Abstract: Somatic hybridization, or protoplast fusion, is a biotechnological tool applicable to citrus improvement. Use of this technique allows addressing objectives that would be impossible to achieve through sexual hybridization or other techniques, both for improvement of existing varieties and rootstocks and to obtain new genotypes of interest. The main objective of this thesis has been the implementation of the somatic hybridization to specific needs of the Spanish citriculture. In addition studies on some technical aspects of the somatic hybridization have been developed, mainly to facilitate its application as a citrus improvement tool. The thesis has been divided into five chapters and one appendix that are described below.
The first chapter was directed towards obtaining new rootstocks of interest using genotypes with complementary traits. Selected genotypes included citrange "Carrizo" and Citrus macrophylla, which are the two most commonly rootstocks used in the Spanish citriculture. The difficulties encountered in obtaining somatic hybrids between the selected parents using the established fusion methods (chemical and electro-chemical) warrant the search for alternative methods that could increase the efficiency of the process. An electrical fusion method employing AC fields and DC pulses was adopted. This new protocol allowed regeneration, for the first time, of symmetrical somatic hybrids from the mentioned parents. It also allowed for direct comparison of the three methods available for citrus somatic hybridization, applying them in parallel to the same genotype combination.
The second chapter shows the comprehensive genetic characterization of the plants obtained, both at nuclear and cytoplasm levels. The results showed that somatic hybrids are not always the perfect combination of the parental genomes and changes at chromosome, subchromosome or cytoplasm level may occur. Application of the electric fusion method to other genotypes allowed regeneration of new hybrids of interest, including those with C. taiwanica and C. macrophylla, or "Encore" with "Rough" lemon. Also these hybrids were fully characterized at nuclear and cytoplasm levels. 
The third chapter shows the study carried out about the effect of the UV radiation on the integrity of the genome of citrus protoplastos. Interest in the application of this kind of radiation resides in the possibility of obtaining asymmetric somatic hybrids. The effect of exposure to different doses of UV light on the viability of both callus and leaves protoplastos, as well as on the multiplication of the callus-derived protoplastos, was studied. Flow cytometry analysis of UV-treated protoplastos demonstrated changes in the structure of the nuclear genome. A direct relationship between the dose of UV light and the degree of observed DNA fragmentation was found. In addition, agarose gels electrophoresis analysis displayed the level of fragmentation that occurs in the protoplasts treated with UV light. 
Clementines are the most important varieties in the Spanish citriculture. However, only a few somatic hybridization references employing members of this group can be found in the literature. The main cause for this is the difficulty to efficiently isolate and purify protoplasts from their leaves applying methodologies applied to other groups of citrus. Chapter fourth describes the development of a protocol specific for the isolation of Clementine protoplasts. Different variables that affect performance in protoplast isolation were studied, especially the physiological status of the plant material used and the conditions of isolation. 
The protocol has allowed to routinely obtaining protoplasts from different varieties of the Clementines group, as well as from some of their hybrids (described in Chapter 5). Implementation of this new approach allowed obtaining new somatic hybrids containing the genome of the "Clemenules" variety. The allotetratploide hybrid obtained between the "Clemenules" and a variety of satsuma can be used as parent to perform hybridizations and obtain new triploid hybrids. Diploid cybrids carrying the "Clemenules" nucleus and the Satsuma cytoplasm were also obtained. Transference of the male sterility trait (determined by the cytoplasm in satsuma varieties) onto the cybrid will be test in later work.
Microsatellite markers (SSR) are a very valuable tool in citrus improvement, helping on varietal identification, characterization of hybrids (both sexual and somatic), genetic mapping and discrimination between zygotic and nuclear individuals. However, not all discovered SSR markers are polymorphic for all citrus genotypes. Appendix 1 shows the results of the application of the SSCP (single strand conformation polymorphism) technique to SSR markers. The SSR-SSCP approach made it possible to detect polymorphisms in markers traditionally considered as monomorphics.