Tissue Culture focused on Micropropagation and the Outplanting of Cypripedium reginae

Cypripedium reginae, commonly known as the “Showy Lady’s Slipper,” is a terrestrial orchid indigenous to Northern New England that is regionally endangered due to factors such as climate change, habitat destruction, and illegal collection. In the wild, C. reginae may take up to ten years to grow from seed to flower because of specific growth requirements.  The research of Faletra et al. (1996) resulted in the development of an in vitro plant tissue culture method for seed germination and propagation of C. reginae, decreasing the time from seed to flower to three years. After sustained success using these techniques for the propagation of C. reginae, research is now investigating micropropagation of this species in an effort to produce large quantities of seedlings most efficiently and to slow, or prevent, further regional population decline. Micropropagation is the separation and independent growth of tissues from one plant. In these experiments, separation of seedling tissue was performed using a sterile blade. Researchers at Kimball Union Academy performed multiple micropropagation experiments that compared the growth of seedling bisections and various seedling cuttings use three different mediums at half and full strength. The health and growth of these seedlings were observed for three months. When bisected, seedlings were cut in two, with each half containing at least one section of rhizome, a shoot, and a root. Seedlings classified cuttings did not meet these parameters. Some seedlings were cut and root sections between ~0.1 cm and ~4 cm were used. The different mediums yielded varying levels of growth, the majority of which occurred in the roots with minimal shoot growth. Marked root growth was seen with full-strength Knudson C Modified Orchid Medium ("Knudson"). Prominent root growth with minimal shoot growth was observed with a half-strength dilution of Phytamax™ Orchid medium with Charcoal and Banana Powder ("Phytamax"). When Knudson was used with bisections, the average length increase was ~2.1 cm. When a half-strength dilution of Phytamax was used, the average length increase was ~2.4 cm. Future experiments could be designed with the addition of specific plant hormones, like kinetin, into the medium mixtures to incite shoot growth. Overall, bisected seedlings showed more growth and oxidized less frequently than seedling cuttings. Cuttings showed an average growth of ~0.3 cm, while bisections grew markedly (7-8x) more in the same time period. Micropropagation is an effective and efficient substitute for in vitro seed inoculation; it can save precious time and effort needed to help repopulate a species with its only drawback being a lack of genetic diversity in the population, which becomes irrelevant in smaller scale repopulation efforts.  Micropropagation can also provide an initial launching point for a full-scale restoration attempt for endangered species.