PROGRESS IN MICROPROPAGATION OF CYPRIPEDIUM REGINAE

The showy lady’s slipper, Cypripedium reginae, is critically endangered in the state of New Hampshire. Our laboratory has extensive experience propagating orchids of the Cypripedium genus including Cyp. reginae. This investigation focuses on efficiently propagating Cyp. reginae. The most common propagation method is using sterile seed culture. Although this method is efficient, it produces one plant from each seed and each seedling takes about 3-4 months to produce primordial shoots and roots. For other orchid genera, micropropagation produces millions of plants from a single plant’s vegetative tissue in a single year. Although attempted many times, micropropagation, or cloning, of Cyp. reginae has made little progress for over 50 years. This investigation hypothesized that dissecting roots, shoots, and rhizomes from about 1-year-old seedlings and transferring these tissues to various media would serve as a productive starting point for micropropagation. Root, shoot, and rhizome specimens were sectioned and grown in the standard seed culture medium published by Faletra et. al. in 1997. A rating system was devised of 0-3 stars that graded specimens based on the length and number of shoots that grew. Although roots and rhizomes showed modest growth, the shoots grew the fastest, averaging 1.4 stars in the first set of experiments, then 1.63 stars in the next set of experiments, as opposed to 1.0 and 0.73 stars for the first set of experiments and 0.93 and 0.73 stars for the second set of experiments for roots and rhizomes respectively. The next hypothesis that was tested was whether the plant hormone indole-3-butyric acid (IBA) would stimulate dissected shoots to produce callus growth. Once dissected shoots were in culture for about a month, some of the specimens were transferred to media containing amounts of IBA ranging from 0.0 to 40.0 mg/liter of medium. The medium containing 20.0 mg/liter of IBA was most effective in producing the growth of shoots, with an average of 2.75 stars, as opposed to 1 star for 40.0 mg/liter, and 1.5 stars for 0.0 mg/liter. In the total experimental time of over half a year, not including the year of raising stock seedlings, micropropagation of Cyp. reginae shoots using 20 mg/liter IBA showed the most promising results although no callus growth was observed.