Cryopreservation of embryogenic cell suspensions of Catharanthus roseus L. (G) Don.

Abstract

An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cultures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium containing 4.52 μM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pretreatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryogenic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 μM 6-benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology.