Abstract
Calcium (Ca(2+)) is a universal signaling cation with a prominent role as second messenger in many different plant processes, including sexual reproduction. However, there is much less knowledge about the involvement of Ca(2+) during in vitro embryogenesis processes. In this work we performed a study of Ca(2+) levels during the different stages of microspore embryogenesis in Brassica napus, with special attention to how Ca(2+) can influence the occurrence of different embryogenic structures with different embryogenic potential. We also performed a pharmacological study to modulate Ca(2+) homeostasis during different stages of the process, using a series of Ca(2+)-altering chemicals (BAPTA-AM, bepridil, chlorpromazine, cyclopiazonic acid, EGTA, inositol 1,4,5-trisphosphate, ionophore A23187, W-7). This study shows that Ca(2+) increase can be considered as an early marker of induction of microspore embryogenesis. Besides, Ca(2+) levels are highly dynamic during microspore embryogenesis, influencing the final embryo yield. Increase of either extracellular or intracellular Ca(2+) levels improves embryo yield without altering the proportion of highly embryogenic structures formed, which suggests that elevated Ca(2+) levels increase the amount of microspores reaching the minimum Ca(2+) threshold required for embryogenesis induction. Conversely, inhibition of Ca(2+) uptake or signaling results in reduced embryogenic response. This allows to modulate embryo yield within a functional range, with lower and upper Ca(2+) thresholds beyond which embryo yield is reduced. There seems to be a relationship between Ca(2+) levels and embryo differentiation.