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Perez M.,University of Oviedo | Perez M.,Intituto Universitario Of Biotecnologia Of Asturias Iuba Asociado Con Csic | Bueno M.A.,Forest Biotechnology | Toorop P.,Royal Botanic Gardens | And 4 more authors.
Trees - Structure and Function | Year: 2013

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures. © 2013 Springer-Verlag Berlin Heidelberg.


Perez M.,University of Oviedo | Perez M.,Intituto Universitario Of Biotecnologia Of Asturias Iuba Asociado Con Csic | Viejo M.,University of Oviedo | Viejo M.,Intituto Universitario Of Biotecnologia Of Asturias Iuba Asociado Con Csic | And 4 more authors.
Journal of Plant Physiology | Year: 2015

Somatic embryogenesis is a powerful alternative to conventional mass propagation of Quercus suber L. However, poor quality and incomplete maturation of somatic embryos restrict any application. Given that epigenetic and hormonal control govern many developmental stages, including maturation of zygotic embryos, global DNA methylation and abscisic acid (ABA) were analyzed during development and maturation of cork oak somatic embryos. Our results indicated that development of somatic embryos concurred with a decrease in 5-mdC. In contrast, endogenous ABA content showed a transient increase with a peak in immature E2 embryos denoting the onset of the maturation phase. A cold stratification phase was necessary for embryos to acquire germination ability, which coincided with a significant decrease in 5-mdC and ABA content. Immunohistochemical analyses showed that there was a specific spatial-temporal regulation during embryogenesis, particularly after the cold treatment. The acquisition of germination capacity concurred with a general low 5-mdC signal in the root meristem, while retention of the 5-mdC signal was mainly located in the shoot meristem and provascular tissues. Conversely, ABA immunolocalization was mainly located in the root and shoot apical meristems. Furthermore, a strong decrease in the ABA signal was observed in the root cap after the stratification treatment suggesting a role for the root cap during development of somatic embryos. These results suggest that, in addition to ABA, epigenetic control appears to play an important role for the correct maturation and subsequent germination of cork oak somatic embryos. © 2014 Elsevier GmbH.


Perez M.,University of Oviedo | Perez M.,Intituto Universitario Of Biotecnologia Of Asturias Iuba Asociado Con Csic | Canal M.J.,University of Oviedo | Canal M.J.,Intituto Universitario Of Biotecnologia Of Asturias Iuba Asociado Con Csic | Toorop P.E.,Royal Botanic Gardens
Plant Cell, Tissue and Organ Culture | Year: 2015

Clonal propagation of Quercus suber via somatic embryogenesis is an alternative to conventional tree propagation methods; however, complete maturation of somatic embryos is considered the major bottleneck for mass propagation of Quercus species. During somatic embryogenesis, embryo development and maturation are controlled by signaling pathways that integrate information from genetic and epigenetic programs as well as hormonal signals. Therefore, in this study genes were identified related to epigenetic regulation and the abscisic acid (ABA) pathway during development and maturation of cork oak somatic embryos. A total of eight expressed sequence tags were obtained of genes encoding a 9-cis-epoxycarotenoid dioxygenase (NCED), two histone deacetylases (HDA6 and HDA19), two histone monoubiquitinases (HUB1 and HUB2), a histone H3 kinase (AUR3) as well as genes related to chromatin remodeling processes PICKLE and VP1/ABSCISIC ACID INSENSITIVE 3-LIKE 1 (VAL1). The analysis of the expression patterns of selected genes during different developmental stages indicated that QsNCED3 may play a role in ABA synthesis during embryogenesis. The change in the expression levels for all seven genes associated with epigenetic regulation showed that QsHUB1 and QsHUB2 may have a role in ABA signalling while QsHDA6 and QsHDA19 could act in different pathways than in Arabidopsis. Furthermore, expression levels of QsAUR3 indicated that histone phosphorylation is an early epigenetic mark in Q. suber somatic embryos while QsPICKLE and QsVAL1 may be necessary for the correct development of cork oak somatic embryos. © 2015, Springer Science+Business Media Dordrecht.

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