Our progress is reviewed on development of somatic embryogenesis in conifers for mass propagation. A distinct embryogenic callus (EC) phenotype, white, mucilaginous, and rapidly growing, has been initiated on modified MS media with 2,4-D or NAA (2-5 mg/L) and BA(0-1 mg/L) from immature embryos of Norway spruce (Picea abies), white spruce (Picea glauca), loblolly pine (Pinus taeda), pond pine (Pinus serotina), and white pine (Pinus strobus). EC has also been initiated from mature embryos of Norway spruce and maintained as rapidly growing (48 hour doubling) liquid suspensions. Initiation of EC in Picea and Pinus differ markedly in several ways. Precotyledonary embryos were optimal in Pinus and EC originated from the suspensor region. In Picea EC originated from the hypocotyl and cotyledon region of predominantly post-cotyledonary embryos. Biochemically, EC of Picea and Pinus were similar and distinctly different from nonembryogenic callus (NEC) in terms of ethylene evolution rates (EC low and NEC high), level of total reductants, including glutathione (EC low and NEC high), and protein synthesis rates (EC high and NEC low). Conifer somatic embryos contained proplastids closely resembling those found in early zygotic embryos. On proliferation medium in the light, EC was white and maintained the proplastid morphology, whereas, NEC was green and contained mature chloroplasts with grana. These biochemical and ultrastructural differences served to both verify and predict embryogenic potential.With Norway spruce somatic embryos, maturation frequencies as high as 25% have been attained. Germination frequencies as high as 82% (mean 56%) have been obtained. Twenty-nine percent of the somatic embryo plantlets survived transfer to the greenhouse, set a dormant terminal bud, overwintered to -5°C, and renewed vegetative growth synchronously with control seedlings. This is the first report of overwintering and renewed vegetative growth from resting buds of conifer somatic embryo plants.

Triglycerides in developing zygotic embryos of Norway spruce and loblolly pine were found to accumulate continuously during the course of development, comprising nearly 50% of the fresh weight of a mature embryo. Embryogenic calli of these two species contained dramatically lower levels of triglycerides. Abscisic acid treatments promoted both embryo production and triglyceride accumulation in Norway spruce cultures. A method used to determine triglyceride levels in human serum, commercially available in kit form, was adapted for use with plant tissues. Low levels of triglycerides in the cultured tissues may be related to difficulties in the development and germination of conifer somatic embryos.

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH4 NO3 and supplemented with 5 mM glutamine, 4.5 pM N6-benzyladenine and 10.7/tM naphthaleneacetic acid or 10 #M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotytedonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos 'germinated' and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.

Whole Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seed embryos were placed on defined medium containing 0.05 or 0.1 mg/ℓ BAP (benzylaminopurine). One vigorous shoot usually grew from the tip of each swollen cotyledon on the 0.05 BAP medium, but small multiple shoots were produced on the 0.1 BAP medium. After transfer of embryos or excised cotyledons to medium without hormones, the vigorous shoots developed normally but the small multiple shoots did not. However, when cotyledons were grown on medium containing auxin and cytokinin, callus was produced that was isolated and subcultured monthly to fresh medium. Shoots were produced from subcultured cotyledon callus in 11 of 35 cultures, for a frequency of 31%. Two of 30–50 excised shoots rooted after treatment with 10 mg/ℓ IBA (indolebutyric acid). A few shoots were also produced from subcultured needle callus and from stem explant callus from young seedling material.