Kellen Boldrini
Transcrição
Kellen Boldrini
KELLEN REGINA BOLDRINI Aspectos da microsporogênese em acessos poliplóides de Brachiaria (Poaceae: Paniceae) Dissertação apresentada ao Programa de Pós-Graduação em Ciências Biológicas (área de concentração – Biologia Celular), da Universidade Estadual de Maringá para obtenção do grau de Mestre em Ciências Biológicas. MARINGÁ- PR 2006 2 KELLEN REGINA BOLDRINI Aspectos da microsporogênese em acessos poliplóides de Brachiaria (Poaceae: Paniceae) Orientadora Dra Maria Suely Pagliarini Co-Orientadora Dra Cacilda Borges do Valle MARINGÁ- PR 2006 3 Dados Internacionais de Catalogação-na-Publicação (CIP) (Biblioteca Central - UEM, Maringá – PR., Brasil) B687a Boldrini, Kellen Regina Aspectos da microsporogênese em acessos poliplóides de Brachiaria (Poaceae: Paniceae) / Kellen Regina Boldrini. -- Maringá : [s.n.], 2006. 51 f. : il. Orientador : Prof. Dr. Maria Suely Pagliarini; Coorientadora: Cacilda Borges do Valle Dissertação (mestrado) - Universidade Estadual de Maringá. Programa de Pós-graduação em Ciências Biológicas, 2006. 1. Biologia celular. 2.Citogenética vegetal. 2. Melhoramento genético. 3.Meiose. 4. Poliploidia. 5. Apomixia. 6. Citomixia. 7. Fusão celular. 8. Gameta 2n. 9. Brachiaria humidicola. 10. Brachiaria decumbens. 11. Brachiaria dura. 12. Gramineae. Universidade Estadual de Maringá. Programa de Pósgraduação em Ciências Biológicas. CDD 21.ed. 571.6845 4 Dedico Para meus pais... Meus mentores e heróis. AGRADECIMENTOS A Deus, luz na minha vida. 5 À professora Dra Maria Suely Pagliarini, pela determinação, dedicação, ensinamentos e profissionalismo, a quem admiro muito. À Dra Cacilda Borges do Valle, pesquisadora do Centro Nacional de Pesquisa de Gado de Corte/Embrapa (Campo-Grande- MS), pela co-orientação e concessão do material analisado. À Coordenação e demais docentes do Programa de Pós-Graduação em Ciências Biológicas/UEM, pela oportunidade de realização desse curso. À Capes, pela bolsa de estudo concedida. Às amigas, Andréa, Eleniza e Neide, pelo carinho e presença constante em todos os momentos e, principalmente, pela amizade. A todos os colegas de curso e de laboratório, pela amizade e convivência. Em especial, agradeço à minha família, cuja força e apoio foram determinantes para a realização deste trabalho e pelo carinho demonstrado nas horas mais difíceis. 6 SUMÁRIO Apresentação..................................................................................................... 1 Resumo.............................................................................................................. 2 Abstract………………………………………………………………...…...… 3 Artigos: 1. A new meiotic mechanism for 2n gamete formation in Brachiaria (Poaceae: Paniceae)………………………………………………………..…. 4 2. Cell fusion and cytomixis during microsporogenesis in Brachiaria humidicola (Poaceae)……………………………………………………….... 17 3. Abnormal timing of cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae)…………………………………….………… 30 Apêndice……………………………………………………………..….......... 40 7 APRESENTAÇÃO Esta dissertação é composta por três artigos originados a partir da análise do comportamento meiótico em acessos de três espécies de Brachiaria (B. humidicola, B. decumbens e B. dura), coletados junto à coleção de germoplasma desta gramínea, alocada na Embrapa Gado de Corte (Campo Grande, MS). Os artigos estão apresentados de acordo com as normas estabelecidas pelas revistas a que foram submetidos. 1. Kellen Regina Boldrini, Patrícia Helena Gallo, Pamela Lonardoni Micheletti, Claudicéia Risso-Pascotto, Maria Suely Pagliarini, and Cacilda Borges do Valle. A new meiotic mechanism for 2n gamete formation in Brachiaria (Poaceae: Paniceae). Sexual Plant Reproduction (submetido) 2. Kellen Regina Boldrini, Maria Suely Pagliarini, and Cacilda Borges do Valle. Cell fusion and cytomixis during microsporogenesis in Brachiaria humidicola (Poaceae). South African Journal of Botany (submetido) 3. Kellen Regina Boldrini, Maria Suely Pagliarini, and Cacilda Borges do Valle. Abnormal timing of cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae). Journal of Genetics (aceito) Aspectos da microsporogênese em acessos poliplóides de Brachiaria (Trin.) Griseb. (Poaceae: Paniceae) Kellen Regina Boldrini Maria Suely Pagliarini Cacilda Borges do Valle RESUMO. A coleção de germoplasma de Brachiaria spp da Embrapa Gado de Corte conta com 475 acessos de 15 espécies. Algumas espécies encontram-se sob estudos citológicos a fim de subsidiar o programa de melhoramento desta forrageira. Anormalidades meióticas têm sido descritas entre acessos já analisados. Acessos poliplóides de três espécies de Brachiaria, incluindo B. humidicola (2n = 4x = 36), B. decumbens (2n = 4x = 36) e B. dura (2n = 6x = 54) apresentaram citocinese anormal. O primeiro sinal de citocinese apareceu somente em metáfase II e não dividiu o meiócito 8 em díade. Ausência total de citocinese também foi detectada entre meiócitos durante a segunda divisão. Em ambos os casos, a proximidade entre as duas placas metafásicas facilitou a reunião dos cromossomos após a anáfase II, formando um núcleo de restituição em telófase II. Na maioria dos meiócitos, a segunda citocinese ocorreu após a telófase II. Mônades, díades e tríades com núcleos n ou 2n foram observados entre os produtos meióticos. Entre 28 acessos poliplóides de B. humidicola analisados, fusões celulares foram encontradas em dois acessos e transferência de cromossomos entre meiócitos em um deles. Nesta espécie, anormalidades meióticas e pós-meióticas relacionadas à citocinese também foram encontradas em um acesso. A primeira citocinese ocorreu após a telófase II, enquanto a segunda citocinese ocorreu por invaginação em micrósporos binucleados após a dissolução da parede de calose. Esta citocinese tardia não afetou a viabilidade do pólen uma vez que o produto meiótico, embora formado tardiamente, foi caracterizado por quatro micrósporos haplóides. A meiose é controlada por um grande número de genes, geralmente dominantes, que são estágio, local e tempo-específicos. As anormalidades meióticas causadas por mutações afetam a viabilidade do pólen e comprometem a produção de sementes. No gênero Brachiaria, acessos poliplóides são, em geral, apomíticos, embora pseudógamos. Conseqüentemente, pólen fértil é essencial para fertilizar o núcleo central do saco embrionário e garantir a produção de sementes viáveis. Assim, acessos com alta freqüência de anormalidades meióticas devem ser eliminados dos programas de melhoramento. Aspects of microsporogenesis in polyploid accessions of Brachiaria (Trin.) Griseb. (Poaceae: Paniceae) Kellen Regina Boldrini Maria Suely Pagliarini Cacilda Borges do Valle ABSTRACT. The Brazilian Brachiaria spp collection at Embrapa Beef Cattle encompasses 475 accessions of 15 species. Some species are under detailed analysis of microsporogenesis. Meiotic abnormalities have been recorded among accessions. Polyploid accessions of three Brachiaria species, including B. humidicola (2n = 4x = 36), B. decumbens (2n = 4x = 36), and B. dura (2n = 6x = 54) presented abnormal cytokinesis. The first sign of cytokinesis appeared only in metaphase II and it did not 9 divid the meiocyte into a dyad. Total absence of cytokinesis was also detected among meiocytes in the second division. In both cases, the two metaphase plates were very close facilitating the rejoining of chromosome sets after anaphase II, forming a restitutional nucleus in telophase II. In the majority of meiocytes, the second cytokinesis occurred after telophase II. Monads, dyads, and triads with n or 2n nuclei were recorded among meiotic products. Among 28 polyploid accessions of B. humidicola analyzed, cell fusions were recorded in two accessions and chromosome transfer among meiocytes in one of them. In this species, meiotic and post-meiotic abnormalities related to cytokinesis were observed in one accession. The first cytokinesis occurred after telophase II, and the second cytokinesis occured by invagination in binucleated microspores only after callose wall dissolution. These late cytokinesis did not affect pollen viability since the meiotic product, although formed late, was characterized by four reduced normal microspores. Meiosis is controlled by a large number of genes, generally dominant, which are stage, site- and time-specific. Meiotic abnormalities caused by mutations compromise pollen viability and impair seed production. In the Brachiaria genus, polyploid accessions are, in general, apomictic, albeit pseudogamous. Consequently, fertile pollen is essential to fertilize the central nucleus of the embryo-sac and ensure viable seed production. Thus, accessions with high frequencies of meiotic abnormalities should be eliminated early from the breeding program. 10 A new meiotic mechanism for 2n gamete formation in Brachiaria (Poaceae: Paniceae) A new meiotic mechanism for 2n gamete formation in Brachiaria (Poaceae: Paniceae) Kellen Regina Boldrini1,Patrícia Helena Gallo1, Pamela Lonardoni Micheletti1, Claudicéia Risso-Pascotto1, Maria Suely Pagliarini1, and Cacilda Borges do Valle2. 1. Department of Cell Biology and Genetics, State University of Maringá, 87020-900 Maringá PR Brazil. 2. Embrapa Beef Cattle, P.O. Box 154, 79002-970 Campo Grande MS Brazil. * Author for correspondence: Maria Suely Pagliarini (E-mail: [email protected]). Abstract. Microsporogenesis of several Brachiaria species of the Brazilian collection at Embrapa Beef Cattle was analysed in detail. Accessions of three species (B. humidicola, 11 2n=4x=36, B. decumbens, 2n=4x=36, and B. dura, 2n=6x=54) presented abnormal cytokinesis. Chromosomes paired in bi-, tri-, and quadrivalents in these accessions, whereas chromosome segregation at meiosis I was characterized by exclusion of laggards as micronuclei. In a high number of meiocytes, the first sign of cytokinesis appeared only in metaphase II and did not divide the meiocyte into a dyad. Total absence of cytokinesis was also detected among meiocytes in the second division. Since in both cases the two metaphase plates were very close, they favored the rejoining of chromosome sets after anaphase II and formed a restitutional nucleus in telophase II. Second cytokinesis occurred after telophase II in most meiocytes. Monads, dyads, and triads with n or 2n nuclei were observed among meiotic products. The 2n gametes observed correspond to the first division restitution (FDR). The number of affected cells in each accession was variable, but the number of microspores with restitutional nucleus, including those scored in tetrads and the released ones, did not exceed 9%. Although polyploidy is common in the genus Brachiaria, its origin is not well known. Current results suggest that 2n gametes may have contributed to the evolutionary history of the genus. Key words: abnormal cytokinesis, Brachiaria, 2n gamete, microsporogenesis, restitutional nucleus, polyploidization. Introduction Polyploidy is a key element in the evolution of higher plants and leads toward the formation of new species. Most flowering species have evolved through one or more rounds of polyploidization, either by the doubling of their chromosome number (autopolyploidy) or by combining chromosome sets from distinct related species (allopolyploidy). Although this behavior suggests that polyploidy must confer some selective advantage (Osborn, 2004), the evolutionary success of polyploidy relies on the ability of polyploid individuals to reproduce and transmit their genes to subsequent generations (Pannell et al., 2004). For nearly 90 years since the discovery of polyploidy, it has not been known what percentage of the angiosperm is polyploid. Estimates of 12 polyploidy among angiosperms range from a liberal 70 – 80% to a conservative 30% (Bennett, 2004). Polyploidization may be asexual through somatic chromosome doubling and sexual through the formation of 2n gametes. The first assumption was seriously questioned by Harlan and De Wet (1975) since it has been shown that 2n gametes are widespread in plants (Harlan and De Wet, 1975; Veilleux, 1985; Bretagnolle and Thompson, 1995). Actually, 2n gametes are considered to be the dominant process involved in the origin of polyploidy in plants. 2n gametes result by modified meiosis affecting specific stages of micro- and megasporogenesis, which lead to the formation of restitutional nucleus. Several mechanisms involved in meiotic nuclear restitution have been reported in several plant species (see Ramanna, 1979; Veilleux, 1985; Bretagnolle and Thompson, 1995). During an extensive study on cytogenetic behavior in several Brachiaria species from a Brazilian collection at Embrapa Beef Cattle (Campo Grande, MS), a new meiotic mechanism for 2n gamete formation was recorded in three different species (B. humidicola, B. decumbens, and B. dura). This mechanism, leading to first division restitution (FDR), is currently discussed. Materials and Methods Accessions of several Brachiaria species from the Embrapa Beef Cattle collection (Campo Grande MS Brazil), comprising B. humidicola (23 accessions), B. decumbens (15 accessions), and B. dura (2 accessions), were cytologically analyzed. All accessions were previously collected in the wild African savannas in the mid-1980s by the Centro Nacional de Agricultura Tropical (CIAT, Colombia) and introduced in Brazil between 13 1986 and 1990. Site’s characteristics in Brazil are: climate type Aw: tropical humid savanna; average annual precipitation = 1526 mm; average temperature = 22°C; altitude 520 m; latitude = 20° 28’ S; longitude = 55° 40’ W; poor Dark Red Latossol (59% sand; 8% silt; 33% clay; pH = 4.2). Inflorescences from several plants representing each accession were collected for meiotic study and fixed in a mixture of ethanol 95%, chloroform and propionic acid (6:3:2) for 24 hours, transferred to 70% alcohol and stored under refrigeration until use. Microsporocytes were prepared by squashing and staining with 0.5% propionic carmine. Photomicrographs were made with a Wild Leitz microscope using Kodak Imagelink – HQ, ISO 25 black and white film. Results and Discussion The Brazilian Brachiaria collection comprises of 475 accessions of 15 species, of which nearly half has been cytologically analyzed. Among these, three of them belonging to distinct species, B. humidicola (H047, 2n=4x=36), B. decumbens (D076, 2n=4x=36) and B. dura (Du001, 2n=6x=54), revealed total absence or an abnormal type of cytokinesis following the first or the second meiotic division. As all accessions were polyploid, characteristica polyploidy abnormalities related to irregular chromosome segregation were also found among meiocytes. However, meiosis proceeded normally in its course till telophase I (Fig. 1 a to c). Brachiaria is a genus of African origin widely used as forage grass in the humid and sub-humid tropical regions, especially in Brazil. As a monocotyledonous species, it shows successive cytokinesis, or rather, the first cytokinesis occurs after telophase I, dividing the cell into a dyad, followed by the second cytokinesis after telophase II, 14 giving rise to microspore tetrads. In the accessions under study, a large number of meiocytes (Table 1) displayed the first signal of cytokinesis in metaphase II (Fig. 1 d), albeit partial, and, it did not divide the meiocyte into a dyad. During prophase II, cytokinesis was not evident in most cells. In metaphase II, anaphase II, and telophase II, the number of meiocytes with partial cytokinesis was much higher than those lacking it. In both cases, the two metaphase plates were very close (Fig. 1 d) favoring the rejoining of chromosome sets after anaphase II (Fig. 1 e, f). Consequently, a restitutional nucleus in telophase II was formed (fig. 1 g, h). The above phenomenon was prevalent and restitutional nucleus formation was increased by moderate convergence of spindles, i.e., tripolar spindles (Fig. 1 e). Depending on its position, the restitutional nucleus occupied either one cell (Fig. 1 p) or shared the cytoplasm with another n nucleus (Fig. 1 h). After telophase II, the start of the second cytokinesis occurred although the first cytokinesis was either not yet complete (Fig. 1 h and j) or totally absent (Fig. 1 k, l). As a consequence, the number of tetrads with incomplete cytokinesis was high, and monads, dyads, and triads with n or 2n nuclei were recorded among meiotic products. Abnormal microspores with different combinations of n and 2n nuclei were observed (Fig. 1 m to p). Table 1. Frequency of meiotic abnormalities in the three accessions analyzed. Phase Abnormalities Accession D076 H047 Analyzed cells Prophase II Total absence of cytokinesis Partial cytokinesis 366 No. of cells affected (%) 227 (62.0) 36 (9,8) Analyzed cells 150 No. of cells affected (%) 85 (56.7) 0 Du001 Analyzed cells 146 No. of cells affected (%) 1 (0.7) 80 (54.8) 15 Metaphase II Total absence of cytokinesis Partial cytokinesis 721 22 (3.1) 83 (11.5) 199 21 (10.6) 23 (11.6) 244 6 (2.5) 134 (54.9) Anaphase II Total absence of cytokinesis Partial cytokinesis Tripolar spindle 177 2 (1.1) 87 (49.2) 21 (11.9) 140 11 (7.9) 34 (24.3) 22 (15.7) 147 3 (2.0) 69 (46.9) 0 Telophase II Total absence of cytokinesis Partial cytokinesis Restitutional nucleus 564 19 (3.4) 210 (37.2) 43 (7.6) 215 13 (6.1) 35 (16.3) 36 (16.7) 206 3 (1.46) 124 (60.2) 12 (5.8) Tetrad Partial cytokinesis Monad trinucleated Dyad binucleated Triad Restitutional nucleus in dyad or triad 519 132 (25.4) 15 (2.9) 13 (2.5) 86 (6.6) 45 (8.7) 1235 146 (11.8) 23 (1.9) 81 (6.6) 0 0 166 24 (14.5) 0 0 4 (2.4) 7 (4.2) Microspores Trinuclead Restitutional nucleus Binucleated Tetranucleated 676 22 (3.3) 3 (0.4) 104 (15.4) 0 620 14 (2.3) 13 (2.1) 60 (9.7) 7 (1.3 ) 539 0 7 (1.3) 60 (11.1) 0 16 Figure 1. Aspects of abnormal cytokinesis and restitutional nucleus formation in Brachiaria. a to c) Regular meiosis I: metaphase I (a), anaphase I (b), and telophase I (c). d) Metaphase II with incomplete cytokinesis. The proximity of the two metaphase plates may be observed. e) Anaphase II with convergent spindles in a cell with total absence of cytokinesis. Chromosomes rejoining in one pole is shown. f) Early telophase II with a restitutional nucleus. g) Telophase II with the formation of a restitutional nucleus. h) Telophase II with the restitutional nucleus sharing the cytoplasm with one n nucleus. i, j) Different aspects of telophase II in relation to cytokinesis. k, l) Telophase II with total absence of cytokinesis. m, n) Triads with one restitutional nucleus in one microspore. o) Tetranucleated monad and two normal n microspores. p) Binucleated microspore and a normal one. (Magnification 400x) 17 Similar mechanism was reported in Hierochloë odorata, a rhizomatous perennial grass (Ferris et al., 1992). The number of affected cells in each accession was variable, but the number of microspores with restitutional nuclei, including those scored in tetrads and the released ones, did not exceed 9%. The trend to form 2n gametes in plants is highly variablea and it varies among individuals within a single taxonomic group or even among flowers of an individual plant (Bretagnolle and Thompson, 1995). Meiotic nuclear restitution may be caused by different mechanisms, including semiheterotypic division, pseudohomotypic division, mitotised meiosis, first division restitution (FDR), second division restitution (SDR), premature cytokinensis 1 and 2, and pre- and post-meiotic doubling of chromosomes (Ramanna, 1979; Veilleux, 1985, Bretagnolle and Thompson, 1995). Most mechanisms have been described in dicotyledons with scanty studies and revealed 2n gamete formation in monocots (Pagliarini et al., 1999; Lim et al., 2001; Barba-Gonzalez et al., 2004). In monocots it is very important to determine how events of chromosomal division and cytokinesis occur to yield restitutional nucleus. Although 2n gametes may result from several different meiotic abnormalities, two types of 2n gametes are the product of one out of two basic processes and depend on the mode of nuclear restitution: FDR and SDR. The 2n gametes in the present accessions resulted from FDR. In spite of correct chromosome pairing and homologous segregation at anaphase I, the homologous chromosomes were rejoined in the second division. Heterozygosis level in 2n gametes is influenced by the timing of nuclear restitution. Although FDR generally conserves heterozygosity of proximal segments, it reduces that of distal segments by half (Veilleux, 1985; Bretagnolle and Thompson, 1995). According to Lim et al. (2001), an important feature of restitutional meiosis with successive cytokinesis, such as that observed in the present Brachiaria accessions, is the manner in wich chromosomal division and cytokinesis 18 events occur. Cytological observations in these accessions clearly indicated that meiosis had been modified: chromosomes paired normally in prophase I, formed bi-, tri-, and quadrivalents and then segregated simultaneously in anaphase I, in spite of certain abnormal segregation due to their polyploid condition. As genetic recombination occurred, FDR nucleus gave rise to 2n gametes with recombinant chromosomes. Recombination is one of the most important events for introgression. Most of the evidence for in using 2n gametes in breeding programs has been focused on their use in autopolyploids or their polysomic polyploids (Mariani and Tavoletti, 1992; Carputo et al., 2000). In these cases, the importance of FDR gametes lies in the transference of heterosis and intact parental gene combinations to sexual polyploids. The 2n gametes allow breeders to broaden the genetic bases of cultivated species. Although the genetic determination of 2n pollen production has been studied in detail, the genetic control of 2n egg formation is still poorly understood. Bretagnolle and Thompson (1995) presented an extensive list of genes responsible for this feature in several plant species, in which monogenic recessive status of mutant alleles is largely predominant. Although the genetic base of abnormal cytokinesis found in these accessions is not known, the fact that accessions of three different Brachiaria species have precisely the same abnormality, has led the authors to hypothesize that the characteristic is genetically controlled. 2n gametes resulting from total absence either of the first or the second cytokinesis have been reported in B. brizantha (Risso-Pascotto et al., 2003), but in much lower frequency. Some exhaustive reviews have shown the influence of seasonal and environmental factors, such as high and low temperature, on 2n gamete production (see Bretagnolle and Thompson, 1995). In the genus Brachiaria, polyploidy is of common occurrence (Valle and Savidan, 1996). Studies performed by flow cytometry on 435 accessions, belonging to 19 13 species, revealed that only 13% are diploids. The ploidy level ranged from 2n = 4x to 2n = 7x, with predominance (58.13%) of tetraploidy (Penteado et al., 2000). The origin of polyploidy in the genus is not well known. Taking into account the meiotic pairing and the meiotic behavior of several accessions of different Brachiaria species studied, there is evidence of autopolyploidy and segmental allopolyploidy (Mendes-Bonato et al., 2002, 2006; Utsunomiya et al., 2005), and even of true allopolyploidy (Mendes et al., 2006). At any rate, 2n gametes may have contributed to the evolutionary history of the genus. In fact, recent studies of natural polyploidy complexes have shown that the production of 2n gametes may have played a role in the creation of new polyploids by unilateral or bilateral sexual polyploidization (see Bretagnolle and Thompson, 1995). The forage potential of Brachiaria species was acknowledged in Brazil about 40 years ago. However, the importance of the genus was felt only in the past three decades when two to three Brachiaria cultivars, were extensively sown in tropical America (Miles et al., 1996, 2004). Brachiaria decumbens cv. Basilisk (D) and B. brizantha cv. Marandu (B) undoubtedly make up the most extensively planted cultivars, covering over 50 million hectares of poor and acid soils of central Brazil and Latin America. To increase genetic variability in the genus, a dynamic breeding program based on intraand interspecies hybridization is underway at the Embrapa Beef Cattle Center since 1988. Hybridization in the genus Brachiaria is rather difficult mainly owing to ploidy differences among accessions and species, and to reproduction by apomixis (Valle and Savidan, 1996). Most accessions in promising species are tetraploid and apomictic. Thus, several tetraploid interspecific hybrids involving different accessions of (D) and (B) were synthesized by the use of artificially tetraploidized sexual accessions of B. ruziziensis as female genitors. The success of such program depends heavily on the high pollen fertility of the apomictic accessions used as the male parent. These crosses 20 obviously involve 2n gametes generated by regular chromosome segregation of both tetraploid genitors. Since 2n gametes from restitutional nucleus have never been explored in the genus for breeding purposes, current results open new perspectives, possibilities and further exploration for Brachiaria breeding. References Barba-Gonzalez R, Lokker AC, Lim KB, Ramanna MS, Tuyl JM (2004) Use of 2n gametes for the production of sexual polyploids from sterile Oriental x Asiatic hybrids of lilies (Lilium). Theor Appl Genet 109:1125-1132 Bennett MD (2004) Perspectives on polyploidy in plants – ancient and neo. Biol J Linn Soc 82:411–423 Bretagnolle F, Thompson JD (1995) Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol 129:1-22 Carputo D, Barone A, Frusciante L (2000) 2n gametes in the potato: essential ingredients for breeding and germplasm transfer. 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Euphytica 108:129-135 Pannell JR, Obbard DJ, Buggs RJA (2004) Polyploidy and the sexual system: what can we learn from Mercurialis annua? Biol J Linn Soc 82:547-560 Penteado MIO, Santos ACM, Rodrigues IF, Valle CB, Seixas MAC, Esteves A (2000) Determinação de poliploidia e avaliação da quantidade de DNA total em diferentes espécies de gênero Brachiaria. Boletim de Pesquisa, 11. Campo Grande-MS, Embrapa Gado de Corte. 19p. Ramanna MS (1979) A re-examination of the mechanisms of 2n gametes formation in potato and its implications for breeding. Euphytica 28:537-561 22 Risso-Pascotto C., Pagliarini MS, Valle CB, Mendes-Bonato AB (2003) Chromosome number and microsporogenesis in pentaploid accession of Brachiaria brizantha (Gramineae). Plant Breed 122:136-140 Utsunomiya KS., Pagliarini MS, Valle CB (2005) Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae). Biocell, (in press). Valle CB, Savidan YH (1996) Genetics, cytogenetics, and reproductive biology of Brachiaria. In: Miles JW, Maass BL, Valle CB (eds) Brachiaria: Biology, Agronomy, and Improvement. Colombia: CIAT, pp 147-163 Veilleux R (1985) Diploid and polyploid gametes in crop plants: mechanisms of formation and utilization in plant breeding. Plant Breed Rev 3:253-288 23 Cell fusion and cytomixis during microsporogenesis in Brachiaria humidicola (Poaceae) 24 Cell fusion and cytomixis during microsporogenesis in Brachiaria humidicola (Poaceae) Kellen Regina Boldrini1, Maria Suely Pagliarini1, and Cacilda Borges do Valle2 1 Department of Cell Biology and Genetics, State University of Maringá, 87020-900 Maringá PR, Brazil; 2 Embrapa Beef Cattle, P.O. Box 154, 79002-970 Campo Grande MS, Brazil. ABSTRACT During cytological analysis for breeding purposes of microsporogenesis in 28 polyploid accessions of Brachiaria humidicola (Poaceae) from the Embrapa Beef Cattle germplasm collection assembled, cell fusions were recorded in two accessions and chromosome transfer among meiocytes in one of them. Cell fusion between two to more than ten cells was recorded from prophase I to telophase II. In the syncyte, each nucleus maintained its integrity. In one of these accessions, cytomixis with characteristics never reported in any other plant species was recorded. It occurred among very small meiocytes that transferred the entire genome or part of it to normal meiocytes. Chromosome transfer occurred preferentially during telophase I and, during migration, chromatin showed structural alteration. Both abnormalities compromise pollen fertility. In the Brachiaria genus, polyploid accessions are, in general, apomictic, albeit pseudogamous. Consequently, fertile pollen is essential to fertilize the central nucleus of the embryo-sac and ensure viable seed production. Thus, accessions with high frequencies of meiotic abnormalities should be eliminated early from the breeding program. Key words: Brachiaria microsporogenesis. humidicola, cell fusion, cytomixis, forage grass, 25 INTRODUCTION Meiosis is a continuous process involving several cytological events that result in the reduction of chromosome number by half, thus ensuring the constancy of ploidy in the species after fertilization. There is ample evidence that meiosis is controlled by a large number of genes (Gottschalk and Kaul, 1974; Baker et al., 1976; Golubovskaya, 1979, 1989). Disruption in any step of meiosis, due to environmental or genetic factors can affect gametic fertility. Depending on the severity of the abnormality, total sterility can be expected. Cytological analyses performed on several Brachiaria species of the Embrapa Beef Cattle collection revealed a large amount of different meiotic (Mendes-Bonato et al., 2001 a, b; 2002 a, b, 2003; Risso-Pascotto et al., 2002, 2003 a; Utsunomiya et al., 2004, 2005) and post-meiotic abnormalities (Junqueira Filho et al., 2003; MendesBonato et al.; 2004, Risso-Pascotto et al., 2005 a) which compromise pollen viability. In spite of the prevalent asexual reproduction by apospoy of the Panicum type (Valle and Savidan, 1996; Araújo et al., 2000), these polyploid accessions in the genus Brachiaria are pseudogamous, i.e. fertile pollen grains are necessary to fertilize the two secondary nuclei of the embryo sac to ensure endosperm development (Alves et al., 2001). B. humidicola is a species natural to Africa and widely used for pastures in the tropics, especially under poorly drained conditions, where its is about the only option available for pasture establishment. New cultivars are urgently needed to minimize the risk of extensive contiguous areas being planted to the only apomictic cultivar available commercially. New varieties are being sought to explore either the natural genetic variability among accessions or to generate novel genetic variability by intraspecific hybridization, since tetraploid sexual accessions have been identified (Valle and 26 Glienke, 1991; Valle and Savidan, 1996). A new cultivar, cv Tupi, is scheduled to be released in Brazil in 2007 and was selected from the germplasm collection. There is an increase in interest in this species by breeders and producers thus justifying the effort to analyze the microsporogenesis of all the accessions of this species and related ones in the Embrapa Beef Cattle collection. During cytological characterization of 28 accessions of B. humidicola, two showed cell fusion and one of these also cytomixis among meiocytes. These abnormalities are reported here. MATERIAL AND METHODS Twenty eight of about 60 accessions of Brachiaria humidicola (Rendle) Schweick from the Embrapa Beef Cattle germplasm collection collected in wild East African savannas in the 1980s were analyzed cytologically. Site characteristics of accessions cultivated at the Embrapa Beef Cattle Research Center at Campo Grande, Mato Grosso do Sul, Brazil were: climate type Aw: tropical humid savanna; average annual precipitation = 1526 mm; average temperature = 22°C; altitude 520 m; latitude = 20° 28’ S; longitude = 55° 40’ W; poor Dark Red Latossol soil composed of 59% sand; 8% silt and 33% clay; pH = 4.2). Inflorescences of each accessions were collected for the meiotic study in plots to represent each accession and fixed in a mixture of 95% ethanol, chloroform and propionic acid (6:3:2) for 24 hours, transferred to 70% alcohol and stored under refrigeration until use. Microsporocytes were prepared by squashing and staining with 0.5% propionic carmine. Photomicrographs were taken in a Wild Leitz microscope using Kodak Imagelink – HQ, ISO 25 black and white film. 27 RESULTS AND DISCUSSION In addition to the expected meiotic abnormalities typical of polyploidy and affecting pollen viability, such as irregular chromosome segregation in the first and the second meiotic divisions, cell fusions were recorded in two accessions (H012, BRA004979 and H003, BRA004812) and chromosome transfer among meiocytes in one of them (H003, BRA004812). Cytological details during microsporogenesis classifies H012 as a hexaploid (2n = 6x = 54), with a basic chromosome number x = 9 and suggests that H003 is a heptaploid (2n = 7x = 42), with a basic chromosome number x = 6. Recent cytological analyses in Brachiaria revealed that the majority of species are polyploid (Penteado et al., 2000), derived from the predominant basic chromosome number in the genus x = 9, followed by x = 7 (Mendes-Bonato et al., 2002 a; Utsunomiya et al., 2005). A new basic chromosome number x = 6 has recently been described in B. dictyoneura (Risso-Pascotto et al., 2006). Table 1 shows the frequency of cells involved in these abnormalities. Cell fusion was detected among some meiocytes in several anthers. The fusions involved from 2 to more than 10 cells (Fig. 1). The majority of fused cells occurred in prophase I (Fig. 1 a, b), but fusions were recorded until telophase I (Fig. 1 e) giving rise to abnormal meiotic products (Figs. 1 f). There was no nuclear fusion in the syncytes. Each genome maintained its integrity. Another interesting aspect observed in H003 was the difference in size of fused cells. Very small cells with an apparently normal genome were found fused with normal cells (Fig. 1 c). Cell fusion had been reported in some Brachiaria species. In some B. brizantha genotypes, this phenomenon was restricted to male flowers of the raceme (Mendes-Bonato et al., 2001 c); however, in accessions of other 28 species they occurred in the hermaphrodite flowers (Mendes-Bonato et al., 2001 a; Risso-Pascotto et al., 2003a; Utsunomyia et al., 2005). In 5 of 22 accessions of B. jubata, cell fusion was found to occur among two cells and, after normal cytokinesis, it produced eight normal microspores (Mendes-Bonato et al., 2003). Cell fusion was also reported in several other plant species (Nirmala and Rao, 1996), and may result from suppression of cell wall formation during premeiotic mitoses. In general, cell fusion leads to abnormal formation of pollen grains. In the present accessions that was also the case. According to Nirmala and Rao (1996), several factors may cause cell fusion such as exposure to chemicals, temperature, culture conditions, and genetic factors. Considering that the present accessions of B. humidicola were cultivated under similar environmental conditions, the results suggest genetic control of cell fusion. Chromosome transfer among meiocytes of H003 was recorded in low frequencies, but showing a pattern never reported before in other plant species. The transfer of chromosomes always occurred between cells of different sizes. Normal microsporocytes received the entire genome, or part of it, from very small cells through large inter-cytomictic channels (Fig. 1 g, h). In general, the genomes of the small cells involved showed chromosome stickiness. A similar process of structural alteration of migrating chromatin was also recorded in B. nigropedata (Utsunomiya et al., 2004). According to Feijó and Pais (1989) such agglutination eases the passage for migrating chromatin. Hyperploid cells involved in cytomixis were also observed in our accession (Fig. 1 h). The result of cytomixis was the increase of the genome in the cells (Fig. 1 i). The phenomenon was found to occur from metaphase I to microspore stage, but most frequently at telophase I. A normal meiocyte could receive chromosomes from two cells in different meiotic stages or one small cell could transfer part of its genome to two 29 normal cells. The origin of the small cells involved in cytomixis is unclear. They were never observed alone in the cytoplasm. Table 1. Frequency of meiocytes affected by cell fusion and cytomixis. Phase No. of cells analyzed Cell fusion Cytomixis No. of affected cells No. of affected cells H003 H012 H003 H012 H003 Zygotene 200 541 100 - - Pachytene 200 322 14 54 - Diplotene 200 122 6 1 - Diakinesis 200 323 46 7 - Metaphase I 200 221 - 17 27 Anaphase I 200 144 2 6 5 Telophase I 200 141 14 6 44 Prophase II 200 172 - 14 - Metaphase II 200 176 - - - Anaphase II 200 149 - - 3 Telophase II 200 166 2 - - Microspores 200 302 5 - 21 30 Figure 1. Aspects of cell fusion and chromosome transfer among meiocytes in B. humidicola H003. a) Fusion of two cells in pachytene. b) Fusion of three cells in diakinesis. c) Fusion of a normal cell and two small cells. d) Fusion of three cells in anaphase I. e) Fusion between two cells in telophase I. f) Abnormal meiotic products resulting from cell fusions. g, h) Chromosome transfer among meiocytes in telophase I (g) and metaphase (h). Observe that in g the normal cell is receiving chromosomes from the smallest cells, and that the receptive cell is hyperploid. i) Telophase II with an extra nucleus resulting from cytomixis. (Scale bar = 1 µm) 31 Cytomixis is commonly reported in meiocytes especially during prophase I, when cytoplasmic channels exist among cells. Heslop-Harrison (1966a, b) demonstrated that cytoplasmic channels initiated in the preleptotene stage, persisted throughout the meiotic prophase and disappeared before meiosis II, when each meiocyte became totally isolated within the enclosing callose wall. Cytomixis has been reported to occur preferentially between genetically unbalanced types such as polyploids, hybrids, and apomictics (Gottschalk 1970; Bahl and Tyagl, 1988). Perhaps the polyploid and the apomictic condition of the accession H003 (Valle, unpublished data) predispose it to chromosome transfer among meiocytes. However, we cannot exclude the possibility of some genetic factor interfering with this phenomenon, because among the 25 accessions of B. humidicola analyzed, only this one was affected. Despite the number of species in which cytomixis has been reported, its origin and significance are still unknown. Its role in the evolutionary process is contradictory, because it results in the formation of hyperploid and hypoploid cells, compromising pollen fertility. The influence of cytomixis on the generation of polyploid gametes can be expected in Brachiaria, a genus where polyploidy is predominant (Valle and Savidan, 1996; Penteado et al., 2000). However, when only a part of the genome is transferred, unbalanced and sterile gametes are formed. In the present accessions not only cell fusion and cytomixis contributed to pollen sterility, but also many other meiotic abnormalities typical of the polyploid condition were recorded. Were it not for apomixis, in wich meiosis is by passed in megagametogenesis, and this genotype evoult probably not be preserved. The Brachiaria breeding program depends on hybridization to produce novel genetic variability using sexual genotypes and the pollen of selected apomictic accessions or hybrids. The hybrids that are produced are then selected, among other 32 traits, for good seed production in order for this technology to be widely adopted. Therefore accessions with high frequencies of meiotic abnormalities such as the ones observed for H003 and H012 present serious problems and should be eliminated early from the breeding program, thus enhancing breeding efficiency and success. References Alves ER, Carneiro VTC, Araújo ACG (2001) Direct evidence of pseudogamy in apomictic Brachiaria brizantha (Poaceae). Sexual Plant Reproduction, 14: 207212. Araújo ACG, Mukhambetzhanov S, Pozzobon MT, Santana EF, Carneiro VTC (2000) Female gametophyte development in apomictic and sexual Brachiaria brizantha (Poaceae). Revue de Cytologie et Biologie Végétales – Le Botaniste, 23: 13-26. Bahl JR, Tyagl BR (1988) Cytomixis in pollen mother cells of Papaver dubium L. Cytologia, 53: 771-775. Baker BS, Carpenter ATC, Esposito MS, Esposito RE, Sandler L (1976) The genetic control of meiosis. Annual Review of Genetics 10: 53-134. Feijó JA, Pais MSS (1989) Cytomixis in meiosis during the microsporogenesis in Ophrys lutea: an ultrastructural study. Caryologia 42: 37-48. Golubovskaya IN (1979) Genetic control of meiosis. International Review of Cytology 58: 247-290. Golubovskaya IN (1989). Meiosis in maize: mei genes and conception of genetic control of meiosis. Advances in Genetics 26: 149-192. Gottschalk W (1970) Chromosome and nucleus migration during microsporogenesis of Pisum sativum. The Nucleus 13: 1-9. 33 Gottschalk W, Kaul MLH (1974) The genetic control of microsporogenesis in higher plants. The Nucleus 17: 133-166. Heslop-Harrison J. (1966 a) Cytoplasmic connections between angiosperm meiocytes. Annals of Botany 30: 221-230. Heslop-Harrison J. (1966 b) Cytoplasmic continuities during spore formation of flowering plants. Endeavour 25: 67-72. Junqueira Filho R G, Mendes-Bonato AB, Pagliarini MS, Bione NCP, Valle CB, Penteado MI.O (2003) Absence of microspore polarity, symmetric divisions and pollen cell fate in Brachiaria decumbens (Gramineae). Genome 46: 83-88. Mendes-Bonato AB, Pagliarini MS, Silva N, Valle CB (2001 a) Meiotic instability in invader plants of signal grass Brachiaria decumbens Stapf (Gramineae). Acta Scientiarum 23: 619-625. Mendes-Bonato AB, Pagliarini MS, Valle CB, Penteado MIO (2001 b) A severe case of chromosome stickiness in pollen mother cells of Brachiaria brizantha (Hochst) Staph (Gramineae). Cytologia 66: 287-291 Mendes-Bonato AB, Pagliarini MS, Valle CB, Penteado MIO (2001 c) Archesporial syncytes restricted to male flowers in a hexaploid accession of Brachiaria brizantha (Hochst) Stapf (Gramineae). The Nucleus 44: 137-140. Mendes-Bonato AB, Pagliarini MS, Forli F, Valle CB, Penteado MIO (2002 a) Chromosome number and microsporogenesis in Brachiaria brizantha (Gramineae). Euphytica 125: 419-425. Mendes-Bonato AB, Junqueira Filho RG, Pagliarini MS, Valle CB, Penteado MIO (2002 b) Unusual cytological patterns of microsporogenesis in Brachiaria decumbens: abnormalities in spindle and defective cytokinesis causing precocious cellularization. Cell Biology International 26: 641-646. 34 Mendes-Bonato A.B., Pagliarini MS, Risso-Pascotto C, Valle CB (2004) Abnormal pollen mitoses (PM I and PM II) in an interspecific hybrid from Brachiaria ruziziensis x Brachiaria decumbens (Gramineae). Journal of Genetics 83: 279-283. Mendes-Bonato AB, Risso-Pascotto C, Pagliarini MS, Valle CB (2003) Normal microspore production after cell fusion in Brachiaria jubata (Gramineae). Genetics and Molecular Biology, 26: 517-520. Nirmala A, Rao PN (1996) Genesis of chromosome numerical mosaicism in higher plants. The Nucleus 39: 151-175. Penteado MIO, Santos ACM, Rodrigues IF, Valle CB, Seixas MAC, Esteves A. (2000) Determinação de poliploidia e avaliação da quantidade de DNA total em diferentes espécies de gênero Brachiaria. Boletim de Pesquisa, 11. Campo Grande-MS, Embrapa Gado de Corte. 19p. Risso-Pascotto C., Pagliarini MS, Valle CB (2002) Abnormal nucleolar cycle in microsporogenesis of Brachiaria decumbens (Gramineae). Cytologia 67: 355-360. Risso-Pascotto C., Pagliarini MS, Valle CB, Mendes-Bonato AB (2003 a) Chromosome number and microsporogenesis in a pentaploid accession of Brachiaria brizantha (Gramineae). Plant Breeding 122:136-140. Risso-Pascotto C, Pagliarini MS, Valle CB (2003 b) A mutation in the spindle checkpoint arresting meiosis in Brachiaria ruziziensis. Genome 46: 724-728. Risso-Pascotto C, Pagliarini, MS, Valle CB (2005 a) Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae). Brazilian Journal of Biological and Medical Research 38: 1603-1608. Risso-Pascotto C, Pagliarini MS, Valle CB (2006) A new basic chromosome number for the genus Brachiaria (Trin.) Griseb. (Poaceae: Panicoideae: Paniceae). Genetic Research and Crop Evolution (in press). 35 Utsunomiya KS, Pagliarini MS, Valle CB (2004) Chromosome transfer among meiocytes in Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae). Cytologia 69:395-398. Utsunomiya KS, Pagliarini MS, Valle CB (2005) Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae). Biocell (in press). Valle CB, Glienke C (1991) New sexual accessions in Brachiaria. Apomixis Newsletter 3: 11-13. Valle CB, Savidan Y (1996) Genetics, cytogenetics, and reproductive biology of Brachiaria. In: Miles JW, Maass BL, Valle CB (eds). Brachiaria: Biology, Agronomy, and Improvement Centro Internacional de Agricultura Tropical – CIAT/Empresa Brasileira de Pesquisa Agropecuária –CIAT pp-147-163. 36 ABNORMAL TIMING OF CYTOKINESIS IN MICROSPOROGENESIS OF Brachiaria humidicola (Poaceae: Paniceae) 37 ABNORMAL TIMING OF CYTOKINESIS IN MICROSPOROGENESIS OF BRACHIARIA HUMIDICOLA (POACEAE: PANICEAE) Running title: Cytokinesis in Brachiaria humidicola Key words: Brachiaria humidicola, microsporogenesis, cytokinesis, 2n gamete, forage grass. Kellen Regina Boldrini1, Maria Suely Pagliarini1, and Cacilda Borges do Valle2 1 Department of Cell Biology and Genetics, State University of Maringá, 87020-900 Maringá PR, Brazil; 2 Embrapa Beef Cattle, P.O. Box 154, 79002-970 Campo Grande MS, Brazil. ([email protected]) Meiosis is controlled by a large number of genes, generally dominant, which are stage, site- and time-specific (Gottschalk and Kaul, 1974, 1980 a, b; Baker et al., 1976; Golubovskaya 1979, 1989). Among genes acting in the meiotic process, those responsible for the partitioning of the cytoplasm after nuclear division play a very important role in the formation of viable gametes. After two rounds of chromosome segregation (karyokinesis) and one simultaneous or two successive cytoplasmic divisions (cytokinesis), the final product of male meiosis in flowering plants emerges as a tetrad of haploid microspores enclosed in a callose wall. The timing of cytokinesis varies among angiosperms. In most monocotyledons, cytokinesis is successive, i.e, one 38 partitioning of the cytoplasm occurs after telophase I and another after telophase II, so that there is a distinct dyad stage; however, in most dicots, it is simultaneous and occurs after telophase II (Peirson et al., 1996). Many meiotic mutants have been reported in higher plants affecting the pattern of cytokinesis (see Peirson et al., 1996). In Brachiaria, a promising African genus of forage grass for the Brazilian savannas, absence of cytokinesis leading to 2n microspores and binucleated or tetranucleated microspores have been reported in B. brizantha (Risso-Pascotto et al., 2003) and B. nigropedata (Utsunomiya et al., 2005). The present study details meiotic and post-meiotic abnormalities related to cytokinesis observed in one accession of B. humidicola. In this accession the first cytokinesis occurred after telophase II, and the second cytokinesis occured by invagination in binucleated microspores only after callose wall dissolution. These late cytokinesis did not affect pollen viability since the meiotic product, although formed lately, was characterized by four reduced normal microspores. Twenty five accessions of Brachiaria humidicola from the Embrapa Beef Cattle germplasm collection (Campo Grande, state of Mato Grosso do Sul, Brazil) originally collected in the wild African savannas in the mid 1980s by CIAT (Colombia) were cytologically analyzed. Inflorescences for meiotic study were fixed in a mixture of ethanol 95%, chloroform and propionic acid (6:3:2) for 24 hours, transferred to 70% alcohol and stored under refrigeration until use. Microsporocytes were prepared by squashing and staining with 0.5% propionic carmine. Photomicrographs were made with a Wild Leitz microscope using Kodak Imagelink – HQ, ISO 25 black and white film. Cytological characterization revealed one accession (H003) presenting several meiotic abnormalities, mainly due to its polyploid condition. Chromosome number in 39 this accession was defined as 2n = 42. Among abnormalities recorded, those related to abnormal timing of cytokinesis are worth discussing. As a monocotyledonous species, B. humidicola was expected to present successive cytokinesis. However, a large number of meiocytes did not show the first cytokinesis after telophase I (Table 1), and the second division occurred in a common cytoplasm (Fig. 1 a to c). The first cytokinesis occurred after telophase II, giving rise to a dyad with two binucleated microspores (Fig. 1 d). Following callose wall dissolution, the binucleated microspores were released (Fig. 1 e) and the second cytokinesis begun to occur discreetly by invagination (Fig. 1 f to h). The furrow of invagination progressed up to the opposite side (Fig. 1 i) dividing the binucleated microspores in two normal uninucleated microspores (Fig. 1 j). A similar pattern of cytokinesis was reported in an intergeneric hybrid between Zea mays x Tripsacum dactyloides (Kindiger, 1993), but affected only a few microspores and was one among several types of abnormal microspore behavior observed. This pattern of cytokinesis is being reported for the first time in Brachiaria. In other Brachiaria species where cytokinesis was absent (Risso-Pascotto et al., 2003; Utsunomiya et al., 2005), the failure of cytokinesis occurred after telophase I or telophase II, but never in released microspores, or by invagination. For this accession of B. humidicola, the first cytokinesis occurred after telophase II, and the second cytokinesis, also programmed by the cell, occurred beyond that time. It is suggested that the genetic control for cytokinesis exists in these meiocytes and was activated, but it was not synchronous with karyokinesis. The absence of the first and/or the second cytokinesis in H003 also predispose it to the occurrence of other abnormalities in the second division. Meiocytes that underwent only one cytokinesis gave rise to binucleated dyads (Fig. 1 k). Some 40 meiocytes that did not suffer the first cytokinesis regrouped the two chromosome sets (Fig. 1 m) originating a restitutional nucleus. Table 1. Percentage of cells with abnormal cytokinesis in the accession H003 of B. humidicola. Phase No. of cells analyzed Abnormalities (%) /No. of abnormal cells Prophase II 104 / 91 Absence of cytokinesis 91 (87.5) Metaphase II 274 / 164 Absence of cytokinesis 164 (58.8) Anaphase II 184 / 132 Absence of cytokinesis 100 (54.3) Tripolar spindle 32 (17.8) Telophase II 564 / 268 Absence of cytokinesis 184 (32.6) Restitutional nucleus 84 (14.9) Meiotic products 450 / 438 Absence of cytokinesis 16 (3.6) Uninucleated dyad 10 (2.2) Binucleated dyad 373 (82.9) Triads 30 (6.8) Monads 9 (2.1) Microspores 2234 / 1403 Binucleated 557 (24.9) Initial cytokinesis 222 (9.9) Advanced cytokinesis 607 (27.2) Trinucleated 10 (0.4) Tetranucleated 17 (0.8) 41 Figure 1. Aspects of abnormal cytokinesis. a to c) Phases of meiosis II with absence of the first cytokinesis: metaphase II (a), anaphase II (b), and telophase II (c). d) Telophase II presenting the first cytokinesis. e) Binucleated microspore released from the dyad. f to j) Progressive stages of the second cytokinesis by invagination in binucleated microspores. Observe that in j one microspore is initiating the cytokinesis whereas the other just completed it. k) Dyad with two binucleated microspores. l) Mononucleated n microspore resulted from the second cytokinesis. m) Meiocytes in the second division without the first cytokinesis (prophase II and metaphase II). Observe that in one meiocyte in metaphase II, the two chromosome sets were rejoined due to absence of cytokinesis, resulting in a restitutional nucleus (arrowhead). n) Trinucleated telophase II with a restitutional nucleus (arrowhead) resulting from tripolar spindle orientation. o) Products of meiosis with different genetic constitution: a binucleated dyad, a triad with a restitutional nucleus in one microspore (arrow), and a future dyad with restitutional nucleus in both microspores (arrowhead). p) Tetranucleated microspore resulted from the absence of both cytokinesis. (Scale bar = 1 µm) 42 Tripolar spindles in anaphase II were detected in a considerable number of meiocytes and gave rise again to restitutional nucleus in telophase II (Fig. 1 n). When these cells underwent cytokinesis, they gave rise to triads with one uninucleated (2n) and two binucleated microspores (Fig. 1 o). However, if these cells did not suffer any cytokinesis, they originated trinucleated microspores with one 2n nucleus. On the other hand, in those meiocytes that lack both cytokinesis, a tetranucleated microspore was formed (Fig. 1 p). Among a large number of microspores analyzed, more than 60% was involved in some event related to irregular cytokinesis, including late but programmed cytokinesis, and total absence of one or both cytokinesis. 2n microspores resulting from abnormal cytokinesis have been reported in B. brizantha (Risso-Pascotto et al., 2003) and B. nigropedata (Utsunomiya et al., 2005). 2n gametes might have contributed to the evolutionary history of the genus Brachiaria. In this genus, the majority of species are polyploid, mainly tetraploid (Valle and Savidan, 1996; Penteado et al., 2000; Mendes-Bonato et al., 2002, 2006; Utsunomiya et al., 2006). The origin of polyploidy is not yet well known. However, evidences from conventional cytological studies point to the hypothesis that some polyploid accessions originated by autotetraploidy, segmental allotetraploidy (Mendes-Bonato et al., 2002, 2006; Risso-Pascotto et al., 2003; Utsunomiya et al., 2005) and, in some cases, by allopolyploidy (Mendes et al., 2006). According to Veilleux (1985) and Bretagnolle and Thompson (1995), 2n gametes play an important role in evolution of higher plants, and even more so in grasses, where polyploidy is widely reported (Hunziker and Stebbins, 1986; De Wet, 1986). Among Brachiaria species available at Embrapa Beef Cattle Research Center, the B. humidicola germplasm collection is represented by 60 accessions (Valle and Savidan, 1996). Determination of ploidy level by flow cytometry showed that all of 43 them are polyploids, with the ploidy level ranging from 4n to 7n (Penteado et al., 2000). Due to their adaptation to poorly drained and infertile acid soils (Keller-Grein et al., 1996), such as those found in the Brazilian “Pantanal” region, some promising apomictic accessions of this species are under careful agronomic and grazing evaluation in hopes of selecting new cultivars. Other accessions, however, may be used in intra- or interspecific hybridization as pollen donors. In this genus polyploidy is correlated with apomixis, but for seed development, the secondary nuclei of the embryo sac need to be fertilized by a male gamete - pseudogamy. Thus, accessions with meiotic abnormalities which severely impair pollen viability need to be discarded from the breeding program. The occurrence of late cytokinesis, per se, did not affect pollen viability because the meiotic product, although lately formed, was characterized by four n normal microspores. However, many other meiotic abnormalities due to polyploidy were recorded in this accession and compromised pollen fertility by generating unbalanced gametes. REFERENCES Baker B. S., Carpenter A.T.C., Esposito M.S., Esposito R.E. and Sandler L. 1976 The genetic control of meiosis. Annu. Rev. Genet. 10, 53-134. Bretagnolle F. and Thompson J. D. 1995 Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol. 129, 1-22. de Wet J.M.J. 1986 Hybridization and polyploidy in the Poaceae. In Grass: Systematics and Evolution. (ed. Soderstrom TR, Hilu WH, Campbell CS, and Barkworth ME), pp 179-187. Smithsonian Institution Press, Washington DC. Golubovskaya I. N. 1979 Genetic control of meiosis. Int. Rev. Cytol. 58, 247-290. Golubovskaya I. N. 1989 Meiosis in maize: mei genes and conception of genetic control of meiosis. Adv. Genet. 26, 149-192. Gottschalk W. and Kaul M. L. H. 1974 The genetic control of microsporogenesis in higher plants. Nucleus 17, 133-166. 44 Gottschalk W. and Kaul M. L. H. 1980 a Asynapsis and desynapsis in flowering plants. I. Asynapsis. Nucleus 23, 1-15. Gottschalk W. and Kaul M.L.H. 1980 b Asynapsis and desynapsis in flowering plants. II. Desynapsis. Nucleus 23, 97-120. Hunziker JH, Stebbins LG. 1986 Chromosomal evolution in the Gramineae. In Grass: Systematics and Evolution (ed. Soderstrom TR, Hilu WH, Campbell CS, and Barkworth ME), pp 179-187. Smithsonian Institution Press, Washington DC. Keller-Grein G., Maass, B. L., and Hanson, J. 1996 Natural variation in Brachiaria and existing germoplasma collections. In Brachiaria: Biology, Agronomy, and Improvement (ed. Miles J. W., Maass, B. L. and Valle, C. B.) CIAT, Colombia. Kindiger B. 1993 Aberrant microspore development in hybrids of maize x Tripsacum dactyloides. Genome 36, 987-997. Mendes D. V., Boldrini K. R., Mendes-Bonato A. B., Pagliarini M. S. and Valle C. B. 2006 Cytological evidence of natural hybridization in Brachiaria brizantha Stapf (Gramineae). Bot. J. Linn. Soc. (in press). Mendes-Bonato A. B., Pagliarini M. S., Forli F., Valle C. B. and Penteado M. I. O. 2002 Chromosome number and microsporogenesis in Brachiaria brizantha (Gramineae). Euphytica 125, 419-425. Mendes-Bonato A. B., Pagliarini M. S., Risso-Pascotto C. and Valle C. B. 2006 Chromosome number and meiotic behavior in Brachiaria jubata (Gramineae). J. Genet. (in press). Peirson B. N., Owen H. A., Feldmann K. A. and Makaroff C. A. 1996 Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis. Sex. Plant Reprod. 9, 1-16. Penteado M. I. O, Santos A. C. M, Rodrigues I. F, Valle C. B, Seixas M. A. C. and Esteves A. 2000 Determinação de poliploidia e avaliação da quantidade de DNA total em diferentes espécies de gênero Brachiaria. Boletim de Pesquisa, 11. Campo Grande-MS, Embrapa Gado de Corte. 19p. Risso-Pascotto C., Pagliarini M. S., Valle C. B. and Mendes-Bonato A. B. 2003 Chromosome number and microsporogenesis in pentaploid accession of Brachiaria brizantha (Gramineae). Plant Breed 122., 136-140. Utsunomiya K. S., Pagliarini M. S. and Valle C. B. 2005 Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae). Biocell (in press). Valle C. B. and Savidan Y. 1996 Genetics, cytogenetics, and reproductive biology of Brachiaria. In Brachiaria: Biology, Agronomy, and Improvment (ed. Miles J. W., Maass, B. L. and Valle, C. B.) CIAT, Colombia. 45 Veilleux R. 1985 Diploid and polyploid gametes in crop plants: mechanisms of formation and utilization in plant breeding. Plant Breed. Rev. 3, 253-288. 46 APÊNDICE
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