Axenic Culture and Histological Analysis of Cypripedium parviflorum

The yellow lady’s slipper, Cypripedium parviflorum, is critically endangered in New Hampshire because of habitat loss, degradation, and low survival rates during its long life cycle in the wild.  These properties make this orchid an ideal plant for a restoration study.  Our ultimate goal is to repopulate the yellow lady’s slipper to its wild habitat.  Before this can be done, we need to establish an efficient means of propagating large numbers of this plant, which has been met with limited success until this study.  This study was conducted to determine the media necessary to improve germination rates.  Our approach was to alter the levels of coconut water, sucrose, and pH in the standard medium we use to efficiently propagate Cypripedium reginae.  The optimal medium for Cyp. parviflorum germination had a pH of 6.0 with 20% coconut water and 15% sucrose by volume, producing a germination rate of 15% compared to the germination rate of less than 1% in the wild.  In an experiment varying only the pH and using our standard Cypripedium reginae medium with 10% sucrose and 10% coconut water, a pH of 7.5 gave the highest germination of 4.6%.  In future experiments, we would like to further test for optimal sucrose, coconut water, and pH levels. We also have begun a histological analysis to better understand the yellow lady’s slipper’s reproductive structures and meristematic tissue locations for future cloning studies.