In vitro germination and low-temperature seed storage of Cypripedium lentiginosum P.J.Cribb & S.C.Chen, a rare and endangered lady's slipper orchid

- Seed development of C. lentiginosum were investigated based on a defined time frame, and the optimum germination was recorded for immature seeds collected at 90-105 days after pollination and cultured on 1/4 or 1/2 MS media.
- The lignified seed coats may cause the coat-imposed dormancy of mature seeds.
- Pretreatments with 0.5 or 1% NaOCl for 45 min were effective in increasing the permeability of seed coats and improving the germination of mature seeds.
- Among cytokinins tested, 2iP stimulated seed germination, furthermore both 2iP and TDZ enhanced the formation of multiple protocorm-like bodies after germination.
- For seed storage, seeds dehydrated to 13.5% of initial water content and stored at 5 °C, both germination and viability decreased slightly after 12 months of storage.

Procedures for asymbiotic seed germination and seed storage were established for Cypripedium lentiginosum, an endangered lady's slipper orchid. Based on a defined time frame, the optimum germination was recorded for immature seeds collected at 90-105 days after pollination (DAP), at which time early globular to globular embryos can be observed. After 120 DAP, as seeds matured, the germination decreased sharply. At maturity, two distinct layers of seed coats enclosed the embryos tightly. Histochemical staining suggests that the lignified seed coats may cause the coat-imposed dormancy. Pretreatments with 0.5 or 1% NaOCl for 45 min were effective in increasing the permeability of seed coats and improving the germination of mature seeds. Among different MS salt concentrations examined, the germination and protocorm formation were higher for seeds cultured on 1/4 and 1/2 MS media. Among cytokinins tested, only 2iP showed a stimulatory effect on germination, while both 2iP and TDZ enhanced the formation of multiple protocorm-like bodies. For seeds dehydrated to 13.5% of initial water content and stored at 5 °C, both germination and viability decreased slightly after 12 months of storage, while the extended storage to 24 months resulted in a sharp decrease of germination and viability. Using this protocol, seedlings with numerous roots were readily acclimatized to greenhouse conditions after 6 months of culture.

The timing of seed collection is critical in asymbiotic seed germination of C. lentiginosum. Collecting immature seeds (the early globular to globular embryos; D and E) at 90-105 days after pollination could maximize the germination. In mature seeds, the lignified seed coats may cause the coat-imposed dormancy. Pretreatments with 0.5 or 1% NaOCl for 45 min were effective in increasing the permeability of seed coats and improving the germination of mature seeds.214