An efficient protocol of protocorm-like bodies regeneration from callus cultures of gastrodia elata blume and the further associations with mycorrhizal fungi

In this study, we reported a reliable protocol of PLBs regeneration from embryogenic callus cultures of Gastrodia elata, a fully mycoheterotrophic orchid and an endangered herbal medicinal plant. The continuous immersion cultures with TDZ (0.5 and 1 mg L-1) and BA (10 mg L-1) improved PLBs regeneration frequency and rate. Histological studies indicated that starch grains were abundant within in the cytoplasm of embryogenic callus and developing PLBs. The formation of PLBs from cell aggregates in the periphery region of embryogenic callus was supposed to be the multicellular origin pathway. After inoculated with suitable Armillaria isolates, PLBs enlarged further and resulted in the formation of tubers. The results of phylogenetic analysis and inoculation experiments indicated that the asymbiotic PLBs could establish the appropriate association with Armillaria mellea complex, including A. mellea and A. gallica. Thus, this protocol of PLBs regeneration from callus cultures is beneficial for the multiplication of selected elites, and could be used as an experimental system for the molecular investigations of the mycoheterotrophic orchid - mycorrhizal fungi interactions.