Cryopreservation of iris pumila shoot tips by vitrification

A protocol for cryopreservation of Iris pumila shoot tips using a PVS2- vitrification procedure was developed. Shoot tips, excised from in vitro grown shoots, were acclimated at 4°C for two days before vitrification procedure. Following this preculture, shoot tips were treated with a loading solution for 30 min at 25°C, PVS2 solution for different times (from 5 to 40 min) at 0°C and then directly plunged into liquid nitrogen for at least one day. Re-warming was performed in water bath at 40°C for 1 min. After re-warming, the PVS2 was drained and replaced with unloading solution containing 1.2 M sucrose for 20 min. Washed explants were cultured on medium for shoot development and multiplication. The best results were obtained after PVS2 treatment for 20 min before immersion in liquid nitrogen. Shoot tips regrowth was observed within 3 weeks and new shoots were regenerated and multiplied (55%) without calli phase within 12 weeks.