Variety improvement in rice (Oryza sativa L.): proteomic, hormonal and in vitro studies

Abstract

Rice (Oryza sativa L.) is without doubt one of the major crops worldwide, as its consumption is continuously increasing, especially in low- and lower-middle-income countries where it is the most important staple. This cereal has been domesticated for a long time, result of which several species and varieties are now available. It was key during the green revolution when its production increased in more than 2-fold times, and in addition has a deep cultural background in all the regions where it is grown. Hence, it is of uttermost importance for researchers and breeders to broaden and expand the knowledge on this cereal on all the study and research areas, especially now that we are living in the 21st century, which is marked by climate change. This phenomenon is one of the most menacing as it will reduce the quantity and quality of arable land due to salinization of soil as well as water scarcity which is the single and most important factor that determines global crop yields. In this sense, this thesis addresses to three important topics on rice that will help researchers and breeders for the improvement of rice varieties in the forthcoming future: (i) Analysis of salinity tolerant rice plants subjected to high salt concentration through a combined approach of shotgun proteomics and physiological characterization for the identification of new key proteins involved in the tolerance to this stress. In this study we provide a huge database for future studies of the salinity-stress response in rice varieties harboring the Saltol region, as we detected more than 2000 proteins involved during the early stages of salinity stress in the shoots and roots of the FL478 rice line. Moreover, this study highlights the importance of examining both the shoots and roots because their salinity tolerance traits are different and respond to different requirements of the rice plant. Finally, the information presented here expands our knowledge on adaptive processes under high salinity in rice plants and, notably, in rice roots. (ii) Phytohormones analysis in dwarf varieties through a broad phytohormone profiling method developed during this thesis for the characterization of phytohormone levels during the development of plants with contrasting heights that will allow to have more information for developing new dwarf mutant varieties. In this work, we reported that GA19 seems to have a crucial role in gibberellin availability in rice as its levels were much higher than all the other gibberellins in all tissues. In addition, it has been demonstrated that the GA20ox-2 mutation is not the only factor affecting height in rice, as a mutated variety had an increased growth during the half of its development period. Finally, we established for the first time a simple and broad phytohormone extraction and detection protocol that allows to identify 13 gibberellins and ABA, JA and IAA in several tissues at different phenological stages. (iii) Improvement and enhancement of anther culture protocols in rice for obtaining higher rates of stabilized green double haploid plants using different cold-pretreatments and additives in the growing media such as hormones and antimitotics in the growing media for their subsequent commercialization. It was evidenced that the best cold-pretreatment was 10ºC for 9 days, and that colchicine addition greatly enhances the production of green double haploid plantlets. Moreover, we have introduced for the first time a post-anther culture treatment with good prospects. Finally, several of the rice lines produced were assayed in rice paddy fields where they displayed good agronomical behavior.

El arroz (Oryza sativa L.) es sin duda uno de los principales cultivos del mundo, especialmente en países en vías de desarrollo donde es el alimento básico, teniendo además un importante componente cultural. Fue clave en la revolución verde cuando su producción aumentó más del doble. En el siglo 21 marcado por el cambio climático, se verá reducida la cantidad y calidad de las tierras cultivables debido a la salinización del suelo y la escasez de agua. Por tanto, es de gran importancia para los investigadores y mejoradores ampliar el conocimiento que se tiene sobre este cereal en todas las áreas de estudio. En este sentido, esta tesis abordó tres temas importantes referentes a este cereal: (i) Un estudio del proteoma, de la parte aérea y radicular, de plantas de arroz tolerantes a la salinidad sometidas a una alta concentración de sal con el objetivo de identificar de nuevas proteínas clave involucradas en la tolerancia a este estrés. Proporcionamos una gran base de datos, más de 200 proteínas involucradas en este estrés y destacamos la importancia de las raíces para la tolerancia a este estrés. (ii) Análisis de fitohormonas en variedades enanas a través de un amplio y rápido método desarrollado durante esta tesis que permite, por primera vez en arroz, la extracción y detección de fitohormonas que posibilida la identificación de 13 giberelinas y ABA, JA e IAA en varios tejidos en diferentes etapas fenológicas. Se observó que la GA19 parece tener un papel crucial en la disponibilidad de giberelinas en el arroz, ya que sus niveles son los más elevados en todos los tejidos. La mutación GA20ox-2 no es el único factor que afecta la altura ya que la variedad mutada alcanza la altura de variedades wild-type. (iii) Por último, se reportan dos métodos de aumento de la eficiencia del protocolo de cultivo de anteras, técnica muy útil para la obtención de plantas de arroz dihaploides estables. El aumento del rendimiento se debió a nuevos pretratamientos de frío, así como la modificación de los medios de cultivo mediante diferentes concentraciones de hormonas y colchicina. Se introdujo, por primera vez, un tratamiento de diploidización para plantas haploides.