Improved cryopreservation procedure for long term storage of synchronised culture of grapevine

Abstract

Anther-derived pre-embryogenic masses (PEMs) of callus, established via suspension cultures, were encapsulated to form synthetic seeds suitable for cryopreservation. The synchronised suspension culture proliferation necessitated the optimisation of plant growth regulators for different cultivars. The growth phase and density of the culture were also important as well as the exposure of cells to vitrification solution containing 0.75 M sucrose with 0.1 M CaCl₂ and 2.0 % sodium alginate (pH 5.7). Pre-treatment of the encapsulated cells for 2 d with Nitsch and Nitsch (NN) medium containing 0.75 M sucrose solution followed by dehydration for 4 h in a laminar flow box provided maximum cell viability, which varied from 0 to 40 %. The embryo proliferation from the cryopreserved beads involved warming them and then transfer to NN medium containing glutamine (50 mg dm⁻³) and activated charcoal (2.5 %). The maximum number of embryos obtained was 31–53 per bead. Subculture into the same medium induced secondary embryogenesis, which was initiated from the meristematic region, radicle, and root cap. Proliferation and maturation of secondary embryos was faster than of primary embryos. No phenotypic variation or abnormal structures compared to the control were observed in the regenerated plantlets.