The expression of Brostm, a KNOTTED1-like gene, marks the cell type and timing of in vitro shoot induction in Brassica oleracea

We studied the early events of de novo formation of adventitious shoot meri stems in stem segments of Brassica oleracea. A regeneration system was used that is efficient, rapid, highly responsive to cytokinins and does not inv olve callus formation, thus allowing studies on a direct developmental swit ch of cells in the stem segment to form adventitious shoot meristem cells. Shoot meristem cells and dividing cells were marked from very early stages using in situ hybridization studies with Brostm, a Brassica homologue of th e Arabidopsis SHOOTMERISTEMLESS (STM) gene, and a cyclin box-derived probe, Brocyc, respectively. We show that the process of developmental switching starts before any cell division occurs in the stem explants. This switching occurs synchronously both longitudinally and transversely in the explant, in groups of 5-7 phloem parenchyma cells subtending vascular bundles in the explant. Brostm is induced specifically in response to a cytokinin, benzyl adenine, within 4 h of treatment and the transcripts persist during cell pr oliferation leading to shoot differentiation. We also show that during adve ntitious shoot formation, cells expressing Brostm are distinct from those e xpressing Brocyc. Lastly, our data suggest that, although developmental swi tching is initiated synchronously within 4 h of treatment, it requires 8 h of treatment for the establishment of organogenic determinance. The latter process is aynchronous, implying that additional factors formed later than Brostm are required to achieve maximal levels of determined cell population s to form adventitious shoots in vitro.