Inseticidas e fungicidas geralmente são estudados quanto a sua eficiência no controle de pragas e doenças, entretanto podem provocar efeitos fisiológicos pouco conhecidos capazes de influenciar o desenvolvimento das culturas. O tiametoxam é um inseticida sistêmico do grupo dos neonicotinóides, da família nitroguanidina, que atua no receptor nicotínico acetilcolina de insetos, danificando o sistema nervoso dos mesmos, levando-os à morte. Este inseticida, largamente utilizado no controle de pragas iniciais e insetos sugadores, apresenta efeito bioativador, uma vez que mesmo na ausência da pragas, promove aumento em vigor e desenvolvimento nas plantas tratadas. Acredita-se que os efeitos do tiametoxam em plantas são indiretos, pois atuam na expressão dos genes responsáveis pela síntese e ativação de enzimas metabólicas, relacionadas ao crescimento da planta, alterando a produção de aminoácidos e precursores de hormônios vegetais. Existem alguns trabalhos com tiametoxam com o objetivo de verificar seus efeitos no metabolismo e desenvolvimento das plantas, mas os resultados ainda não são claros, evidenciando forte interação entre cultivares, épocas, condições de estresse e disponibilidade de nutrientes. Em função da grande utilização do inseticida tiametoxam na agricultura brasileira, o estudo deste agroquímico, no sentido de alterar o metabolismo e desenvolvimento de plantas, constitui-se de grande importância na agregação de informações à literatura biológica e agronômica. Os objetivos deste trabalho foram verificar o efeito da aplicação de tiametoxam em plantas de cana-de-açúcar, feijoeiro, soja, laranjeira e cafeeiro, em diferentes doses e formas de aplicação (tratamento de sementes, pulverização foliar e aplicação no solo), sobre parâmetros de desenvolvimento (área foliar, massa seca de folhas e raízes, comprimento radicular) e aspectos bioquímicos (teores foliares de nutrientes e atividade enzimática). Os experimentos foram realizados em vasos, no Horto Experimental do Departamento de Ciências Biológicas da ESALQ/USP. Concluiu-se que a aplicação de tiametoxam em pulverização foliar, em cana-deaçúcar, aumenta a área foliar e o comprimento das raízes, amplia a espessura do córtex da raiz, incrementa o diâmetro do cilindro vascular e aumenta o número de metaxilemas; em feijoeiro, em tratamento de sementes, aumenta a área foliar, a massa seca da parte aérea e a atividade da nitrato redutase em folhas e em pulverização foliar aumenta a atividade da nitrato redutase e a atividade da fenilalanina amônia-liase em folhas; em soja, em tratamento de sementes, aumenta a área foliar, massa seca e comprimento das raízes e os teores foliares de cálcio e magnésio, e em pulverização foliar, reduz os teores de fósforo e cálcio, mas aumenta os teores de potássio; em mudas de laranjeira, aplicado no solo, aumenta a área foliar, a massa seca das folhas, a massa seca do caule e ramos e a área foliar média, mas reduz os teores foliares de nitrogênio, fósforo e enxofre; em mudas de café arábica, aplicado na raiz, aumenta o número de folhas, a massa seca de raízes finas e o comprimento das raízes. / Insecticides and fungicides are often studied to their efficiency in controlling pests and diseases, however can cause physiological effects little known that can affect crop growth. Thiamethoxan is a systemic insecticide from the neonicotinoid group, nitroguanidine family, which acts on nicotinic acetylcholine receptor of insects, damaging their nervous system, leading them to death. This insecticide, that is widely used for controlling pests and sucking insects, has bioactivator effect, since even in the absence of pests, promotes an increase in vigor and development in treated plants. It is believed that the effects of thiamethoxan in plants are indirect, because it acts in the genes expression responsible for metabolic enzymes activation and synthesis, related to plant growth, by modifying amino acids and plant hormones precursors production. There is a number of studies with thiamethoxan in the way to determine the effects on metabolism and development of plants, but the results are not clear, showing strong interaction between cultivars, stress conditions and nutrient availability. Due to the wide use of the insecticide thiamethoxan in Brazilian agriculture, the study of this insecticide in order to know the metabolism changes in the plants, brings a great importance in the collection of knowledge to the agronomic and biological literature. The objectives of this study was to evaluate the effect of thiamethoxan on sugar cane, common beans, soy, orange and coffee plants with different doses and forms of application (seed treatment, foliar spray and soil application) on developmental parameters (leaf area, dry mass of leaves and roots, root length) and biochemical aspects (nutrient content and enzymatic activity). The experiments were conducted in pots in the ESALQ / USP, Experimental Field of Biological Sciences Department. It was concluded that the application of thiamethoxan as foliar spray on sugar cane, increases leaf area and root length, increases the thickness of the cortex of the root, increases the diameter of the vascular cylinder and increases the number of vessels; in bean for seed treatment, increases the leaf area, dry mass of shoots and the activity of nitrate reductase in leaves, and foliar spray increases the activity of nitrate reductase and phenylalanine ammonia-lyase in leaves; in soybean, in seeds treatment, increases leaf area, dry weight of roots, root length and foliar calcium and magnesium, and foliar spray, reduces the amount of phosphorus and calcium, but increased levels of potassium; in orange trees, applicated on the soil, increases the leaf area, leaf area average, leaves dry mass, stems and branches dry mass, but reduces the amount of nitrogen, phosphorus and sulfur in the leaf; in arabica coffee seedlings, applied at the root, increases the number of leaves, fine roots dry mass and length of roots.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

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[ES] Los dobles haploides son una gran herramienta para la mejora genética de híbridos debido a que se puede alcanzar la homocigosis completa en una sola generación. Entre las técnicas usadas para obtener estas plantas, la inducción de la embriogénesis de microsporas, mediante el cultivo de anteras o de microsporas, es la más eficiente y la más usada. La embriogénesis de microsporas es también un ejemplo de totipotencia de las células vegetales gracias a su habilidad de reprogramar su desarrollo gametofítico hacia una ruta esporofítica, donde las células proliferan de forma organizada para crear un nuevo organismo. Como en muchos otros procesos in vitro, las condiciones de cultivo deben ser optimizadas para incrementar la eficiencia. En la presente Tesis Doctoral, hemos usado dos especies como sistemas experimentales para estudiar y optimizar el cultivo de microsporas. Por un lado, usamos berenjena (Solanum melongena) como un ejemplo de cultivo de importancia económica en el que los protocolos todavía tienen mucho margen de mejora. La optimización de la densidad celular y los reguladores de crecimiento han demostrado ser útiles para modificar la eficiencia del cultivo de microsporas de berenjena. Por otra parte, hemos utilizado el cultivo de microsporas de Brassica napus para estudios básicos puesto que ha sido ampliamente usado como modelo para entender procesos celulares que ocurren durante este cambio en el desarrollo. Se detalla un protocolo estandarizado para el cultivo de microsporas de B. napus, el cual ha sido utilizado en todos los cultivos en esta Tesis para explorar una serie de procesos y estructuras celulares potencialmente implicados en el cambio de desarrollo hacia embriogénesis. Estos procesos incluyen estrés del retículo endoplásmico, muerte celular programada, autofagia y estructura y composición de la pared celular. Estudiamos en paralelo el cultivo de microsporas de dos genotipos de B. napus con diferente respuesta androgénica en condiciones estándar y añadiendo Tricostatina A, un modulador epigenético que ha mostrado ser beneficioso para la respuesta androgénica en algunos casos. En conjunto, esta Tesis representa un avance en la optimización del cultivo de microsporas en estas especies y arroja luz sobre el papel de algunos procesos en el contexto de embriogénesis de microsporas. / [CA] Els dobles haploides són una gran eina en millora vegetal per a la producció d'híbrids, a causa de la seua total homozigosi, que es pot aconseguir en només una generació in vitro. Entre les diverses tècniques que s'utilitzen per tal d'obtenir aquestes plantes, la inducció de l'embriogènesi de microspores, mitjançant cultiu d'anteres o microspores, és la més comuna i eficient. L'embriogènesi de microspores també és un exemple de la totipotència de les cèl·lules vegetals, capaços de reprogramar-se d'una via gametofítica a una via esporofítica, on proliferen de manera organitzada per crear un nou organisme. Com en moltes altres tecniques in vitro, s'han d'optimitzar les condicions del cultiu per tal d'augmentar l'eficiència. En la present Tesi Doctoral, hem utilitzat dues espècies de plantes com a sistemes experimentals per estudiar i optimitzar el cultiu de microspores. Per una banda, hem utilitzat l'albergínia (Solanum melongena) com a exemple de cultiu d'importància econòmica on els protocols encara tenen marge per a l'optimització. La optimització de la densitat de cèl·lules en cultiu i la concentració de reguladors de creixement van demostrar ser útils per modificar l'eficiència de la resposta dels cultius de microspores d'albergínia. D'altra banda, hem utilitzat cultius de microspores de Brassica napus principalment per a estudis bàsics, ja que s'utilitza àmpliament com a model per entendre els processos cel·lulars que es produeixen durant aquest canvi de desenvolupament. Es detalla un protocol estandarditzat per al cultiu de microspores de B. napus, que s'ha utilitzat en tots els cultius inclosos en aquesta Tesi per explorar una sèrie de processos i estructures cel·lulars potencialment implicades en el canvi de desenvolupament cap a l'embriogènesi. Aquests inclouen l'estrès del reticle endoplasmàtic, la mort cel·lular programada, l'autofàgia i l'estructura i composició de la paret cel·lular. Vam estudiar en paral·lel cultius de microspores de dos genotips de B. napus amb diferent resposta androgènica, cultivats en condicions estàndard i afegint-hi Tricostatina A, un modulador epigenètic que s¿ha demostrat beneficiós per a la resposta androgènica en alguns casos. En conjunt, aquesta Tesi representa un avanç en l'optimització dels cultius de microsporas en aquestes espècies i aporta llum sobre el paper d'alguns processos en el context de l'embriogènesi de microspores. / [EN] Doubled haploids are a great tool for hybrid breeding due to their complete homozygosity achievable in only one in vitro generation. Among the several techniques used to obtain these plants, induction of microspore embryogenesis, via anther or microspore culture, is the most common and efficient approach. Microspore embryogenesis is also an example of totipotency of plant cells due to their ability to reprogram themselves from a gametophytic to a sporophytic pathway, where cells proliferate in an organized way to create a new organism. As in many other in vitro procedures, culture conditions must be optimized in order to increase efficiency. In the present Doctoral Thesis, we used two plant species as experimental systems to study and optimize microspore culture. On one hand, we used eggplant (Solanum melongena) as an example of economically important crop where protocols have still room for optimization. Optimization of cell density and growth regulators demonstrated to be useful to modify the efficiency of eggplant microspore cultures. On the other hand, we used B. napus microspore cultures principally for basic studies since it is widely used as a model to understand cellular processes occurring during this developmental switch. A standardized protocol for Brassica napus microspore culture is detailed, which was used in all the cultures included in this Thesis to explore a series of processes and cellular structures potentially involved in the developmental switch towards embryogenesis. These included endoplasmic reticulum stress, programmed cell death, autophagy, and cell wall structure and composition. We studied in parallel microspore cultures from two B. napus genotypes with different androgenic response cultured in standard conditions and adding Trichostatin A, a epigenetic modulator shown to be beneficial for the androgenic response in some cases. Together, this Thesis represents an advance in the optimization of microspore cultures in these species, and sheds light on the role of some processes within the context of microspore embryogenesis. / Thanks are due to the Electron Microscopy Service of Universitat Politècnica de València, Marisol Gascón (IBMCP Microscopy Service). This work was supported by grant AGL2017-88135-R to JMSS from MICINN jointly funded by FEDER and by a Marie Skłodowska-Curie Individual Fellowship (656579) to PC-M This work was supported by grant AGL2017-88135-R to JMSS from MINECO jointly funded by FEDER. / Camacho Fernández, C. (2021). Microspore embryogenesis: cell wall dynamics and reprogramming of cell fate [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/163698