O Brasil é o maior produtor de laranja doce do mundo, mas a história da citricultura brasileira é marcada por sucessivas perdas devido a pragas e doenças que atacam os pomares. Entre as doenças que afetam os pomares de citros, o huanglongbing (HLB) tem merecido destaque nos últimos anos. O HLB já é conhecido desde 1900 na China, mas no Brasil essa doença está presente desde 2004 e tem causado perdas significativas a citricultura. Essa doença é associada a três espécies de \"Candidatus Liberibacter\", mas no Brasil a espécie Candidatus Liberibacter asiaticus (CLas) é a mais comum. Devido à ausência de plantas de laranja doce resistentes a essa doença, a busca por plantas transgênicas que apresentem resistência a essa doença têm se intensificado nos últimos anos. Uma estratégia para projetar uma construção gênica visando a CLas é a utilização de promotores floema-específico, pois a CLas coloniza o floema das plantas de citros infectadas. Entretanto, para provar que uma sequência promotora funciona é preciso desafiar a construção gênica na presnça da CLas. Portanto, conduziu-se este trabalho com o objetivo de multiplicar plantas de Citrus sinensis Osbeck (L.) cv. \'Hamlin\' transgênicas contendo o gene uidA sob controle dos promotores floema-específicos Arabidopsis thaliana phloem protein 2 (AtPP2), Citrus phloem protein 2 (CsPP2) e Arabidopsis thaliana sucrose transporter 2 (AtSUC2), inocular com CLas por Diaphorina citri, avaliar a interdependência entre a expressão do transgene (gene uidA) e a concentração de CLas, para poder inferir sobre o controle da expressão dos promotores quando as plantas são inculadas com CLas. Para isso, cinco eventos de transformação de cada construção gênica, contendo apenas uma cópia do transgene foram selecionados, multiplicados e inoculados com CLas por Diaphorina citri, dezoito meses após a inoculação, o DNA e o RNA das plantas foram coletados. Foi realizada análise para verificar a concentração de CLas em todas as plantas inoculadas e a expressão do gene uidA foi realizada em uma linhagem transgênica de cada construção gênica. Com o auxílio da análise de coeficiente de correlação de Person, foi possível classificar qualitativamente a interdependência entre a concentração bacteriana e a expressão gênica. A frequência média de inoculação de CLas por Diaphorina citri foi de 30 %, demostrando que é possível incular CLas via Diaphorina citri. A análise de coeficiente de correlação de Person mostrou que nas construções com promotores AtPP2 e CsPP2 há baixa interdependência entre o controle da expressão gênica e a concentração de CLas. A construção sob controle do promotor AtSUC2 apresentou correlação forte e positiva, mostrando que quanto maior a concentração de CLas maior a expressão do transgene. / Brazil is the largest producer of sweet oranges in the world, however the Brazilian citrus history is marked by successive losses due to pests and diseases attacking orchards. Among the diseases affecting citrus groves, the huanglongbing (HLB) has been highlighted in recent years. The HLB is known in China since 1900, although in Brazil this disease has been present since 2004 and has caused significant losses to citrus industry. This disease is associated with three species of \"Candidatus Liberibacter\", however, in Brazil Candidatus Liberibacter asiaticus species (CLas) is the most common. Due to the absence of sweet orange plants resistant to this disease, the search for transgenic plants with resistance to this disease have intensified in recent years. One strategy to design a genetic construct targeting the CLas includes the use of phloem-specific promoters, because the CLas colonizes the phloem of infected citrus plants. On the other hand, to prove that a promoter sequence works it is necessary to challenge the genetic construct in the presence of CLas. Therefore, we conducted this study with the objectives of multiplying transgenic Citrus sinensis Osbeck (L.) cv. \'Hamlin\' bearing the uidA gene under the control of the phloem-specific promoters Arabidopsis thaliana phloem protein 2 (AtPP2), Citrus phloem protein 2 (CsPP2) and Arabidopsis thaliana sucrose transporter 2 (AtSUC2), inoculate CLas with Diaphorina citri, and evaluate the interdependence between the expression of the transgene (uidA gene) and the concentration of CLas, to infer the promoter expression when the plants are inoculated with CLas. Five events processing of each gene construct, containing only one copy of the transgene, and inoculated with CLas by Diaphorina citri. The DNA and RNA of plants were collected after eighteen months of inoculation. The concentration of CLas in the inoculated plants and the expression of the uidA gene were performed in each transgenic line of each gene construct, with the aid of the analysis of Person correlation coefficient. Thus it was possible to classify qualitatively the interdependence between bacterial concentration and gene expression. The average frequency of CLas inoculation by Diaphorina citri was 30%, demonstrating that it was possible to inoculate CLas via Diaphorina citri. Person correlation coefficient values suggested low interdependence between the control of gene expression and the concentration of CLas in constructions with promoters AtPP2 and CsPP2. The construction under AtSUC2 promoter control showed strong positive correlation, indicating that the higher the concentration of CLas the greater is the transgene expression.

O Brasil é o maior produtor de laranja doce do mundo, mas a história da citricultura brasileira é marcada por sucessivas perdas devido a pragas e doenças que atacam os pomares. Entre as doenças que afetam os pomares de citros, o huanglongbing (HLB) tem merecido destaque nos últimos anos. O HLB já é conhecido desde 1900 na China, mas no Brasil essa doença está presente desde 2004 e tem causado perdas significativas a citricultura. Essa doença é associada a três espécies de \"Candidatus Liberibacter\", mas no Brasil a espécie Candidatus Liberibacter asiaticus (CLas) é a mais comum. Devido à ausência de plantas de laranja doce resistentes a essa doença, a busca por plantas transgênicas que apresentem resistência a essa doença têm se intensificado nos últimos anos. Uma estratégia para projetar uma construção gênica visando a CLas é a utilização de promotores floema-específico, pois a CLas coloniza o floema das plantas de citros infectadas. Entretanto, para provar que uma sequência promotora funciona é preciso desafiar a construção gênica na presnça da CLas. Portanto, conduziu-se este trabalho com o objetivo de multiplicar plantas de Citrus sinensis Osbeck (L.) cv. \'Hamlin\' transgênicas contendo o gene uidA sob controle dos promotores floema-específicos Arabidopsis thaliana phloem protein 2 (AtPP2), Citrus phloem protein 2 (CsPP2) e Arabidopsis thaliana sucrose transporter 2 (AtSUC2), inocular com CLas por Diaphorina citri, avaliar a interdependência entre a expressão do transgene (gene uidA) e a concentração de CLas, para poder inferir sobre o controle da expressão dos promotores quando as plantas são inculadas com CLas. Para isso, cinco eventos de transformação de cada construção gênica, contendo apenas uma cópia do transgene foram selecionados, multiplicados e inoculados com CLas por Diaphorina citri, dezoito meses após a inoculação, o DNA e o RNA das plantas foram coletados. Foi realizada análise para verificar a concentração de CLas em todas as plantas inoculadas e a expressão do gene uidA foi realizada em uma linhagem transgênica de cada construção gênica. Com o auxílio da análise de coeficiente de correlação de Person, foi possível classificar qualitativamente a interdependência entre a concentração bacteriana e a expressão gênica. A frequência média de inoculação de CLas por Diaphorina citri foi de 30 %, demostrando que é possível incular CLas via Diaphorina citri. A análise de coeficiente de correlação de Person mostrou que nas construções com promotores AtPP2 e CsPP2 há baixa interdependência entre o controle da expressão gênica e a concentração de CLas. A construção sob controle do promotor AtSUC2 apresentou correlação forte e positiva, mostrando que quanto maior a concentração de CLas maior a expressão do transgene. / Brazil is the largest producer of sweet oranges in the world, however the Brazilian citrus history is marked by successive losses due to pests and diseases attacking orchards. Among the diseases affecting citrus groves, the huanglongbing (HLB) has been highlighted in recent years. The HLB is known in China since 1900, although in Brazil this disease has been present since 2004 and has caused significant losses to citrus industry. This disease is associated with three species of \"Candidatus Liberibacter\", however, in Brazil Candidatus Liberibacter asiaticus species (CLas) is the most common. Due to the absence of sweet orange plants resistant to this disease, the search for transgenic plants with resistance to this disease have intensified in recent years. One strategy to design a genetic construct targeting the CLas includes the use of phloem-specific promoters, because the CLas colonizes the phloem of infected citrus plants. On the other hand, to prove that a promoter sequence works it is necessary to challenge the genetic construct in the presence of CLas. Therefore, we conducted this study with the objectives of multiplying transgenic Citrus sinensis Osbeck (L.) cv. \'Hamlin\' bearing the uidA gene under the control of the phloem-specific promoters Arabidopsis thaliana phloem protein 2 (AtPP2), Citrus phloem protein 2 (CsPP2) and Arabidopsis thaliana sucrose transporter 2 (AtSUC2), inoculate CLas with Diaphorina citri, and evaluate the interdependence between the expression of the transgene (uidA gene) and the concentration of CLas, to infer the promoter expression when the plants are inoculated with CLas. Five events processing of each gene construct, containing only one copy of the transgene, and inoculated with CLas by Diaphorina citri. The DNA and RNA of plants were collected after eighteen months of inoculation. The concentration of CLas in the inoculated plants and the expression of the uidA gene were performed in each transgenic line of each gene construct, with the aid of the analysis of Person correlation coefficient. Thus it was possible to classify qualitatively the interdependence between bacterial concentration and gene expression. The average frequency of CLas inoculation by Diaphorina citri was 30%, demonstrating that it was possible to inoculate CLas via Diaphorina citri. Person correlation coefficient values suggested low interdependence between the control of gene expression and the concentration of CLas in constructions with promoters AtPP2 and CsPP2. The construction under AtSUC2 promoter control showed strong positive correlation, indicating that the higher the concentration of CLas the greater is the transgene expression.

O Huanglongbing (HLB) é uma das doenças mais ameaçadoras para citricultura mundial e, até o momento, não foi encontrada resistência na base genética do gênero Citrus. A doença é causada pela bactéria Candidatus Liberibacter spp., endêmica de floema. Portanto, na busca por uma planta transgênica resistente ao HLB é desejável avaliar construções gênicas em que o gene de interesse se expresse preferencialmente na região em que a bactéria coloniza a planta, ou seja, no floema. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas via Agrobacterium tumefaciens, de citrange Carrizo [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] e de laranja doce [Citrus sinensis (L.) Osbeck] cultivares Hamlin, Valência e Pêra, contendo o gene uidA (GUS) sob o controle dos promotores Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) e Arabidopsis thaliana sucrose transporter 2 (AtSuT2), para verificar se esses promotores regulam a expressão do gene repórter na região do floema. Foram utilizados segmentos de epicótilo de plântulas germinadas in vitro e como agente de seleção de regeneração de plantas transgênicas foi utilizado o gene nptII, que confere resistência ao antibiótico canamicina. Dos brotos regenerados foi coletada uma amostra de material para a realização do ensaio histoquímico com X-GLUC. Os brotos que formaram coloração azulada confirmaram a integração do transgene, sendo esses enxertados em porta enxertos previamente germinados e estiolados in vitro. A partir do número de explantes introduzidos, número explantes responsivos, número de brotos regenerados e número de brotos regenerados GUS positivos calculou-se a eficiência de transformação genética dos experimentos. Para a confirmação da transformação genética de laranja Hamlin foram realizadas análises de PCR e Southern blot de três plantas GUS positivas aclimatizadas. Também foram feitos cortes histológicos manuais para melhor visualização da reação histoquímica de GUS das plantas de laranja Hamlin Southern blot positivas. Todos os experimentos de transformação regeneraram pelo menos um broto GUS positivo. As plantas de laranja Hamlin analisadas por PCR e Southern blot analisadas foram confirmadas como transformadas com uma inserção do transgene. Plantas nas quais foram realizados cortes histológicos indicaram expressão diferencial das construções gênicas no floema. / Huanglongbing (HLB) is one of the most threatening diseases to worldwide citriculture and till the present moment, no resistance has been found in citrus genetic basis. This disease has Candidatus Liberibacter spp. as the pathogenic agent, an endemic phloem bacterium. Therefore, in the search for a transgenic plant resistant to HLB, it is desirable to evaluate constructions in which the gene of interest is expressed, preferentially, in tissues where the bacteria grow, i.e., in the phloem. Therefore, this work aimed to obtain transgenic plants of Carrizo citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck], and of Hamlin, Valencia, and Pera sweet oranges [Citrus sinensis (L.) Osbeck], via Agrobacterium tumefaciens, containing uidA (GUS) gene, controlled by the promoters Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) and Arabidopsis thaliana sucrose transporter 2 (AtSuT2), in order to check if these promoters drive the reporter gene expression in the phloem. For the transformation, in vitro germinated seedlings epicotil segments were used. The gene nptII, which confers resistance to the antibiotic kanamycin, was used as the selective system. From the regenerated shoots, a tissue sample was collected to perform the X-GLUC histochemical analysis. The regenerated shoots colored in blue were considered transgenic, and these were grafted on in vitro grown rootstocks. The transformation efficiency of each experiment was calculated based on the number of introduced explants, responsive explants, number of regenerated shoots, and number of GUS positive regenerated shoots. In order to confirm the genetic transformation of Hamlin sweet orange, PCR and Southern blot analyses of three positive GUS acclimatized plants were performed. Anatomic slices for better visualization of blue color formed by GUS reaction were also made. All transformation experiments regenerated at least one GUS positive shoot. Plants of Hamlin sweet orange analyzed by PCR and Southern blot are transformed and have one transgene insertion. Anatomical analyses indicated preferential expression of the transgenes in the phloem.

O Huanglongbing (HLB) é uma das doenças mais ameaçadoras para citricultura mundial e, até o momento, não foi encontrada resistência na base genética do gênero Citrus. A doença é causada pela bactéria Candidatus Liberibacter spp., endêmica de floema. Portanto, na busca por uma planta transgênica resistente ao HLB é desejável avaliar construções gênicas em que o gene de interesse se expresse preferencialmente na região em que a bactéria coloniza a planta, ou seja, no floema. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas via Agrobacterium tumefaciens, de citrange Carrizo [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] e de laranja doce [Citrus sinensis (L.) Osbeck] cultivares Hamlin, Valência e Pêra, contendo o gene uidA (GUS) sob o controle dos promotores Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) e Arabidopsis thaliana sucrose transporter 2 (AtSuT2), para verificar se esses promotores regulam a expressão do gene repórter na região do floema. Foram utilizados segmentos de epicótilo de plântulas germinadas in vitro e como agente de seleção de regeneração de plantas transgênicas foi utilizado o gene nptII, que confere resistência ao antibiótico canamicina. Dos brotos regenerados foi coletada uma amostra de material para a realização do ensaio histoquímico com X-GLUC. Os brotos que formaram coloração azulada confirmaram a integração do transgene, sendo esses enxertados em porta enxertos previamente germinados e estiolados in vitro. A partir do número de explantes introduzidos, número explantes responsivos, número de brotos regenerados e número de brotos regenerados GUS positivos calculou-se a eficiência de transformação genética dos experimentos. Para a confirmação da transformação genética de laranja Hamlin foram realizadas análises de PCR e Southern blot de três plantas GUS positivas aclimatizadas. Também foram feitos cortes histológicos manuais para melhor visualização da reação histoquímica de GUS das plantas de laranja Hamlin Southern blot positivas. Todos os experimentos de transformação regeneraram pelo menos um broto GUS positivo. As plantas de laranja Hamlin analisadas por PCR e Southern blot analisadas foram confirmadas como transformadas com uma inserção do transgene. Plantas nas quais foram realizados cortes histológicos indicaram expressão diferencial das construções gênicas no floema. / Huanglongbing (HLB) is one of the most threatening diseases to worldwide citriculture and till the present moment, no resistance has been found in citrus genetic basis. This disease has Candidatus Liberibacter spp. as the pathogenic agent, an endemic phloem bacterium. Therefore, in the search for a transgenic plant resistant to HLB, it is desirable to evaluate constructions in which the gene of interest is expressed, preferentially, in tissues where the bacteria grow, i.e., in the phloem. Therefore, this work aimed to obtain transgenic plants of Carrizo citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck], and of Hamlin, Valencia, and Pera sweet oranges [Citrus sinensis (L.) Osbeck], via Agrobacterium tumefaciens, containing uidA (GUS) gene, controlled by the promoters Citrus pholem protein 2 (CsPhP2), Arabidopsis thaliana pholem protein 2 (AtPhP2) and Arabidopsis thaliana sucrose transporter 2 (AtSuT2), in order to check if these promoters drive the reporter gene expression in the phloem. For the transformation, in vitro germinated seedlings epicotil segments were used. The gene nptII, which confers resistance to the antibiotic kanamycin, was used as the selective system. From the regenerated shoots, a tissue sample was collected to perform the X-GLUC histochemical analysis. The regenerated shoots colored in blue were considered transgenic, and these were grafted on in vitro grown rootstocks. The transformation efficiency of each experiment was calculated based on the number of introduced explants, responsive explants, number of regenerated shoots, and number of GUS positive regenerated shoots. In order to confirm the genetic transformation of Hamlin sweet orange, PCR and Southern blot analyses of three positive GUS acclimatized plants were performed. Anatomic slices for better visualization of blue color formed by GUS reaction were also made. All transformation experiments regenerated at least one GUS positive shoot. Plants of Hamlin sweet orange analyzed by PCR and Southern blot are transformed and have one transgene insertion. Anatomical analyses indicated preferential expression of the transgenes in the phloem.

Water quality in the Vaalharts region in the Northern Cape Province, South Africa, decreased over the past few years and there was a need for the microbiological and physico-chemical assessment. This problem was identified through discussions with Vaalharts Water (Vaalharts Water User Association) in 2010 when the issue of the impact of deteriorating water quality on drinking water production was raised. It was thus important to investigate concerns of the water users association pertaining to water quality issues. The aim of this study was to assess the physico-chemical and microbiological quality of household water in the Vaalharts irrigation scheme. The main residential areas were Hartswater, Pampierstad, Jan Kempdorp and Warrenton. Faecal coliforms were detected in the raw water of all the drinking water distribution systems during 2011 and 2012. No faecal coliforms were detected in the household water during 2011. This was a very positive result, because not only did the household water comply with the SANS 241 (2011) standard (0 CFU/100ml), but the purification processes were successful by removing all the E. coli’s from the raw water. However, during March 2012 faecal coliforms were detected in the household water of Jan Kempdorp (191CFU/100ml). This could be due to point pollution and possible breakage of faecal coliforms in the distribution system. Low amounts of total coliforms were detected in the raw water of some of the drinking water distribution systems. This could be due to high amounts of other colonies (pink and purple) growing on the m-Endo agar which suppress the growth of the metallic green sheen (total coliform) colonies. The total coliform numbers complied with the SANS 241 (2011) standard of ≤10 CFU/100ml at most of the distribution systems, except for Hartswater during July 2011 (14CFU/100ml) and Warrenton during March 2012 (256 CFU/100ml). Heterotrophic plate count bacteria were very high in the household water of some of the distribution systems during 2011 and 2012 which exceeded the SANS 241 (2011) standard of ≤1000 CFU/ml. A large number of pigmented (yellow, orange, pink) and non-pigmented (white) colonies were isolated on R2A agar. This can be an indication of some failure in treatment processes. Other microbiological parameters that were tested such as faceal streptococci, Clostridia, Pseudomonas aeruginosa and fungi did not indicate any danger, but there were high levels of total anaerobic bacteria in the raw water during 2011 and 2012. A high level of anaerobic bacteria was detected in the household water of Hartswater during July 2011. Clostridia were also present in the household water of some of the distribution systems during 2011 and 2012. Sequencing results of the mdh, lacZ and uidA genes indicated that one of the isolates was identified as Enterobacter cloacae and the other isolates were E. coli. Four of the isolates were identified as Escherichia coli O104:H4. This is a pathogenic strain and raised concern. The physicochemical parameters that were measured complied with the SANS 241 (2011) standards during 2011 and 2012, but some of the parameters increased gradually from 2011 to 2012. Statistical analysis indicated that physico-chemical parameters had an influence on microbiological parameters and that deteriorating raw water may have an impact on drinking water quality. Another concern currently is that there is no SANS 241 (2011) for faecal streptococci, Clostridia, Pseudomonas aeruginosa, fungi and anaerobic bacteria. These are all opportunistic pathogenic bacteria and consuming water with high levels of these bacteria may cause health problems. This study indicated good progress in the treatment processes of the distribution systems over the two years. This may be due to the feedback given to Vaalharts Water during this study regarding the water quality of the residential areas. The physico-chemical and microbiological results of the present study indicated possible biofilm formation in the distribution systems. This may have impacts on the drinking water quality of the distribution systems. It was also evident that deteriorating raw water sources may have an impact on drinking water production. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Water quality in the Vaalharts region in the Northern Cape Province, South Africa, decreased over the past few years and there was a need for the microbiological and physico-chemical assessment. This problem was identified through discussions with Vaalharts Water (Vaalharts Water User Association) in 2010 when the issue of the impact of deteriorating water quality on drinking water production was raised. It was thus important to investigate concerns of the water users association pertaining to water quality issues. The aim of this study was to assess the physico-chemical and microbiological quality of household water in the Vaalharts irrigation scheme. The main residential areas were Hartswater, Pampierstad, Jan Kempdorp and Warrenton. Faecal coliforms were detected in the raw water of all the drinking water distribution systems during 2011 and 2012. No faecal coliforms were detected in the household water during 2011. This was a very positive result, because not only did the household water comply with the SANS 241 (2011) standard (0 CFU/100ml), but the purification processes were successful by removing all the E. coli’s from the raw water. However, during March 2012 faecal coliforms were detected in the household water of Jan Kempdorp (191CFU/100ml). This could be due to point pollution and possible breakage of faecal coliforms in the distribution system. Low amounts of total coliforms were detected in the raw water of some of the drinking water distribution systems. This could be due to high amounts of other colonies (pink and purple) growing on the m-Endo agar which suppress the growth of the metallic green sheen (total coliform) colonies. The total coliform numbers complied with the SANS 241 (2011) standard of ≤10 CFU/100ml at most of the distribution systems, except for Hartswater during July 2011 (14CFU/100ml) and Warrenton during March 2012 (256 CFU/100ml). Heterotrophic plate count bacteria were very high in the household water of some of the distribution systems during 2011 and 2012 which exceeded the SANS 241 (2011) standard of ≤1000 CFU/ml. A large number of pigmented (yellow, orange, pink) and non-pigmented (white) colonies were isolated on R2A agar. This can be an indication of some failure in treatment processes. Other microbiological parameters that were tested such as faceal streptococci, Clostridia, Pseudomonas aeruginosa and fungi did not indicate any danger, but there were high levels of total anaerobic bacteria in the raw water during 2011 and 2012. A high level of anaerobic bacteria was detected in the household water of Hartswater during July 2011. Clostridia were also present in the household water of some of the distribution systems during 2011 and 2012. Sequencing results of the mdh, lacZ and uidA genes indicated that one of the isolates was identified as Enterobacter cloacae and the other isolates were E. coli. Four of the isolates were identified as Escherichia coli O104:H4. This is a pathogenic strain and raised concern. The physicochemical parameters that were measured complied with the SANS 241 (2011) standards during 2011 and 2012, but some of the parameters increased gradually from 2011 to 2012. Statistical analysis indicated that physico-chemical parameters had an influence on microbiological parameters and that deteriorating raw water may have an impact on drinking water quality. Another concern currently is that there is no SANS 241 (2011) for faecal streptococci, Clostridia, Pseudomonas aeruginosa, fungi and anaerobic bacteria. These are all opportunistic pathogenic bacteria and consuming water with high levels of these bacteria may cause health problems. This study indicated good progress in the treatment processes of the distribution systems over the two years. This may be due to the feedback given to Vaalharts Water during this study regarding the water quality of the residential areas. The physico-chemical and microbiological results of the present study indicated possible biofilm formation in the distribution systems. This may have impacts on the drinking water quality of the distribution systems. It was also evident that deteriorating raw water sources may have an impact on drinking water production. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Tesis por compendio / [EN] Cannabis sativa L. (Cannabaceae) is an angiosperm, allogamous and dicotyledonous species that includes short and neutral-day varieties with dioecious specimens (males and females), and monoecious plants. Among its many applications, its industrial and medicinal uses stand out. Despite the fact that cannabis has been used by humans since ancient times and the growing interest that the C. sativa therapeutic properties have aroused in researchers around the world, the psychoactivity of some of its varieties, derived from its ¿9-tetrahydrocannabinol (THC) content, has motivated the prohibition of its cultivation for almost sixty years. The strict control to which cannabis has been subjected has prevented professionals from all over the world from carrying out genetic breeding programs for this species, which has resulted in the absence of uniform varieties. In this Doctoral Thesis, different biotechnological tools for cannabis genetic improvement have been developed. In the first place, given the lack of reproducibility of some cannabis plant in vitro regeneration protocols and the great influence that the genotype exerts on their effectiveness, plant in vitro regeneration competence of different explants was evaluated. As a result, an hormone-free protocol from C. sativa hypocotyls that presents high regeneration rates (ranging from 32.26% to 71.15%) in all the genotypes evaluated, also presenting a 17.94% of spontaneous rooting rate of regenerants has been developed. At the same time, the polysomatic pattern of different cannabis explants has been studied, and it has been possible to regenerate, from them, a significant percentage of mixoploid specimens (17.65% from cotyledons and 13.33% from hypocotyls) that, as described in the existing literature, could show a greater capacity for cannabinoid synthesis. On the other hand, given the absence of scientific publications in this regard, and the potential that this technique presents to alleviate the intrinsic variability of this species, the most in-depth study to date on the male floral biology of C. sativa has been developed. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% have been found. Furthermore, all stages of development of the microgametophyte have been correlated with an easily measurable floral morphological marker such as the bud length, identifying bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains in all the phenotypes evaluated. In this way, and although the starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis, it has been possible to address the induction of microspore embryogenesis in this species, obtaining for the first time microspore-derived multicellular structures after one week long cold-shock bud pretreatment. Finally, as a prerequisite for the genetic editing of C. sativa by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, and taking advantage of the in vitro plant regeneration protocol which resulted from this Doctoral Thesis, it has been possible to develop for the first time a protocol for the production of stably transformed cannabis plants, which represents a historical milestone in the genetic improvement of the species. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyls achieved 23.1% and 5.0% of regeneration and transformation rates respectively. As a whole, the present Doctoral Thesis provides a range of biotechnological tools that will allow the development of a new generation of high-yield cannabis varieties with uniform traits, resistant to multiple biotic and abiotic stresses, and therefore being suitable for both industrial and medicinal use. / [ES] Cannabis sativa L. (Cannabaceae) es una especie angiosperma, alógama y dicotiledónea compuesta por variedades de día corto y día neutro que presentan ejemplares dioicos (machos y hembras), y plantas monoicas. Entre sus múltiples aplicaciones destacan tanto su uso industrial como su uso medicinal. A pesar de que el cannabis ha sido empleado por el ser humano desde tiempos ancestrales, la psicoactividad que presentan algunas de sus variedades, derivada de su contenido en ¿ 9 -tetrahidrocannabinol (THC), ha motivado la prohibición de su cultivo durante casi sesenta años. La estricta fiscalización a la que ha sido sometido el cannabis, ha impedido llevar a cabo programas de mejora genética de esta especie, lo que se ha traducido en la ausencia de variedades uniformes. En esta Tesis Doctoral se han desarrollado diferentes herramientas biotecnológicas para la mejora genética del cannabis. En primer lugar, dada la falta de reproducibilidad de algunos protocolos de cultivo in vitro de cannabis y la gran influencia que el genotipo ejerce en la efectividad de los mismos, se evaluó la capacidad de regeneración in vitro de diferentes explantes. Como resultado, se ha desarrollado un protocolo libre de hormonas a partir de hipocótilos de C. sativa que presenta altas tasas de regeneración (las cuales oscilan del 32,26% al 71,15%) en todos los genotipos evaluados, presentando además un 17,94% de tasa de enraizado espontáneo de los regenerantes. A su vez, se ha estudiado el patrón polisomático de diferentes explantes de cannabis y se ha conseguido regenerar, a partir de los mismos, un porcentaje significativo de ejemplares mixoploides (17,65% procedentes de cotiledones y 13,33% de hipocotilos) que, tal y como describe la bibliografía existente, podrían mostrar una mayor capacidad de síntesis de cannabinoides. Por otro lado, dada la ausencia de publicaciones científicas al respecto y el potencial que esta técnica presenta para paliar la variabilidad intrínseca de esta especie, se ha desarrollado el estudio más profundo hasta la fecha relativo a la biología floral masculina de C. sativa. Se han descrito hasta 476.903 microsporas y granos de polen por flor masculina, con tasas de viabilidad in vivo de las microsporas del 53,71 al 70,88%. Además, se han correlacionado todas las etapas de desarrollo del microgametofito con la longitud de la yema, identificando intervalos de longitud de yema que contienen mayoritariamente microsporas vacuoladas y granos de polen joven bicelular en todos los fenotipos evaluados. De este modo, y aunque la presencia de almidón en las microsporas y granos de polen de C. sativa sigue un patrón similar al observado en especies recalcitrantes a la androgénesis, ha sido posible abordar la inducción de la embriogénesis de microsporas en esta especie, consiguiendo producir por primera vez estructuras multicelulares derivadas de las microsporas tras aplicar sobre las yemas un pretratamiento de frío de una semana de duración. Finalmente, como requisito previo para la edición genética de C. sativa mediante los sistemas CRISPR/Cas, y haciendo uso del protocolo de regeneración in vitro de plantas surgido de la presente Tesis Doctoral, se ha conseguido desarrollar por primera vez un protocolo para producir plantas de cannabis transformadas genéticamente de forma estable, lo que supone un hito histórico en la mejora genética de la especie. Después del cocultivo con A. tumefaciens y el posterior cultivo en medio de regeneración selectiva con antibióticos, los hipocótilos lograron respectivamente un 23,1% y un 5,0% de tasas de regeneración y transformación. En su conjunto, la presente Tesis Doctoral proporciona un abanico de herramientas biotecnológicas que permitirán el desarrollo de una nueva generación de variedades de cannabis de alto rendimiento, que presenten caracteres homogéneos, resistentes a múltiples estreses tanto bióticos como abióticos, y siendo así aptas tanto para un uso industrial como medicinal. / [CAT] Cannabis sativa L. (Cannabaceae) és una espècie angiosperma, alógama i dicotiledònia composta per varietats de dia curt i dia neutre que presenten exemplars dioics (mascles i femelles), i plantes monoiques. Entre les seues múltiples aplicacions destaquen tant el seu ús industrial com el seu ús medicinal. Tot i que el cànnabis ha sigut emprat per l'ésser humà des de temps ancestrals, la psicoactivitat que presenten algunes de les seues varietats, derivada del seu contingut en ¿9-tetrahidrocannabinol (THC), ha motivat la prohibició del seu cultiu durant gairebé seixanta anys. L'estricta fiscalització a la qual ha sigut sotmés el cànnabis, ha impedit que professionals de tot el món puguen dur a terme programes de millora genètica d'aquesta espècie, la qual cosa s'ha traduït en l'absència de varietats uniformes. En aquesta Tesi Doctoral s'han desenvolupat diferents eines biotecnològiques per a la millora genètica del cànnabis. En primer lloc, donada la falta de reproducibilitat d'alguns protocols de cultiu in vitro de cànnabis i la gran influència que el genotip exerceix en l'efectivitat d'aquests, es va avaluar la capacitat de regeneració in vitro de diferents explants. Com a resultat, s'ha desenvolupat un protocol lliure d'hormones a partir de hipocòtils de C. sativa que presenta altes taxes de regeneració (les quals oscil·len del 32,26% al 71,15%) en tots els genotips avaluats, presentant a més un 17,94% de taxa d'arrelat espontani dels regenerants. Al mateix temps, s'ha estudiat el patró polisomàtic de diferents explants de cànnabis i s'ha aconseguit regenerar, a partir d'aquests, un percentatge significatiu d'exemplars mixoploids (17,65% procedents de cotilèdons i 13,33% de hipocòtils) que, tal com descriu la bibliografia existent, podrien mostrar una major capacitat de síntesi de cannabinoids. D'altra banda, donada l'absència de publicacions científiques sobre aquest tema i el potencial que aquesta tècnica presenta per a pal·liar la variabilitat intrínseca d'aquesta espècie, s'ha desenvolupat l'estudi més profund fins hui relatiu a la biologia floral masculina de C. sativa. S'han descrit fins a 476.903 microspores i grans de pol·len per flor masculina, amb taxes de viabilitat in vivo de les microspores del 53,71 al 70,88%. A més, s'han correlacionat totes les etapes de desenvolupament del microgametòfit amb la longitud de la gemma, identificant intervals de longitud de gemma que contenen majoritàriament microspores vacuolades i grans de pol·len jove bi-cel·lular en tots els fenotips avaluats. D'aquesta manera, i encara que la presència de midó en les microspores i grans de pol·len de C. sativa segueix un patró similar a l'observat en espècies recalcitrants a la androgènesi, ha sigut possible abordar la inducció de la embriogènesi de microspores en aquesta espècie, aconseguint produir per primera vegada estructures multicel·lulars derivades de les microspores després d'aplicar sobre les gemmes un pretractament de fred d'una setmana de duració. Finalment, com a requisit previ per a l'edició genètica de C. sativa mitjançant els sistemes CRISPR/Cas, i fent ús del protocol de regeneració in vitro de plantes sorgit de la present Tesi Doctoral, s'ha aconseguit desenvolupar per primera vegada un protocol per a produir plantes de cànnabis transformades genèticament de manera estable, la qual cosa suposa una fita històrica en la millora genètica de l'espècie. Després del cocultiu amb A. tumefaciens i el posterior cultiu en medi de regeneració selectiva amb antibiòtics, els hipocòtils van aconseguir respectivament un 23,1% i un 5,0% de taxes de regeneració i transformació. En el seu conjunt, la present Tesi Doctoral proporciona un ventall d'eines biotecnològiques que permetran el desenvolupament d'una nova generació de varietats de cànnabis d'alt rendiment, que presenten caràcters homogenis, resistents a múltiples estressos tant biòtics com abiòtics, i sent així aptes tant per a un ús industrial com medicinal. / Galán Ávila, A. (2021). Development of biotechnological tools for the genetic improvement of Cannabis sativa L [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/176013 / TESIS / Compendio