Brachiaria brizantha (Poaceae) as a selection tool for breeding

The genus Brachiaria comprises more than 100 species and is the single most important genus of forage grass in the tropics. Brachiaria brizantha, widely used in Brazilian pastures for beef and dairy production, is native to tropical Africa. As a subsidy to the breeding program underway in Brazil, cytological studies were employed to determine the chromosome number and to evaluate microsporogenesis in 46 accessions of this species available at Embrapa Beef Cattle (Brazil). Thirty-four accessions presented 2n = 36; seven had 2n = 45, and five had 2n = 54 chromosomes. Based on the higher level of chromosome association observed in diakinesis, in tetra-, penta-, and hexavalents, respectively, it was concluded that they are derived from x = 9; consequently, these accessions are tetra- (2n = 4x = 36), penta- (2n = 5x = 45), and hexaploids (2n = 6x = 54). The most common meiotic abnormalities were irregular chromosome segregation due to polyploidy. Chromosome stickiness, abnormal cytokinesis, non-congressed bivalents in metaphase I and chromosomes in metaphase II, and chromosome elimination were recorded at varying frequencies in several accessions. The mean percentage of meiotic abnormalities ranged from 0.36 to 95.76%. All the abnormalities had the potential to affect pollen viability by generating unbalanced gametes. Among the accessions, only the tetraploid ones with less than 40% of abnormalities are suitable as pollen donors in intra- and interspecific crosses. Currently, accessions with a high level of ploidy (5 and 6n) cannot be used as male genitors in crosses because of the lack of sexual female genitors with the same levels of ploidy.     

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae)

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.     

Evidence of programmed cell death during microsporogenesis in an interspecific Brachiaria (Poaceae: Panicoideae: Paniceae) hybrid

Morphological changes have been investigated during plant programmed cell death (PCD) in the last few years due to the new interest in a possible apoptotic-like phenomenon existing in plants. Although PCD has been reported in several tissues and specialized cells in plants, there have been few reports of its occurrence during microsporogenesis. The present study reports a typical process of PCD during meiosis in an interspecific Brachiaria hybrid leading to male sterility. In this hybrid, some inflorescences initiated meiosis but it was arrested in zygotene/pachytene. From this stage, meiocytes underwent a severe alteration in shape showing substantial membrane blebbing; the cytoplasm became denser at the periphery; the cell nucleus entered a progressive stage of chromatin disintegration, and then the nucleolus disintegrated, and the cytoplasm condensed and shrunk. The oldest flowers of the raceme showed only the callose wall in the anthers showing obvious signs of complete sterility.     

Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis

In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.     

Understanding spikelet orientation in Paniceae (Poaceae)

Spikelet structure and grouping are key characters to identify grasses. Here we tested the possibility that spikelet pairs, a distinctive morphological structure of many Andropogoneae and Paniceae, are the starting point for a secondary single spikelet condition that can also explain the change of spikelet orientation among Paniceae genera. As a first approach, we studied the inflorescence development of Paspalum simplex, P. stellatum, and Axonopus sufultus to clarify the origin of the spikelet orientation and other basic homologies. The results support that solitary spikelets of A. suffultus are homologous to the subsessile spikelets of P. simplex and that solitary spikelets of P. stellatum are homologous to the pedicellate spikelet of P. simplex. This last homology supports that spikelet orientation results from a differential reduction/abortion of either the pedicellate or the subsessile spikelet primordia. We also discuss the possibility that the RAMOSA and polar auxin pathways could play a role in the abortion of the lateral subsessile spikelets in P. stellatum. However, the apical meristem inhibition observed in A. suffultus and P. stellatum seems to depend on a very different genetic control, suggesting that the single spikelet condition is homoplasic within Paniceae and derived from at least two different genetic mechanisms.     

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.     

Karyotype analysis in Brachiaria (Poaceae) species

This is the first karyotype characterization of Brachiaria species. Twelve accessions belonging to five species were analysed. The basic chromosome number was x = 9 and 7, the same reported for the tribe Paniceae. Variations in the chromosome number were observed in B. decumbens (2n = 18; 36) and B. humidicola (2n = 36; 42; 54). Chromosome numbers of 2n = 18 in B. ruziziensis and 2n = 36 in B. brizantha and B. jubata were recorded. Inter- and intraspecific karyotype differentiation of the accessions analysed was facilitated by variations in karyotypic symmetry. The karyotypes were generally considered symmetrical, with a tendency to asymmetry in the direction of the polyploids. It is suggested that addition, deletions and mainly polyploidy have been the most direct causes involved in the chromosome evolution of this genus.     

Reproductive biology of Setaria magna Griseb. (Poaceae: Panicoideae: Paniceae)

The genus Setaria is economically important because many species are cultivated for grains or forage. Setaria magna is an American species, native to North America and introduced in South America. The morphological aspect of this species is similar to S. italica (foxtail millet), suggesting its potential value as a crop. The purpose of this work was to understand the breeding system (self-pollination vs. open pollination) of S. magna; additionally, the floral development was described. The results of the breeding system analysis indicated that S. magna is mainly autogamous and does not appear to have a self-incompatibility mechanism. The floral development observed was similar to that described for other Paniceae; in most of the spikelets only the upper anthecium developed fruit, but a small number of spikelets presented bisexual lower florets. These spikelets produced two caryopses per spikelet. Thus, S. magna can produced two types of dispersal units; a few whitish naked caryopses from the lower anthecium that fall without their lemma and palea at maturity; and a high number of brown caryopses protected by the lemma and palea from the upper anthecium. The presence of a normal embryo sac in the upper anthecium suggests that S. magna would present normal sexual reproduction, although we can not reject the formation of apomitic seeds in the lower anthecium.     

Diversification of inflorescence development in the PCK clade (Poaceae: Panicoideae: Paniceae)

In grasses, inflorescence diversification and its correlation with species evolution are intriguing and not well understood. Part of this problem lies in our lack of comprehension about the inflorescence morphological complexity of grasses. We focused our study on the PCK clade (named for phosphoenol pyruvate carboxykinase), a well-supported monophyletic group for which the relationships among its taxa are not well resolved. Interestingly, the PCK clade has an extensive diversity of adult inflorescence forms. A comparative developmental approach can help us to understand the basis of such morphological differences as well as provide characters that can be used in phylogenetic studies of the group. Using SEM studies, we demonstrate that inflorescence morphology in this clade is even more complex than what is typically observed in adult forms. We describe a number of new characters, and some classical features previously used for taxonomic purposes are redefined on the basis of development. We also define four morphological groups combining adult inflorescence form and development, and we discuss some of the evolutionary aspects of inflorescence diversification in the PCK clade. Taxonomic delimitation among genera in the PCK clade remains confusing and unclear where molecular and morphological studies support different classifications.     

Cytological evidence of natural hybridization in Brachiaria brizantha Stapf (Gramineae)

Two accessions of Brachiaria brizantha under cytological analysis showed 2 n  = 5x = 45 chromosomes. Pentaploidy probably resulted from natural hybridization between two species that were not closely related: an apomictic tetraploid male (2 n  = 4x = 36), and a sexual diploid female (2 n  = 2x = 18). The lack of affinity between genomes was clearly indicated by asynchrony during meiosis. The haploid genome ( n  = 9) showed unique behaviour, remaining univalent during prophase I and metaphase I, and undergoing sister-chromatid segregation and lagging at anaphase I. The laggard genome did not always reach the poles in time to be included in the telophase nucleus. However, when the inclusion was effective, this genome was distributed peripherally, changing the otherwise spherical nucleus shape. In the second division, the haploid genome behaved similarly, but as there was sister-chromatid segregation during the first division, the chromatids were slow to reach the poles, forming several micronuclei at telophase II. The two accessions were characterized as allo-autopentaploids, with the tetraploid genome (2 n  = 4x = 36) designated as B (from B. brizantha ) and the haploid genome as X, representing a species with a distinct genome having little affinity with the B genome. Thus, the hybrids' genome composition is represented by BBBBX. By comparing their meiotic behaviour with that observed in synthetic hybrids between B. brizantha and B. ruziziensis analysed previously, B. ruziziensis is the putative diploid sexual parent species in these pentaploid accessions.  © 2006 The Linnean Society of London , Botanical Journal of the Linnean Society , 2006, 150 , 441–446.     

Inflorescence diversity and evolution in the PCK Clade (Poaceae: Panicoideae: Paniceae)

The PCK Clade, represented by six to nine genera, is a monophyletic group situated within the Paniceae tribe. The highly diverse inflorescences within the PCK Clade provide an interesting system for the study of morphological evolution and also may aid in better understanding its unclear systematics. The inflorescence structure of 110 members of the PCK Clade has been investigated. Inflorescences are polytelic showing different levels of truncation. At least 21 different inflorescence subtypes were identified. Fourteen variable inflorescence characters were found, among which some have suprageneric or infrageneric value and others are polymorphic. A key for the identification of inflorescence types is presented. Nine processes have been identified as responsible for inflorescence diversification. Highly branched inflorescences with different internode lengths are present in the basal genus whereas truncated inflorescence morphologies appear late in the history of the clade. The precise timing of morphological changes is impossible to assess until we have a well supported phylogeny for the PCK Clade.     

Histochemical Localization of Citral Accumulation in Lemongrass Leaves (Cymbopogon citratus(DC.) Stapf., Poaceae)

Lemongrasses (Cymbopogonspp., Poaceae) are a group of commerciallyimportant C₄tropical grasses. Their leaves contain up to 1.5%(d.wt) essential oils with a typical lemon-like aroma, consistingmainly of citral (a mixture of the isomeric acyclic monoterpenealdehydes geranial and neral). To specifically locate the sitesof citral accumulation in lemongrass we employed Schiff's reagent,which reacts with aldehydes and gives a purple-red colorationwith citral. Using this technique, single oil-accumulating cellswere detected in the adaxial side of leaf mesophyll, commonlyadjacent to non-photosynthetic tissue, and between vascularbundles. Cell walls of these oil cells are lignified. Our resultssuggest that citral accumulation takes place in individual oilcells within the leaf tissues.Copyright 1998 Annals of BotanyCompany Lemongrass;Cymbopogon citratus; Poaceae; oil cells; histochemistry; citral; aldehydes; Schiff's-reagent.     

Direct evidence of pseudogamy in apomictic Brachiaria brizantha (Poaceae)

Brachiaria brizantha is a forage grass that has several apomictic accessions. B. brizantha cv. Marandu is a natural tetraploid aposporous apomict widely cultivated in Brazil. Pseudogamy was detected in this species by observation that seed set is suppressed in plants that have had the stigmas excised from the flowers. The egg cell develops parthenogenetically in the apomictic plants, meaning that fertilisation is necessary for the formation of the endosperm. A thorough knowledge of all the events of seed formation in natural apomictic plants is essential for a complete understanding of this mode of reproduction. In this paper, we show direct evidence of pseudogamy in B. brizantha through the cytological analysis of polar nucleus fertilisation and the determination of triploid level of the endosperm tissue. The development of the male gametophyte gives rise to a reduced tri-celled pollen, the viability of which varies throughout the year, reaching 88% in the peak of the flowering period. Discharge of the male gamete takes place around 10 h after pollination and monospermy is the predominant system observed. Precocious embryony was also observed in these plants; embryos arise from egg cells. Endosperm development followed the free nucleus model and was associated with the presence of an embryo. Cellularisation and reserve uptake occurred 2 days after pollination (DAP) and mature endosperm was observed 8 DAP. The triploid level of the endosperm in the apomictic accession confirmed the 2:1 maternal:paternal ratio of genome contribution in the tissue.     

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.