EFFECTS OF FREEZING CYPRIPEDIUM REGINAE SEEDS ON GERMINATION

 Cypripedium reginae, commonly known as the showy lady’s slipper, is a terrestrial orchid that is critically endangered in northern New England. In an effort to help prevent this plant’s extinction and possibly help similar terrestrial orchids, our lab has been raising these plants from seeds in sterile media. Although our lab obtains reasonable germination rates of between 30% and 50%, there is room for improvement. According to Chu and Mudge’s research at Cornell University, pretreatment of yellow lady’s slipper seeds produced increased germination in axenic seed culture. Our lab has pretreated seeds at 5ºC with no improvement in germination. Our hypothesis was that inoculating showy lady’s slipper seeds into culture medium followed by storage at -20ºC would improve germination since this is somewhat similar to the type of cold and moisture that seeds experience in the wild. A 15-week experiment focusing on the effects of a cold treatment on the seeds was conducted. Cyp. reginae seeds were inoculated into culture tubes containing Murashig and Skoog basal salts with minimal organics (Sigma Co.) and supplemented with 10 grams of sucrose per liter of medium and 100 mls of coconut water per liter of medium. The experiment consisted of 4 groups (one control and 3 experimental) that were each inoculated with surface-sterilized seeds. The control group was not frozen after inoculation. The three experimental groups were all subsequently frozen at -20ºC for 5, 6, and 7 weeks. In the groups where seeds were frozen after inoculation for 5, 6 or 7 weeks, no seeds germinated over the following 9 weeks. The control group showed 5% germination at the end of week 6 and steadily increased over the following 9 weeks to 37% by week 15. Freezing of the medium degraded the semisolid consistency and caused many of the seeds to float and be deposited on the culture tube side walls, preventing seeds from having access to the medium. This data suggests that this method of freezing is not efficient partly because of the poor consistency of the medium. To improve germination efficiency, future experiments will test the efficacy of freezing seeds for varying lengths of time followed by inoculation into tissue culture medium.