Abstract
Cremastra appendiculata var. variabilis (Blume) I.D. Lund), also known as single-leaf cremastra (SLC), is a rare and threatened species native to Korea, and it has the potential to be grown as a beautiful flowering pot or garden plant. There is still no reliable strategy to multiply SLC. Thus, an effective method for propagating single-leaf cremastra was needed for its conservation and mass production. In the present study, we examined the effects of culture media, seed age, activated charcoal, and plant growth regulators on in vitro asymbiotic seed germination, secondary protocorm induction, and seedling formation. Asymbiotic seed germination of SLC was influenced by culture media, seed age, and their interaction. The addition of activated charcoal (500 mg/L) to the Murashige and Skoog (MS) medium increased the rate of germination. The seeds were best germinated (91.9%) by culturing on MS medium supplemented with activated charcoal (500 mg/L), alpha-naphthaleneacetic acid (3 mu M), and kinetin (1 mu M). The highest number (28.9) of secondary protocorms were produced when protocorms were cultured on MS medium containing 6-benzyladenine (4 mu M) and kinetin (2 mu M). When the protocorms were cultivated in a medium containing gibberellic acid (1 mu M), they were able to transform into SLC with the highest success rate (78.7%). The propagation protocol described here may be helpful for SLC restoration programs and large-scale production.