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During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.

This paper is part of a series considering the impact of activated carbon (AC) on the composition of plant tissue culture media. Using liquid culture media for initiation of Norway spruce embryogenic tissue and eight different ACs, we present a method for achieving target plant growth regulator (PGR) levels in AC-containing medium based on sorption isotherms for individual PGRs. Linear relationships were found between PGR adsorption and specific BET (Brunauer, Emmett, Teller theory) surface area and specific total pore volume of AC. When using a new AC, this linear relationship allows one to achieve multiple PGR levels similar to historic levels through adjustment of the mass of AC based on its relative BET surface area or relative total pore volume. Target levels of PGRs and an initiation success similar to that in medium without AC were achieved with several different AC types when AC mass was adjusted on the basis of pore volume.

Somatic embryogenesis (SE), the most promising technology to multiply high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests in the United States. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (LP; Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation of LP, slash pine (SP; Pinus elliottii), Douglas-fir (DF; Pseudotsuga menziesii), and Norway spruce (NS; Picea abies) were improved through the use of paclobutrazol, a gibberellin synthesis inhibitor. Paclobutrazol was effective at concentrations ranging from 0.25 mg/l to 3.0 mg/l (0.85-10.2 microM) and optimal in LP at 1.0 mg/l. Using control media (no paclobutrazol) and 0.33-1.0 mg/l paclobutrazol, initiation percentages in LP, SP, DF, and NS were improved from 37.7% to 44.2% (across experiments), 19.3% to 28.5%, 16.9% to 23.7%, and 38.8% to 48.5%, respectively. Other gibberellin inhibitors such as flurprimidol, chlormequat-Cl, and daminozide also caused statistically significant increases in LP initiation when added to the medium at concentrations of 0.34, 10.0, and 1.0 microM, respectively. No detrimental effects on subsequent embryo development were observed when 29 new initiations from medium without GA inhibitor and 28 new initiations from medium containing paclobutrazol were tracked through culture capture, liquid culture establishment, cotyledonary embryo development, and germination.