In vitro culture of immature M. truncatula grains under conditions permitting embryo development comparable to that observed in vivo

Abstract

Working with the model legume species Medicago truncatula, we have developed an in vitro strategy for the culture of immature embryos that permits their development in a way comparable to that observed in planta. Thus, seeds (8 and 12 days after pollination, DAP) and 12 DAP embryos were harvested and cultured on a basal salts medium with 130 g/L added sucrose, and with or without a nitrogen source. With an exogenous nitrogen supply, both 12 DAP seeds and embryos developed, with storage protein synthesis comparable to that observed in vivo as revealed by two-dimensional gel electrophoretic profiles. Conversely, in the absence of added nitrogen, seeds and embryos responded differently; with entire seeds there was a remobilisation of endogenous nitrogen during the initial stages of embryo development from tissues surrounding the embryo, thereby ensuring initial storage protein accumulation, whereas isolated embryos rapidly ceased synthesizing de novo proteins, and their development appeared arrested, presumably reflecting a shortage of nitrogen. This system is therefore useful for investigating the embryo's response to nitrogen.