Evaluation of Vacuum Infiltration Vitrification and Cryo-mesh Cryopreservation Techniques with Arabidopsis Thaliana Shoot Tips

Abstract

BACKGROUND:Novel cryo-techniques are continuously being developed that may better improve cryogenic survival in plants, with the aim of reducing exposure times to otherwise toxic cryoprotective agents whilst maximising regeneration rates. OBJECTIVE:This study used cryo-mesh and vacuum infiltration vitrification with two vitrification solutions (PVS2 and PVS3) to develop an optimised cryopreservation protocol for Arabidopsis thaliana . MATERIALS AND METHODS:Shoot tips from 10-day old seedlings of wild type A. thaliana were cryopreserved using either vacuum infiltration vitrification or the cryo-mesh technique. Shoot tips were treated for up to 60 min in increments of 10 min with PVS2 and PVS3, and for an additional 180 and 300 min incubation for cryo-mesh prior to exposure to liquid nitrogen. RESULTS:Both methods resulted in very high regeneration rates, but which decreased after longer exposure to the vitrification solutions. The highest regeneration rate for vacuum-infiltration vitrification was attained after only 30 min incubation in PVS2 (92.5%) and 50 min incubation in PVS3 (93.55%). In the case of cryo-mesh the highest regeneration was observed after 180 min incubation in either PVS2 (100%) or PVS3 (92.2%). CONCLUSION: Vacuum- infiltration vitrification is more effective than cryo-mesh by reducing exposure times to cryoprotective solutions whilst achieving very high regeneration rates of shoot tips of A. thaliana .