Protoplasts allow tracking of early somatic embryo development in the conifer

Abstract

The purpose of the study was to follow early steps in the initiation of somatic embryos in hybrid larch ( Larix x eurolepis Henry) by using protoplasts of different lineages and studying their cryptic potential to simulate zygotic embryogenesis. Protoplasts of embryonal-suspensor tissue, separated by discontinuous density gradient centrifugation into three fractions, were immobilised in thin films of alginate and cultured on a medium sustaining embryo development. The fractions consisted mainly of (1) vacuolated non-cell regenerating suspensor protoplasts and cytoplasts, (2) mononucleate cell- and somatic embryo-regenerating protoplasts, and (3) multinucleate non-cell regenerating protoplasts. Concerning the latter fraction, it was hypothesised that compartmentalisation of the nuclei by cell wall formation (in analogy to the partitioning of free nuclei during early prophase in zygotic embryogeny) might result in the direct inception of an embryo or embryogenic tissue. Partitioning of nuclei by cell wall formation was not observed. However, a protoplast containing two, three or even four nuclei could undergo mitosis and cytokinesis, but only as a result of one of the nuclei dividing and entering the cell cycle and only when nuclear division had been preceded by cell wall resynthesis. Given that there are developmental differences between somatic and zygotic embryos, more detailed information on the cytology of somatic embryo development will be required in order to determine whether or not somatic embryogenesis truly mimics that of its zygotic counterpart.