La embriogénesis derivada de microsporas es la ruta androgénica más empleada para la obtención de individuos dobles haploides (DHs), 100% homocigotos, que pueden ser utilizados como líneas puras. Este proceso es una alternativa biotecnológica a los programas clásicos de mejora que permite reducir el tiempo y los recursos necesarios para la obtención de híbridos comerciales. Gracias a los avances realizados en los últimos 30 años, hay determinadas especies que pueden ser consideradas modelo debido a la elevada eficiencia que presentan en la obtención de individuos DHs. Sin embargo, aun existen muchas especies, interesantes desde un punto de vista agrícola y comercial, que permanecen recalcitrantes a la embriogénesis de microsporas. Debido al elevado potencial biotecnológico de esta técnica es fundamental mejorar el proceso en este tipo de especies en las que la androgénesis no está puesta a punto. Para ello es esencial realizar estudios en los que se combine un enfoque más aplicado, tratando de conseguir las condiciones experimentales más adecuadas, y en paralelo, un enfoque más básico, orientado a investigar las bases de la reprogramación de las microsporas y de este modo, tener una mayor posibilidad de influir en el proceso. En esta Tesis se han empleado ambos enfoques, empleando la colza como especie modelo y el pimiento como especie recalcitrante a la androgénesis. En pimiento, el estudio de factores clave para la inducción de la androgénesis nos ha permitido optimizar un protocolo de cultivo de anteras aplicable a diferentes genotipos. Proponemos el uso combinado de la relación sépalo/yema (80-90% de la yema cubierta por los sépalos) junto con la pigmentación de las anteras (extremo apical morado) como marcadores morfológicos fiables y fáciles de medir para determinar qué yemas y anteras contienen las microsporas en su estadio idóneo para la inducción. También se ha demostrado que la presencia de callos de origen somático es más dependiente de las condiciones de cultivo que del genotipo, lo cual nos ha permitido obtener un protocolo que reduzca la presencia de estos callos e incremente el número de embriones obtenidos. En los estudios de investigación básica realizados con colza, el uso de la fijación por alta presión combinada con la criosustitución ha permitido observar cambios ultraestructurales en las microsporas inducidas nunca antes descritos. Se ha estudiado la arquitectura y composición de las paredes celulares que se forman de novo en las microsporas embriogénicas y se han observado paredes celulares incompletas y deformes que podrían estar favoreciendo fenómenos de fusión nuclear, que a su vez da lugar a la duplicación cromosómica típica de los DHs. Se ha comprobado que estas paredes presentan elevados niveles de calosa y ausencia de celulosa. También se ha estudiado la arquitectura de diferentes orgánulos presentes en las microsporas recién inducidas y se ha demostrado que los plastidios presentes en las microsporas embriogénicas actúan como plastolisomas. Estos estudios ultraestructurales han servido para extraer dos nuevos marcadores de embriogénesis: la presencia de calosa en las nuevas paredes y en las placas celulares en formación y plastolisomas que posiblemente actúen como parte de un mecanismo más general de limpieza del citoplasma, en paralelo a la reprogramación. / Parra Vega, V. (2015). Aspectos básicos y aplicados de la inducción de embriogénesis en microsporas de pimiento y colza [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48518 / TESIS

In this study, buckwheat (Fagopyrum esculentum Moench) and alyssum (Lobularia maritima (L.)) flowers were used to examine the effect of floral resources on the efficacy of the leafroller parasitoid Dolichogenidea tasmanica (Cameron) in vineyards. This was done by assessing the influence of these flowers on parasitoid abundance and parasitism rate, and by investigating the consequences of this for leafroller abundance. In laboratory experiments, alyssum flowers were used to investigate the effect of floral food on the longevity, fecundity and sex ratio of D. tasmanica. Dolichogenidea tasmanica comprised more than 95 % of parasitoids reared from field collected leafrollers in this study. The abundance of D. tasmanica during the 1999-2000 growing season was very low compared with previous studies, possibly due to the very low abundance of its leafroller hosts during the experiment. The number of males of this species on yellow sticky traps was increased (although not significantly) when buckwheat flowers were planted in a Marlborough vineyard; however, the number of female D. tasmanica on traps was no greater with flowers than without. The abundance of another leafroller parasitoid, Glyptapanteles demeter (Wilkinson)(Hymenoptera: Braconidae), on traps was also not significantly affected by the presence of buckwheat flowers, although females of this species were caught in greater numbers in the control than in buckwheat plots. Naturally-occurring leafrollers were collected from three vineyard sites in Marlborough, and one in Canterbury during the 2000-2001 season to assess the effect of buckwheat and alyssum flowers on parasitism rate. Parasitism rate more than doubled in the presence of buckwheat at one of the Marlborough vineyards, but alyssum had no effect on parasitism rate in Canterbury. A leafroller release/recover method, used when naturally-occurring leafrollers were too scarce to collect, was unable to detect any effect of buckwheat or alyssum on parasitism rate. Mean parasitism rates of approximately 20 % were common in Marlborough, although rates ranged from 0 % to 45 % across the three vineyard sites in that region. In Canterbury in April, mean parasitism rates were approximately 40 % (Chapter 4). Rates were higher on upper canopy leaves (40-60 %) compared with lower canopy leaves and bunches (0-25 %). Leafroller abundance was apparently not affected by the presence of buckwheat in Marlborough, or alyssum in Canterbury. Buckwheat did, however, significantly reduce the amount of leafroller evidence (webbed leafroller feeding sites on leaves or in bunches) in Marlborough, suggesting that the presence of these flowers may reduce leafroller populations. Leafrollers infested less than 0.1 % of Cabernet Sauvignon leaves throughout the 1999-2000 growing season, but increased in abundance in bunches to infest a maximum of 0.5 % of bunches in late March in Marlborough. In Pinot Noir vines in the 2000-2001 season, leafroller abundance was also low, although sampling was not conducted late in the season when abundance reaches a peak. In Riesling vines in Canterbury, between 1.5 % and 2.5 % of bunches were infested with leafrollers in April. In the laboratory, alyssum flowers significantly increased the longevity and lifetime fecundity of D. tasmanica compared with a no-flower treatment. However, daily fecundity was not increased by the availability of food, suggesting that the greater lifetime fecundity was related to increases in longevity. Parasitoids were also able to obtain nutrients from whitefly honeydew, which resulted in similar longevity and daily fecundity to those when alyssum flowers were present. The availability of food had a significant effect on the offspring sex ratio of D. tasmanica. Parasitoids reared from naturally-occurring leafrollers produced an equal sex ratio, assumed to be the evolutionarily stable strategy (ESS) for this species. In the laboratory, this ESS was observed only when parasitoids had access to alyssum flowers. Without food, or with honeydew only, sex ratios were strongly male-biased. In the field, floral resources affected the sex ratio of D. tasmanica only when this species was reared from leafrollers released and recovered in Marlborough. In that experiment, buckwheat shifted the sex ratio in favour of female production from the equal sex ratio found in control plots. No firm explanations can be given to account for these results, due to a lack of research in this area. Possible mechanisms for the changes in sex ratio with flowers are discussed. This study demonstrated that flowers are an important source of nutrients for D. tasmanica, influencing the longevity, fecundity and offspring sex ratio of this species. However, only some of the field experiments were able to show any positive effect of the provision of floral resources on parasitoid abundance or parasitism rate. More information is needed on the role these parasitoids, and other natural enemies, play in regulating leafroller populations in New Zealand vineyards, and on how they use floral resources in the field, before recommendations can be made regarding the adoption of this technology by growers.

Arthropod species that have the potential to damage crops are food resources for communities of predators and parasitoids. From an agronomic perspective these species are pests and biocontrol agents respectively, and the relationships between them can be important determinants of crop yield and quality. The impact of biocontrol agents on pest populations may depend on the availability of other food resources in the agroecosystem. A scarcity of such resources may limit biological control and altering agroecosystem management to alleviate this limitation could contribute to pest management. This is a tactic of ‘conservation biological control’ and includes the provision of flowers for species that consume prey as larvae but require floral resources in their adult stage. The use of flowers for pest management requires an understanding of the interactions between the flowers, pests, biocontrol agents and non-target species. Without this, attempts to enhance biological control might be ineffective or detrimental. This thesis develops our understanding in two areas which have received relatively little attention: the role of flowers in biological control by true omnivores, and the implications of flower use by fourth-trophic-level life-history omnivores. The species studied were the lacewing Micromus tasmaniae and its parasitoid Anacharis zealandica. Buckwheat flowers Fagopyrum esculentum provided floral resources and aphids Acyrthosiphon pisum served as prey. Laboratory experiments with M. tasmaniae demonstrated that although prey were required for reproduction, providing flowers increased survival and oviposition when prey abundance was low. Flowers also decreased prey consumption by the adult lacewings. These experiments therefore revealed the potential for flowers to either enhance or disrupt biological control by M. tasmaniae. Adult M. tasmaniae were collected from a crop containing a strip of flowers. Analyses to determine the presence of prey and pollen in their digestive tracts suggested that predation was more frequent than foraging in flowers. It was concluded that the flower strip probably did not affect biological control by lacewings in that field, but flowers could be significant in other situations. The lifetime fecundity of A. zealandica was greatly increased by the presence of flowers in the laboratory. Providing flowers therefore has the potential to increase parasitism of M. tasmaniae and so disrupt biological control. A. zealandica was also studied in a crop containing a flower strip. Rubidium-marking was used to investigate nectar-feeding and dispersal from the flowers. In addition, the parasitoids’ sugar compositions were determined by HPLC and used to infer feeding histories. Although further work is required to develop the use of these techniques in this system, the results suggested that A. zealandica did not exploit the flower strip. The sugar profiles suggested that honeydew had been consumed by many of the parasitoids. A simulation model was developed to explore the dynamics of aphid, lacewing and parasitoid populations with and without flowers. This suggested that if M. tasmaniae and A. zealandica responded to flowers as in the laboratory, flowers would only have a small effect on biological control within a single period of a lucerne cutting cycle. When parasitoids were present, the direct beneficial effect of flowers on the lacewing population was outweighed by increased parasitism, reducing the potential for biological control in future crops. The results presented in this thesis exemplify the complex interactions that may occur as a consequence of providing floral resources in agroecosystems and re-affirm the need for agroecology to inform the development of sustainable pest management techniques.

Landscape heterogeneity is thought to differ among farm management types (i.e. organic and conventional), and this difference is hypothesized to result in variations in pest control by natural enemies. However, it is unclear if these variations in pest control are driven by landscape structure or by farm management practices themselves. Remotely sensed datasets were used to describe the landscape structure surrounding a group of organic and conventional caneberry farms in Oregon and Washington that have different leafroller parasitism rates attributed to farm management type. A finer scale survey was done at one of the farms using the remotely sensed data as well as field surveys. Landscape metrics of diversity, richness and percent non-crop were used to describe the landscapes surrounding the farm fields at scales ranging from 0.05 km to 5.00 km for the large scale study, and 0.05 km to 0.20 km for the fine scale study. In the fine scale study, data on parasitoid species assemblages, diversity, and parasitism rate were collected and analyzed against the calculated landscape metrics spatially and seasonally. The purpose of this study was to quantify effects of farm management type on habitat structure, effect of habitat structure on leafroller parasitism rate, and to access correlations between landscape metrics calculated at the landscape and field scale. Overall, the farms were embedded in a landscape that was broadly similar, with very few differences in landscape structure occurring between organic and conventional farms. Organic farms had higher vegetation height class diversity at the largest scale compared to conventional farms, while conventional farms had significantly higher percent non-crop area compared to organic farms. There was no significant effect of any of the calculated landscape metrics on parasitism rates. In the field scale study, no correlations were found between habitat metrics and parasitism rates, or between field based metrics and those calculated at the landscape scale. The results of this study suggest that conventional and organic caneberry farms in the Willamette Valley are broadly similar in the habitat conditions they provide parasitoids. This suggests that management changes to pesticide use alone could increase levels of leafroller biological control on conventional farms to levels that are comparable to those seen on organic farms. Our comparisons of the landscape scale and field scale landscape metrics showed no connection, this suggests that direct comparisons cannot be made with these particular metrics at these very different scales. Rather than comparing these types of data, it may be more useful to combine them in order to increase the resolution and predictive power of remotely sensed data for describing landscapes at broad scales. / Graduation date: 2013