In situhybridization

Developing tubers (8 mm in length) of G. elata were fixed in the FAA solution (18 : 1 : 1 of ethanol (50%), glacial acetic acid and formalin) for 24 h at 4 °C. Samples were
then dehydrated through an ethanol series and embedded in Histoplast paraffin wax (Thermo Scientific). Sections of 8 μm were cut on a Leica RM2235 rotary microtome. Digoxigenin-labeled sense and antisense RNA probes, transcribed from 1053-1353 bp downstream from the start codon, were synthesized following the manufacturer’s instructions (Roche Applied Science, USA). The procedures for hybridization and immunological detection of signals with alkaline phosphatase were performed as described previously (Schwarzacher & Heslop-Harrison 2000).