(191y) New Approaches in Engineering Somatic Embryogenesis in Loblolly Pine Suspension Cultures

Many industries, including agriculture and healthcare, require efficient methods for replication of plants with optimal traits. The loblolly pine (Pinus taeda) is a valuable crop in the timber industry, occupying 30 million acres of U.S. land, and breeding efforts aim to produce a crop with ideal phenotypic traits, including superior growth and wood quality. One method to large-scale clonal crop propagation is somatic embryogenesis (SE), the process through which aggregated plant cells undergo differentiation in vitro, resulting in a germination-competent embryo. We seek engineering strategies in the earliest stage of growth to improve yield and more efficiently use resources. There are three stages of growth and development that lead to the production of embryos: 1) aggregated cells that form embryonic suspensor masses (ESMs) are grown and scaled up in maintenance cultures; 2) ESMs are plated on solid media to initiate SE; and 3) embryos are separated from the ESM and germinated to generate plants. However, the process is not fundamentally understood, leading to large, unpredictable variability in embryo yield, a number only determined 8-12 weeks downstream of SE initiation. Here, we present on a variety of process manipulations (both biochemical and biophysical) we have evaluated and their effects on SE process outcomes and performance. We have demonstrated that induction of stress-related pathways through exogenous addition of plant stress hormones prior to moving a culture into development improves the rate of SE, and have used this knowledge to explore a means for more efficient embryo production. We have separated cellular communities at different stages of development within maintenance cultures and determined how the different populations progress to form embryos. We will also comment on recent advances in culture protein modification without genetic transformation, by inhibiting or supplementing endogenous arabinogalactan proteins (AGPs), which we have found to significantly influence SE in loblolly pine. We will present the influence of these treatments on embryo yield as well as the molecular response of the cultures, including culture stress (determined by phenolic content), growth (using the Coulter Counter), as well as total and specific protein biomarkers. These are the first studies to consider process engineering as a simple and cost effective means to improve the overall feasibility of SE on both an academic and industrial scale.