High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant Emmenopterys henryi Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25A degrees C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25A degrees C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS2) for 30 min at 0A degrees C and followed by 100% PVS2 for 40 min at 0A degrees C. After changing the solution with fresh 100% PVS2, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40A degrees C for 2 min, the shoot tips were washed for 20 min at 25A degrees C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l(-1), alpha-naphthaleneacetic acid 0.1 mg l(-1), 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75-85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.