Desiccation‐Tolerant Somatic Embryos of Norway Spruce (Picea abies) Can Be Produced in Liquid Cultures and Regenerated into Plantlets

Abstract

A rapid and efficient culture system for Picea abies somatic embryos grown in liquid culture has been developed. Embryogenic cultures grown on semisolid medium (K‐medium) supplemented with auxin (10 μM naphthaleneacetic acid) and cytokinin (5 μM N6‐benzyladenine) for 2–7 yr were used as inoculum for liquid cultures. Liquid cultures were established after 1 mo in liquid K‐medium and were then subcultured to a liquid culture medium supplemented with 16 μM abscisic acid and 7.5% polyethylene glycol (LAP medium) for further development of the somatic embryos. After ca. 2 wk, a large number of well‐formed somatic embryos could be observed. Within 5 wk the embryos had initiated both cotyledons, shoot apical meristem, and radicle. They also accumulated storage proteins, as revealed by immunoblot analysis of a 42‐kDa storage protein and by immunogold labeling of sectioned embryos. By 8 wk, mature somatic embryos had developed. The cell lines were maintained in liquid LAP medium for up to 9 mo. Simultaneously, cultures multiplied and produced embryos that matured. A cell density of 2–3 mL of packed cells to 30 mL culture medium or of 6–8 mL packed cells to 300 mL culture medium was necessary for successful culture. Regeneration capacity of the investigated cell lines was generally stable. Reinitiating embryogenic tissue from mature embryos could also restore this capacity. Embryos that had matured in liquid medium could withstand a high degree of desiccation when subjected to successive reduction of relative humidity, from 90% to 31% over 3 wk. About 40% of the desiccated embryos grew into plantlets after transfer to semisolid medium without added plant growth regulators. They also exhibited an improved morphology of the regenerated plantlets compared to those derived from nondesiccated embryos.