Age-dependent biological characteristics of embryonal mass of maritime pine in relation to the embryogenic potential

Somatic embryogenesis (SE) in conifers is at the base of nearly allbiotechnology products that potentially might be developedincluding transgenic trees. SE is a powerful tool for clonalpropagation that has opened avenues for deployment of superiorclonally replicated planting stock in forest plantations. Althoughthe SE process can be managed for high efficiency there are stillseveral problems that require better understanding of underlyingcauses. One phenomenon, which occurs after the long-termculture of embryogenic tissue, particularly of pines, is the loss ofability to produce mature somatic embryos and consequentlyplants. Such unproductive cultures must be replaced, which is bothexpensive and undesirable. In an attempt to get an insight into thedifferences between young, primary lines of embryonal mass ofmaritime pine that produced somatic embryos and the same linesof significantly increased age that stopped producing somaticembryos, we analyzed in both types of material the levels ofendogenous hormones and their metabolites, polyamines, globalDNA methylation and DNA methylation patterns. DNAmethylation plays an important role in regulation of geneexpression and may cause epigenetic change leading to suppressedembryogenic potential. To determine if the DNA methylationcould be altered, we exposed the cultures to differentconcentrations of hypomethylating agents such as 5-aza-2'-deoxycytidine, 5-aza-cytidine and zebularine and tested theembryogenic potential of a line after the treatment. Forcomparison, we induced secondary SE from a few mature somaticembryos of the same lines and carried out the same as aboveanalyzes. Secondary SE has been implicated in restoring/enhancingthe embryogenic potential of a given line and thereforetheoretically the tissue was expected to be biologically similar tothe primary young line. Our results indicated that embryogeniccultures of different ages varied with respect to the polyamineslevels and DNA methylation pattern but not with respect to thehormone concentration. Treatment of the cultures with thehypomethylating agents resulted in different methylation patternsand somatic embryo production ability.