Evaluation of the stability of embryogenic cultures and of emblings in maritime pine

Abstract

Large scale propagation of selected plant genotypes can be efficiently achieved by using in vitro culture methodologies. However, changes in chromosome structure, number and DNA alterations have already been detected in plants (including conifers) regenerated from in vitro cultures. Somaclonal variation can also involve mechanisms of gene silencing or gene activation that are not due to chromosomal aberrations or sequence change. The integrity evaluations during early stages of regeneration should be done at the phenotypic, genetic and epigenetic levels to prevent potential losses, especially in long-living trees.
This work aimed at the establishment of an efficient marker system to evaluate the genetic and epigenetic stability of embryogenic cultures and regenerated emblings in Pinus pinaster. Genetic stability was studied by SSR molecular markers and by Flow Cytometry. Epigenetic analyses were also performed through the quantification of global DNA methylation by HPCE (High Performance Capillary Electrophoresis) and through immunolocalization of 5-methylcytosine in zygotic and somatic embryo tissues. Genetic variation at seven SSR loci was detected in embryogenenic cell lines under proliferation conditions and in up to 9% of the regenerated emblings. The estimation of the DNA ploidy level by flow cytometry of embryogenic cell lines, somatic and zygotic embryos and emblings, showed a ploidy level consistent with the diploid status. Regarding the epigenetic analyses, the level of 5-methyl 2’-deoxycytidine (mdC) obtained in emblings was similar to the values obtained for seedlings with the same age under field conditions (17% mdC). However, during the zygotic and somatic embryogenesis process some oscillations in the mdC were observed, especially during maturation and germination stages. Results will be discussed based on comparative analysis of somatic versus zygotic embryogenesis.
  

Short CV:

Since November 2004 - PhD student at Forest Biotech lab (IBET/ITQB-UNL) under the supervison of Prof. Margarida Oliveira and Dr. Célia Miguel.
2003- Master in Plant Production by Instituto Superior de Agronomia, Universidade Técnica de Lisboa.
1999-2003- Research fellowship at Forest Biotech Lab (IBET/ITQB) working in the areas of tissue culture and molecular biology of Pinus pinaster within the scope of several projects.
1995- Degree in Biology by Universidade de Aveiro.