A hibridação somática de citros tem sido extensivamente aplicada, favorecendo o desenvolvimento de híbridos somáticos em programas de melhoramento genético, como fonte de germoplasma ou como variedades copa e porta-enxerto. Neste contexto, este trabalho foi desenvolvido com o objetivo de selecionar plantas de toranja (Citrus grandis L. Osbeck) tolerantes à Phytophthora sp. e utilizá-las como parentais no processo de hibridação somática com outras espécies do gênero Citrus, a fim de produzir híbridos somáticos para o melhoramento de porta-enxertos. Plantas de 20 variedades de toranja tolerantes à Phytophthora spp. foram selecionadas, após serem cultivadas em solo infestado. Experimentos de fusão de protoplastos foram realizados envolvendo laranjas doces, tangerinas e o tangor ‘Murcote’, como parentais embriogênicos, e variedades de toranja selecionadas e plantas enxertadas de 12 variedades de toranja, como parentais não-embriogênicos, utilizando-se a técnica de fusão química, via polietilenoglicol (PEG). Microcolônias foram transferidas para meio de cultura MT semi-sólido, suplementado com 500 mg.l -1 de extrato de malte para indução da embriogênese somática. A confirmação da hibridação somática das plantas regeneradas e aclimatizadas foi realizada por meio de análises de morfologia foliar, de citologia, pela contagem do número de cromossomos, e moleculares, por marcadores do tipo RAPD. As metodologias utilizadas para seleção de plantas matrizes, fusão de protoplastos, regeneração de plantas e confirmação da hibridação somática foram adequadas, e permitiram a obtenção de híbridos somáticos de laranja ‘Hamlin’ com toranja enxertada ‘Indian Red’ e com ‘seedling’ selecionado de toranja ‘Singapura’, que apresentam potencial para serem incorporados em programas de melhoramento de porta-enxertos. / Citrus somatic hybridization has been extensively applied assisting the development of the somatic hybrids which can be used in improvement programs, indirectly as germoplasm source or directly as scion and rootstock varieties. In this context, this research was developed with the objective of selecting plants of pummelo (C. grandis L. Osb) tolerant to Phytophthora sp. and use these plants as parents in the somatic hybridization process with other species of Citrus. Plants of 20 pummelo varieties, tolerant to Phytophthora sp., were selected after being grown in infested soil. Protoplast fusion experiments were induced by chemical method, with polyethylene glicol (PEG), involving sweet oranges, mandarins and Murcott tangor, as embryogenic parents, selected pummelo varieties and grafted plants of 12 pummelo varieties, as non-embryogenic parents. Microcolonies were transferred to EME semi-solid MT containing 500 mg.l -1 of malt extract for somatic embryogenesis. Somatic hybridization was confirmed by analysis of leaf morphology, citology by chromosome counting and molecular analysis by RAPD markers. The protocols used to select plants to be used as protoplast source, protoplast fusion, plant regeneration and somatic hybridization confirmation were adequate, allowing to produce somatic hybrids of Hamlin sweet orange with Indian Red grafted pummelo and Singapura pummelo selected seedling, which may be used as rootstocks and incorporated in rootstocks improvement programs.

O conhecimento da herança dos caracteres de interesse é essencial para programas de melhoramento genético. Entretanto, a herança é altamente influenciada pelo ambiente e pela constituição genotípica da população e, assim, o acúmulo de informações é de grande importância para um melhor conhecimento das heranças dos caracteres. A maior parte dos estudos de herança de caracteres quantitativos é baseada em genótipos que não são mais usados em programas de melhoramento. Este trabalho teve como objetivo o estudo da herança da produção de grãos (PG) e dos componentes de produção em soja, isto é, número de vagens por planta (VP), número de sementes por planta (SP) e número de sementes por vagem (SV), em uma população derivada do cruzamento entre os cultivares Embrapa 60 e MG/BR 46, de alta divergência genética. Os genitores e as gerações F1, F2 e os dois retrocruzamentos foram avaliados experimentalmente no ano agrícola de 2007/8 para os quatro caracteres no Departamento de Genética da ESALQ, em Piracicaba, SP. Os dados experimentais foram submetidos às análises genéticas segundo o modelo aditivodominante, proposto por Mather e Jinks. As estimativas da heterose foram positivas para três dos caracteres (PG, VP e SP), e em torno de 50%, em relação à média dos genitores, enquanto que para SV a heterose foi nula. A herdabilidade entre plantas foi mediana para os componentes da produção (entre 33% e 42%) e baixa para PG (16%). As estimativas das variâncias aditivas foram sempre superiores às da variância dominante, de forma que os graus médios de dominância para todos os caracteres foram em torno de 1,0 (0,96 a 1,21), indicando a ocorrência de dominância completa para os caracteres avaliados. / The knowledge of inheritance of traits is very important for breeding purposes. However, the inheritance is highly influenced by the environment and the genotypic composition of the population and thus, the accumulative information of estimates is very important for a better knowledge of the inheritance of quantitative traits. Most of the studies of inheritance of quantitative traits in soybeans are based on populations which nowadays have no more interest in soybean breeding programs. The objective of this study was to investigate the inheritance of grain yield (PG) and yield components in soybeans, i.e., number of pods per plant (VP), number of seeds per plant (SP), and number of seeds per pod (SV), in a population derived from two genetically divergent soybean cultivars: Embrapa 60 and MG/BR 46. The parents, F1, F2 and two backcross generations were evaluated in the 2007/8 growing season for the four traits, at the Department of Genetics (ESALQ), in Piracicaba, SP. Experimental data were submitted to genetic analyses according to the additive-dominant model proposed by Mather and Jinks. Heterosis estimates were positive for three out of four traits (PG, VP, SP), with magnitudes around 50%, based on the parent means, while for SV heterosis was null. The heritability estimates on a plant basis were medium for yield components (between 33% and 42%) and low for PG (16%). Additive variance estimates were always higher than the dominance estimates, and the degree of dominance (gmd) for all the traits were around 1.0 (0.96 to 1.21), indicating the occurrence of complete dominance for the traits assessed.

O Brasil é o maior produtor de laranja doce do mundo. O histórico da citricultura brasileira é marcado por uma sucessão de doenças causadas por diferentes agentes etiológicos. Entre as principais doenças que afetam a cultura, têm levado a maiores prejuízos, as bacterianas, com destaque para o cancro cítrico causado por Xanthomonas citri subsp. citri e o Huanglongbing associado a três espécies de \"Candidatus Liberibacter\". Devido à ausência de cultivares de laranja doce resistentes a estas doenças, a transformação genética é uma alternativa promissora para obtenção de plantas resistentes. Uma das estratégias no uso da transgenia para conferir ação contra bactérias é a inserção de genes que codificam peptídeos antimicrobianos como o D4E1, um peptídeo sintético, que tem apresentado eficiência no controle de doenças fúngicas e bacterianas de várias culturas, in vivo e in vitro. Este trabalho foi realizado com o objetivo de obter plantas transgênicas de laranja doce das cultivares \'Hamlin\', \'Pêra\' e \'Valência\', via Agrobacterium tumefaciens, expressando o gene D4E1, dirigido pelos promotores CaMV35S (Cauliflower mosaic virus 35S promoter) de expressão constitutiva ou pelo AtPP2 (Arabidopsis thaliana phloem protein 2) com expressão preferencial no floema, visando obter plantas resistentes a doenças bacterianas. Foram obtidas 13 plantas transgênicas da cultivar \'Hamlin\', 10 da cultivar \'Pêra\' e 8 da cultivar \'Valência\', contendo a construção gênica CaMV35S/D4E1 e 19 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Pêra\' e 15 da cultivar \'Valência\' contendo a construção gênica AtPP2/D4E1. As plantas transgênicas apresentaram um a três eventos de inserção do T-DNA no genoma. Os níveis de expressão do transgene dirigido pelo promotor de expressão preferencial no floema foi menor comparado ao das plantas contendo o transgene dirigido pelo promotor de expressão constitutiva. Os resultados da expressão do transgene permitem selecionar plantas com maior expressão de cada uma das construções gênicas, para que, futuramente, estas sejam multiplicadas e avaliadas quanto à resistência ao cancro cítrico e ao HLB. / Brazil is the largest sweet orange producer in the world. The history of the Brazilian citrus industry is marked by a series of diseases caused by different etiologic agents. Among the diseases affecting the culture, those caused by bacteria are the ones that have caused more significant losses, especially the citrus canker caused by Xanthomonas citri subsp. citri, and huanglongbing (HLB) associated with three \"Candidatus Liberibacter\" bacteria species. Due to the absence of genetic resistance to these diseases in commercial sweet orange cultivars, the genetic transformation is a promising alternative to produce resistant plants. One of the strategies to produce transgenic resistant plants to bacteria is the use of genes that code for antimicrobial peptides, such as D4E1, a antimicrobial synthetic peptide, which has shown efficient results controlling diseases caused by bacteria and fungi in several crops, through in vitro and in vivo experiments. The aim of this study was to produce \'Hamlin\', \'Pêra\' and \'Valencia\' sweet orange transgenic plants, via Agrobacterium tumefaciens, expressing the D4E1 gene driven by the constitutive promoter Cauliflower mosaic virus (CaMV35S) or Arabidopsis thaliana phloem protein 2 (AtPP2), a promoter preferentially expressed in the phloem. It was possible to regenerate 13 \'Hamlin\' transgenic lines, 10 \'Pêra\' transgenic lines and 8 \'Valencia\' transgenic lines bearing the gene construct CaMV35S/D4E1, whereas 19 \'Hamlin\' transgenic lines, 6 \'Pêra\' transgenic lines and 15 \'Valencia\' transgenic lines bearing the AtPP2/D4E1 gene construct were regenerated. The transgenic plants had one to three T-DNA insertion events in the genome. The transgene expression levels in transgenic plants for D4E1 gene driven by the phloem preferential promoter were lower than the transgenic expression levels of the transgene driven by the constitutive promoter. Transgene expression levels results may allow the selection of those plants with higher expression levels of each genetic construct for future multiplication and evaluation for citrus canker and HLB resistance.

A hibridação somática de citros tem sido extensivamente aplicada, favorecendo o desenvolvimento de híbridos somáticos em programas de melhoramento genético, como fonte de germoplasma ou como variedades copa e porta-enxerto. Neste contexto, este trabalho foi desenvolvido com o objetivo de selecionar plantas de toranja (Citrus grandis L. Osbeck) tolerantes à Phytophthora sp. e utilizá-las como parentais no processo de hibridação somática com outras espécies do gênero Citrus, a fim de produzir híbridos somáticos para o melhoramento de porta-enxertos. Plantas de 20 variedades de toranja tolerantes à Phytophthora spp. foram selecionadas, após serem cultivadas em solo infestado. Experimentos de fusão de protoplastos foram realizados envolvendo laranjas doces, tangerinas e o tangor ‘Murcote’, como parentais embriogênicos, e variedades de toranja selecionadas e plantas enxertadas de 12 variedades de toranja, como parentais não-embriogênicos, utilizando-se a técnica de fusão química, via polietilenoglicol (PEG). Microcolônias foram transferidas para meio de cultura MT semi-sólido, suplementado com 500 mg.l -1 de extrato de malte para indução da embriogênese somática. A confirmação da hibridação somática das plantas regeneradas e aclimatizadas foi realizada por meio de análises de morfologia foliar, de citologia, pela contagem do número de cromossomos, e moleculares, por marcadores do tipo RAPD. As metodologias utilizadas para seleção de plantas matrizes, fusão de protoplastos, regeneração de plantas e confirmação da hibridação somática foram adequadas, e permitiram a obtenção de híbridos somáticos de laranja ‘Hamlin’ com toranja enxertada ‘Indian Red’ e com ‘seedling’ selecionado de toranja ‘Singapura’, que apresentam potencial para serem incorporados em programas de melhoramento de porta-enxertos. / Citrus somatic hybridization has been extensively applied assisting the development of the somatic hybrids which can be used in improvement programs, indirectly as germoplasm source or directly as scion and rootstock varieties. In this context, this research was developed with the objective of selecting plants of pummelo (C. grandis L. Osb) tolerant to Phytophthora sp. and use these plants as parents in the somatic hybridization process with other species of Citrus. Plants of 20 pummelo varieties, tolerant to Phytophthora sp., were selected after being grown in infested soil. Protoplast fusion experiments were induced by chemical method, with polyethylene glicol (PEG), involving sweet oranges, mandarins and Murcott tangor, as embryogenic parents, selected pummelo varieties and grafted plants of 12 pummelo varieties, as non-embryogenic parents. Microcolonies were transferred to EME semi-solid MT containing 500 mg.l -1 of malt extract for somatic embryogenesis. Somatic hybridization was confirmed by analysis of leaf morphology, citology by chromosome counting and molecular analysis by RAPD markers. The protocols used to select plants to be used as protoplast source, protoplast fusion, plant regeneration and somatic hybridization confirmation were adequate, allowing to produce somatic hybrids of Hamlin sweet orange with Indian Red grafted pummelo and Singapura pummelo selected seedling, which may be used as rootstocks and incorporated in rootstocks improvement programs.

O Brasil é o maior produtor de laranja doce do mundo. O histórico da citricultura brasileira é marcado por uma sucessão de doenças causadas por diferentes agentes etiológicos. Entre as principais doenças que afetam a cultura, têm levado a maiores prejuízos, as bacterianas, com destaque para o cancro cítrico causado por Xanthomonas citri subsp. citri e o Huanglongbing associado a três espécies de \"Candidatus Liberibacter\". Devido à ausência de cultivares de laranja doce resistentes a estas doenças, a transformação genética é uma alternativa promissora para obtenção de plantas resistentes. Uma das estratégias no uso da transgenia para conferir ação contra bactérias é a inserção de genes que codificam peptídeos antimicrobianos como o D4E1, um peptídeo sintético, que tem apresentado eficiência no controle de doenças fúngicas e bacterianas de várias culturas, in vivo e in vitro. Este trabalho foi realizado com o objetivo de obter plantas transgênicas de laranja doce das cultivares \'Hamlin\', \'Pêra\' e \'Valência\', via Agrobacterium tumefaciens, expressando o gene D4E1, dirigido pelos promotores CaMV35S (Cauliflower mosaic virus 35S promoter) de expressão constitutiva ou pelo AtPP2 (Arabidopsis thaliana phloem protein 2) com expressão preferencial no floema, visando obter plantas resistentes a doenças bacterianas. Foram obtidas 13 plantas transgênicas da cultivar \'Hamlin\', 10 da cultivar \'Pêra\' e 8 da cultivar \'Valência\', contendo a construção gênica CaMV35S/D4E1 e 19 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Pêra\' e 15 da cultivar \'Valência\' contendo a construção gênica AtPP2/D4E1. As plantas transgênicas apresentaram um a três eventos de inserção do T-DNA no genoma. Os níveis de expressão do transgene dirigido pelo promotor de expressão preferencial no floema foi menor comparado ao das plantas contendo o transgene dirigido pelo promotor de expressão constitutiva. Os resultados da expressão do transgene permitem selecionar plantas com maior expressão de cada uma das construções gênicas, para que, futuramente, estas sejam multiplicadas e avaliadas quanto à resistência ao cancro cítrico e ao HLB. / Brazil is the largest sweet orange producer in the world. The history of the Brazilian citrus industry is marked by a series of diseases caused by different etiologic agents. Among the diseases affecting the culture, those caused by bacteria are the ones that have caused more significant losses, especially the citrus canker caused by Xanthomonas citri subsp. citri, and huanglongbing (HLB) associated with three \"Candidatus Liberibacter\" bacteria species. Due to the absence of genetic resistance to these diseases in commercial sweet orange cultivars, the genetic transformation is a promising alternative to produce resistant plants. One of the strategies to produce transgenic resistant plants to bacteria is the use of genes that code for antimicrobial peptides, such as D4E1, a antimicrobial synthetic peptide, which has shown efficient results controlling diseases caused by bacteria and fungi in several crops, through in vitro and in vivo experiments. The aim of this study was to produce \'Hamlin\', \'Pêra\' and \'Valencia\' sweet orange transgenic plants, via Agrobacterium tumefaciens, expressing the D4E1 gene driven by the constitutive promoter Cauliflower mosaic virus (CaMV35S) or Arabidopsis thaliana phloem protein 2 (AtPP2), a promoter preferentially expressed in the phloem. It was possible to regenerate 13 \'Hamlin\' transgenic lines, 10 \'Pêra\' transgenic lines and 8 \'Valencia\' transgenic lines bearing the gene construct CaMV35S/D4E1, whereas 19 \'Hamlin\' transgenic lines, 6 \'Pêra\' transgenic lines and 15 \'Valencia\' transgenic lines bearing the AtPP2/D4E1 gene construct were regenerated. The transgenic plants had one to three T-DNA insertion events in the genome. The transgene expression levels in transgenic plants for D4E1 gene driven by the phloem preferential promoter were lower than the transgenic expression levels of the transgene driven by the constitutive promoter. Transgene expression levels results may allow the selection of those plants with higher expression levels of each genetic construct for future multiplication and evaluation for citrus canker and HLB resistance.

O conhecimento da herança dos caracteres de interesse é essencial para programas de melhoramento genético. Entretanto, a herança é altamente influenciada pelo ambiente e pela constituição genotípica da população e, assim, o acúmulo de informações é de grande importância para um melhor conhecimento das heranças dos caracteres. A maior parte dos estudos de herança de caracteres quantitativos é baseada em genótipos que não são mais usados em programas de melhoramento. Este trabalho teve como objetivo o estudo da herança da produção de grãos (PG) e dos componentes de produção em soja, isto é, número de vagens por planta (VP), número de sementes por planta (SP) e número de sementes por vagem (SV), em uma população derivada do cruzamento entre os cultivares Embrapa 60 e MG/BR 46, de alta divergência genética. Os genitores e as gerações F1, F2 e os dois retrocruzamentos foram avaliados experimentalmente no ano agrícola de 2007/8 para os quatro caracteres no Departamento de Genética da ESALQ, em Piracicaba, SP. Os dados experimentais foram submetidos às análises genéticas segundo o modelo aditivodominante, proposto por Mather e Jinks. As estimativas da heterose foram positivas para três dos caracteres (PG, VP e SP), e em torno de 50%, em relação à média dos genitores, enquanto que para SV a heterose foi nula. A herdabilidade entre plantas foi mediana para os componentes da produção (entre 33% e 42%) e baixa para PG (16%). As estimativas das variâncias aditivas foram sempre superiores às da variância dominante, de forma que os graus médios de dominância para todos os caracteres foram em torno de 1,0 (0,96 a 1,21), indicando a ocorrência de dominância completa para os caracteres avaliados. / The knowledge of inheritance of traits is very important for breeding purposes. However, the inheritance is highly influenced by the environment and the genotypic composition of the population and thus, the accumulative information of estimates is very important for a better knowledge of the inheritance of quantitative traits. Most of the studies of inheritance of quantitative traits in soybeans are based on populations which nowadays have no more interest in soybean breeding programs. The objective of this study was to investigate the inheritance of grain yield (PG) and yield components in soybeans, i.e., number of pods per plant (VP), number of seeds per plant (SP), and number of seeds per pod (SV), in a population derived from two genetically divergent soybean cultivars: Embrapa 60 and MG/BR 46. The parents, F1, F2 and two backcross generations were evaluated in the 2007/8 growing season for the four traits, at the Department of Genetics (ESALQ), in Piracicaba, SP. Experimental data were submitted to genetic analyses according to the additive-dominant model proposed by Mather and Jinks. Heterosis estimates were positive for three out of four traits (PG, VP, SP), with magnitudes around 50%, based on the parent means, while for SV heterosis was null. The heritability estimates on a plant basis were medium for yield components (between 33% and 42%) and low for PG (16%). Additive variance estimates were always higher than the dominance estimates, and the degree of dominance (gmd) for all the traits were around 1.0 (0.96 to 1.21), indicating the occurrence of complete dominance for the traits assessed.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology and Food Technology, Durban University of Technology, 2007 iv, 75 leaves / The development of transgenic plants resulted in the need to utilize the various molecular methods (e.g., ELISA, real - time PCR etc.) for the detection or analysis of the presence or absence of a specific trait in a particular plant (Bt in this study). The overall aim of this study was to optimize a half – seed extraction technique as part of a laboratory protocol for transgenic maize plants and to explore the possibility of using the following molecular techniques: horizontal isoelectric focusing, real - time PCR and ELISA, as methods for detection of the Bt trait for incorporation into the half – seed extraction protocol.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation. / AFRIKAANSE OPSOMMING: Die ooglopende voordele wat, vanuit 'n landboukundige én ekonomiese oogpunt, uit siekte- en stresbestande plante spruit, het gedurende die laaste dekade aanleiding gegee tot die ontwikkeling van plantpatogeen-interaksies as "n baie belangrike studieveld. Dit was dus ook te verwagte dat fundamentele navorsing in hierdie area nie net beperk gebly het tot modelorganismes soos Arabidopsis en tabak (ook natuurlik van landboukundige belang) nie, maar ook na meer tradisionele landbougewasse soos mielies, boontjies, sojaboontjies, appels, druiwe, ens. oorgevloei het. Verskeie siekteweerstands-verwante gene is byvoorbeeld al vanuit wingerd geïsoleer. Een só "n geen wat vir "n poligalakturonase-inhiberende proteïen (PGIP) kodeer, vorm deel van hierdie groep gene. Die funksie en regulering van PGIP's is baie goed bestudeer. Hierdie proteïene word normaalweg in die selwande van die meeste dikotiele plante aangetref. Leusienryke herhalings is algemeen in PGIP's en hierdie tipe van herhalings is kenmerkend van proteïene betrokke by proteïen-proteïen-interaksies. Verder word pgip-gene gewoonlik in klein multigeenfamilies aangetref, waar in die meeste gevalle die uitdrukking weefselspesifiek en die regulering spesifiek ten opsigte van die ontwikkelingsfase is. Verskeie faktore kan tot die induksie van pgip-gene lei, soos onder andere patogeen-infeksie, elisitoor-, salisiensuur-, jasmoonsuur- en kouebehandeling, asook verwonding. Differensiële regulering word in baie gevalle tussen lede van dieselfde multigeenfamilie aangetref. Hierdie differensiële regulering kan selfs bemiddel word deur onafhanklike reguleringsweë in reaksie op dieselfde induksiestimulus. PGIP's is in staat om te reageer met poligalakturonases (PGs), wat selwande afbreek en wat gedurende die infeksieproses deur swamme of fungi afgeskei word. Die effek van hierdie interaksie is tweeledig: (i) Die fisiese interaksie tussen PGIP en PG moduleer die aktiwiteit van die PG deur die ensiemaksie te inhibeer, en (ii) PGinhibisie lei tot die verhoogde stabiliteit van langketting-oligogalakturonades, molekules wat daartoe in staat is om die weerstandsrespons van plante te ontlok. Die inhibisie van die patogeen-PG's, tesame met die geïnduseerde weerstandrespons, stel die plant dan in staat om verdere infeksie te vertraag of te verhoed. Die doel van hierdie studie was om die onderliggende aspekte van PGIPregulering in wingerd te bestudeer. In teenstelling met die meeste plantspesies waar pgip-gene in klein multigeenfamilies aangetref word, is daar nie 'n pgip-multigeenfamilie in wingerd nie. Veelvuldige kopieë van In enkele pgip-geen word egter in die wingerdgenoom aangetref. Daar is onlangs in ons laboratorium In genoom-DNAfragment vanaf Vitis vinifera cv Pinotage geïsoleer wat die oopleesraam en 5'-stroomopsekwense van In PGIP-enkoderende geen (Vvpgip1) bevat. In hierdie studie is die uitdrukkingspatroon van Vvpgip1 ten opsigte van weefselspesifisiteit, korrelontwikkelingsfase, asook die effek van verskeie omgewings en patogeenverwante stres-stimuli ontleed. Die regulatoriese meganismes van Vvpgip1 bevat spesifieke in planta-ontwikkelingsfaseseine wat verder deur spesifieke faktore, insluitende omgewings- en patogeenstres, gereguleer word. In lyn hiermee is mRNS-transkripte van Vvpgip1 tot wortel- en korrelweefsels beperk, terwyl die mRNS-vlakke ook tussen verskillende korrelontwikkelingsfases wissel. Kumulatiewe uitdrukking kon waargeneem word in veráison-korrels in reaksie op verwonding en osmotiese stres. Die weefselspesifieke uitdrukkingspatroon tipies van wingerd-PGIP is in blare opgehef in reaksie op Botrytis cinerea-infeksie, verwonding, osmotiese stres, ouksien (indoolasynsuur) en salisiensuur. PGIP-uitdrukking word ook onderdruk deur In staurosporien-sensitiewe proteïenkinase, wat In goeie aanduiding is van die betrokkenheid van proteïenfosforilasie in die seintransduksiekaskade wat tot PGIPuitdrukking aanleiding gee. Die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare kan ook nageboots word in tabak wat met die Vvpgip1-geen en -promotor getransformeer is. PG-inhibisie-eksperimente met membraan-geassosieerde proteïenekstrakte van geïnduseerde wingerdblare het ook dieselfde profiel getoon as dié van PGIP wat deur die Vvpgip1-geen geënkodeer is. Die uitdrukkingsprofiel van PGIP in die transgeniese tabakplante het ook bewys dat die promotor van die Vvpgip1-geen vir die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare verantwoordelik is. In silica-analise van die promotorarea dui op die teenwoordigheid van verskeie cis-werkende elemente. Die kern promotor en transkripsie-aanvangsgedeelte is gevolglik eksperimenteel bepaal. Verder het uitdrukkingseksperimente met promotorfragmente verskeie dele van die promotor geïdentifiseer wat by stimulis-geassosieerde uitdrukking betrokke is. Posisioneel is hierdie fragmente in goeie konteks met die voorspelde cis-werkende elemente en kan dus die basis vorm vir verdere studies oor Vvpgip-regulering. Met hierdie studie word die eerste data verskaf waar die regulering van PGIP deur omgewingsverwante faktore verbind kan word met onwikkelingspesifieke toestande in die plant. Verder verskaf die resultate verdere bewyse vir die rol van PGIP in plant-patogeen-interaksies en lewer spesifieke bydraes tot die onderliggende prosesse wat by die regulering van siekteweerstandverwante gene betrokke is.

Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016. / Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

Considerado um dos aspectos mais complexos da genética quantitativa, a epistasia tem sido ignorada pelos melhoristas nos estudos de herança dos caracteres, principalmente os da herança da resistência de plantas a insetos, que são de difícil obtenção. No milho, a principal praga de grãos é o Sitophilus zeamais (Coleoptera: Curculionidae), devido a sua capacidade de atacar grãos tanto no campo quanto em silos. Contudo, as estimativas dos componentes aditivo e de dominância envolvidos na herança dessa resistência podem estar viesadas pela presença do efeito da epistasia. Utilizando o delineamento triple testcross, os objetivos deste trabalho foram: (i) verificar a presença da epistasia para os caracteres relacionados à resistência do milho ao S. zeamais; (ii) estimar os efeitos epistáticos em cada planta F2; e (iii) estimar o efeito da interação epistasia x ambientes para estes caracteres. As 300 progênies de retrocruzamentos utilizadas nesse estudo foram avaliadas em dois ambientes no município de Piracicaba/SP, em delineamento alfa-látice 15 x 20, no esquema fatorial com duas repetições por ambiente. Os caracteres avaliados foram: número de insetos mortos (NM); número de insetos emergidos (EM); tempo médio de desenvolvimento dos insetos (TM); índice de suscetibilidade (IS) e perda percentual de massa seca dos grãos (PE). No ambiente E. E. Anhumas a presença da epistasia foi detectada para todos os caracteres; porém, no ambiente Caterpillar o efeito da epistasia não foi detectado para nenhum dos caracteres. Na análise conjunta, os efeitos epistáticos foram detectados para os caracteres NM, EM, IS e PE. O efeito da epistasia do tipo aditivo x dominante e/ou dominante x dominante foi mais importante para todos os caracteres que a epistasia do tipo aditiva x aditiva. A interação da epistasia com os ambientes foi significativa apenas para os caracteres TM e PE. Identificaram-se efeitos epistáticos bidirecionais significativos em plantas F2 para todos os caracteres no ambiente E. E. Anhumas e para os caracteres NM, EM, IS e PE na análise conjunta. O grande número de plantas F2 que apresentaram epistasia para mais de um caráter simultaneamente sugere a presença de epistasia pleiotrópica. As estimativas da variância aditiva e da interação aditiva com ambientes foram significativamente maiores que as das variâncias de dominância para a maioria dos caracteres. As magnitudes das estimativas dos coeficientes de herdabilidade para todos os caracteres variaram de baixas a medianas. A alta correlação genética entre os caracteres EM e PE sugere que o caráter PE, que é de difícil avaliação, pode ser selecionado indiretamente através do caráter EM que é de fácil avaliação para programas de melhoramento visando resistência ao S. zeamais. Os resultados obtidos na análise conjunta sugerem que, na população estudada, a epistasia constitui um componente importante da variância genética, de forma que as estimativas da variância aditiva, de dominância, graus médios de dominância e coeficientes de herdabilidade estão viesadas. / Considered one of the most complex components in quantitative genetics, the epistasis has been ignored by plant breeders, especially in inheritance studies of plant resistance, because the traits are laborious to evaluate. Sitophilus zeamais (Coleoptera: Curculionidae) is an important grain pest, due to its ability to attack in both field and silo conditions. However, the estimation of additive and dominance components in the inheritance of resistance to this pest may be biased due to epistatic effects. With the use of the triple test cross design, this research was aimed to: (i) verify the role of epistasis in the inheritance of maize resistance to S. zeamais, (ii) estimate the epistatic effects in the resistance traits of each F2 plant; (iii) estimate the epistasis x environment interaction. The 300 backcross progenies of this study were evaluated in two environments at Piracicaba, Brazil, in 2008/2009 growing season, using an alpha-lattice 15 x 20 design in a factorial arrangement with two replications per environment. The recorded traits were: number of dead weevils (NDW); number of emerged weevils (EW); mean development period (DP); index of susceptibility (IS) and the percentage of dry grain weight loss (DGWL). The epistatic effects were detected in Anhumas Experimental Station (AES) environment for all traits although they were absent in Caterpillar Experimental Station environment. In the combined analysis epistasis was detected for NDW, EW, IS and DGWL traits. The additive x dominance and/or dominance x dominance epistasis were more important than the additive x additive epistasis for all traits. The epistasis x environment interaction was significant only for traits DP and DGWL. Significant epistatic effects, which were not unidirectional, were detected in F2 plants for all traits in AES and for the traits NDW, EW, IS and DGWL in the combined analysis. Several F2 plants presented epistasis for more than one trait simultaneously suggesting the presence of pleiotropic epistasis. Estimates of additive, dominance and the additive by environment interaction variances were significant for all traits. Estimates of additive and additive by environment interaction variances were significantly higher than those of dominance variance for most of the traits. The magnitudes of the heritability coefficients estimates ranged from low to intermediate. The genetic correlation between EW and DGWL traits suggests that DGWL, which is difficult to evaluate, can be indirectly selected by the EW trait, which is easier to evaluate, in a breeding program. The results from the combined analysis suggests that, in the population studied, the epistasis is an important component of the genetic variance; therefore, estimates of additive, dominance variance, the average levels of dominance and heritability coefficients are biased.