Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Abstract

Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N6-benzyladenine (BAP) each (20×10−6 M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10−6 M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10−6 M) and 2,4-D (5×10−6 M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10−6 M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability.