Somatic embryogenesis in southern and tropical pine species: Loblolly pine (Pinus taeda), Longleaf pine (P. palustris) and Oocarpa pine (P. oocarpa)

The focus of the current project was to establish an improved and reliable protocol for somatic embryogenesis in 1) Pinus taeda and Pinus palustris; pine species of high value for commercial applications and germplasm conservation supported through breeding programs at The Virginia Department of Forestry (Chapter III); and 2) Pinus oocarpa; an economically important pine species in the southern half of Mexico and Central America (Chapter IV). In addition, 3) the study of the gene expression analysis of developmental stages of both somatic and zygotic embryos of P. taeda was compared to assess developmental fidelity at the molecular level (Chapter V). By testing four basal media combined with different plant growth regulator combinations, we have established stable embryogenic cultures from high value families of P. taeda and P. palustris using the tissue culture medium 1218 (Pullman et al 2005) in combination with an auxin:citokinin ratio at 10:5 (molar). However, optimization of the protocols for the maturation and further conversion of somatic embryos to seedlings requires further work. For P. oocarpa, we hypothesized that somatic embryo induction may be possible by mimicking natural seed-embryo developmental conditions, and a new tissue culture medium, based on the mineral content of the seed nutritive tissue (megagametophyte), was formulated. The novel culture medium (PO) was tested in combination with different plant growth regulator concentrations for the initiation of somatic embryogenesis from fresh collections of P. oocarpa immature zygotic embryos. Additionally, the established embryogenic cultures were able to mature and germinate, to our knowledge resulting in the first report of the production of P. oocarpa plantlets through somatic embryogenesis. PO medium also has the potential to be used successfully for other tropical pine species which today suffer from suboptimal somatic embryogenesis protocols. The fundamental study of molecular regulation of embryo development showed that under the current maturation conditions, P. taeda somatic embryos were temporally similar in gene expression to zygotic embryos of the same species. However, potentially important differences were found and results could potentially explain the low germination success during somatic embryogenesis. More research is still needed to further explore the natural environment of developing seed embryos to improve the somatic embryogenesis protocols and to enable full integration of this clonal propagation method into the breeding programs for pines.