cryopreservation of shoot tips of Picrorhiza kurroa Royle ex Benth., an indigenous endangered medicinal plant, through vitrification

The cryopreservation of shoot tips of Picrorhiza kurroa Royle ex Benth (IC 266698), an endangered medicinal plant of India was investigated. Shoot tips (about 1 mm in length) excised from four-week-old proliferating shoot cultures were precultured on MS medium supplemented with various osmotica before dehydrating with PVS2 solution at 0ºC. The dehydrated shoot tips were directly immersed in LN2. Following cryopreservation, and after rapid rewarming at 45ºC, shoot tips were quickly washed with 1.2 M sucrose solution and then plated on solidified shoot culture medium. Shoot tips were successfully cryopreserved by vitrification, when they were precultured on medium supplemented with 5% DMSO at 4ºC for two days before dehydrating in PVS2 for 10-20 minutes at 0ºC. Average survival in terms of normal shoot formation after 4 wks of plating was about 20% without callus formation. Cold hardening of shoot cultures for four weeks at 4ºC significantly improved the survival and shoot regeneration of cryopreserved shoot tips to 70% and 35%, respectively.