Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 134-145) Also available in print.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

Monopoloid potato (2n = 1x = 12) can be derived either paternally through anther/microspore culture or maternally through crossing with a haploid-inducing pollinator. Evidence from other genera suggests that androgenic and gynogenic haploid populations derived from the same parent may differ due to gametic selection and/or epigenetic factors. Our objectives were to derive androgenic and gynogenic monoploid populations from each of two diploid (2n = 2x = 24) Solanum phureja clones and compare their phenotypic characteristics in a greenhouse study. A haploid-inducing pollinator, S. phureja IVP101, was crossed to two selections of S. phureja (PP5 and BARD1-3). A total of 185 fruit was obtained from PP5 and 398 from BARD1-3, resulting from 85% and 65% fruit set, respectively. Seed lacking the dominant embryo spot marker carried by IVP101 were selected and germinated in vitro. From 29,300 PP5 x IVP101 seeds, 278 were spotless, resulting in 27 monoploids. Approximately 37 monoploids were obtained from the 99,500 BARD1-3 x IVP101 seeds of which 500 were spotless. In anther culture, PP5 and BARD1-3 yielded 0.16 and 1.67 embryos per anther respectively of which 51% and 44% of the regenerants were monoploid. A total of 32 anther-derived monoploids has been obtained from PP5 and 130 from BARD1-3. Rooted cuttings of 21 androgenic and 21 gynogenic PP5 monoploids were established in a greenhouse in each of three randomized complete blocks. Although the anther-derived monoploids appeared more vigorous, none of the differences between the two populations were significant. Flow cytometry revealed that anther derived progeny of PP5 and BARD1-3 had 3-8% greater nuclear DNA content than gynogenic monoploids derived from the same parental clones. / Master of Science

Apyrases (NTP-diphosphohydrolases, EC 3.6.1.5) are a family of enzymes that catalyze the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di- phosphates in the presence of divalent cations. In Arabidopsis, AtAPY1 and AtAPY2 function in part as ectoapyrases and have been shown to play important roles in controlling the concentration of extracellular nucleotides, which, in turn, can regulate pollen germination and growth, and cell expansion in diverse plant tissues. We used an NCBI nucleotide blast keyed to Apyrase Conserved Regions (ACRs) to identify five other AtAPYs (3-7). To assess the biological function of each of these five AtAPY genes, the phenotypes of their T-DNA insertion mutants were analyzed. We did not observe any obvious phenotypes from the T-DNA insertion single knockout of any of these genes. However, double knockout mutants of AtAPY6 and 7 exhibited late anther dehiscence and low male fertility. Transmission and scanning electron microscopy revealed that the pollen grains of double knockout mutant of AtAPY6 and 7 are largely deformed in shape and in most cases, the cell walls of the pollen grains are interconnected. Using an AtAPY6-YFP fusion protein in transgenic Arabidopsis, AtAPY6 was localized to intracellular vesicles. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and promoter:GUS fusion analysis demonstrated that the transcripts of both AtAPY6 and 7 are expressed in mature pollen grains, AtAPY6 is also expressed in the veins and hydathode regions of rosette leaves, and AtAPY7 is expressed in more diverse tissues such as roots, leaves, stems, pistils and sepals. The tissue specificity of AtAPYs 3, 4 and 5 expression was also determined using qRT-PCR assays and promoter:GUS fusion analysis. AtAPY3 and AtAPY4 were primarily expressed in roots but not in rosette leaves. AtAPY5 was expressed primarily in rosette leaves but only weakly in roots. AtAPY5 and AtAPY7 were upregulated when the rosette leaves are wounded or exposed to drought stress. RNA interference (RNAi) was performed to simultaneously suppress the gene expression of AtAPYs 3, 4, 5 to around 10% of that of the wild type. However, we did not observe any obviously altered phenotypes in the RNAi lines. The suppression of AtAPYs 3, 4, 5 by RNAi in the background of AtAPY6 single knockout did not cause any phenotype either. A possible explanation for this lack of phenotype in the RNAi lines is that functional redundancy exists between AtAPYs 3-5 and AtAPYs 1-2 and/or AtAPYs 6-7. / text

Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-15Bitstream added on 2014-06-13T20:54:01Z : No. of bitstreams: 1 pereirajunior_ej_me_sjrp.pdf: 1176824 bytes, checksum: ea26888e0c90e98ae7bc9367c03e1901 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Caracteres embriológicos possuem valor sistemático e sua utilidade foi demonstrada por diversos autores para elucidar o posicionamento filogenético de certas famílias de angiospermas. Este estudo visa analisar a ontogenia das estruturas reprodutivas de Cybistax antisyphilitica, com o propósito de acrescentar dados embriológicos relevantes ao delineamento filogenético da família, ou de categorias taxonômicas infrafamiliares. As características embriológicas observadas demonstraram similaridade com espécies pertencentes à ―Tabebuia alliance‖ cuja embriologia já foi investigada. Observou-se durante a ontogenia do óvulo de C. antisyphilitica que apenas o ginósporo calazal se desenvolve e, durante sua diferenciação em célula-mãe do saco embrionário, a face micropilar de sua parede celular assume uma conformação côncava, na qual há grande deposição de calose. No estádio octonuclear, há acúmulo de uma substância fibrogranular entre o endotélio e a parede do ginófito fazendo com que o saco embrionário apresente um característico afunilamento mediano. Em relação a ontogenia da antera da espécie estudada, verificou-se que as camadas tapetais são dimórficas, embora ao final do estádio pré-meiótico se tornem similares; embora, após a meiose, o dimorfismo se acentua novamente nas camadas tapetais. Os amiloplastos das células-mãe dos andrósporos são herdados pelos andrósporos e grãos de pólen deles resultantes. Os amiloplastos dos grãos de pólen gradualmente aumentam em número e tamanho em um único ciclo de amilogênese/amilólise. Com base nos dados obtidos conclui-se que a configuração da parede distal do ginósporo calazal e o acúmulo de secreção na porção mediana do saco embrionário são características não relatadas para outras espécies da família e podem... / Embryological characters have systematic value and its usefulness has been demonstrated by several authors to elucidate the phylogenetic position of certain angiosperms families. This study aims to analyze the ontogeny of reproductive structures of Cybistax antisyphilitica, with the purpose of adding relevant embryological data to the phylogenetic design of the family or, infra-familiar taxonomic categories. The embryological features observed was similar to species belonging to ―Tabebuia alliance‖ whose embryology has been investigated. During the ontogey of C. antisyphilitica ovule, only the chalazal megaspore develops, and in the course of its differentiation in the embryo sac mother cell, the micropylar side of its cell wall assumes a concave conformation in which there is an expressive deposition of callose. In the octonuclear stage, there is an accumulation of a fibrogranular substance between the endothelium and megagametophyte wall causing a bottleneck in the middle portion of embryo sac. For anther ontogeny of the species studied, the tapetal layers are dimorphic, although becoming similar at the late pre-meiotic stage. After meiosis, the dimorphism is accentuated between the two tapetal layers. The microspore mother cell amyloplasts are inherited by the microspores and the resulting pollen grains. The pollen grain amyloplasts gradually increasing in number and size in a single amylogenesis/amylolyse cycle. Based on the obtained data we concluded that the configuration of the distal wall of the chalazal megaspore and the accumulation of secretion in the median portion of embryo sac are characters not reported for other species of the family, and may possibly be autapomorphic characters. Regarding the ontogeny of the anther, Cybistax antisyphilitic showed a multistratified fibrous endothecium... (Complete abstract click electronic access below)

Anther culture experiments were conducted to construct a monoploid family. The donor plants used were hybrids between high leptine producing selections of Solanum chacoense Bitt. and anther culture responsive selections of Solanum phureja Juz. et Buk. Several steps of the anther culture process were studied. The results indicated that genotype remains the main factor affecting anther culture response. Growing anther-donor plants in higher greenhouse temperatures (30 degrees C day/20 degrees C night) increased the number of embryos per anther by 40 percent. A heat shock given to anthers in culture for 12h at 35 degrees C was also found to be beneficial resulting in an increase of the anther culture response by 40 percent. However the effect of the high temperature shock resulted in lower regeneration rates. In all experiments a highly significant "date" effect was observed with one or two days differing from the others by showing higher response rates in all hybrids tested. The majority of the regenerated plants was diploid, probably resulting from unreduced gametes. Simple sequence repeat analysis with eight polymorphic primer pairs was used successfully to identify the homozygous diploid plants that were added to the monoploids. In total 34 monoploid plants and 14 homozugous diploids were obtained. The degree of heterozygosity revealed by SSR analysis indicated that the diploid plants originated from unreduced gametes formed by first division restitution (FDR) mechanism. The SSR marker data were used to map the genes with respect to the centromeres by half tetrad analysis. SSR-containing sequences from the public databases, as well as sequences obtained from a genomic library enriched for SSRs, were used to generate 48 primer pairs. Only 12 of them were found to be polymorphic in the monoploid family. Ten primer pairs did not amplify any specific fragment. The monoploid population showed distorted segregation at four of the polymorphic loci, showing overrepresentation of the chacoense alleles in three of them. One of the loci showing distorted segregation (STSTP, amplified by primer pair RV 11+12) is most probably linked to lethal alleles, whereas another one (ST13ST, amplified by primer pair RV 21+22) could be linked to genes affecting anther-culture response. The location of the SSR loci on the potato chromosomes is not known except for one (waxy, primer pair 3+4), but statistical analysis on the segregation data obtained from 70 heterozygous anther-derived diploids showed no linkage between them. The SSR primer pairs developed in this study might be useful in studying genetic relationships among cultivars and accessions in breeding programs. Randomly amplified polymorphic DNA (RAPD) analysis was used in association with bulked segregant analysis to detect linkage with genes controlling leptine biosynthesis. With all the limitations imposed by the population size and contamination from foreign pollen, a band amplified by primer OPA-16 could differentiate the bulks contrasting for leptine content. It is possible that this band is linked to genes suppressing leptine biosynthesis, since it appears only in the plants that do not synthesize leptines. Further investigation with larger populations is needed to confirm this possibility. / Ph. D.

Colchicine, an antimicrotubule agent, has enhanced the androgenic response of cruciferous and graminaceous species. Our objectives were to determine if colchicine treatments enhanced androgenic embryo production in diploid potato, and if colchicine treatments during anther culture affected the ploidy of anther-derived plants. An additional study, analysis of genetic identity among regenerated anther-derived plants based on RAPD markers, was also conducted. Anther culture of a hybrid between <i>Solanum chacoense</i> and <i>S. phureja</i> (clone CP2) was conducted with five colchicine treatments (0, 25, 50, 100, and 200 mg/L) for 24 h. Anthers containing late uninucleate pollen were cultured on modified liquid LS medium. A mean of 0.25 embryos per anther was obtained; however, there was no significant difference among the colchicine treatments, which ranged from a mean of 0.17 to 0.28 embryos per anther for 200 mg/L colchicine and control, respectively. In total 56 plants were regenerated of which 11% were monoploid. The second experiment using the same colchicine treatments was conducted with 54 hybrids derived by crossing six anther-derived doubled monoploids with three heterozygous pollinators. A mean of 1.78 embryos per anther was obtained; again, there was no significant difference among the colchicine treatments, which ranged from 1.27 to 2.75 for 50 mg/L colchicine and control, respectively. In total 679 plants were regenerated. The 312 plantlets for which ploidy was determined, 75% were monoploid. The third experiment was conducted to study various durations (0, 90 sec vacuum infiltration, 24, 48, and 72 h) of high colchicine treatment (200 mg/L) applied to <i>S. phureja</i> family DM 13-14 202 x ID 5. There was nearly significant difference among treatments (α = 0.05). Mean embryos per anther ranged from 0.96 to 1.90 for 48 h and 90 sec vacuum infiltration, respectively. In an additional study, 26 regenerated anther-derived monoploid plants, from the DM 13-14 202 x ID 5 family, from the second experiment, were genetically characterized based on RAPD markers. Forty-three loci were scored from 13 primers for groups of monoploids derived from the same flasks of anther culture. From one flask, two pairs of monoploids were found to be genetically identical. From a second flask 6 of 7 were genetically identical, and from a third 3 of 7 were genetically identical. The presence of genetically identical individuals within three monoploid populations indicates the prevalence of secondary embryogenesis during anther culture such that a single embryogenic microspore can generate many anther-derived plants. / Master of Science

Em Mimosoideae (Leguminosae), glândulas em anteras são estruturas de destaque, pois ocorrem em muitos de seus representantes e provêm caracteres com valor taxonômico no clado. O conhecimento a respeito destas glândulas era baseado principalmente em observações de morfologia externa, faltando dados a respeito de sua anatomia, ontogenia, seu funcionamento e seus aspectos evolutivos, o que contribuiria para uma caracterização mais detalhada e uma classificação mais natural dessas estruturas, além da inferência de funções ecológicas mais objetivas. Assim, o objetivo do estudo foi revisitar a estrutura das glândulas nas anteras, investigar sua origem meristemática e o seu desenvolvimento, acompanhar seu funcionamento, inferir funções ecológicas e, então, reconstruir sua evolução em Mimosoideae. Para contemplar os objetivos propostos, o presente estudo investigou aspectos das glândulas em anteras em 25 espécies de Mimosoideae, que representam grupos com divergências mais antigas e recentes no clado. Foram encontrados quatro tipos principais de glândulas: (1) O tipo Piptadenia, caracterizado por uma glândula pedunculada com ápice esférico e que inclui três subtipos: Piptadenia, com ápice composto de células isodiamétricas; Adenanthera, com ápice composto de uma camada de células epidérmicas alongadas que delimitam células isodiamétricas; e Entada, com ápice composto de uma camada de células epidérmicas volumosas que delimitam um lúmen. (2) O tipo Pentaclethra, uma glândula robusta composta de uma camada de células epidérmicas em paliçada e células centrais isodiamétricas, com pedúnculo vascularizado. (3) O tipo Prosopis africana, uma glândula robusta, séssil e vascularizada. (4) Finalmente o tipo Gagnebina, uma glândula acuminada com células alongadas. Apesar da diversidade morfológica, as glândulas de anteras compartilham uma origem meristemática comum. Elas surgem de divisões simultâneas nas primeiras três camadas de células meristemáticas que cobrem a porção apical da antera jovem, sendo classificadas como emergências. Os tipos de glândulas podem apresentar diferenças quanto a: (1) estrutura na pré-antese e antese; (2) longevidade; (3) aspecto e composição química do exsudato e (4) forma de liberação do exsudato. Baseados na caracterização e forma de liberação do exsudato, e no comportamento dos visitantes florais, nossos dados apontam que as glândulas atuam principalmente (a) na produção de substâncias que atuam na adesão do pólen ao corpo do polinizador ou à antera e (b) na produção de fragrância floral atuando como osmóforos. A presença de glândulas em anteras parece ter uma origem única em Mimosoideae, com alguns eventos de perda. O estado ancestral da estrutura das glândulas provavelmente corresponde ao tipo Piptadenia subtipo Adenanthera. Ainda, parece existir uma relação positiva entre ausência de glândulas e a presença de inflorescências de eixo curto, de estames numerosos e de estames unidos. Nossos dados indicam que em Mimosoideae, as glândulas em anteras são caracteres com potencial significado filogenético. A origem meristemática conservada das glândulas além da origem única da presença de glândulas em anteras Mimosoideae favorece a hipótese de homologia dessas estruturas e apoia a monofilia do clado. / In Mimosoideae (Leguminosae), anther glands are prominent structures because they occur in many taxa and provide characters with taxonomic value in the clade. The knowledge about the anther glands was mainly based on external morphological observations, lacking data concerning the anatomy, ontogeny, function and evolutionary aspects of the glands, which would contribute to a more detailed characterization and a more natural classification of these structures, as well as the inference of more objective ecological functions. The aims of this study were to revisit the structure of the anther glands, to investigate their meristematic origin, and their developmental stages, their functioning mechanisms, to infer ecological functions, and thus reconstruct the evolutionary history of these glands in Mimosoideae. For this, the present study investigated several aspects of the anther glands in 25 species of Mimosoideae representing groups with early and late divergences in the clade. We found four major types of glands in anther Mimosoideae: (1) Piptadenia type, characterized by a stalked gland with a spherical apex, and that includes three subtypes: Piptadenia, with the apex composed of isodiametrical cells; Adenanthera, with the apex composed a layer of elongated epidermal cells which delimits isodiametric cells; and Entada with the apex composed of a layer of epidermal bulky cells delimiting a lumen. (2) Pentaclethra type,a robust gland comprised of a layer of epidermal and palisade cells isodiametric central cells with vascularized peduncle. (3) The Prosopis africana type, a robust sessile and vascularized gland. (4) Finally, the Gagnebina type, an acuminate gland with elongated cells. Despite the morphological diversity, the anthers glands share a common meristematic origin. They arise from simultaneous divisions in the first three layers of meristematic cells covering the apical portion of the young anther, being classified as emergences. The types of glands may differ as to: (1) structure in the pre-anthesis and anthesis; (2) longevity; (3) aspect and chemical composition of exudate and (4) release form of exudate. Considering the characterization and release form of exudate, and behavior of floral visitors, our data show that the glands act mainly (a) in the production of substances that act on the accession of pollen to the bee body or anther and (b) in production of floral fragrance acting as osmophores. The presence of anther glands appears to have a single origin in Mimosoideae, with some loss events. The ancestral state of the structure of the glands probably corresponds to the type Piptadenia subtype Adenanthera. Still, there seems to be a negative relation between the presence of the glands, the presence of inflorescences with short axis, numerous stamens and united stamens. Our data indicate that in Mimosoideae, the glands in anthers provide characters with potential phylogenetic significance. The conserved meristematic origin of the anther glands beyond the single evolutionary origin of the presence of glands in anthers favor the hypothesis of homology of these structures and support the monophyly of Mimosoideae.

Bromeliaceae owns a huge diversity of species in the Neotropical region, comprising about 3140 species, in which 551 consist of the genus Tillandsia. The article aims the descritption, of the anther development and ovule as the esporogenesis and gametogenesis characterization as well, in order to make easier both the taxonomy and philogeny of the family, which is still in process of change. Tillandsia aeranthos (Lois.) L. B. Sm. contains six stamens flowers; superior, tricarpellate and trilocular ovary. The pattern of development of the anther wall is characterized as mixed-type. Androsporangium is formed by epidermis, middle layers, endothecium and tapetum. The tapetum is the secretor-type. Meiosis is the successive-type with cleavage of the centrifugal type. The tetrads formed are decussate or isobilateral. The first division of the pollen is preceded by vacuolation, it is assymmetric and produce both generative and vegetative cells. The two cells formed are separated by a callose wall. The mature grain pollen is bicelullar. The ovule of Tillandsia aeranthos is anatropus, bitegmic and crassinucellate, with axial placentation. The ovule originates in the subdermal layer (zone II) of the placentae. The integument nucellar epidermis is originated by divisions in the dermal layers. One to three ginospore mother celss, which are originated by divisions in archesporial cell, undergo meiotic divisions development a linear tetrad, with presence callosic wall. Only the chalazal ginospore becomes functional. The functional ginospore differs in that a gametophyte uninucleate after mitosis and yields a two-nucleate and four-nucleate gametophyte. The female gametophyte has a monosporic origin and a Polygonum-type development. The female gametophyte consists of two synergids, an egg cell, three antipodes and two polar nuclei. Polar nuclei fuse prior to fertilization. / Bromeliaceae possui grande diversidade de espécies na região neotropical, compreendendo cerca de 3140 espécies, sendo 551 do gênero Tillandsia. O presente trabalho teve como objetivo descrever o desenvolvimento da antera e do rudimento seminal, além da caracterização da espogênese e gametogênese, com a finalidade de auxiliar na taxonomia e filogenia da família, ainda em fase de mudança. Tillandsia aeranthos, apresenta flores com estames em número de seis e ovário súpero, tricarpelar. Na antera, o desenvolvimento dos estratos parietais é do tipo misto , formado por epiderme, endotécio, camada média e tapete. A epiderme é papilada e o tapete é do tipo secretor. A microsporogênese é sucessiva com clivagem do tipo centrífuga, onde são formadas tétrades isobilaterais e decussadas. Após a liberação do andróspro da tétrade, ocorre a primeira divisão do grão de pólen e é precedida por vacuolação. Após mitose, é formada a célula generativa e a célula vegetativa, separadas por uma parede de calose. O andrófito é liberado bicelular. O rudimento seminal de Tillandsia aeranthos é anátropo, bitegumentado e crassinucelado, com placentação axial. O rudimento seminal tem origem na camada subdérmica da placenta. Os tegumentos são de origem dérmica. A inicial arquesporial dá origem á célula mãe de ginósporo (CMG). A CMG sofre o primeiro ciclo meiótico que origina uma díade de ginósporo com presença de calose ao redor. O segundo ciclo meiótico dá origem a tétrade linear de ginósporo, tornando-se o ginósporo calazal funcional. O ginósporo funcional diferencia-se em um gametófito uninucleado que após mitoses origina um gametófito bi e tetranucleado. Esse tipo de desenvolvimento do ginófito é do tipo monospórico e Polygonum, onde o ginófito apresenta sete células e oito núcleos, com a presença de duas sinérgides, uma oosfera, formando o aparelho oosférico e três antípodas. Os núcleos polares de fusionam antes da fecundação.