Cryopreservation and plant regeneration of anther callus in Hevea by vitrification

Abstract

Callus induced from anther of Hevea brasiliensis was successfully cryopreserved in liquid nitrogen (LN) by vitrification method and subsequently regenerated into plants. The effects of different preculture time, loading and dehydration duration on callus viability after cryopreservation were evaluated. The effective cryopreservation protocol involved preculturing on modified Murashige and Skoog (MS) medium containing 5 % sucrose (w/v) and 5% dimethyl sulfoxide (DMSO) (v/v) for 3 days, loading with 60% plant vitrification solution 2 (PVS2) for 20 min at 0°C and dehydration with ice-cold PVS2 for 40 min. Dehydrated samples were directly immersed into LN, stored for 24 h and re-warmed in a water bath at 40°C. Using this protocol, H. brasiliensis callus showed 71.7% viability after cryopreservation. In conclusion, we developed a simple and effective method for the cryopreservation of H. brasiliensis callus, which allows long-term maintenance of valuable genotypes.

Key words: Callus, cryopreservation, Hevea brasiliensis, regeneration, vitrification.