Cryopreservation In Vitro of Blackberry 'Cacanska Bestrna' Shoot Tips by Encapsulation Dehydration

Abstract

In the present study, in vitro grown shoot tips of blackberry (Rubus subg. Rubus Watson.) 'Cacanska Bestrna' were tested for regrowth after cryopreservation using slightly modified encapsulation dehydration method described by Dereuddre et al. (1990). Apical shoot tips 2-3 mm length were encapsulated in alginate beads composed of 3, 5 and 10% (w/v) alginic acid sodium salt in calcium-free liquid MS medium supplemented with 1 mg L-1 BA, 0.1 mg L-1 IBA and 0.1 mg L-1 GA(3). Polymerization was done in liquid MS medium with 100 mM CaCl2 for 30 min at room temperature. Encapsulated shoot tips were pre-treated in liquid MS medium with 0.75 or 1 M sucrose for 24 h in growth room and dehydrated for 4 and 8 h to 29 and 20% moisture content, resp. (fresh weight basis) before rapid immersion in liquid nitrogen (LN) for 1 h. Upon thawing performed by placing the cryovials in the air currant of the laminar airflow cabinet for 2 min the beads were directly transferred on a regrowth medium. Explants showing normal development 28 days after bead burst were considered be regrowing. The osmotic dehydration in 1 M sucrose followed by 8 h of desiccation gave the highest survival rate (22.2%) and regrowth (16.7%) after freezing in LN for explants encapsulated in 5% alginate beads. Cryopreserved shoot tips multiplied in the three subcultures had normal morphology with similar multiplication capacity in comparison with non-cryopreserved shoots.