Effects of explant position and dark treatment on bud formation in floret culture of Ponerorchis graminifolia Rchb.f

An efficient method of micropropagation was investigated for Ponerorchis graminifolia. Shoot apexes and florets with inflorescence nodes were used as explants for in vitro cultures. A high efficiency of bud formation was observed on half-strength MS medium containing 4.44 μM N6-benzyladenine (BA) and 0.54 μM α-naphthaleneacetic acid (NAA) using florets from upper positions of inflorescences as explants. However, the explants frequently exuded browning compounds into the media during culture. Dark treatment, a countermeasure, decreased exudation of the browning compounds during floret culture. When shoot apexes from dark treated florets were used as an explant material after the floret culture, the dark-pretreated shoot apexes developed 2.2 buds per explant, compared with 1.2 buds per explant from light pretreated shoot apexes, and showed decreased exudation of browning compounds. These results suggest that the most suitable explants for bud formation in P. graminifolia are top florets and shoot apexes derived from the floret culture with dark treatment.