2018 ASHS Annual Conference

Reproducible propagation protocols are needed to develop genetically diverse ex situ collections of U.S. native orchids, but do not exist for many species of conservation concern. Approximately 45 species of the taxonomically difficult genus Spiranthes are found worldwide; 23 species occur primarily in the eastern United States and several are considered rare, threatened or endangered by federal and state agencies. Using the Pennsylvania endangered Spiranthes casei as a model species, a series of experiments were designed to determine optimal conditions for in vitro seed germination, in vitro seedling development and greenhouse acclimatization. Seeds were collected in November 2015 from 10 individual plants found in three subpopulations in Elk and McKean counties, Pennsylvania, cleaned and air-dried for six weeks. Seeds were surface sterilized and scarified for 10 and three minutes in 10% bleach solution, then plated on five replicates each of three different commercially available terrestrial orchid seed germination media: P723, M551 and K400 (Phytotechnology Labs, Shawnee Mission, KS) with pH adjusted to 5.8. Seed germination ranged from 24 to 60 % and occurred on all three media only after the 3-minute treatment; none of the seeds germinated after the 10-minute treatment and visual inspection revealed badly damaged embryos. After shoot initiation, 150 seedlings were transferred to individual test tubes on one of two media (P723 and P658) and each was given one of the three photoperiod treatments for 10 months: 24/0 hour L/D, 18/6 hour L/D and 0/24 hour L/D. Seedling survival and growth occurred in all treatments, but incubated on P723 in the 24/0 and 16/8 treatments had a significantly greater fresh weight, leaf length, number of roots and root length than light treatments on P658 and dark treatments, indicating that light is essential for optimal seedling growth. Seedlings were removed from the test tubes and given a 100-day vernalization period at 2 °C. Seedlings were then randomly planted in one of four soilless media (Longwood Gardens research mix (LGRM), New Zealand sphagnum (NZ), Fafard 3b and Good Earth BC5). The survival rate (95%) and highest incidence of flowering (20%) occurred on NZ. The lowest survival rate (55%) and incidence of flowering (10%) was on LGRM. Results support anecdotal evidence that Spiranthes seeds are degraded by extended chemical scarification times. The propagation techniques described above are being applied to additional Spiranthes species to determine the efficacy of the protocol for propagating a greater range of taxa.