Academic literature on the topic 'Staminode'

Although mostly resident in the bone marrow (BM), few circulating HSPC (cHSPC) regularly traffic in the peripheral blood (PB) of un-mobilized subjects. Mainly descriptive studies have been published so far about this rare population in humans, and a complete evaluation of their composition, functional features and hierarchical relationship with respect to BM HSPC is still missing. In the present study, we phenotypically characterized cHSPC composition during aging, by applying multi-parametric flow cytometry on 114 PB and, as control, 48 BM samples of healthy donors (HD) of diverse age. These analyses were integrated with single-cell transcriptome profiling (scRNAseq), and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC with respect to BM counterpart. Moreover, to study circulating vs. resident HSPC relationships and differentiation potential in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from patients treated with HSPC-gene therapy (GT). We observed that cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation capability. cHSPC are endowed with multilineage differentiation potential both in vitro and in vivo, with comparable BM homing capability but reduced long-term survival after transplantation in immune-deficient mice than BM HSPC. This latter finding can be explained by the low primitive HSC content and the transcriptional pre-activated state observed in steady-state PB HSC. Indeed, applying scRNA-seq, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. The enrichment of lymphoid phenotypic and transcriptional signatures found in PB HSPC, together with their higher IS sharing with PB lymphoid than myeloid mature lineages suggest that cHSPC could have a role in seeding lymphoid organs. Moreover, the higher expression of erythroid marker genes detected in trafficking than resident HSPC was consistent with cHSPC erythroid differentiation bias observed after transplantation and in single-cell in vitro differentiation assay. These findings suggest cHSPC as a source of erythroid-committed progenitors, able to sustain stress-responsive extramedullary erythropoiesis. Finally, our preliminary data on a cohort of HSPC-GT patients suggest that cHSPC may sustain clonal redistribution to distant BM sites, both during active hematopoietic reconstitution and, at a lower extent, during steady-state conditions. Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which continuously recirculate among multiple BM sites and are poised for promptly sustaining activation and in situ local hematopoietic differentiation in case of demand.<br>Nonostante risiedano principalmente nel midollo osseo (BM), poche HSPC circolanti (cHSPC) ricircolano regolarmente nel sangue periferico (PB) di donatori sani in assenza di mobilizzazione. Soprattutto studi di tipo descrittivo sono stati pubblicati sino ad ora su questa rara popolazione nell’uomo e al momento manca una valutazione completa della loro composizione, caratteristiche funzionali e relazione gerarchica rispetto alle HSPC di BM. In questo studio abbiamo caratterizzato fenotipicamente la composizione delle cHSPC durante l'invecchiamento fisiologico, applicando la citometria a flusso multiparametrica su 114 campioni di PB e, come controllo, 48 campioni di BM di donatori sani (HD) di età diversa. Queste analisi sono state integrate con l’analisi del profilo trascrizionale a livello di singola cellula (scRNAseq) e studi in vitro e in vivo, al fine di studiare rispettivamente le proprietà trascrizionali e funzionali delle cHSPC in condizioni fisiologiche rispetto alla controparte di BM. Inoltre, per studiare la relazione tra HSPC circolanti e residenti nel midollo osseo e il loro potenziale di differenziamento in vivo nell'uomo, abbiamo effettuato il monitoraggio clonale dei siti d’integrazione (IS) di cHSPC, BM HSPC e cellule mature di PB isolate da pazienti trattati con terapia genica (GT) basata su HSPC. Abbiamo osservato che le cHSPC si riducono progressivamente durante l'invecchiamento e sono caratterizzate da una composizione diversa rispetto alla controparte di BM, mostrando una più alta capacità di ricircolo dei progenitori linfoidi. Le cHSPC sono in grado di differenziare nelle varie popolazioni ematopoietiche sia in vitro che in vivo, mostrando una simile capacità di migrazione nel BM ma una ridotta sopravvivenza a lungo termine dopo trapianto in topi immunodeficienti rispetto a HSPC di BM. Quest'ultima scoperta può essere spiegata dal basso contenuto di HSC primitive e da uno stato trascrizionale pre-attivato presente nelle HSC di sangue periferico in condizioni fisiologiche. Applicando scRNA-seq, abbiamo identificato un profilo trascrizionale unico nelle sottopopolazioni di cHSPC, sia primitive che più differenziate, caratterizzato da una minore attività replicativa, metabolica e trascrizionale, ma una maggiore capacità di differenziamento, adesione e risposta immunitaria rispetto alla controparte di BM. L'arricchimento dei profili fenotipici e trascrizionali di tipo linfoide osservato nelle HSPC di PB, in associazione con la loro più elevata condivisione di IS con cellule di PB di lignaggio linfoide rispetto a quello mieloide, suggeriscono che le cHSPC potrebbero seminare organi linfoidi. Inoltre, la maggiore espressione di geni di differenziamento eritroide rilevati nelle HSPC circolanti rispetto a quelle residenti è coerente con il bias di differenziamento eritroide osservato nelle cHSPC post-trapianto e nel test di differenziamento in vitro. Questi risultati suggeriscono che le cHSPC costituiscono una fonte di progenitori eritroidi, in grado di sostenere l'eritropoiesi extramidollare in risposta a stress. Infine, i nostri dati preliminari su una coorte di pazienti trattati con HSPC-GT suggeriscono che il ricircolo di HSPC può sostenere la ridistribuzione clonale di cellule staminali in siti midollari distanti, sia durante la ricostituzione ematopoietica attiva che, in misura minore, in condizioni fisiologiche. Complessivamente, i nostri risultati indicano le HSPC circolanti come una peculiare popolazione di progenitori ematopoietici in stato pre-attivato, che ricircolano continuamente tra diversi siti di midollo osseo e sono pronti per sostenere rapidamente un'attivazione e un differenziamento ematopoietico locale in situ in caso di necessità.

The influence of annual rainfall and a Mediterranean climate in structuring the indigenous vegetation in the Southwest Botanical Province of Western Australia has long been recognised, especially in relation to seedling germination during the cooler wet season. However, over the past decade numerous authors have hypothesised that a number of climatic factors, including variability in the timing and intensity of rainfall events, may be drivers of germination. Verticordia staminosa subsp. staminosa (Myrtaceae) is a naturally rare narrow range endemic shrub that occurs on only one granite inselberg near Wongan Hills in the „wheatbelt‟ of southwest Western Australia. Smoke and fruit wall weathering have been claimed to be specific dormancy breakers for the seeds of many Australian genera, including Verticordia. However, I found no evidence of smoke and artificial weathering influencing germination of Verticordia staminosa. Using data from a long-term (12 year) investigation into field germination of Verticordia staminosa, my thesis applies logistical regression techniques to model the impact of climatic variables on germination within the only recorded Verticordia staminosa subsp. staminosa meta-population. My analyses reveal that a complex interplay of the amount of rainfall, number of rainy days, diurnal temperature range, and storms related to tropical cyclones/lows, under a traditional four seasons or six Noongar seasons climate, best describes the observed germination of Verticordia staminosa seedlings. Impact of climatic factors on germination of the rare shrub V. staminosa.

Made available in DSpace on 2014-06-11T19:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-28Bitstream added on 2014-06-13T21:01:48Z : No. of bitstreams: 1 paula_oc_dr_rcla.pdf: 2531374 bytes, checksum: a92e198da4df4bf433952e2e684de112 (MD5)<br>Croton agrupa cerca de 1.200 espécies portadoras de flores bissexuais e representa um grupo complexo, sob o ponto de vista taxonômico e morfológico. Astraea, recentemente separada de Croton e com quem divide inúmeras características morfológicas, é um gênero pouco estudado. A natureza dos verticilos florais, especialmente nectários e estruturas filamentosas das flores pistiladas, é um assunto bastante controvertido, nos dois gêneros. Estudos com as flores de Croton mostram o desenvolvimento do óvulo e da semente e outros apenas descrevem a morfologia do grão de pólen maduro. Entretanto, para o gênero Astraea nada se conhece até o momento. Analisando o desenvolvimento, a vascularização e a morfologia de flores estaminadas e pistiladas, de Croton e Astraea, verificou-se que as estruturas filamentosas das flores pistiladas correspondem às pétalas presentes nas flores estaminadas, e que os nectários representam estames transformados; observou-se, inclusive, que flores pistiladas podem desenvolver estames em substituição aos nectários. Também foi possível apontar caracteres que apóiem a segregação de Astraea e Croton, embora os dois gêneros compartilhem inúmeras características morfológicas. O estudo do desenvolvimento da antera e óvulo de duas espécies de Astraea e de sete de Croton mostrou a ocorrência de cristais estilóides, no tapete, e de idioblastos portadores de drusa, no endotécio; mostrou também, a presença de óvulos com os dois tegumentos de origem epidérmica e a diferenciação de megásporo funcional em posição micropilar. Esses aspectos, descritos pela primeira vez para a família, precisam ser melhor investigados a fim de se determinar sua ocorrência e distribuição, dentro do grupo, e avaliar melhor seu potencial taxonômico.<br>Croton comprises approximately 1,200 species with bisexual flowers and is a taxonomically and morphologically complex group. Astraea, which was recently separated from Croton and shares innumerous morphological characteristics, is still a poorly studied genus. The nature of the floral whorls, especially the nectaries and the filamentous structures of the pistillate flowers, is a controversial subject in both genera. Some studies with Croton flowers have examined the development of ovules and seeds, while others have only described the morphology of mature pollen grains. Very little is currently known about Astraea. Analyses of the development, vascularization, and morphology of the staminate and pistillate flowers of Croton and Astraea demonstrated that the filamentous structures of the pistillate flowers represent transformed petals, and that nectaries represent transformed stamens in pistillate flowers; stamens were occasionally observed in place of nectaries. Many characteristics were observed that support the segregation of Astraea from Croton, although the two genera do share numerous morphological features. An examination of the development of the anthers and ovules of two species of Astraea and seven species of Croton demonstrated the presence of styloid crystals in the tapetum as well as idioblasts with druses in the endothecia; ovules with two teguments of epidermal origin and the differentiation of a functional megaspore in a micropylar position were also observed. These aspects, described here for the first time for the family, need to be further investigated to determine their occurrence and distribution within the taxon, and to evaluate their taxonomic potential.

Resumo: Croton agrupa cerca de 1.200 espécies portadoras de flores bissexuais e representa um grupo complexo, sob o ponto de vista taxonômico e morfológico. Astraea, recentemente separada de Croton e com quem divide inúmeras características morfológicas, é um gênero pouco estudado. A natureza dos verticilos florais, especialmente nectários e estruturas filamentosas das flores pistiladas, é um assunto bastante controvertido, nos dois gêneros. Estudos com as flores de Croton mostram o desenvolvimento do óvulo e da semente e outros apenas descrevem a morfologia do grão de pólen maduro. Entretanto, para o gênero Astraea nada se conhece até o momento. Analisando o desenvolvimento, a vascularização e a morfologia de flores estaminadas e pistiladas, de Croton e Astraea, verificou-se que as estruturas filamentosas das flores pistiladas correspondem às pétalas presentes nas flores estaminadas, e que os nectários representam estames transformados; observou-se, inclusive, que flores pistiladas podem desenvolver estames em substituição aos nectários. Também foi possível apontar caracteres que apóiem a segregação de Astraea e Croton, embora os dois gêneros compartilhem inúmeras características morfológicas. O estudo do desenvolvimento da antera e óvulo de duas espécies de Astraea e de sete de Croton mostrou a ocorrência de cristais estilóides, no tapete, e de idioblastos portadores de drusa, no endotécio; mostrou também, a presença de óvulos com os dois tegumentos de origem epidérmica e a diferenciação de megásporo funcional em posição micropilar. Esses aspectos, descritos pela primeira vez para a família, precisam ser melhor investigados a fim de se determinar sua ocorrência e distribuição, dentro do grupo, e avaliar melhor seu potencial taxonômico.<br>Abstract: Croton comprises approximately 1,200 species with bisexual flowers and is a taxonomically and morphologically complex group. Astraea, which was recently separated from Croton and shares innumerous morphological characteristics, is still a poorly studied genus. The nature of the floral whorls, especially the nectaries and the filamentous structures of the pistillate flowers, is a controversial subject in both genera. Some studies with Croton flowers have examined the development of ovules and seeds, while others have only described the morphology of mature pollen grains. Very little is currently known about Astraea. Analyses of the development, vascularization, and morphology of the staminate and pistillate flowers of Croton and Astraea demonstrated that the filamentous structures of the pistillate flowers represent transformed petals, and that nectaries represent transformed stamens in pistillate flowers; stamens were occasionally observed in place of nectaries. Many characteristics were observed that support the segregation of Astraea from Croton, although the two genera do share numerous morphological features. An examination of the development of the anthers and ovules of two species of Astraea and seven species of Croton demonstrated the presence of styloid crystals in the tapetum as well as idioblasts with druses in the endothecia; ovules with two teguments of epidermal origin and the differentiation of a functional megaspore in a micropylar position were also observed. These aspects, described here for the first time for the family, need to be further investigated to determine their occurrence and distribution within the taxon, and to evaluate their taxonomic potential.<br>Orientador: Maria das Graças Sajo<br>Coorientador: Inês Cordeiro<br>Banca: Adelita Aparecida Sartori Paoli<br>Banca: Denise Maria Trombert Oliveira<br>Banca: Silvia Rodrigues Machado<br>Banca: Lygia Dolores Ribeiro de Santiago Fernandes<br>Doutor

Negli ultimi anni, l’editing genetico nelle cellule staminali/progenitrici ematopoietiche umane (HSPC) per il trattamento di malattie genetiche del sangue è migliorato drasticamente trasformando inserzioni genetiche casuali in precise e mirate modificazioni del genoma. La modifica mirata dei geni mutati ereditati consente la correzione in situ e la ricostituzione funzionale con il mantenimento del controllo endogeno dell'espressione. Recentemente abbiamo dimostrato che sia le rotture del DNA a doppio filamento indotte dall’editing che il genoma stesso dell’Adeno-Associated Virus 6 (AAV) innescano una risposta dipendente da p53 nell'HSPC che risulta in un ritardo della proliferazione con conseguente diminuzione della ricostituzione ematopoietica dopo il trapianto delle cellule editate in animali immuno-compromessi. Per cui, abbiamo quindi dimostrato come la soppressione di questa risposta mediante l’espressione transitoria della forma negativa dominante di p53 preservi la ricostituzione del lineage ematopoietico. Tuttavia, la biologia sottostante è rimasta sconosciuta, così come l'impatto dell'editing genetico sulle dinamiche clonali dell'HSPC modificate con riparo diretto per omologia (Homology Directed Repair, HDR) al momento del trapianto. Inoltre, lo stato quiescente delle HSC primitive costituisce un limite per l’editing genetico mediato da HDR, riducendo le sue possibili applicazioni cliniche. In questo lavoro, abbiamo prima superato tale limite esprimendo transitoriamente la proteina dell'adenovirus 5 E4orf6/7, che regola il principale controllore del ciclo cellulare, E2F, insieme alla nucleasi. Mediante un'analisi dell'espressione genica globale e mirata, abbiamo dimostrato come E4orf6/7 spinga le cellule in fase S/G2 con concomitante sovra-regolazione di tutti i principali componenti del macchinario HDR, aumentando così l'efficienza dell'inserimento del transgene in cellule precedentemente quiescenti. Nel contesto dello xenotrapianto, l'espressione combinata di E4orf6/7 e l'inibizione di p53 hanno migliorato l'efficienza del HDR (>50%) all'interno dell'innesto umano totale, superando i livelli riportati fino ad ora in letteratura. Tale risultato è stato riprodotto in diversi donatori da diverse fonti di HSPC e sono stati modificati più loci genomici, dimostrando la maggior versatilità di questa piattaforma se paragonata ad altre strategie di editing. In parallelo, abbiamo ideato una nuova tecnologia (BAR-seq) che consente il monitoraggio clonale di singole HSC modificate con HDR. Questo approccio prevede l’introduzione di un codice a barre ereditabile univoco (BAR) nel templato AAV6 necessario al HDR. Il sequenziamento ad alta copertura di tali sequenze negli xenotrapianti ha mostrato come l’editing genetico risulti in un attecchimento di pochi cloni dominanti. Mentre l'inibizione transitoria di p53 durante l’editing ha consentito un aumento sostanziale della composizione clonale dell'innesto senza alterare la capacità ripopolante delle HSC. Inoltre, questi dati suggeriscono come la risposta mediata da p53 sia responsabile di un'ematopoiesi oligoclonale. È importante sottolineare che il BAR-seq ha fornito la prima prova diretta che le HSC umane modificate con HDR mantengono un potenziale multilineage e subiscono più cicli di divisioni simmetriche e asimmetriche nei trapianti primari e secondari. In conclusione, auspichiamo che i miglioramenti messi a punto nel nostro protocollo di editing possano ampliare le possibili applicazioni cliniche dell’editing genetico.<br>The scope of genome engineering in hematopoietic stem/progenitor cells (HSPCs) has broadened from random to precise genome insertions for treating genetic diseases of the blood lineages. Targeted editing of inherited mutant genes allows in situ correction and functional reconstitution with preserved expression control. We recently showed that both the induced double-strand DNA breaks and the AAV6 genome trigger a p53-dependent DNA damage response in HSPC delaying proliferation and decreasing hematopoietic reconstitution after xenotransplantation. Suppression of this response by transient expression of a dominant negative p53 released cell-cycle block and rescued hematopoietic reconstitution. Yet, the underlying biology remained unknown as well as the impact of gene editing on clonal dynamics of HDR-edited HSPC upon transplantation. Moreover, it has long been contended that the quiescence of primitive HSC constrains HDR-mediated gene editing, thus limiting its perspective clinical applications in several diseases. Here, we first overcame such constraints by transiently expressing the adenovirus 5 protein E4orf6/7, which operates the major cell cycle controller E2F, together with the nuclease. By global and targeted gene expression analysis we showed engagement of targeted cells in S/G2 phases with concomitant upregulation of all major components of the HDR machinery, thus increasing the efficiency of targeted transgene insertion. Combined E4orf6/7 expression and p53 inhibition enhanced >50% HDR efficiency within human graft surpassing the levels reported until now in the literature. Such outcome was reproducible across several HSPC donors and sources, genomic loci and conceivably portable to most types of editing platforms. In parallel, we devised a novel technology (BAR-seq) which enables clonal tracking of individual HDR-edited HSC by introducing a unique heritable barcode in the AAV6 template. Deep sequencing of integrated BARs in human hematochimeric mice showed that only few (5-10) dominant clones of edited HSC robustly contributed to the hematopoietic graft long-term after transplant. Transient p53 inhibition during editing enabled substantial increase in polyclonal graft composition without altering individual HSC output, thus explaining the improved engraftment and highlighting the p53-mediated response as culprit of an otherwise oligoclonal hematopoiesis. Importantly, BAR-seq provided the first direct evidence that human HDR-edited HSC maintain multilineage potential and undergo multiple rounds of symmetric and asymmetric divisions in primary and secondary xenogeneic hosts. Altogether, we expect that the substantial gains obtained in HDR efficiency and polyclonal repopulation by our improved editing protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

Flores díclinas originam-se da ausência funcional/estrutural de um dos verticilos envolvidos na reprodução, seja desde o início ou no decorrer do desenvolvimento dos primórdios. Este trabalho visa ao estudo do desenvolvimento floral de Parkia multijuga e Stryphnodendron adstringens, leguminosas andromonoicas, a fim de verificar se as flores estaminadas se formam por ausência do carpelo desde o início do desenvolvimento ou por seu aborto no decorrer do desenvolvimento. Botões florais de vários tamanhos e flores foram coletados e processados para observações em microscopia eletrônica de varredura (MEV) e microscopia de luz (ML). O desenvolvimento dos órgãos florais inicia-se com o surgimento de cinco primórdios de sépalas no meristema floral, a partir do lado abaxial em P. multijuga e adaxial em S. adstringes: em alguns botões, um sexto primórdio de sépala inicia-se tardiamente; cinco primórdios de pétalas simultaneamente em P. multijuga e bidirecionalmente em S. adstringes; um primórdio de carpelo concomitante aos cinco primórdios de estames antessépalos em P. multijuga, e após a formação das pétalas em S. adstringes; e cinco primórdios de estames antepétalos. As etapas finais do desenvolvimento são similares entre as espécies. Os estames iniciados em dois verticilos, um mais externo (antessépalo) e outro mais interno (antepétalo), encontram-se arranjados em um único verticilo. Entretanto, os estames antessépalos, que se iniciaram primeiro, apresentam-se um pouco mais alongados que os antepétalos. Em P. multijuga os filetes unem-se na base durante seu alongamento, formando um tubo que, posteriormente, torna-se adnato às pétalas. Nas flores hermafroditas de ambas as espécies, o primórdio carpelar inicia-se, alonga-se, a fenda carpelar se fecha e, logo após, começa a diferenciação do estilete. Já nas flores estaminadas, o primórdio carpelar inicia-se e alonga-se, mas sua fenda não se fecha e o carpelo não termina seu desenvolvimento. Na fase final, as flores hermafroditas apresentam estigmas completamente diferenciados, enquanto que nas flores estaminadas, o primórdio carpelar permanece como um rudimento na base do botão floral. Não há iniciação de óvulos neste caso. Conclui-se que as flores estaminadas em ambas as espécies surgem por aborto do carpelo e não por sua ausência desde o início, semelhante a outros membros de Mimosoideae.<br>Diclinous flowers originate by functional/structural absence of one of the reproductive whorls, from the inception or by abortion in the development. This study aims to compare the floral development of two andromonoecious legumes, Parkia multijuga and Stryphnodendron adstringens, in order to verify which process acts in the staminate flower formation (carpel absence from inception or by abortion). Flowers and flower buds of various sizes were collected and processed for observations in scanning electron microscopy (SEM) and light microscopy (LM). The development of floral organs begins with the emergence of five sepal primordia in the floral meristem from the abaxial side (P. multijuga) and from the adaxial side (S. adstringens) in which in some buds, a sixth primordia arises late; five petal primordial simultaneously in P. multijuga and bidirectionally in S. adstringens, a carpel primordium emergence concomitantly to five antesepalous stamens primordia in P. multijuga, and after the petal formation in S. adstringens, and five antepetalous stamen primordium. In the final stages of the development, the stamens initiated in two whorls, one outer (antesepalous) and the other innermost (antepetalous), are arranged in a single whorl. However, the antesepalous stamens, which emerged first, are a little more elongated than the antepetalous ones. In P. multijuga the fillets join in the base while its elongation, forming a tube, which thereafter becomes adnate to the petals. In hermaphrodite flowers, in both species, the carpel primordium emerges, stretches, the carpel cleft closes and, soon after, the style begins its differentiation. In staminate flowers, the carpel primordium begins and stretches, but the carpel cleft does not close and the carpel does not end its development. In the mature stage, the hermaphrodite flowers present fully differentiated stigmas, while in the staminate flowers, the carpel primordium remains as a rudiment at the base of the bud. In this case, there is no initiation of any ovule. We conclude that staminate flowers in both species arise by carpel abortion and not from inception, alike the other members of Mimosoideae.

碩士<br>國立臺灣大學<br>植物學系<br>86<br>Abstract The changes of staminate flower and pollen during different developmental stages of Luffa cylindrica (L.) Roem were studied with light microscope, scanning and transmission electron microscope. Floral primodium initiated from infloresence primodium in a spiral order. Five sepal primodia intiated from the edge of a floral primodium in a spiral order, too. Then five petal primodia arised simultaneously in a whorl arrangement, followed by five stamen primodia. Consequently, the stamen grew up and developed a curved anther. The anther wall layers occurred as dicotyledonous type and were composed of epidermis, endothecium, middle layers (2 ~ 3 layers) and tapetum. Sporogenous tissue was derived from the inner layer, which was resulted from periclinal division of the hypodermal layer. Callose wall was deposited between the plasma membrane and cell wall of microspore mother cells before meiosis. The cytokinesis after meiosis occurred simultaneously by the centripetal invasion of callose wall, then a tetrahedral tetrad enclosed in the callose wall was produced. After callose wall was dissolved, four microspores were released into anther loculus. At the same time, tapetal cell wall was disintegrated, and deposition of lipid droplets started in tapetal cytoplasm. Deposition of lipid droplets also occurred in the cytosol of microspores during the middle and late microspore stage. Haploidal mitosis of microspore took place, which resulted in a big vegetative cell and a small generative cell. Mature pollen grains contained two kinds of reserves, lipid droplet and starch grain; the former was produced in the cytosol and the latter was deposited inside the amyloplasts. The secretory tapetum reached its optimal activity from the late tetrad to early microspore stage. Two organelles involved in production of lipid droplets were smooth endoplasmic reticulum (SER) and elaioplast. As tapetal cytoplasm degenerated, clusters of lipid droplets that were deposited within the cytosol and elaioplast moved towards the pollen grain. At anthesis, pollenkitt derived from tapetum were coated on the surface of the mature pollen grain. Development of pollen wall started during the early tetrad stage. At the beginning, a fibrillar material was deposited between the callose wall and plasma membrane of microspores. The primexine was then accumulated on the position where fibrillar material possessed. Subsequently, exine material secreted by microspore itself was accumulated on the same position to replace primexine. The tectum and bacule had been formed before the discontinuous foot layer was visible. There were two distinguishable laye in endexine. The outer one seemed to be formed after the appearance of white lines which was just beneath the foot layer at the late tetrad stage, and the inner one was formed after dissolution of callose wall and composed of a number of sporopollenin-like granules. At the late microspore stage that was just before the haploidal mitosis of microspore, the intine was formed under the endexine at aperatural zone. After mitosis, the formation of intine finished around the pollen grain beneath the inner endexi . There were three layers in the intine of aperatural zone. Fibrillar material between the bacules and intine showed PAS positive reaction. Besides PAS, the intine of aperatural zone also showed Coomassie blue positive reaction. The results of this study present a detailed information about the microsporogenesis and pollen wall development of Luffa cylindrica (L.) Roem, and ascertained that exine was derived from a combined contribution from tapetum and microspore, rather than from either of them. We presumed that the control of exine pattern has been determined in the microspore mother cell. IV

碩士<br>國立臺灣大學<br>生態學與演化生物學研究所<br>95<br>Koelreuteria henryi Dummer (Sapindaceae) is a deciduous tree and endemic to Taiwan. This species has been widely cultivated as side tree. The inflorescence is thyrse mixed rhipidium. The flower is polygamous-andromonoecious with staminate and hermaphrodite flowers on the same plant, fertile and steril pollen grains are produced respectively. In this study, with the aids of LM, TEM, SEM and histochemical observations, it is attempted to reveal (1) the floral initiation, flowers'' sex differentiation and sex expression in the inflorescence; (2) the similarity and dissimilarity between pollen morphology, pollen viability and cytoplasmic content in staminate and hermaphrodite flowers; (3) the cellular and organellar transformations during the microsporogenesis; and (4) the anther wall configuration at anthesis. The results show that thyrse primordium of K. henryi was emerged from the shoot apical meristem in middle August and subtended conspicuously by bracts, which possesses numerous pedicellate flowers. Five sepal primodia intiated from the floral primodium in a spiral order. Then five petal primordia arised simultaneously in a whorl arrangement, followed by eight stamen primodia arised and last by pistil primodium. The anther wall development of K. henryi conforms to the basic type, consisting of four different tissues: epidermis, endothecium, 2~3 middle layer and tapetum The anthers in staminate flower of K. henryi are opened by longitudinal splits to release 2-celled pollen with the help of insects-pollinators. Although the pollen grains from hermaphrodite flowers have ca. 15.3 % viability when tested with FCR reaction, no pollen grain germinated in vitro. In constrast, those pollen grains from the staminate flowers have higher germination rate (76.8 %) tested in vitro. The results of histochemical staining show that there is a remarkable difference in storage compounds between sterile and fertile pollen grains. In the former, lipids globules with fewer starch grains are detected in the cytoplasm, while in the latter, only lipid globules are detectable. Cytokinesis in microspore mother cells is simultaneous, forming tetrahedral tetrads. The secretory tapetum has two organelles involved in production of lipid bodies were endoplasmic reticulum (ER) and elaioplast. During early-vacuolated stage, the tapetum has lipids in the endoplasmic reticulum. The elaioplasts of the tapetum produce numerous saturated and unsaturated lipids. At maturation stage, the plastoglobules released from elaioplasts coalesced with unsaturated lipids from endoplasmic reticulum to form the pollenkitt mass deposited on the exine. At the time of pre-dehiscence, the epidermal and endothecium are still intact. Fibrous thickenings are formed in the cells of the endothecium. In staminate flowers, the septum separating anther locules was cleaved and the stomium was degenerated to release the pollen grains. In hermaphrodite flowers, all of these structures remained intact without any release of pollen grains. All two pollen grains possess a well-developed pollen wall (exine and intine). The exine is composed of tectum, columellae, foot layer and endexine. The intine (I) of aperture in fertile pollen grains was opened with conspicuous three strata. Sterile pollen grains display a thickness intine and exine in the aperture which was not opened. According to this study, the flowers'' sex differentiation in K. henryi are potentially developed as bisexual and only at the final stage (ovules formed) of development sex organs could be defined. Not only the staminate and hermaphrodite flowers have functionally and structurally difference, but also the fertile and steril pollen grains produced respectively.