"NEW APPROACHES IN ENGINEERING SOMATIC EMBRYOGENESIS IN LOBLOLLY PINE SUSPENSION CULTURES"
ELIZABETH MORGAN CUMMINGS BENDE
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 asexual (somatic) plant cells undergo differentiation in vitro, resulting in germination-competent embryos. There are three main 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, this process is not fundamentally understood, leading to large, unpredictable variability in embryo yield, a number only determined 8-12 weeks downstream of SE initiation. This work aims to glean fundamental insight into culture dynamics and correlations with outcomes while simultaneously providing engineering strategies to improve embryo yield.
Here, a variety of process manipulations and their effects on SE process outcomes and performance are presented. Induction of stress-related pathways through exogenous addition of plant stress hormones prior to moving a culture into development was shown to improve the rate of SE, and this knowledge was used to explore a means for more efficient embryo production. A clear link between the total volume of small cell aggregates and embryo yield was demonstrated, enabling yield prediction at a timepoint 12 weeks before previously possible. Finally, methods for culture protein modification without genetic transformation were developed through inhibition or supplementation of endogenous extracellular arabinogalactan proteins (AGPs), which were found to significantly influence SE in loblolly pine. Techniques were developed to characterize 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 a Coulter counter), and total and specific protein biomarkers. The work presented here is among the first studies that consider process engineering as a simple and cost-effective means to improve the overall feasibility of SE on both an academic and industrial scale.