Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem

Somatic embryogenesis in Arabidopsis is achieved by culturing bending-cotyledon embryos on a 2,4-D-containing induction medium for 14 d followed by a transfer on to a hormone-free development medium. Several genes orthologous to Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were isolated from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn), and ectopically expressed in Arabidopsis to assess their effects on somatic embryogenesis. Ectopic expression of BoSTM, BrSTM, and BnSTM increased the number of somatic embryos, whereas a different effect was observed in lines overexpressing BnCLV1 in which somatic embryo formation was severely repressed. The introduction of BnZLL did not have any effects on Arabidopsis somatic embryogenesis. The increased embryo-forming capacity observed in lines overexpressing Brassica STM was associated with a lower requirement for the inductive signal 2,4-D, and a higher expression of WUSCHEL (WUS) which demarcates the formation of embryogenic cells. This was in contrast to the 35S::BnCLV1 lines which showed the highest requirement for exogenous 2,4-D and a reduced WUS expression. Microarray studies were conducted to monitor global changes in transcript levels during Arabidopsis somatic embryogenesis between the wild-type (WT) line and a BoSTM-overexpressing line, which showed the most pronounced enhancement of somatic embryo yield. The introduction of BoSTM affected the expression of many genes involved in hormone perception and signalling, as well as genes encoding DNA methyltransferases and enzymes of glutathione metabolism. Pharmacological experiments performed to confirm some of the microarray results showed that Arabidopsis somatic embryogenesis is encouraged by a global hypomethylation of the DNA during the induction phase and by a switch of the glutathione pool towards an oxidized state during the subsequent development phase. Both events occurred in the 35S::BoSTM line, but not in the WT line. Altered expression of Brassica STM also had profound effects on B. napus microspore-derived embryogenesis. The yield of microspore-derived embryos increased in lines overexpressing BnSTM and significantly decreased in antisense lines down-regulating BnSTM.