Embriogénesis somática en Quercus suber L. Optimización de la proliferación en sistemas de inmersión temporal y regulación epigenética de la maduración

Abstract

Somatic embryogenesis is a powerful alternative to conventional culture techniques like vegetative propagation or seeds storage. In addition, it forms an important experimental system to study processes concerning embryo development, cell division and morphogenic competence. In the Quercus genus it is considered the most effective in vitro regeneration method for oak species. Nevertheless, high efficient embryo proliferation and especially poor quality and incomplete maturation of somatic embryos seem to be the most important factors that limit the production that this technique offers. During embryo development a coordinated action of hormonal signals and epigenetic mechanisms regulate the activation and repression of different gene expression programs. The use of temporary immersion systems has been demonstrated to be an efficient method for the proliferation of cork oak embryogenic cultures because the fresh weight increase was much higher when compared with semi-solid cultures. Additionally, the use of different immersion frequencies could determine the cellular fate of the cells; more frequent immersions favoured the production of cotyledonary embryos while 1 min every 6 h enhanced the production of proliferative stages. It has also been demonstrated that amount and type of initial inoculum had to be optimized both for RITA® systems and semi-solid cultures to improve the multiplication of the cultures. During development of cork oak somatic embryos variations in ABA levels and a decrease on embryos moisture content appeared to play an important role for the correct maturation and subsequent germination of embryos. Cold stratification concurred with decreased ABA levels and was associated with increased germination capacity. ABA immunolocalization analysis revealed that not only a variation was observed in endogenous ABA levels along the different developmental stages but also that the hormone showed a differential pattern in the embryo axes and was mainly located in the root and shoot apical meristems possibly controlling the germination arrest and correct meristem organization. Furthermore, a huge decrease in the signal was observed in the root cap after the stratification treatment suggesting a role for the root cap during development. Epigenetic control seemed to form an important regulatory factor during cork oak somatic embryos development. During the early development of somatic embryos no differences in DNA methylation levels were observed. However, differences in the distribution of the methylation signal recorded for the different stages of Q. suber indicated that differential epigenetic regulation must take place. Embryos maturation concurred with a decrease on DNA methylation levels and seems necessary for germination. This differential expression was also recorded in 5-mdC marks distribution. In the most mature stages DNA methylation concurred with low activity of meristems and the highest signal was mainly distributed in the shoot meristem and provascular tissues. This could indicate that during embryo maturation shoot growth is strongly repressed, whereas a strong decline in the root meristem facilitates growth during germination. Sequences from genes related to the maturation process and also to epigenetic regulation were isolated in cork oak somatic embryos. The differential expression levels observed in the candidate genes QsNCED3, QsHUB1, QsHUB2, QsAUR3, QsHDA19, QsHDA6, QsPICKLE and QsVAL1 indicated that there is epigenetic regulation of development and maturation of somatic embryos. Moreover, these changes in expression were related to the changes observed in the global DNA methylation levels as well as ABA endogenous levels.