Survival and genetic stability of Picea abies embryogenic cultures after cryopreservation using a pregrowth-dehydration method

A vitrification method enabled efficient cryopreservation of embryogenic tissue (ETs) of Norway spruce (Picea abies L.) at −196 °C in liquid nitrogen (LN). Correctly formed, normal somatic embryos were generated from ETs that had been thawed after removal from LN. The pregrowth-dehydration method involved preculture of ETs with sucrose (0.25–1.00 M) in the presence or absence of 10 μM abscisic acid (ABA), followed by air-drying for 2 h and rapid freezing in LN. Pretreatment of ETs with both sucrose and ABA promoted ET growth after preculture and thawing more effectively than treatment with sucrose alone. Survival of ETs after thawing from LN using both sucrose and ABA was 54.4 % compared to pretreatment with sucrose alone which was 20 %. Addition of ABA in the preculture medium also improved the ability of ETs to form cotyledonary stage somatic embryos. The somatic embryos, which had normal shoot and root apices and the correct number of cotyledons, were indistinguishable from regenerants obtained from control cultures. Genetic analysis of control and cryopreserved ETs, as well as somatic embryos derived from cryopreserved ETs, indicated that the cryopreservation method had no effect on any of the five microsatellite loci (SpAGC1, SpAGC2, SpAGG3, SpAC1H8, and SpAC1F7) tested. The cryopreservation protocol outlined should enable the long-term storage of valuable clones of Norway spruce in LN, potentially for hundreds of years.