title Embriogénese somática em genótipos de Quercus suber: análise bioquímica e histológica de produtos de reserva
authors Rodrigues S
supervisors Jorge Canhoto, Sandra Correia
author full name Sara Catarina Reis Rodrigues
nationality nacional
language Portuguese
author keywords cork-oak, embryogenic calli, plant growth regulators, repetitive embryogenesis, SDS-PAGE
abstract Quercus suber (cork oak) is a plant with a big economic interest due to the production of cork, Portugal being the main producer. Traditional propagation of this species through vegetative multiplication techniques ensures clonal propagation, although presenting strong limitations. Because of that, in vitro culture techniques have been developed for the propagation of cork oak. Somatic embryogenesis was induced in this species through the culture of explants of adult trees. First, embryogenic calli were obtained on an auxin (2,4-D and NAA) and cytokinin (KIN and BAP) containing medium, from leaves sprouted in the greenhouse. Then they were transferred to media containing lower concentrations of plant growth regulators (NAA and BAP) and then to an auxin-free medium, where somatic embryos appeared. These somatic embryos showed the capacity to develop repetitive embryogenesis in media containing NAA and BAP, with the secondary embryos developing in PGR free media. The best PGR combination for the induction of somatic embryos was 50 μM NAA + 10 μM BAP. Calli from other genotypes, previously induced with the same protocol, originated somatic embryos which were grouped in 4 different development stages according to their appearance (from a premature with a globular aspect embryo, until a mature embryo stage) when subcultured to PGR free media on 16h:8h light: dark conditions, on 6 weeks intervals. To better understand somatic embryo development in this species, the 4 development stages were biochemically and histochemically analyzed for storage product accumulation (proteins, lipids and starch), and compared with zygotic embryos (cotyledons and embryonary axis). The results showed that reserve compounds in somatic embryos were always lower than in zygotic embryos. During somatic embryo development, reserves changed according to the developmental stage of the somatic embryo, with the starch and protein content being greater on the mature stages, whereas lipid content showed little variation, although being particularly high during the embryogenic calli stage. Protein profiles of the samples obtained with SDS-PAGE showed sets of protein bands (with 60 kDa, 46 kDa and 26 kDa) with an increasing expression throughout the maturation of the embryos, and another sets of protein bands (with 30 kDa, 40 kDa and 50 kDa) with reduced expression throughout the maturation of the somatic embryos. Also, the protein profile obtained in mature somatic embryos was similar to that obtained in zygotic embryos, although with some differences that may explain the difficulty of the somatic embryos to involve into plantlets. The results also showed that it is possible to obtain embryogenic and non-embryogenic material from somatic embryos. This will permit in the future to do proteomic and genomic comparisons between the two types of calli so that the factors involved on the control of somatic embryogenesis can be identified.
e-mail address scrrodrigues13@gmail.com
publication date September 5
year published 2014
page count 81
subject category Somatic embryogenesis
CFE authors
Sara Catarina Reis Rodrigues