(518c) Elucidating and Engineering the Role of Arabinogalactan Proteins in Somatic Embryogenesis

There is a critical need for efficient reproduction of high-value crops including timber crops, biofuel feedstocks and medicinal plants. Our work focuses on the loblolly pine (Pinus taeda), a critical crop to the timber industry, currently occupying 30 million acres of U.S. timberlands and making up one half of the U.S. standing pine volume. Somatic embryogenesis (SE) provides a means to create a clonal crop and enables selection for plants with ideal characteristics (e.g. fast growth rate, disease resistance, superior wood quality), and our goal is to identify and manipulate predictors of embryo yield in the earliest stage of development to streamline SE and enable process commercialization. SE is an in vitro cellular process that leads to the formation of a fully developed and viable embryo from a single cell. The three main stages that lead to the formation of seedlings from a plant cell culture are: 1) embryonic suspensor masses (ESMs) are propagated and maintained in liquid suspension culture; 2) ESMs are plated onto solid development media to induce SE; and 3) embryos are isolated and germinated to generate seedlings. The primary limitation to large-scale implementation of SE is that the process is not fundamentally understood, leading to large variability in embryo yieldâ??a number that cannot be determined until 3-4 months from SE initiation. Arabinogalactan proteins (AGPs) are a promising biomarker that have been implicated in other SE systems, providing many avenues for manipulation to improve yields. In the work presented here, targeted treatments were applied to maintenance cultures to evaluate influence on yield. For example, we demonstrated that AGPs are influential on loblolly pine somatic embryo yield by inhibiting AGP function in vitro using the AGP-binding β-glucosyl Yariv reagent. We also introduced generic, exogenous AGPs (e.g., gum arabic, from the Acacia tree) to cultures, supplementing the endogenous AGPs to quantify the molecular response and effect on SE. We further extend this approach using novel characterization techniques to elucidate the role of AGPs in culture, including new colorimetric assays, ELISAs using antibodies with AGP subclass specificity and UPLC characterization and fractionation. Understanding the complex properties of AGPs and their influence on SE will enable a better fundamental understanding of SE and identification of further strategies to promote high embryo yield.