Gerald S. Pullman

Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination, and gene expression

Clonal production of loblolly pine (Pinus taeda L.) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage. A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2xa0P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5xa0mg/l abscisic acid (ABA), and 2.5xa0g/l Gelrite. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described. A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed. The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression. All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.

Expressed Sequence Tags from loblolly pine embryos reveal similarities with angiosperm embryogenesis

The process of embryogenesis in gymnosperms differs in significant ways from the more widely studied process in angiosperms. To further our understanding of embryogenesis in gymnosperms, we have generated Expressed Sequence Tags (ESTs) from four cDNA libraries constructed from un-normalized, normalized, and subtracted RNA populations of zygotic and somatic embryos of loblolly pine (Pinus taeda L.). A total of 68,721 ESTs were generated from 68,131 cDNA clones. Following clustering and assembly, these sequences collapsed into 5,274 contigs and 6,880 singleton sequences for a total of 12,154 non-redundant sequences. Searches of a non-identical amino acid database revealed a putative homolog for 9,189 sequences, leaving 2,965 sequences with no known function. More extensive searches of additional plant sequence data sets revealed a putative homolog for all but 1,388 (11.4%) of the sequences. Using gene ontologies, a known function could be assigned for 5,495 of the 12,154 total non-redundant sequences with 13,633 associations in total assigned. When compared to ∼72,000 sequences in a collated P. taeda transcript assembly derived from >245,000 ESTs derived from root, xylem, stem, needles, pollen cone, and shoot ESTs, 3,458 (28.5%) of the non-redundant embryo sequences were unique and thereby provide a valuable addition to development of a complete loblolly pine transcriptome. To assess similarities between angiosperm and gymnosperm embryo development, we examined our EST collection for putative homologs of angiosperm genes implicated in embryogenesis. Out of 108 angiosperm embryogenesis-related genes, homologs were present for 83 of these genes suggesting that pine contains similar genes for embryogenesis and that our RNA sampling methods were successful. We also identified sequences from the pine embryo transcriptome that have no known function and may contribute to the programming of gene expression and embryo development.

Genetic control of somatic embryogenesis initiation in loblolly pine and implications for breeding

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.

Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation with abscisic acid and silver nitrate

Loblolly pine (Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7xa0µM), silver nitrate (20xa0xa0µM), and guanosine 3′,5′-cyclic monophosphate, 8-bromo-, sodium salt (10xa0µM) to the medium and by raising cytokinin levels in the presence of 50xa0mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50xa0mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500xa0mg/l casamino acids, 450xa0mg/l glutamine, 2xa0mg/l α-naphthaleneacetic acid (NAA), 0.55xa0mg/l 6-benzylaminopurine (BA), 0.53xa0mg/l kinetin, and 2xa0g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63xa0mg/l BA and 0.61xa0mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4–6xa0months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4xa0months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.

The impact of Gelrite and activated carbon on the elemental composition of two conifer embryogenic tissue initiation media

The elemental composition of plant tissue culture media was studied in response to (1) different levels of Gelrite and activated carbon (AC) in semisolid media and (2) different levels and types of AC in liquid media. Doubling the level of Gelrite from 2xa0g/l to 4xa0g/l reduced available magnesium (20%), calcium (16%), zinc (17%) and manganese (24%) and increased potassium (6%). AC adsorbed copper (90–95%) and zinc (35–51%) from both liquid and semisolid media. Two significantly different ACs gave minor differences in adsorption. No adsorption was indicated for inorganic anions. Nonacid-washed AC released significant levels of magnesium (44% increase), calcium (16% increase) and silica (a 75% increase to 1.8xa0mg/l). The elemental composition of media may need to be adjusted when increasing the Gelrite level or adding AC.

Somatic embryogenesis in loblolly pine (Pinus taeda) and douglas fir (Pseudotsuga menziesii): improving culture initiation and growth with MES pH buffer, biotin, and folic acid

Loblolly pine (Pinus taeda L.) somatic embryogenesis initiation was improved by supplementing the initiation medium with the pH buffer agent 2(n-morpholino)ethanesulphonic acid (MES) at 250 mg l−1, folic acid at 0.5 mg l1, and biotin at 0.05 mg l−1. MES and vitamins increased the percentage of explants with extruded tissue that continued the initiation process to form embryogenic tissue. The increase in initiation was about 12%. Initiation of 12 open-pollinated families averaged 38.5%, which is 16% higher than initiation on medium without these additives. When tested with 18 control-pollinated families, initiation averaged 26.3%. Basal medium contained a combination of modified 1/2 P6 salts, activated carbon (AC) at 50 mg −1, Cu and Zn adjusted to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg l−1 casamino acids, 450 mg l−1 glutamine, 2 mg l−1 NAA, 0.63 mg l−1 BAP, 0.61 mg l−1 kinetin, 3.4 mg l−1 silver nitrate, 10 μM 8-Br-cGMP, 0.1 μM brassinolide, and 2 g l−1 Gelrite. Early-stage embryo growth and initiation in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were also improved in the presence of these additives.

Loblolly pine (Pinus taeda L.): stage-specific elemental analyses of zygotic embryo and female gametophyte tissue

Abstract Stage-specific analyses for key elements (metals) for full-term seed and across seed development were completed for zygotic embryo and female gametophyte (FG) tissues of loblolly pine ( Pinus taeda L.). Tissue was analyzed in replicate for major and minor elements by the use of inductively coupled plasma emission spectroscopy (ICPES). Variation in elemental composition among five full-term seed sources was minimal providing elemental composition targets for somatic embryos. Seed tissues from two open-pollinated families, grown in different locations, were analyzed across the sequence of development. Significant changes in elemental composition occurred over time. Phosphorus, potassium, magnesium, calcium and zinc contents in the FG were highest during early seed development while sulfur and iron were relatively constant. The lowest copper levels were detected during early FG development. Zygotic embryos attained maximum potassium, calcium, manganese and zinc content during early embryo development. Embryo phosphorus, magnesium, and iron generally increased across embryo development. Both seed collections showed minimal values of embryo iron content at stages 4–7 followed by gradual increases until embryos stopped growth. Sulfur and copper were fairly constant; lowest copper occurred during early embryo stages. Patterns for sodium and boron were not consistent in the two open-pollinated families analyzed for either FG or embryo tissues. One family showed peaks in sodium and boron during early embryo development. The second family showed similar levels for sodium and boron through embryo development. These data provide stage-specific targets for somatic embryos and suggest element media composition changes for each step in the somatic embryogenesis protocol.

Gibberellin inhibitors improve embryogenic tissue initiation in conifers

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 0.25xa0mg/l to 3.0xa0mg/l (0.85–10.2xa0μM) and optimal in LP at 1.0xa0mg/l. Using control media (no paclobutrazol) and 0.33–1.0xa0mg/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 1.0xa0μM, 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.

The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium

This study considered the elemental composition of plant tissue culture media in response to pH and two different types of activated C (AC; tissue culture and non acid-washed grades) in liquid media. When tissue culture medium is supplemented with AC the method of AC addition and pH adjustment can greatly impact the final medium pH, in turn, altering mineral availability. Over the pH range of 4–7, Cu and Zn adsorbed (95% and 50%) onto the two physically different ACs to the same extent. As the pH exceeded 5.8, precipitation became pronounced, resulting in 50% reductions in Mn and Fe and smaller reductions in Ca (20%), and P (15%), independent of AC. Non acid-washed AC released significant levels of Mg (65% increase) and Ca (10% increase) at pHxa05.8 compared to the no-AC control. No adsorption was indicated for inorganic anions. Low levels for Cu and Zn are a concern when using AC, and low levels of Fe and Mn are a concern when the pH of the medium exceeds 5.8. Due to its impurity content and difficulty associated with its neutralization, non-acid-washed AC may be a poor choice for use in tissue culture medium.

Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers

Somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future US forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (Pinus taeda L.), the main commercial US forest species, is often recalcitrant for desirable genotypes. Liquid initiation medium with no or low gelling agent or placement of the explant on gelled medium followed later by a liquid medium overlay during the initiation process increased initiation for loblolly pine and Norway spruce (Picea abies). Loblolly pine liquid medium required reduction of NAA from 2xa0mg/l in gelled medium to 0.3xa0mg/l in liquid medium. Once the NAA concentration was adjusted, loblolly pine initiation occurred in liquid medium with fully immersed megagametophytes, explants supported at the liquid medium surface, or on gelled medium overlaid with liquid medium. Liquid overlays (0.25xa0ml) consisting of medium with NAA reduced to 0.3 mg/l, 9xa0mg/l ABA and no gelling agent applied to explants on 2xa0ml of gelled medium provided excellent initiation results. Greatest initiation percentages occurred when the liquid overlay was applied 14xa0days after placement of the megagametophyte on gelled medium. Initiation increases ranged from +8.5% with high-value cross-pollinated seed sources to +6.5 to +9.9% with open-pollinated and often recalcitrant seed sources. Liquid medium addition allows rapid replenishment of nutrients and adjustment or change of pH, hormones, or other parameters without disturbing the tissue.