| Cultured Brassica napus microspores undergo a shift from pollen development to embryo formations when incubated at 30°C. Since intracellular calcium increases coincide with fertilization in other species, it was hypothesized that intercellular calcium signaling contributes to embryogenic induction in B. napus microspores. To test this hypothesis, cultured microspores were treated with modulators that affect calcium-signaling pathway. The treatments' impacts were evaluated using flow cytometric profiles, microscopic observations and three-week embryo counts. Inhibition of diacylglycerol kinase (DAGK) enhanced embryogenic potential in 25°C microspore cultures while inhibition of IP3 synthesis and association with intracellular receptors abolished the embryogenic response. Three putative RopGAP gene fragments were cloned and sequenced after RT-PCR with RNA from embryogenic microspores and degenerate PCR primers from two Arabidopsis thaliana RopGAPs. One of the sequences was transcriptionally up-regulated in embryogenic microspores. Understanding intracellular calcium's role during embryogenesis may allow the development of novel approaches to increase the regenerative potential of cultured microspores. |
