Immature zygotic embryo explants (isolated or with intact megagametophytes) from 10 loblolly pine (Pinustaeda L.) clones (7-34, 7-56, 11-9, 11-16, 11-25, 10-1003, 10-1007, 10-1011, 10-1018, and 10-1019) were surveyed for their potential to form embryogenic tissue from the suspensor region of zygotic embryos. After over 14 000 explants were cultured, embryogenic cultures were initiated from explants of 8 of the 10 clones; only explants from clones 11-25 and 10-1019 were not responsive. Embryogenic tissue was initiated from zygotic embryos with intact megagametophytes on MSG basal medium with no exogenous plant growth regulators or with 2–5 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0–1 mg/L N6-benzyladenine (BA). The highest initiation frequency (5%) was obtained from isolated zygotic embryos of clone 7-34 less than 0.5 mm in length just prior to cotyledon primordia development on DCR basal medium with 3 mg/L 2,4-D and 0.5 mg/L BA. Two types of embryogenic cultures were maintained on medium with 2,4-D and BA: (i) those that contained pre-embryonal masses of cells interspersed with unaggregated suspensorlike cells, but which rarely contained well-formed somatic embryos, and (ii) those that frequently contained well-formed somatic embryos. Somatic embryo development from both types of cultures progressed to a precotyledonary stage on medium with 2.6 mg/L abscisic acid.

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.

The spermidine synthesis inhibitors methylglyoxal bis-(guanylhydrazone) (MGBG) and dicyclohexylammordum sulfate (DCHA) were found to reduce growth and embryogenesis in wild carrot cultures. Cellular polyamine levels were also affected by the inhibitors, with spermidine levels being especially reduced by DCHA. Similarly, MGBG reduced organogenetic development of shoots on excised aspen hypocotyls. These data suggest that the polyamines, especially spermidine, play an important role in the growth and development of plants.

Both embryogenic and nonembryogenic calli of Picea abies (L.) Karst. were initiated from the hypocotyl region of immature embryos. The two callus phenotypes were manually separated and subsequently maintained independently, but under identical culture conditions. Biochemical analysis of the two phenotypes revealed significant differences in ethylene evolution rate and in concentrations of glutathione and total reductants. Due to the constancy of the genetic background, age and growth conditions of the two callus types, differences in the measured quantities are not likely to be traceable to the genetic origin of the callus and serve to highlight biochemical changes associated with somatic embryogenesis in Norway spruce.

A method for quantitative determination of the level of somatic embryogenesis in Norway spruce embryogenic callus is described. Embryogenic callus was dispersed in liquid by agitation and plated in a thin layer of medium containing 0.6% low melting point agarose. The number of embedded somatic embryos per mg of callus ranged from 0.2 to 1.5 among 11 embryogenic callus lines surveyed. Each callus line was derived from an individual immature embryo explant. Further development occurred as somatic embryos grew out of the agarose layer. This method was useful for identifying highly embryogenic callus lines among phenotypically similar lines, and should be useful for quantitatively determining the effect of medium and growth regulator modifications on somatic embryo density and developmental capacity.