Early Stress Activation via Methyl Jasmonate and Loblolly Pine Somatic Embryo Yield

Somatic embryogenesis (SE) provides a means for large scale production of a clonal crop, thereby enabling the propagation of significant crops with ideal phenotypes. This is particularly important in crops with large time investments prior to crop maturity, including pine trees. The loblolly pine is an important timber crop that can be produced through SE, but variable yields of viable embryos are often reported and the process is not fundamentally understood. The process of SE involves a response to change in the culturing conditions of cells from maintenance conditions to conditions associated with embryo development, and some of the changes are associated with a stress on the cultures. Here, we explore the early activation of stress on the cultures prior to moving to embryo-producing media. We show that early addition of methyl jasmonate (MeJA), a largely conserved activator of specialized metabolism in numerous plant systems, initiates a response in the cultures that is different between cultures that produce a high yield and those that produce a low yield. In high yielding cultures, low MeJA concentrations improve SE yield and high concentrations impede SE. In low yielding cultures, little response to MeJA is observed, suggesting that the culture metabolism is not as responsive in these low yielding cultures, and providing a potential way to screen cultures prior to plating. We present a robust culture engineering strategy and characterization platform using the Coulter Counter to quantify biomass and cell types, an assay using the Folin-Ciocalteu reagent to measure total phenolic content, as well as methods to quantify culture growth using both weight and image processing techniques.