Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine)

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz.,ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semiquantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.

Biochemical and morphological changes during the growth kinetics of Araucaria angustifolia suspension cultures

Embryogenic cultures of Araucaria angustifolia were established in a BM liquid medium supplemented with 2 µM 2,4-dichlorophenoxyacetic acid, 1 µM 6-benzylaminopurine and 1 µM kinetin (BM2) and in a BM medium free of growth regulators (BM0). During 42 days in culture, the cell growth pattern of both cultures was similar. The pH of the culture medium of both BM0 and BM2 underwent progressive reduction during culture time. For both the embryogenic cultures a preferential uptake of glucose in the late stages of cell growth kinetics was observed. The extracellular protein content was similar for both the embryogenic cultures. Acetocarmine and Evan's blue double stain showed major differences for early somatic embryo organisation, in which only the embryogenic culture grown in a liquid culture medium free of plant growth regulators showed the presence of bipolar somatic pro-embryos.

Culturas embriogênicas de Araucaria angustifolia foram estabelecidas em meio de cultura líquido BM suplementado com 2 µM Ácido 2,4 Diclorofenoxiacético, 1 µM 6-Benzilaminopurina e 1 µM Cinetina (BM2) e em meio BM isento de reguladores de crescimento (BM0). Durante 42 dias de cultivo, o padrão de crescimento celular em ambas as culturas foi similar. O pH do meio de cultura BM0 e BM2 sofreu uma progressiva redução durante o período de cultivo. Em ambas as culturas embriogênicas foram observadas um consumo preferencial de glicose no período final da curva de crescimento celular. O nível de proteínas extracelulares foi similar para ambas as culturas embriogênicas. A dupla coloração com carmin acético e azul de Evans revelou diferenças na organização das linhagens celulares embriogênicas, sendo que a presença de proembriões somáticos bipolares foi apenas evidenciada nas culturas embriogênicas mantidas em meio de cultura líquido sem reguladores de crescimento.