La croissance hétérotrophe de l’hypocotyle est une étape clé pour la réussite de la levée. La présente étude est focalisée sur le déterminisme génétique de l’allongement de cet organe à l’obscurité chez Medicago truncatula en analysant le nombre et la longueur des cellules de l’épiderme, tissu gouvernant l’allongement des organes. Une grande variabilité génétique du nombre de cellules a été révélée dans les graines de 15 génotypes représentatifs de la diversité génétique de l’espèce. La stabilité de ce caractère dans des graines provenant de différentes productions suggère qu’il est sous contrôle génétique fort. Il a été montré que ce nombre de cellules, préétabli dans les graines, est le principal déterminant de la variation génotypique de la longueur de hypocotyle en conditions optimales de croissance. Par contre, l'élongation cellulaire devient le déterminant majeur des différences génotypiques observées sous stress abiotiques (basse température, déficit hydrique).Des loci contrôlant le nombre de cellules de l’épiderme et leur longueur maximale à basse température ont ensuite été identifiés dans une population de lignées recombinantes. Ceux ayant un impact sur l’élongation de l’hypocotyle à basse température ont été mis en évidence. Enfin, deux génotypes présentant un nombre de cellules similaire mais des capacités d’allongement cellulaire contrastées ont été plus finement comparés. Des protéines ayant un rôle dans la formation et l’organisation du cytosquelette et dans la modification des parois cellulaires ont été révélées en lien avec les différences d’allongeme / The heterotrophic growth of hypocotyl is a crucial process for successful seedling emergence. The present study is focused on the genetic determinism of its elongation in darkness in Medicago truncatula by analyzing the number and the length of cells of epidermis, the tissue controlling organ elongation.A large genetic variability of the epidermal cell number of the hypocotyl in seeds of 15 genotypes representative of the genetic diversity of the species was revealed. The stability of this trait in the seeds collected from different productions suggests it is under strong genetic control. This cell number was shown to be the main contributor of genotypic variation of hypocotyl length in optimal conditions. On the other hand, cell elongation becomes the major determinant of the genotypic differences observed under abiotic stresses (low temperature, water deficit).Quantitative Trait Loci (QTLs) controlling the number of epidermal cells and their maximal length at low temperature were then identified using a recombinant inbred lines population, and those impacting hypocotyl elongation at low temperature were highlighted.Finally, two genotypes sharing a similar cell number but contrasted capacities of cell elongation were compared more in detail. Proteins playing a role in the formation and organization of cytoskeleton and in the modification of the cell wall were revealed in connection with the differences in cellular elongation between genotypes. Moreover, differences in the cell wall sugar composition, in the degree of methylation of pectins and in a potential inhibito

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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 / Compendio

During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.

During embryogenesis and post-embryonic patterning, active transport of the phytohormone auxin, reflected in the expression of the Arabidopsis PIN family of auxin efflux mediators, generates local auxin distributions that are crucial for correct organ and tissue specification. Polar auxin transport routes have also long been postulated to regulate vein formation in the leaf. The molecular identification of PIN proteins has made it possible to investigate this hypothesis further by visualizing auxin transport routes in developing leaves. In Arabidopsis leaf primordia, PIN1 is expressed before the earliest known markers of vascular identity, in domains that are gradually restricted to sites of vein formation. PIN1 polarity indicates that auxin is directed towards distinct “convergence points” (CPs) in the marginal epidermis, from which it defines the sites of major vein formation. Within incipient veins, PIN1 polarity indicates drainage of auxin into preexisting veins, such that veins connected at both ends display two divergent polarities. Local auxin application triggers the formation of ectopic CPs and new veins, demonstrating the sufficiency of auxin as a vein-specifying signal. However, not all PIN1-labeled auxin transport routes differentiate as veins: Minor veins are initially unstable, suggesting local competition for auxin transport. Expression of ATHB8, a marker of vascular cell selection, correlates with enhanced PIN1 expression domain (PED) stability and vascular differentiation. Auxin application and auxin transport inhibition reveal that both CP formation in the epidermis and subepidermal PED dynamics are auxin-dependent and self-organizing. Furthermore, normal auxin perception through the ARF-Aux/IAA signaling pathway is required for the restriction of PIN1-mediated auxin transport to narrow subepidermal domains. ARF-Aux/IAA signaling is known to control auxin transport through the regulation of PIN1 dynamics, but the mechanism of this regulation is unclear. It is here shown that two redundantly acting AUXIN RESPONSE FACTOR (ARF) transcription factors, ARF5/MONOPTEROS (MP) and ARF7/NPH4, jointly regulate both PIN1 expression and localization during lateral root patterning in Arabidopsis, in part through the direct transcriptional activation of PIN1 by MP. Taken together, these results indicate that feedback between PIN-mediated auxin transport and ARF-Aux/IAA signaling regulates the patterning of root and shoot organs.

No description available.