Cryopreservation of Digitalis lanata Shoot Tips

Abstract

A method for the preservation in liquid nitrogen of shoot tips (meristems) of D. lanata is described. It includes the following steps: (a) hardening of shoots by cultivation at 4° C for 8 weeks, (b) treatment of the explanted shoot tips with cryoprotectors, e.g., 2 mol DMSO l^-1 for 2 h, (c) either ultrarapid cooling (ca. 4000 K min^-1) of the shoot tips by submerging in liquid nitrogen or slow cooling (ca. 0.5 K min^-1) of the shoot tips to -40° C using a suitable freezer, (d) storage of the shoot tips at -196° C in liquid nitrogen, (e) ultrarapid rewarming of the ultrarapidly cooled shoot tips by placing them directly into nutrient medium or rapid rewarming of the ampoules containing the slowly cooled shoot tips with water at 40° C, and (f) recultivation of the shoot tips at the surface of a solidified nutrient medium containing 2.5 µmol BA 1^-1. About 70% of the shoot tips survived this procedure and about 30% of the shoot tips regenerated shoots.