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).