Caracterización y análisis de la expresión de genes durante la embriogénesis y el enraizamiento adventicio en fagáceas

La embriogénesis somática y el enraizamiento adventicio son procesos morfogenéticos de gran interés para la producción clonal de árboles forestales que requieren una etapa de desdiferenciación celular y reprogramación genética para la adquisición de la competencia embriogénica, o en el caso del enraizamiento adventicio, el destino de células iniciales de raíz. De momento se desconocen en gran medida los mecanismos moleculares que regulan dichos procesos. En esta Tesis se han caracterizado genes implicados en la inducción y desarrollo de los embriones somáticos y en la formación y/o desarrollo de raíces adventicias en tres especies forestales: Castanea sativa, Quercus robur y Q. suber. Los genes SCL1 y CPE parecen están implicados en adquisición y/o mantenimiento de la competencia embriogénica y en procesos de histodiferenciación durante el desarrollo embrionario. El gen SCL1 parece estar involucrado en el establecimiento del patrón radial del embrión y de la raíz y en la iniciación del meristemo de la raíz adventicia. El papel del gen CPE está asociado a desarrollo apical del brote y especificación de las células de la columela. El gen TCTP se expresa en tejidos en división activa durante la embriogénesis y el crecimiento del brote y de la raíz, participando en la formación de órganos laterales y el crecimiento y desarrollo de la planta

Caracterización y análisis de expresión del gen TCTP en fagáceas

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Análisis de la expresión del gen CPE en embriones somáticos de alcornoque y estudio filogenético en diferentes especies.

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Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northen blot and in situ hybridization were used to determine the specific localization of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype- dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene 2 was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localized expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved the fate of root cap cells.Peer reviewe

Using in vitro culture for conservation of genetic resources: micropropagation of a monumental Prunus dulcis tree

The need to conserve biodiversity has been granted increasing political and social attention in the last years. Monumental or emblematic trees, both those >wild> trees living in forests and the century-old, >domesticated> agricultural trees, should be preserved in situ for its intrinsic value, its cultural legacy, the rich diversity of microhabitats they generate, and the quantity of organisms that depend on them to life. Also, they should be preserved ex situ for the study and the conservation of their genetic resources, for educational issues and for reintroducing plant material of high quality in their natural areas, most of them currently degraded or threatened. Within ex situ conservation methods, in vitro culture presents special advantages in the case of emblematic trees, such as the small quantity of plant material needed to begin the micropropagation procedure and the possibility of implementing long-term conservation techniques as cryopreservation. The aim of this study was to micropropagate mature material from an ancient almond tree, named ¿Gladiador¿, located in Membrilla (Central Spain), together with juvenile material proceeding from its seeds. This monumental tree, probably 300-years-old, dominates a landscape formed by hundreds of olive trees, and has a special emblematic meaning for the population of the area. For establishment of axillary shoot cultures, plant material was provided by the FIRE foundation. Three types of material were used: 1) shoots flushed at the tree at the beginning of spring, 2) shoots forced to flush in a phytotron from branch segments collected in late winter, and 3) seeds collected in autumn and stored at a cool place for six months before being germinated in vitro. Murashige and Skoog medium supplemented with 0.5 mg L-1 N6-benzyladenine and 0.5 mg L-1 indole-3-butyric acid was used for culture establishment and stabilization. Different combinations of plant growth regulators were evaluated for shoot proliferation, elongation and adventitious root formation. Rooted shoots from cultures obtained from the Gladiador mother tree and from lines originated from seeds were successfully acclimatized in the phytotron and the greenhouse. For mid-term conservation, shoots from mature and juvenile origins were submitted to cold storage at 4-6 ºC. So far, we have obtained 78 plantlets that are currently being acclimated, 70 corresponding to clonal material from the mother tree and 10 corresponding to clonal material from seeds

Recent Advances in Adventitious Root Formation in Chestnut

The genus Castanea includes several tree species that are relevant because of their geographical extension and their multipurpose character, that includes nut and timber production. However, commercial exploitation of the trees is hindered by several factors, particularly by their limited regeneration ability. Regardless of recent advances, there exists a serious limitation for the propagation of elite genotypes of chestnut due to decline of rooting ability as the tree ages. In the present review, we summarize the research developed in this genus during the last three decades concerning the formation of adventitious roots (ARs). Focusing on cuttings and in vitro microshoots, we gather the information available on several species, particularly C. sativa, C. dentata and the hybrid C. sativa x C. crenata, and analyze the influence of several factors on the achievements of the applied protocols, including genotype, auxin treatment, light regime and rooting media. We also pay attention to the acclimation phase, as well as compile the information available about biochemical and molecular related aspects. Furthermore, we considerate promising biotechnological approaches that might enable the improvement of the current protocols.This research was funded by the Xunta de Galicia of grant number IN607 2017/6 and the APC was partially funded by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Recent Advances in Adventitious Root Formation in Chestnut

The genus Castanea includes several tree species that are relevant because of their geographical extension and their multipurpose character, that includes nut and timber production. However, commercial exploitation of the trees is hindered by several factors, particularly by their limited regeneration ability. Regardless of recent advances, there exists a serious limitation for the propagation of elite genotypes of chestnut due to decline of rooting ability as the tree ages. In the present review, we summarize the research developed in this genus during the last three decades concerning the formation of adventitious roots (ARs). Focusing on cuttings and in vitro microshoots, we gather the information available on several species, particularly C. sativa, C. dentata and the hybrid C.sativa × C. crenata, and analyze the influence of several factors on the achievements of the applied protocols, including genotype, auxin treatment, light regime and rooting media. We also pay attention to the acclimation phase, as well as compile the information available about biochemical and molecular related aspects. Furthermore, we considerate promising biotechnological approaches that might enable the improvement of the current protocols

Análisis de la expresión de un gen que codifica una proteína rica en residuos de glicina e histidina durante el desarrollo embrionario en roble y castaño

Trabajo presentado en la XI Reunión de Biología Molecular de Plantas, celebrada en Segovia (España),del 14 al 16 de junio de 2012Peer reviewe

Transcriptomics Analysis Reveals a Putative Role for Hormone Signaling and MADS-Box Genes in Mature Chestnut Shoots Rooting Recalcitrance

20 páginas, 6 figurasMaturation imposes several changes in plants, which are particularly drastic in the case of trees. In recalcitrant woody species, such as chestnut (Castanea sativa Mill.), one of the major maturation-related shifts is the loss of the ability to form adventitious roots in response to auxin treatment as the plant ages. To analyze the molecular mechanisms underlying this phenomenon, an in vitro model system of two different lines of microshoots derived from the same field-grown tree was established. While juvenile-like shoots root readily when treated with exogenous auxin, microshoots established from the crown of the tree rarely form roots. In the present study, a transcriptomic analysis was developed to compare the gene expression patterns in both types of shoots 24 h after hormone and wounding treatment, matching the induction phase of the process. Our results support the hypothesis that the inability of adult chestnut tissues to respond to the inductive treatment relies in a deep change of gene expression imposed by maturation that results in a significant transcriptome modification. Differences in phytohormone signaling seem to be the main cause for the recalcitrant behavior of mature shoots, with abscisic acid and ethylene negatively influencing the rooting ability of the chestnut plants. We have identified a set of related MADS-box genes whose expression is modified but not suppressed by the inductive treatment in mature shoots, suggesting a putative link of their activity with the rooting-recalcitrant behavior of this material. Overall, distinct maturation-derived auxin sensibility and homeostasis, and the related modifications in the balance with other phytohormones, seem to govern the outcome of the process in each type of shoots.This work was funded by Xunta de Galicia (Spain) through the projects IN607A and “Contrato Programa” 2021 (AGI/CSIC I + D + I 2021, Ref-ACAM 20210200033), and also by the CSIC program I-COOP + 2020 (RefCOOPB20584).Peer reviewe

A Temporary Immersion System to Improve Cannabis sativa Micropropagation

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, β-A and β-H, all supplemented with 2-μM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in β-A medium supplemented with 2-μM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species.This work was supported by a research contract from Phytoplant Research (Ref. 20190548) and by the Xunta de Galicia (Spain) through the projects IN607A 2021/06 and Contrato Programa 2021 (AGI/CSIC I+D+I 2021, Ref- ACAM 20210200033)