An improved dehydration protocol for cryopreservation of Brassica napus somatic embryos

Abstract

This study compared three predehydration methods for cryopreservation of somatic embryos of oilseed rape (Brassica napus L.). These methods were (1) fast-drying in a laminar airflow cabinet at 25°C and 60 to 70% RH, (2) slow-drying for two days in closed jars with constant relative humidities (85%, 75%, 64% and 51%), and (3) stepwise slow-drying over six days using four humidity conditions (97% → 92% → 85% → 75%). Desiccation tolerance of oilseed rape somatic embryos increased significantly after preculture for 7 days in the presence of 10 mg/L abscisic acid (ABA). Under the fast-drying condition, the critical water content of desiccation tolerance (50% survival) for embryos decreased from 1.16 to 0.24 g/g dw after ABA preculture for 7 days. However, fast-dried somatic embryos with water content ranging from 2.51 to 0.16 g/g dw did not survive after freezing and storage in liquid nitrogen. Slow-drying improved the survival after cryopreservation. The highest survival rate (55 ± 3 %) was observed with 75% RH. The best survival rate (81 ± 3 %) was obtained, using the stepwise slow-drying procedure in which RH was progressively lowered from 97 to 75% RH over a period of six days. The data suggest that the drying rate during dehydration greatly affects the survival of somatic embryos after cryopreservation.