We investigated direct and indirect formation of somatic embryogenesis in Brassica oleracea var. botrytis (cauliflower), a very important vegetable crop worldwide. Direct somatic embryogenesis, which is rather rare, was achieved in culture of 2-week-old hypocotyl explants of Brassica oleracea var. botrytis on MS medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5; 1.0; and 1.5 mg/l kinetin. Initial induction of embryogenic callus was achieved on MS supplemented with very low concentrations of 2,4-D (0.05 mg/l and 0.1 mg/l). Indirect somatic embryogenesis from leaf sections was obtained on MS supplemented with 0.05 or 0.1 mg/l 2,4-D. We examined various stages of somatic embryos (globular, heart, torpedo, cotyledonary). More embryos per explant were produced through the indirect pathway (23-25) than through the direct pathway (14-19). The number of embryos produced was high. There is a potential for recurrent, repeated or secondary somatic embryogenesis, possibly an unlimited source for mass propagation and ideal for synthetic seed production in this species. Plant regeneration was achieved on half-strength MS medium without any hormones.
To keep genetic diversity, flowering plants have developed a self-incompatibility system, which can prevent self-pollination. It has been reported that calcium concentration in pistil papilla cells was increased after self-pollination in transformed self-incompatible Arabidopsis thaliana. In this study, we found that CML27 changed its expression level for both mRNA and protein when compared to transcriptome and proteome. At the same time, CML27 was expressed in the anther and pistil at a high level and reached up to 5-fold up-regulated expression in the pistil at 1 h post-pollination when compared to 0 min. In order to find out potential proteins that may interact with BoCML27, BoCML27 was expressed in and isolated from E. coli. After its co-incubation with Brassica oleracea pistil proteins, the products were separated on SDS-PAGE gels. We found a specific band at the position between 130–180 kDa. Through LC-MS-MS (Q-TOF) analysis, eight proteins were identified from the band. The proteins include 26S proteasome non-ATPase regulatory (26S), Phospholipase D, alpha 2 (PLDα2) involved in Ca2+ binding and Coatomer subunit alpha-2-like (Coatomer) involved in vesicle mediated transport. All of these identified proteins provide new insights for the self-incompatibility response in B. oleracea, specific for increasing Ca2+ concentration in pistil papilla cells.