Molybdenum experiments: In Australia young Merlot vines sometimes suffer from vegetative disorders such as slow, zigzagged growth and leaf distortion. Merlot is also particularly known as a low- and inconsistent-yielding grape variety. Previous research showed that when foliar applications of molybdenum (Mo) were applied to Merlot vines the vegetative symptoms improved. More recently, when sodium molybdate was applied to Mo-deficient Merlot, yield improved; a function of increased bunch weight brought about by bigger berries. It has also been reported that at high concentrations, molybdenum might be detrimental to yield. Experiments were conducted on own-rooted Merlot (clone D3V14) vines in commercial vineyards in the Adelaide Hills (Hills) and at McLaren Vale, South Australia. Effects of molybdenum deficiency on the vegetative growth and yield of Vitis vinifera cv. Merlot: The aims of the current study were to: a) elucidate the mechanism by which molybdenum affects yield of Merlot; b) to monitor the effects of Mo-treatment on the balance between vine reproductive and vegetative growth; c) to monitor the residual effects of Mo-treatment on growth and yield of Merlot and; d) to determine whether high concentrations of molybdenum are detrimental to yield. Three rates of sodium molybdate were applied to vines in springtime (control = 0g, rate 1 = 0.101g and rate 2 = 0.202g sodium molybdate per vine). Vine molybdenum status was measured prior to treatment and again at flowering time using petiole, shoot tip and inflorescence analysis. The effects on vegetative growth were monitored at veraison, during dormancy and at budburst in the seasons following Mo-treatment. At flowering time, pollen vitality, pollen tube growth and flower structure were examined. Bunch number per vine, fruitset, berry weight and berry composition were measured at harvest. In the Hills, the controls had adequate molybdenum however, at McLaren Vale petiolar molybdenum concentration fell within the suggested deficiency range of 0.05-0.09 mg/kg in the petioles at flowering time. No visual symptoms of Mo-deficiency were observed on the experimental vines. At McLaren Vale, Mo-treatment reduced pruning weight and improved vine balance. Mo-treated vines in the Hills and at McLaren Vale were affected by delayed budburst in the season following Mo-treatment irrespective of their Mo-status. However, no seasonal carryover of molybdenum could be detected in tissue analysis at flowering time. Juice total soluble solids, pH and titratable acidity were not affected by Mo-treatment at McLaren Vale or in the Hills. However, juice from Mo-treated vines in the Hills had a significantly higher concentration of molybdenum than the controls. At McLaren Vale there was no significant difference in juice molybdenum concentration between treatments. In the Hills, yield was not affected by Mo-treatment. However, Mo-treated vines at McLaren Vale had significantly higher yields (approximately double) than the Mo-deficient controls. Bunch number per vine was not affected by Mo-treatment, either in the year that treatments were applied or in the following season. However, bunches from Mo-treated vines had significantly better fruitset resulting in more berries per bunch. Berry weight was affected by Mo-treatment in one season only. Yield was not detrimentally affected on vines that received the higher rate of sodium molybdate. In the Hills, Mo-treatment did not affect pollen numbers, pollen vitality or pollen tube growth. At McLaren Vale, where the controls were Mo-deficient, pollen vitality was not affected by Mo-treatment. However, pollen tube growth was significantly enhanced by Mo-treatment. Significantly more pollen tubes penetrated the ovules from Mo-treated vines and a higher proportion of ovaries had at least one penetrated ovule. Structural observations revealed that a significantly higher proportion of ovules from Mo-deficient vines were defective. The absence of an embryo sac in those ovules is probably the cause of pollen tube growth inhibition and subsequent poor fruitset. Effects of mode of pollination on yield of Merlot and the interacting effects of sodium molybdate sprays: Pollination experiments were conducted on field-grown own-rooted Merlot (clone D3V14) vines in commercial vineyards in the Adelaide Hills and at McLaren Vale in 2003-04 and in 2004-05. Inflorescences were supplied with supplementary Merlot pollen (self-pollination), with pollen from another variety (cross-pollination) or they were left to pollinate naturally (open pollination). In the Hills, mode of pollination did not affect fruitset or berry weight. In 2003-04 fruitset increased significantly at McLaren Vale when inflorescences were cross-pollinated with Semillon. Applying supplementary Merlot pollen also tended to improve fruitset, however none of the treatments affected berry weight. In 2004-05 there was no significant difference between treatments. These results indicate that Merlot may be a poor producer of pollen and may suffer from self-incompatibility. Given the significant improvements in yield gained by spring foliar applications of sodium molybdate to Mo-deficient Merlot vines, in 2005-06 a reciprocal experiment was conducted to separate the effects of Mo-treatment and mode of pollination on the male and female flower parts. The aims of this experiment were to: a) determine whether the male or female reproductive organs are more important in determining the success of fruitset of Merlot and; b) determine which remedial measure, Mo-treatment or pollination, is more effective at overcoming poor fruitset. Supplementary pollination treatments—cross-pollination (Semillon); self-pollination (Mo-deficient pollen); self-pollination (Mo-treated pollen) and; open-pollination—were applied to Mo-treated and Mo-deficient vines. Cross-pollinating Mo-deficient vines with Semillon significantly improved fruitset of Merlot compared to other pollination treatments on those vines, however applying molybdenum to the vines in springtime was more effective at improving fruitset. Within the Mo-treated vines the effects of supplementary pollination on fruitset were not thought to be of any practical significance. The results of this experiment provide further evidence that Mo-deficiency affects the female flower parts more than the male reproductive organs of Merlot. The occurrence of ‘star’ flowers in Australia: In 2003 faulty flowers were discovered on Canada Muscat grown in the Coombe Vineyard at the University of Adelaide’s Waite Campus. The Canada Muscat flowers opened from the top in ‘star’ formation in contrast to normal grape flowers, which shed the calyptra from its base. Star flowers were reported in French literature in the late 1800s. They were reported to as a symptom of a ‘disease’ that caused ‘coulure’, the cure for which was vine removal. The current report is the first known report of star flowers occurring in Australia. Through dissemination of the news of this discovery, several star flower variants were found in other varieties in South Australia. The association of star flowers with poor berry development and the frequency of the occurrence of star flowers suggest that this flower aberration may be affecting yield to a greater extent than previously recognised. This study provides a detailed description of two types of star flowers: those that occur in response to environmental conditions and those that occur every season. Other star flower variants are also documented. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1280856 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

The olive (Olea europaea L.) is one of the most ancient fruit trees and has been cultivated for its oil in the Mediterranean area for thousands of years. Today, the consumption of olive oil and table olives is increasing both in traditional producing countries and the entire world. Most olive cultivars are self-incompatible and do not produce a commercial yield after self pollination. In this thesis, inflorescence architecture and sexual compatibility relationships of some olive cultivars, and gene expression in olive pistils during flowering were studied. To study the inflorescence architecture of olive, 45 inflorescences in each of the cultivars Manzanillo, Mission, and Frantoio were checked every morning from flower opening to petal fall. The flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than flowers located on the secondary branches. Flower position also had a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal halves, on which the flowers tend to be perfect, of 120 inflorescences in three trees of Manzanillo were removed about one month before full bloom. This resulted in a highly significant increase in the percentage of perfect flowers on the proximal halves. The effects of shoot orientation and inflorescence location on inflorescence characteristics in the cultivars Frantoio, Kalamata, and Koroneiki were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle, and base) and four sides of the three selected trees (north, south, east, and west) were recorded. The statistical analysis showed that basal inflorescences were shorter and with fewer flowers but with the same percentage of perfect flowers. Shoot orientation did not have any influence on these characteristics in any of the cultivars. Sexual compatibility was assessed using two methods. In the first method, controlled crossings were performed in the cultivars Frantoio, Koroneiki, and Kalamata. The pistils were harvested one week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol, and observed under a fluorescence microscope. In Frantoio and Koroneiki, the number of ovules penetrated by a pollen tube was used to estimate the level of sexual compatibility. In Kalamata, the numbers of ovules penetrated by pollen tubes were not significantly different between treatments; therefore, the number of pollen tubes in the lower style was used. All the cultivars studied were self- incompatible. Frantoio (as a host) was incompatible with Koroneiki and Barnea but partially compatible with Mission. Koroneiki (as a host) was incompatible with Barnea but partially compatible with Frantoio and Mission. Kalamata (as a host) was compatible with Barnea, incompatible with Mission and Koroneiki in 2004, but partially compatible with them in 2005. In the second method, eight microsatellite markers were used for genotyping three Kalamata mother trees, 40 embryos per mother tree, and all the potential pollen donors. Genotyping data were analysed using FaMoz software, and the number of embryos assigned to each putative pollen donor was determined. Paternity analysis showed that Kalamata (as a host) was self-incompatible, compatible with Barnea, Benito, and Katsourela, but incompatible with Arbequina, Azapa, and Picual. To study the gene expression in olive pistils during flowering, a genomic approach was initiated using cDNA subtractive array analysis. Total RNA was isolated from olive pistils at two developmental stages, where self-incompatibility (SI) genes are expected to be differentially expressed: 1) small green flower buds (expression of SI genes not expected) and 2) large white flower buds containing receptive pistils just prior to opening (expression of SI genes expected). From each stage, cDNA libraries were prepared and put through forward and reverse subtractive hybridisations to enrich for differentially expressed cDNAs in stage 2. Macroarrays were prepared by printing 2304 differentially expressed cDNAs onto nylon membranes and hybridised with forwardand reverse-subtracted probes. The analysis identified 90 up-regulated cDNA clones highly expressed in receptive pistils. Further subtracted and unsubtracted hybridisations confirmed up-regulation of the majority of these cDNAs. Gene expression profiles across different tissues showed that most of the genes were pistil-specific. The expression pattern of the genes showed high similarity in Kalamata, Frantoio, Barnea, and Pendolino. All the screened genes were sequenced and their similarities were searched in the NCBI database. The most redundant and interesting up-regulated clones were those similar to a receptor protein kinase-like protein. Some versions of this protein play a role in the sporophytic SI system of Brassica and the gametophytic SI system of Papaver and rye. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325369 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Molybdenum experiments: In Australia young Merlot vines sometimes suffer from vegetative disorders such as slow, zigzagged growth and leaf distortion. Merlot is also particularly known as a low- and inconsistent-yielding grape variety. Previous research showed that when foliar applications of molybdenum (Mo) were applied to Merlot vines the vegetative symptoms improved. More recently, when sodium molybdate was applied to Mo-deficient Merlot, yield improved; a function of increased bunch weight brought about by bigger berries. It has also been reported that at high concentrations, molybdenum might be detrimental to yield. Experiments were conducted on own-rooted Merlot (clone D3V14) vines in commercial vineyards in the Adelaide Hills (Hills) and at McLaren Vale, South Australia. Effects of molybdenum deficiency on the vegetative growth and yield of Vitis vinifera cv. Merlot: The aims of the current study were to: a) elucidate the mechanism by which molybdenum affects yield of Merlot; b) to monitor the effects of Mo-treatment on the balance between vine reproductive and vegetative growth; c) to monitor the residual effects of Mo-treatment on growth and yield of Merlot and; d) to determine whether high concentrations of molybdenum are detrimental to yield. Three rates of sodium molybdate were applied to vines in springtime (control = 0g, rate 1 = 0.101g and rate 2 = 0.202g sodium molybdate per vine). Vine molybdenum status was measured prior to treatment and again at flowering time using petiole, shoot tip and inflorescence analysis. The effects on vegetative growth were monitored at veraison, during dormancy and at budburst in the seasons following Mo-treatment. At flowering time, pollen vitality, pollen tube growth and flower structure were examined. Bunch number per vine, fruitset, berry weight and berry composition were measured at harvest. In the Hills, the controls had adequate molybdenum however, at McLaren Vale petiolar molybdenum concentration fell within the suggested deficiency range of 0.05-0.09 mg/kg in the petioles at flowering time. No visual symptoms of Mo-deficiency were observed on the experimental vines. At McLaren Vale, Mo-treatment reduced pruning weight and improved vine balance. Mo-treated vines in the Hills and at McLaren Vale were affected by delayed budburst in the season following Mo-treatment irrespective of their Mo-status. However, no seasonal carryover of molybdenum could be detected in tissue analysis at flowering time. Juice total soluble solids, pH and titratable acidity were not affected by Mo-treatment at McLaren Vale or in the Hills. However, juice from Mo-treated vines in the Hills had a significantly higher concentration of molybdenum than the controls. At McLaren Vale there was no significant difference in juice molybdenum concentration between treatments. In the Hills, yield was not affected by Mo-treatment. However, Mo-treated vines at McLaren Vale had significantly higher yields (approximately double) than the Mo-deficient controls. Bunch number per vine was not affected by Mo-treatment, either in the year that treatments were applied or in the following season. However, bunches from Mo-treated vines had significantly better fruitset resulting in more berries per bunch. Berry weight was affected by Mo-treatment in one season only. Yield was not detrimentally affected on vines that received the higher rate of sodium molybdate. In the Hills, Mo-treatment did not affect pollen numbers, pollen vitality or pollen tube growth. At McLaren Vale, where the controls were Mo-deficient, pollen vitality was not affected by Mo-treatment. However, pollen tube growth was significantly enhanced by Mo-treatment. Significantly more pollen tubes penetrated the ovules from Mo-treated vines and a higher proportion of ovaries had at least one penetrated ovule. Structural observations revealed that a significantly higher proportion of ovules from Mo-deficient vines were defective. The absence of an embryo sac in those ovules is probably the cause of pollen tube growth inhibition and subsequent poor fruitset. Effects of mode of pollination on yield of Merlot and the interacting effects of sodium molybdate sprays: Pollination experiments were conducted on field-grown own-rooted Merlot (clone D3V14) vines in commercial vineyards in the Adelaide Hills and at McLaren Vale in 2003-04 and in 2004-05. Inflorescences were supplied with supplementary Merlot pollen (self-pollination), with pollen from another variety (cross-pollination) or they were left to pollinate naturally (open pollination). In the Hills, mode of pollination did not affect fruitset or berry weight. In 2003-04 fruitset increased significantly at McLaren Vale when inflorescences were cross-pollinated with Semillon. Applying supplementary Merlot pollen also tended to improve fruitset, however none of the treatments affected berry weight. In 2004-05 there was no significant difference between treatments. These results indicate that Merlot may be a poor producer of pollen and may suffer from self-incompatibility. Given the significant improvements in yield gained by spring foliar applications of sodium molybdate to Mo-deficient Merlot vines, in 2005-06 a reciprocal experiment was conducted to separate the effects of Mo-treatment and mode of pollination on the male and female flower parts. The aims of this experiment were to: a) determine whether the male or female reproductive organs are more important in determining the success of fruitset of Merlot and; b) determine which remedial measure, Mo-treatment or pollination, is more effective at overcoming poor fruitset. Supplementary pollination treatments—cross-pollination (Semillon); self-pollination (Mo-deficient pollen); self-pollination (Mo-treated pollen) and; open-pollination—were applied to Mo-treated and Mo-deficient vines. Cross-pollinating Mo-deficient vines with Semillon significantly improved fruitset of Merlot compared to other pollination treatments on those vines, however applying molybdenum to the vines in springtime was more effective at improving fruitset. Within the Mo-treated vines the effects of supplementary pollination on fruitset were not thought to be of any practical significance. The results of this experiment provide further evidence that Mo-deficiency affects the female flower parts more than the male reproductive organs of Merlot. The occurrence of ‘star’ flowers in Australia: In 2003 faulty flowers were discovered on Canada Muscat grown in the Coombe Vineyard at the University of Adelaide’s Waite Campus. The Canada Muscat flowers opened from the top in ‘star’ formation in contrast to normal grape flowers, which shed the calyptra from its base. Star flowers were reported in French literature in the late 1800s. They were reported to as a symptom of a ‘disease’ that caused ‘coulure’, the cure for which was vine removal. The current report is the first known report of star flowers occurring in Australia. Through dissemination of the news of this discovery, several star flower variants were found in other varieties in South Australia. The association of star flowers with poor berry development and the frequency of the occurrence of star flowers suggest that this flower aberration may be affecting yield to a greater extent than previously recognised. This study provides a detailed description of two types of star flowers: those that occur in response to environmental conditions and those that occur every season. Other star flower variants are also documented. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1280856 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

The olive (Olea europaea L.) is one of the most ancient fruit trees and has been cultivated for its oil in the Mediterranean area for thousands of years. Today, the consumption of olive oil and table olives is increasing both in traditional producing countries and the entire world. Most olive cultivars are self-incompatible and do not produce a commercial yield after self pollination. In this thesis, inflorescence architecture and sexual compatibility relationships of some olive cultivars, and gene expression in olive pistils during flowering were studied. To study the inflorescence architecture of olive, 45 inflorescences in each of the cultivars Manzanillo, Mission, and Frantoio were checked every morning from flower opening to petal fall. The flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than flowers located on the secondary branches. Flower position also had a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal halves, on which the flowers tend to be perfect, of 120 inflorescences in three trees of Manzanillo were removed about one month before full bloom. This resulted in a highly significant increase in the percentage of perfect flowers on the proximal halves. The effects of shoot orientation and inflorescence location on inflorescence characteristics in the cultivars Frantoio, Kalamata, and Koroneiki were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle, and base) and four sides of the three selected trees (north, south, east, and west) were recorded. The statistical analysis showed that basal inflorescences were shorter and with fewer flowers but with the same percentage of perfect flowers. Shoot orientation did not have any influence on these characteristics in any of the cultivars. Sexual compatibility was assessed using two methods. In the first method, controlled crossings were performed in the cultivars Frantoio, Koroneiki, and Kalamata. The pistils were harvested one week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol, and observed under a fluorescence microscope. In Frantoio and Koroneiki, the number of ovules penetrated by a pollen tube was used to estimate the level of sexual compatibility. In Kalamata, the numbers of ovules penetrated by pollen tubes were not significantly different between treatments; therefore, the number of pollen tubes in the lower style was used. All the cultivars studied were self- incompatible. Frantoio (as a host) was incompatible with Koroneiki and Barnea but partially compatible with Mission. Koroneiki (as a host) was incompatible with Barnea but partially compatible with Frantoio and Mission. Kalamata (as a host) was compatible with Barnea, incompatible with Mission and Koroneiki in 2004, but partially compatible with them in 2005. In the second method, eight microsatellite markers were used for genotyping three Kalamata mother trees, 40 embryos per mother tree, and all the potential pollen donors. Genotyping data were analysed using FaMoz software, and the number of embryos assigned to each putative pollen donor was determined. Paternity analysis showed that Kalamata (as a host) was self-incompatible, compatible with Barnea, Benito, and Katsourela, but incompatible with Arbequina, Azapa, and Picual. To study the gene expression in olive pistils during flowering, a genomic approach was initiated using cDNA subtractive array analysis. Total RNA was isolated from olive pistils at two developmental stages, where self-incompatibility (SI) genes are expected to be differentially expressed: 1) small green flower buds (expression of SI genes not expected) and 2) large white flower buds containing receptive pistils just prior to opening (expression of SI genes expected). From each stage, cDNA libraries were prepared and put through forward and reverse subtractive hybridisations to enrich for differentially expressed cDNAs in stage 2. Macroarrays were prepared by printing 2304 differentially expressed cDNAs onto nylon membranes and hybridised with forwardand reverse-subtracted probes. The analysis identified 90 up-regulated cDNA clones highly expressed in receptive pistils. Further subtracted and unsubtracted hybridisations confirmed up-regulation of the majority of these cDNAs. Gene expression profiles across different tissues showed that most of the genes were pistil-specific. The expression pattern of the genes showed high similarity in Kalamata, Frantoio, Barnea, and Pendolino. All the screened genes were sequenced and their similarities were searched in the NCBI database. The most redundant and interesting up-regulated clones were those similar to a receptor protein kinase-like protein. Some versions of this protein play a role in the sporophytic SI system of Brassica and the gametophytic SI system of Papaver and rye. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325369 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Orientador: Simone de Pádua Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T06:00:00Z (GMT). No. of bitstreams: 1 Costa_MarinaFernandaBortolin_M.pdf: 6136363 bytes, checksum: 7ebcccef8efb53a979a8fa6e1e6d3a96 (MD5) Previous issue date: 2011 / Resumo: Indigofera L., terceiro maior gênero de Leguminosae, possui cerca de 700 espécies, tropicais e subtropicais, descritas como melitófilas, com hábitos variados, e ampla ocorrência em áreas degradadas e de Cerrado. Estudos prévios mostraram que o estigma de I. lespedezioides e I. suffruticosa é secretor e cuticularizado e que suas flores não produzem frutos por autopolinização espontânea. Assim, este trabalho avaliou a morfologia da interação pólen-pistilo em quatro espécies relacionadas e neotropicais de Indigofera: I. hirsuta, I. lespedezioides, I. suffruticosa e I. truxillensis. Pretendeu-se compreender o comportamento do grão de pólen e do tubo polínico no pistilo, em especial no estigma destas espécies, caracterizado como semi-seco. Grão de pólen, estigma, estilete e ovário de botões florais e flores foram observados em microscopias eletrônica e de luz. Testes para a detecção de substâncias foram realizados em grão de pólen e estigma. Polinizações induzidas foram realizadas visando à compreensão do sistema reprodutivo das espécies. O grão de pólen é harmomegático, tricolporado, prolato, com exina perfurada e pouco "pollenkitt". O estigma é revestido por cutícula sem poros, constituído por células secretoras e parcialmente delimitado por tricomas tectores. A secreção, hidrofílica e lipofílica, fica retida nos espaços intercelular e subcuticular. Após autopolinização e polinização cruzada, indivíduos das quatro espécies apresentaram grãos de pólen germinando no estigma e tubos polínicos crescendo pelo estilete até o ovário. Indigofera hirsuta foi a única espécie que produziu frutos por autopolinização espontânea. Nas quatro espécies, o grão de pólen adere-se ao estigma receptivo, reidrata-se e germina emitindo o tubo polínico que percorre os espaços intercelulares do tecido transmissor, cresce pelo espaço central ou pela margem do canal estilar, penetrando o óvulo pela micrópila, 6 a 24h após a polinização. A escassez de "pollenkitt", a secreção estigmática abundante e as visitas frequentes de abelhas sugerem que, nas espécies que não produzem frutos por autopolinização espontânea, a germinação polínica depende do polinizador para romper a cutícula do estigma possibilitando o contato do pólen com a secreção. Em I. hirsuta, a ruptura da cutícula deve ocorrer devido à pressão exercida pela secreção estigmática ou pelo contato com as anteras. Este trabalho confirma a presença de estigma semi-seco em Indigofera e sugere que I. hirsuta, I. lespedezioides, I. suffruticosa e I. truxillensis são espécies autocompatíveis, além de fornecer dados reprodutivos para este gênero e para a tribo Indigoferae, grupos pouco estudados sob este enfoque / Abstract: Indigofera is the third largest genus of Leguminosae with tropical and subtropical distribution, is considered melittophilous, with varied habits and wide occurrence in degraded areas and Cerrado ecosystems of Brazil. Previous research demonstrated that I. lespedezioides and I. suffruticosa have secretory stigma covered by cuticle and fruit are not produced by spontaneous self-pollination. Thus, this work evaluated the morphology of pollen-pistil interaction of four related and neotropical species of Indigofera: I. hirsuta, I. lespedezioides, I. suffruticosa and I. truxillensis. Pollen grain, stigma, style and ovary of flower buds and flowers were examined under electron and light microscopy. Tests to detect substances were made with pollen grain and stigma. Controlled pollinations were realized aiming at helping to understand the reproductive systems of these species. The pollen grain is harmomegathic, tricolporate, prolate, with perforated exine and a small quantity of pollenkitt. The stigma, considered semi-dry, is covered by a cuticle, partially covered by trichomes and has secretory cells. The hydrophilic and lipophilic secretion is retained in intercellular and subcuticular spaces. Pores were not found at the stigmatic cuticle. After self-pollination and cross-pollination experiments, individuals of four species had germinating pollen grains on stigma and pollen tubes growing by style toward ovary. Indigofera hirsuta was the only species that produced fruit after spontaneous self-pollination. The pollen grain adheres to the receptive stigma, rehydrates and germinates, emitting the pollen tube through the stigmatic cells spaces. The pollen tube passes through the intercellular spaces of the transmitting tissue and it grows through the central space of the style or around the edge of the style channel. The pollen tube penetrates the micropyle about 6 to 24h after pollination. The scarcity of "pollenkitt", the abundant stigmatic secretion, and the frequent visits of stingless bees suggest that, in the species that do not have spontaneous self-pollination, the pollen germination depends on the pollinator to break the stigmatic cuticle allowing the contact between pollen and stigmatic secretion. In I. hirsuta, the cuticle rupture may occur due to stigmatic secretion pressure. This study confirms the presence of a semi-dry stigma in Indigofera and suggests that I. hirsuta, I. lespedezioides, I. suffruticosa and I. truxillensis are self-compatible, providing reproductive data for these genera and for the tribe Indigoferae, groups that are poorly studied under this approach / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal

Chez les plantes à fleurs, la communication entre les grains de pollen et les cellules épidermiques du stigmate, aussi appelées papilles, est cruciale pour le succès de la reproduction. Lorsqu’il est accepté, le grain de pollen germe et émet un tube pollinique qui transporte les gamètes mâles jusqu’aux ovules. La rencontre et la fusion entre les gamètes mâles et femelles reposent par conséquent sur la bonne trajectoire des tubes polliniques lors de leur progression dans les différents tissus du partenaire femelle pour atteindre les ovules. Les tubes polliniques croissent dans la paroi cellulaire des papilles stigmatiques et génèrent une pression sur ces dernières. De telles forces sont connues pour modifier le réseau de microtubules corticaux (MTC) ainsi que le comportement de la cellule. La première partie de ma thèse a consisté à étudier le rôle des MTC du stigmate dans le contrôle de la croissance du tube pollinique. En combinant imagerie cellulaire et approches génétiques, nous avons mis en évidence que chez le mutant katanin1-5 (ktn1-5) d’Arabidopsis, les papilles ont un réseau de MTC très isotrope, associé à une forte tendance des tubes polliniques sauvages à faire des spires autour des papilles. Ce phénotype a pu être partiellement reproduit par traitement des papilles avec un agent dépolymérisant les MTC, l’oryzaline. Compte tenu que le réseau de MTC est fortement lié à l’organisation des fibres de cellulose, et donc potentiellement à la rigidité de la paroi, nous avons mesuré la rigidité des papilles du mutant ktn1-5 grâce au microscope à force atomique. L’ensemble de ces résultats suggère que la KATANIN, en régulant l’organisation des MTC et conférant des propriétés mécaniques particulières à la paroi cellulaire, joue un rôle primordial dans le guidage des tubes polliniques lors de leur croissance dans les papilles stigmatiques. De façon similaire à la croissance des tubes dans les papilles, les hyphes des pathogènes filamenteux pénètrent les tissus épidermiques de leur hôte. Lors d’une attaque par un pathogène, les cellules de l’épiderme de l’hôte réagissent rapidement pour mettre en place une réponse appropriée, décisive sur le résultat de l’interaction plante-pathogène. La seconde partie de ma thèse a eu pour objectif de comparer la réponse cellulaire des papilles stigmatiques suite à l’invasion par deux types d’organismes, le tube pollinique lors de la pollinisation et les hyphes de deux Oomycètes pathogènes, Phytophtora parasitica et Hyaloperonospora arabidopsidis durant leurs processus d’infection. Nos résultats démontrent que la papille stigmatique est capable d’adapter sa réponse en fonction de l’identité de l’envahisseur. / The epidermis is the first cellular barrier in direct contact with the environment in both animal and plant organisms. In plants, the result of the cell-to-cell communication that occurs between the pollen grain and the epidermal cells of the stigma, also called papillae, is crucial for successful reproduction. When accepted, the pollen grain germinates and emits a pollen tube that transports the male gametes towards the ovules. Effective fertilization in angiosperms depends on the proper trajectory that pollen tubes take while progressing within the pistil tissues to reach the ovules.Pollen tubes grow within the cell wall of the papilla cells, applying pressure to the wall. Such forces are known to alter the cortical microtubule (CMT) network and cell behaviour. The first part of my PhD thesis aimed at investigating the role of the microtubule cytoskeleton of stigmatic cells in pollen tube growth. By combining cell imaging and genetic approaches, we found that in the Arabidopsis katanin1-5 (ktn1-5) mutant, papillae have a highly isotropic CMT array, associated with a marked tendency of wild-type (WT) pollen tube to turn around the papillae. We could partially phenocopy this coiled growth of pollen tubes by treating WT papillae with the microtubule-depolymerizing drug oryzalin. As CMT pattern is linked to cellulose microfibrils organisation, and hence possibly to cell-wall stiffness, we assessed the stiffness of ktn1-5 and aged papillae using Atomic Force Microscopy. Altogether, our results suggest that both organisation of CMT and cell wall properties dependent on KATANIN have a major role in guiding early pollen tube growth in stigma papillae.Similarly to pollen tube growth within the stigmatic papilla, hypha of filamentous pathogens penetrates the epidermal tissue of the host. During pathogen attacks, epidermal cells promptly react to the invading organisms to adjust the most relevant response. Early response of the first cell layers including epidermal cells is decisive for the result of plant-pathogen interactions. The second part of my PhD work aimed at comparing the cellular response of stigmatic cells challenged by two types of invaders, the pollen tube during pollination and hyphae of two oomycete filamentous pathogens, Phytophtora parasitica and Hyaloperonospora arabidopsidis, during the infection process. We demonstrate that a stigmatic cell challenged by a pollen tube or an oomycete hypha adapts its response to the invader’s identity.

Dynamic actin remodeling is at the core of a number of fundamental cellular processes in a variety of organisms ranging from animal neuronal outgrowth to pollen tube growth during plant reproduction and asymmetrical cell division in budding yeast. Such dynamism results from a concerted effort of a number of temporally and spatially regulated actin binding proteins. The polymerization and depolymerization of elaborate F-actin networks takes place by the addition and removal of actin subunits at the two filament ends with each end possessing distinct properties, making the filaments themselves polar structures with a fast growing “barbed end” and a slow growing “pointed end”. Although actin polymerization at the barbed end is an efficient and favorable process, the initial filament nucleation step is a much more inefficient and thermodynamically unfavorable process requiring the need for a variety of actin nucleating proteins. These nucleating proteins not only determine the precise location of actin assembly at the cell membrane during polarized cell growth but also directly control the number of force producing filaments. Here we have concentrated our efforts in characterizing a number of Arabidopsis thaliana , Group I membrane bound, formin homologues AFH1, AFH5, AFH6, AFH7, AFH8 and AFH11 using a range of functional genomics tools. Consequently we have shown that AFH5 is a pollen expressed actin nucleating protein localized at the tip of the polarized tube. Genetic alterations of the AFH5 gene using T-DNA gene knockout and gene overexpression both show distinct deformities at the tip of the rapidly growing pollen tube leading to inefficient fertilization during plant reproduction and reduced silique length. In the tip growing root hair cells of Arabidopsis the afh5 mutants show a wavy and bulgy phenotype at the hypocotyl region while the afh1 mutants project branched, split root hairs along the primary root. Although our results do indicate that AFH1 and AFH5 are expressed in the polarized pollen tubes and root hairs their, expression and hence activity is spatially controlled and restricted to different parts of the cell.

The overall aim of this PhD study was to develop protocols to improve the efficiency of eucalypt controlled pollinations (CPs) in order to make it more cost-effective for forestry companies to perform them on a commercial scale on small-flowered species. To achieve this, three research areas were explored, namely pollen handling, breeding systems and controlled pollination technique. Study species were Eucalyptus grandis, E. dunnii, E. smithii, E. nitens, E. urophylla and E. macarthurii. The first specific aim of the study was to identify a suitable liquid in vitro germination medium for reliably testing pollen viability of all six study species. Six levels of sucrose [0, 10, 20, 30, 40 and 50% (w/v)] were tested, both with (0.15 mg l-1) and without boric acid. The optimal sucrose concentration was found to be 30% (w/v), with boric acid stimulating pollen tube growth. A second aim was to determine temperatures suitable for the short-, medium- and long-term storage of E. smithii, E. nitens and E. grandis pollen. Pollen samples were stored at room (25oC), refrigerator (4oC), freezer (-10oC) and liquid nitrogen (-196oC) temperatures, and pollen viability tested every two months over a 12-month period. There was a rapid decline in the germination of pollen stored at 25oC, while temperatures cooler than 4oC appeared to maintain pollen viability for the duration of the 12-month study. Recommendations were thus to use a refrigerator for short-term (< 2 months), a freezer for medium-term (up to 10 months) and cryopreservation for longer-term storage. In the second part of the study, breeding systems of E. urophylla and E. grandis were examined by studying pollen-tube growth in the style after single-donor self- and cross-pollinations. Results showed that, in addition to both species exhibiting reduced seed yields following self-pollination, pollen tubes from self-pollen took significantly longer than those from cross-pollen to grow to the base of the style. This suggested the presence of both late-acting self-incompatibility and cryptic self-incompatibility (CSI) as possible mechanisms responsible for outcrossing in these two species. In a follow-up study, the siring ability of self- and cross-pollen was examined after single- and mixed-donor pollinations were performed on E. grandis. Once again, single-donor cross-pollinations resulted in a significantly higher number of seeds compared to self-pollinations. In addition, microsatellite molecular markers revealed that 100% of the progeny from mixed (self + outcross) pollinations were outcrossed, confirming the competitive advantage of cross-pollen. To date, CSI has never been associated with Eucalyptus, making this the first study to suggest its presence in the genus. For the final study area, three CP-techniques were compared, namely the Conventional method, One Stop Pollination (OSP) and Artificially Induced Protogyny (AIP), in E. grandis, E. smithii and E. macarthurii maternal parents. Although the AIP technique produced the highest seed yields in all three species, it also resulted in high self- and foreign-pollen contamination (determined using microsatellite markers). This necessitated exploration of different methods of isolating the pollinated flower, and this led to the identification of a novel method which uses sodium alginate gel. Flowers hand-pollinated and isolated with sodium alginate produced progeny that were 100% outcrossed with the applied pollen, confirming the superiority of this innovative isolation technique compared to the currently used exclusion bag. Sodium alginate isolation also increased the efficiency of CPs as the gel was naturally shed, removing the need for operators to return to the tree to remove the isolation material, and thereby reducing the cost per seed. Application of these results should make commercial CP-seed production of small-flowered eucalypts a practical reality. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Submitted by Luis Ricardo Andrade da Silva (lrasilva@uefs.br) on 2016-02-22T22:50:07Z No. of bitstreams: 1 Disserta??o_Alex_RGV_Final2.pdf: 2862200 bytes, checksum: e222cf5496c76484ec34f8fdd9012d23 (MD5) / Made available in DSpace on 2016-02-22T22:50:07Z (GMT). No. of bitstreams: 1 Disserta??o_Alex_RGV_Final2.pdf: 2862200 bytes, checksum: e222cf5496c76484ec34f8fdd9012d23 (MD5) Previous issue date: 2015-11-16 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / In Brazil, research on Arecaceae pollen grains are related to melissopalynology, the palynotaxonomy, being scarce the physiology of pollen. Reproductive biology studies pollination are of great importance in different cultures, from their results can obtain parameters to be used to obtain a considerable increase in the production of croops, as well as the size and quality of fruits, helping for good economic income of the producers. In vitro pollen germination also allows you to check its viability and reproductive strength, and are important tools in breeding programs of plants, assisting in the selection of the most effective genotypes and training hybrids. The chapter one of this work consists of evaluation of the type of culture medium and implications on the choice of micronutrients which form part of its composition, as well as the pretreatment of specimens. It was observed that oil palm pollen (Elaeis guineensis Jacq.) Showed an increase in germination rates after being dissected for a period of four hours and the culture medium with only boric acid and calcium nitrate, in addition to the power source (sucrose) are efficient to display their germination potential. Chapter two contained in the evaluation glycoconjugate (lectins) on the pollen germination of oil palm, which was discussed metabolic interactions of these processes. Lectins are important regulators and operate in recognition and acknowledgment of pollen on the pistil and can act as genetic incompatibility factors. Germination was evaluated in BCa means (boron and calcium) with the addition of lectins: Crotalaria pallida lectin (CPL), Concanavalin A (ConA) and Jacalin (JAC) in two concentrations. An increase of the germination rate in the presence of ConA and CPL lectins and inhibition in the presence of JAC. The results of this study were valuable, since aggregate informative values and extend discussions on the processes involving the germination of pollen grains and formation of pollen tubes of oil palm. / No Brasil, pesquisas com gr?os de p?len da fam?lia Arecaceae est?o relacionados ? melissopalinologia e palinotaxonomia, sendo escassos aqueles voltados ? fisiologia do p?len. Estudos de biologia reprodutiva relacionados ? poliniza??o s?o de grande import?ncia nas diversas culturas, visto que a partir dos seus resultados pode se obter par?metros a serem utilizados visando o aumento consider?vel da produ??o de plantas cultivadas, bem como no tamanho e na qualidade dos frutos, contribuindo para bons rendimentos econ?micos dos produtores. A germina??o de gr?os de p?len in vitro tamb?m permite verificar seu vigor reprodutivo, sendo importante ferramentas em programas de melhoramento gen?tico de plantas, auxiliando na sele??o de gen?tipos mais eficazes para cruzamentos e forma??o de h?bridos. O cap?tulo um deste trabalho teve como prop?sito avaliar o tipo de meio de cultura e as implica??es da escolha dos micronutrientes que fazem parte da sua composi??o, bem como do tratamento pr?vio das amostras. Foi observado que o p?len de dendezeiro (Elaeis guineensis Jacq.) apresentou aumento nas taxas de germina??o depois de ser desidratado por um per?odo de quatro horas e que o meio de cultura com apenas ?cido b?rico e nitrato de c?lcio, al?m da sacarose como fonte de energia, s?o eficientes para exibir o potencial germinativo. O cap?tulo dois consta da avalia??o de prote?nas glicoconjugadas (lectinas) sobre a germinabilidade pol?nica do dendezeiro. Foram discutidas as intera??es metab?licas desses processos, bem como o papel das lectinas que s?o importantes reguladores sobre a recogni??o e reconhecimento do p?len no pistilo, podendo atuar como fatores de incompatibilidade gen?tica. Foi avaliada a germina??o em meio BCa (boro e c?lcio) com adi??o das lectinas: Lectina de Crotalaria pallida Aiton (CPL), Concanavalina A (ConA) e Jacalina (JAC), em duas concentra??es. Verificou-se aumento das taxas de germina??o na presen?a das lectinas CPL e ConA e inibi??o da germina??o na presen?a da JAC. Os resultados deste trabalho agregam valores informativos sobre a compreens?o da biologia reprodutiva de dendezeiro (Arecaceae) ocorrente na ?Costa do Dend??/Bahia e ampliam as discuss?es sobre os processos que envolvem a germina??o de gr?os de p?len e forma??o dos tubos pol?nicos.

of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...