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Potato is one of the main targets for genetic improvement by gene transfer. The aim of this study was to compare the efficacy of genetic transformation of potato, cultivar Bintje, using two methods: Agrobacterium tumefaciens mediated transformation and microprojectile bombardment. The same plasmid p35SGUSint, which cosists of 35S CaMV promoter, gus and nptII genes, was used for both transformations of internodal potato explants. Kamamycin selection, transient and stable expressions of {$\beta$}-glucuronidase and PCR amplification of gus and nptII transgenes were used for transgenic plant selection, identification and analysis.

ResumenEn esta tesis se ha optimizado un protocolo de regeneración a partir de material varietal de 'Helena' y 'Canino'. Mediante el estudio de los diversos factores que afectan la transformación de material adulto, se ha establecido por primera vez un protocolo eficiente de transformación mediada por Agrobacterium tumefaciens de una variedad comercial de albaricoquero.El diseño de una estrategia de selección gradual con paromomicina ha permitido la regeneración de plántulas transformadas con los genes marcadores nptII y sgfp o gus, con las eficiencias más elevadas que se han publicado hasta el momento para transformar material varietal en especies del género Prunus, aunque la baja viabilidad de las yemas transformadas redujo el número final de plantas obtenidas.El protocolo establecido en esta tesis sienta las bases que permitirán la introducción de genes de interés agronómico y comercial, modificando de manera discreta variedades élite aceptadas y establecidas en el mercado. / In this thesis a protocol of regeneration has been optimized from leaf explants of the cultivars 'Helena' and 'Canino'. By means of the study of the diverse factors that affect the transformation of adult material, an efficient protocol of Agrobacterium tumefaciens-mediated transformation has been established for the first time for a commercial cultivar of apricot.The design of a gradual selection strategy with paromomycin has permitted the regeneration of transformed shoots with the marker genes nptII and sgfp or gus, with the highest efficiencies that have been published up to now from adult material in Prunus, although the low viability of the transformed buds reduced the final number of plants obtained. This protocol establishes the bases that will permit the introduction of agronomic and commercial interesting genes, modifying discreetly commercial cultivars accepted and established in the market.

Plant promoters are the principle cis-acting regulatory sequences responsible for the temporal and spatial expression of genes. One method for isolating plant promoters is based on the ability of a common soil bacterium, <i> Agrobacterium tumefaciens </i>, to transfer a specific segment of DNA (T-DNA) into plant cells. This specific T-DNA has been shown to integrate stably into the recipient plant genome. If the T-DNA contains a promoterless marker gene, then T-DNA integration events occurring adjacent and downstream to a promoter region can be detected by the activation of the marker gene. These T-DNA-mediated gene fusions, consisting of an unknown plant promoter sequence and the coding sequence of a marker gene, can be isolated using the marker gene as a promoter tag. The key objective of this work was to develop a novel, bifunctional selectable marker gene and assess its use as: a selectable marker gene in bacterial and plant transformation systems, and as a promoter tag for T-DNA promoter-tagging studies in dicots. A bifunctional fusion gene was produced between phosphinothricin acetyltransferase and neomycin phosphotransferase (PAT::NPT II), by fusing an NPT II coding sequence to the 3' terminus of the PAT gene. The PAT gene product confers tolerance to a non-selective herbicide L-phosphinothricin (Ignite, Hoechst AG). The neomycin phosphotransferase ('npt II') gene allows for direct selection of transformed cells with the antibiotic, kanamycin. Using an <i>in vivo Escherichia coli </i> selection system, a translational fusion gene between these two reporter genes was achieved. The resulting protein had activities of both parent enzymes. This was demonstrated both in transformed <i>Escherichia coli</i> and in transformed <i>Nicotiana tabacum</i> and <i>Brassica napus</i> plants. Using this bifunctional selectable marker gene, a T-DNA promoter tagging vector, pBAU2, was constructed and its utility was demonstrated in <i>Nicotiana tabacum</i>. One of the <i>N. tabacum</i> promoter tagged events was selected for subsequent promoter isolation studies. The promoter from this regenerant was isolated by screening a Lambda subgenomic library and also by thermal asymmetric interlaced (TAIL-)PCR. The isolated upstream regulatory sequence was fused to a reporter gene, â-glucuronidase ('gus'), and subjected to a preliminary evaluation in <i> Nicotiana tabacum</i> and in <i>Brassica napus</i>.

An efficient system for or 'Agrobacterium'-mediated transformation of <i>Brassica carinata</i> was used together with a promoterless <i> gus</i>::<i>nptII</i> gene fusion to isolate putative promoter sequences. Cotyledonary petioles were transformed using the promoterless gene fusion construct. Only transformation events in which the promoterless gene fusion had integrated downstream from plant regulatory sequences were expected to produce viable tissue under kanamycin selection. Forty-two transgenic plants were recovered. Transformation efficiency was approximately 0.6%. Regenerated plants were screened for GUS expression in different tissues and organs by histological and fluorometric assays. Tissue-specific GUS expression was detected (stigmas, seed coat, leaf edges and vascular tissue) in some plants, while strong constitutive GUS expression was detected in others (based on GUS histological assays). Using subgenomic libraries, putative promoter fragments were isolated from the plants which exhibited GUS expression in stigmas, leaf edges and constitutively. A putative promoter fragment from a plant which exhibited GUS expression only in the stigma was fused with the gus gene and reintroduced by <i>Agrobacterium </i> -mediated transformation into <i>B. napus, B. carinata, Arabidopsis' and tobacco </i>. GUS expression was observed in the stigma of <i>B. napus </i> but not in ' B. carinata'. In <i>Arabidopsis </i> and tobacco GUS expression. was not tissue specific (weakly constitutive or restricted to two or more tissues). The 3' DNA sequence (15 kb) flanking the <i> gus</i>::<i>nptII </i> insert in the plant with GUS expression in the stigma was also isolated using a subgenomic library. A gene for a cytochrome P450 like protein was discovered on the minus DNA strand of the 3' sequence with a start codon approximately 6.5 kb from the T-DNA left border.

The aim of this study was to analyze number of incorporated T-DNA harbouring pmi and nptII transgenes and expression of the pmi gene in tomato transformants using Southern blot and Northern blot methods. The presence of a functional PMI protein was assesed using PMI-assay with Chlorophenol red dye. Mannose selection of tomato seeds was established and segregation patterns in T1 progeny were studied.

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