Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Summary Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory. Published with approval of the Director of Arkansas Agricultural Experimental Station.     

Water status and growth of loblolly pine (Pinus taeda L.) callus

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl⁻¹ sucrose was used to produce a high water potential (Ψ_w) of −0.4 MPa (H), and the same medium was used to create a moderate Ψ_w of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψ_w of both H and M media remained constant throughout the culture period. On H medium, callus Ψ_w and osmotic potential (Ψ_s) both increased 0.05 MPa/week with the callus Ψ_w approaching that of the external medium. On M medium, callus Ψ_w and Ψ_s both decreased more than 0.1 MPa/week with the callus Ψ_w decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψ_p) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψ_w increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.     

Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.)

The development and mapping of genetic markers based upon expressed sequence tag polymorphisms (ESTPs) in loblolly pine (Pinus taeda L.) are reported. The new markers were generated by PCR-amplification of loblolly pine genomic DNAs with primers designed from sequenced cDNAs. The cDNA libraries were constructed from RNAs expressed in the needles of loblolly pine seedlings or in the xylem from young trees. DNA polymorphisms were identified by analyzing the amplified products for differences in fragment size or restriction sites, or by examining mobility differences using denaturing gradient gel electrophoresis (DGGE). DGGE revealed more DNA polymorphisms than the other two methods. Fifty six ESTPs were mapped using either of two mapping populations and positioned onto a loblolly pine consensus genetic map. Unlike many other markers commonly used in forestry, ESTPs can be used as orthologous markers for comparative mapping, to map genes of known function, or to identify candidate genes affecting important traits in loblolly pine. Received: 10 April 2000 / Accepted: 13 July 2000     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> 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) (3.7 µM), silver nitrate (20 µM), and guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt (10 µM) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l -naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/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 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4–6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months 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.Abbreviations ABA Abscisic acid - AC Activated carbon - BA 6-Benzylaminopurine - 8-Br-cGMP Guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt - NAA -Naphthaleneacetic acid Communicated by G.C. Phillips     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Nutrient proportions in foliage of semi-mature loblolly pine

Summary Nitrogen and phosphorus limitations to growth are common in many loblolly pine (Pinus taeda) stands. Interactions of these nutrients may complicate interpretation of foliar nutrient analysis for predicting response to forest fertilization. Proportions of foliar nutrient concentrations (and the changes in these proportions following fertilization) were examined in 36 semi-mature loblolly pine plantations in the southeastern United States. Mean proportions of nutrient concentrations (NPKCaMg) for non-fertilized stands were 1009.336.517.29.2. Potassium and phosphorus were higher. Nitrogen fertilization generally decreased the PN ratio and enhanced growth, indicating a nitrogen deficiency in most stands under study. Additions of nitrogen and phosphorus together yielded a significant increase in the PN ratio. Effects of fertilization effects on other nutrient concentration ratios were also examined.Paper No. 9401 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695.     

The effects of ultraviolet-B radiation on loblolly pine

Summary Depletion of stratospheric ozone and the resulting increase in ultraviolet-B (UV-B) radiation may negatively impact the productivity of terrestrial ecosystems. This concern has led to a number of studies that report the influence of supplementing UV-B radiation on plant growth and development. However, only two of these field studies have included tree species and both were singleseason experiments. In this study, loblolly pine (Pinus taeda L.) from seven seed sources was grown under natural and supplemental levels of UV-B radiation. Irradiation treatments were continued for three seasons on plants from four of the seven groups and for 1 year only for three groups. The supplemental irradiances simulated those that would be anticipated with stratospheric ozone reductions of 16% and 25% over Beltsville, Md. The effects of UV-B radiation during the 1st year on plant growth varied among the seed sources. The growth of plants from two of the seven seed sources tested showed significant reductions following a single irradiation season and plants from one group tended to be larger under increased UV-B radiation. However, after 3 years of supplemental irradiation, plant biomass was reduced in all four groups by 12–20% at the highest simulated ozone depletion. These results suggest that the effects of UV-B radiation may accumulate in trees and that increased UV-B radiation could significantly reduce the growth of loblolly pine over its lifetime. However, they also point to a need for multiple season research in any analysis of potential consequences of global change on the long-term growth of trees.     

Estimation of gene flow into two seed orchards of loblolly pine (Pinus taeda L.)

Summary Multilocus allozyme markers, which were present in background stands but not in two seed orchards, enabled estimation of gene flow into two seed orchards of loblolly pine. Estimates averaged 36% in two orchards over three years. When pollen pool frequencies in the background stands were used rather than gene frequencies in the non-orchard trees, the estimates averaged 60%. In one year, trees in the row on the edge of one orchard received significantly more pollen from background stands. Estimates of gene flow between the two 2-ha orchards average 10%. Implications of the observed levels of gene flow are discussed in terms of population genetics and seed orchard management.Scientific Contribution No. 1285 from the New Hampshire Agricultural Experiment Station. This work was part of McIntire-Stennis Project 11 Allozyme studies of coniferous forest tree populations     

The effects of annual fertilization and complete competition control on fascicle morphology of different aged loblolly pine stands

The goal of this study was to determine how increased nutrient availability affects foliage morphology of loblolly pine (Pinus taeda L.) without introducing the confounding influence of light availability. Morphology of fascicles from the terminal leader (radius, length, specific needle area, density, and needles per fascicle) and terminal leader traits (length, foliated length, total leaf area, and total fascicles) were measured for different aged (ranged from 5-year-old to 12-year-old) loblolly pine stands growing on the Piedmont and lower Coastal Plain of Georgia, USA. A factorial combination of annual fertilization and complete interspecific competition control was applied as stand level treatments. Competition control and stand age generally did not affect fascicle morphology. In contrast, annual fertilization significantly increased fascicle length (5%), needles per fascicle (4%), and total leaf area of the terminal (18%), while decreasing specific needle area (4%). Fertilization also increased terminal leader growth (5%) and total number of fascicles (6%). Therefore, loblolly pine foliage morphology does have plasticity to respond to increased nutrient availability. However, the magnitude of these morphological changes is small compared to changes in total canopy leaf area.     

Induced defenses of loblolly pine, Pinus taeda: potential impact on Dendroctonus frontalis within-tree mortality

Loblolly pine (Pinus taeda L.) produces an induced defensive hypersensitive response in inner bark colonized by the southern pine beetle, Dendroctonus frontalis Zimm. (Coleoptera: Scolytidae), and its associated fungi. Adult beetles forced to colonize the induced response tissue in a laboratory study constructed galleries that were the same length as adults boring in normal phloem. However, each female laid fewer eggs in the induced tissue. Larval and pupal mortality were also higher in this tissue when compared to surrounding phloem. Beetles colonizing trees in response to pheromone baits constructed less gallery and laid fewer eggs in induced tissue than in surrounding normal phloem. These results suggest that the lesions produced by the induced defense system in conifers may not only contain the growth of fungi inoculated into trees during the attack phase of beetle colonization, but may also affect survival of bark beetle progeny.

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Résumé Une réaction hypersensible de défense a été induite dans les couches profondes de l'écorce de P. taeda colonisée par D. frontalis et le champignon qui lui est associé. Au laboratoire, des adultes, contraints de coloniser les tissus où une réaction hypersensible e été induite, ont foré des galeries de même longueur que celles creusées dans du phloème sain. Cependant les femelles ont pondu moins d'oeufs dans les tissus induits; les mortalités larvaires et nymphales étaient aussi plus fortes dans ce tissu que dans le phloème voisin. Les scolytes, ayant colonisé les arbres après attraction par des pièges à phéromones, forent moins de galeries et pondent moins d'oeufs dans le tissu induit que dans le phloème sain voisin. Ces résultats suggèrent que les lésions, provoquées par le système de défense induit des conifères, peuvent non seulement limiter la croissance du champignon inoculé dans l'arbre au cours de la colonisation des scolytes, mais aussi affecter la survie des descendants dans l'écorce.

     

Growing season temperatures limit growth of loblolly pine (Pinus taeda L.) seedlings across a wide geographic transect

We grew potted loblolly pine (Pinus taeda L.) seedlings from a single provenance under well watered and fertilized conditions at four locations along a 610 km north–south transect that spanned most of the species range to examine how differences in the above-ground environment would affect growth rate, biomass partitioning and gas exchange characteristics. Across the transect there was an 8.7°C difference in average growing season temperature, and temperature proved to be the key environmental factor controlling growth rate. Biomass growth was strongly correlated with differences in mean growing season temperature (R ² = 0.97) and temperature sum (R ² = 0.92), but not with differences in mean daily photosynthetic photon flux density or mean daily vapor pressure deficit. Biomass partitioning between root and shoot was unchanged across sites. There was substantial thermal acclimation of leaf respiration, but not photosynthesis. In mid-summer, leaf respiration rates measured at 25°C ranged from 0.2 μmol m⁻² s⁻¹ in seedlings from the warmest location to 1.1 μmol m⁻² s⁻¹ in seedlings from the coolest site. The greatest biomass growth occurred near the middle of the range, indicating that temperatures were sub- and supra-optimal at the northern and southern ends on the range, respectively. However, in the middle of the range, there was an 18% decrease in biomass increment between two sites, corresponding to 1.4°C increase in mean growing season temperature. This suggests that thermal acclimation was insufficient to compensate for this relatively small increase in temperature.     

Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms

The Waxy (Wx) gene product controls the formation of a straight chain polymer of amylose in the starch pathway. Dominance/recessiveness of the Wx allele is associated with amylose content, leading to non-waxy/waxy phenotypes. For a total of 113 foxtail millet accessions, agronomic traits and the molecular differences of the Wx gene were surveyed to evaluate genetic diversities. Molecular types were associated with phenotypes determined by four specific primer sets (non-waxy, Type I; low amylose, Type VI; waxy, Type IV or V). Additionally, the insertion of transposable element in waxy was confirmed by ex1/TSI2R, TSI2F/ex2, ex2int2/TSI7R and TSI7F/ex4r. Seventeen single nucleotide polymorphims (SNPs) were observed from non-coding regions, while three SNPs from coding regions were non-synonymous. Interestingly, the phenotype of No. 88 was still non-waxy, although seven nucleotides (AATTGGT) insertion at 2,993 bp led to 78 amino acids shorter. The rapid decline of r ² in the sequenced region (exon 1–intron 1–exon 2) suggested a low level of linkage disequilibrium and limited haplotype structure. K _s values and estimation of evolutionary events indicate early divergence of S. italica among cereal crops. This study suggested the Wx gene was one of the targets in the selection process during domestication. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Adventitious root formation in cuttings of loblolly pine (Pinus taeda L .): developmental sequence and effects of maturation

 Adventitious root formation in cuttings from fascicular shoots in loblolly pine (Pinus taeda L.) consists of four more or less discontinuous stages: (1) proliferation of cells at the base of the cutting, (2) differentiation of wound vascular tissue and periderm, (3) dedifferentiation of a zone near the wound cambium and wound phloem to form a root initial, and (4) formation of a root meristem. Anatomical changes during adventitious root initiation are described in cuttings from donors of different types and ages. Cuttings from seedlings and 3- to 7-year-old hedged stock plants rooted better than cuttings from 3-year-old tree form donors. It is concluded that the loss of rooting capacity in loblolly pine can be arrested by shearing loblolly pine stock plants to low hedges. The process of root initiation, however, was similar in cuttings from all sources and is apparently not the cause for the rapid decline of rooting potential with increasing age of the donor plant. Received: 3 June 1997 / Accepted: 15 August 1997     

Identification of quantitative trait loci influencing annual height- and diameter-increment growth in loblolly pine (Pinus taeda L.)

 Quantitative trait loci (QTLs) for annual height- and diameter-increment growth were estimated in each of two three-generation loblolly pine pedigrees. Annual height-increment was measured in three consecutive years in the BASE pedigree and in four consecutive years in the QTL pedigree. Annual diameter-increment was measured only in the QTL pedigree for two consecutive years. An interval mapping-approach was used to estimate the number of QTLs, the magnitude of QTL effects, and their position on genetic linkage maps. Thirteen different height-increment and eight different diameter-increment QTLs were detected, suggesting that the these traits are, at least in part, controlled by a few genes of large effect. Little evidence was found for the expression of individual QTLs in multiple years or in multiple genetic backgrounds. These results were discussed in terms of the power of the experiment and their consequences for marker-assisted breeding. Received: 12 July 1998/Accepted: 5 August 1998     

cDNA isolation,functional expression,and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis

The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.