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.

Major advances have been achieved in somatic embryogenesis (SE) of loblolly pine, making it a promising method for the implementation of clonal forestry. However, the frequency of initiation of SE cultures, which is highly variable among loblolly pine families, needs improvement to further advance the implementation of this technology in conjunction with tree breeding. Genetic control of SE initiation was investigated using a diallel mating design with six parent trees. The results showed that SE initiation is under the control of strong genetic additive effects, as 42% of the total variance was explained by the variation due to general combining ability effects. The variation due to maternal effects explained a moderate proportion of the total variance, whereas other components of variance had small but significant effects. The conclusions regarding the strong genetic control of SE initiation were drawn from two independent experiments in which consistent results were obtained with seed from the same controlled pollinations but using entirely different procedures. Practical implications for breeding and clonal propagation were tested in independent experiments with targeted matings. Our results indicated that large improvement in SE culture initiation could be achieved in a predictable manner by selecting the most favorable female parent, or in some cases, a favorable male parent.

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Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 μM abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.