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.     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

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.     

Anatomy of seedling tap roots of loblolly pine (Pinus taeda L.)

The development of tap root anatomical features was investigated in seedlings of loblolly pine (Pinus taeda L.) under both pot and pouch growth regimes. The roots possessed the three anatomical zones previously observed in jack pine (Pinus banksiana Lamb) and Eucalyptus pilularis Sm. - white, condensed tannin (CT), and cork - suggesting that this developmental sequence is preserved over species and growth conditions. Xylem development was centripetal and similar to that found earlier in P. sylvestris. Tracheids with lignified, secondary walls were detected distal to the point of endodermal Casparian band deposition. However, tests for ability to conduct fluid indicated that the protoxylem was capable of transport only proximal to the Casparian bands. Detailed examination of suberin lamella deposition in the endodermis demonstrated that passage cells were present through the white and CT zones. Progressive, centripetal cortical death in the CT zone did not include the endodermis, which remained alive until the cork layer formed, at which point the endodermis was crushed. Therefore, passage cells remain as functional portals for nutrient and water uptake in the CT zone even though the central cortex is dead. Tracer tests indicated that the endodermis provides an apoplastic barrier to tracer diffusion into the stele and that this function was taken over by the young cork layers. Results of this study point to a strong role for the endodermis in the regulation of nutrient and water uptake until the maturation of the first cork layer.     

Polyethylene glycol-promoted development of somatic embryos in loblolly pine (Pinus taeda L.)

Summary The effect of polyethylene glycol (PEG) combined with abscisic acid (ABA) and KCl on somatic embryo development in loblolly pine was investigated. Two embryogenic cell lines, which had not produced cotyledonary stage embryos using previously published methods, were employed in this study. As a maturation medium, basal medium was supplemented with 0 to 10% PEG (MW 3350), 10 to 40 mg/l (37.8 to 151.3 μM) ABA, 0 or 10 mM KCl, 1.5 g/l activated charcoal, 30 g/l sucrose, and 6 g/l agar. Without PEG in the maturation medium, most somatic embryos at stage 1 could not mature further. Embryogenic tissues on the maturation medium with 5 to 7.5% PEG consistently produced stage 2 and stage 3 somatic embryos. PEG at 10% significantly decreased the number of stage 2 and 3 embryos compared to PEG at 5 to 7.5% ABA at 40 mg/l (151.3 μM), combined with 7.5% PEG and 10 mM KCl, gave the maximum number of stage 3 embryos in both cell lines. ABA at 10 mg/l (37.8 μM) induced an over-proliferation of embryogenic tissues and generally failed to produce mature embryos. KCl also significantly enhanced initial stage embryo formation and subsequent embryo maturation.     

Natural hybridization within seed sources of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf and loblolly pine trees (n = 93 and 102, respectively) from 22 seed sources of the Southwide Southern Pine Seed Source Study plantings or equivalent origin were evaluated for amplified fragment length polymorphism (AFLP) variation. These sampled trees represent shortleaf pine and loblolly pine, as they existed across their native geographic ranges before intensive forest management. Using 17 primer pairs, a total of 96 AFLPs between shortleaf pine and loblolly pine were produced and scored on the sample trees and two control-pollinated F1 interspecies hybrids and their parents. In addition, the well known isocitrate dehydrogenase (IDH) isozyme marker was scored for all trees. IDH detected two putative hybrids among the loblolly pine samples and two among the shortleaf pine samples, while either 13 or 12 putative hybrids were detected using all AFLP markers and IDH and either NewHybrids or Structure software, respectively. Results of this study show that later generation hybrids can be reliably identified using AFLP markers and confirmed that IDH is not a definitive marker for detecting hybrids; that is, at least in some seed sources, the alternative species’ IDH allele resides in the source species. Based on all the markers, hybridization frequency varied geographically, ranging from 30% in an Arkansas seed source to 0% in several other seed sources. The hybridization level was higher in populations west of the Mississippi River than in populations east of the river; the shortleaf pine hybridization rates were 16.3% and 2.4% and the loblolly pine rates were 4.5% and 3.3%, west and east of the river, respectively. The results suggest that hybridization between these two species is significant but varies by seed source and species, and the potential for the unintended creation of hybrids should be considered in forest management decisions regarding both natural and artificial regeneration.     

Changes in leaf expansion and epidermal screening effectiveness in Liquidambar styraciflua and Pinus taeda in response to UV-B radiation

Absorption or screening of ultraviolet-B (UV-B) radiation by the epidermis may be an important protective method by which plants avoid damage upon exposure to potentially harmful UV-B radiation. In the present study we examined the relationships among epidermal screening effectiveness, concentration of UV-absorbing compounds, epidermal anatomy and growth responses in seedlings of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.). Seedlings of each species were grown in a greenhouse at the University of Maryland under either no UV-B radiation or daily supplemental UV-B radiation levels of 4, 8 or 11 kJ m^?2 of biologically effective UV-B (UV-B_BE) radiation. Loblolly pine seedlings were subsequently grown in the field under either ambient or supplemental levels of UV-B radiation. At the conclusion of the growing season, measurements of epidermal UV-B screening effectiveness were made with a fiber-optic microprobe. In loblolly pine, less than 0.5% of incident UV-B radiation was transmitted through the epidermis of fascicle needles and about 1% was transmitted in primary needles. In contrast, epidermal transmittance in sweetgum ranged from about 20% in leaves not preconditioned to UV-B exposure, to about 10% in leaves grown under UV-B radiation. The concentration of UV-absorbing compounds was unaffected by UV-B exposure, but generally increased with leaf age. Increases in epidermal thickness were observed in response to UV-B treatment in loblolly pine, and this accounted for over half of the variability in UV-B screening effectiveness. In spite of the low levels of UV-B penetration into the mesophyll, delays in leaf development (both species) and final needle size (loblolly pine) were observed. Seedling biomass was reduced by supplemental UV-B radiation in loblolly pine. We hypothesize that the UV-induced growth reductions were manifested by changes in either epidermal anatomy or epidermal secondary chemistry that might negatively impact cell elongation.     

Electrophoretic analysis of genetic linkage in Scots pine (Pinus sylvestris L.)

Eighty megagametophytes from each of 24 Scots pines (Pinus sylvestris L.) were subjected to horizontal starch gel electrophoresis. The trees were from crosses among widely separated provenances, and each was polymorphic for 8 to 14 loci. Evidence for linkage among 275 two-locus combinations was tested using chi-square analysis. Data from different trees were pooled to calculate map distances for the species. Nineteen of the twenty-nine loci tested were linked in one of six groups; the groups varied in size from two to seven loci. Similarities in linkage relationships among Scots pine, other pines, and other species within the Pinaceae support karyological research that suggests extensive conservation of the conifer genome.     

Photosynthetic electron transport adjustments in overwintering Scots pine (Pinus sylvestris L.)

As shown before [C. Ottander et al. (1995) Planta 197:176-183], there is a severe inhibition of the photosystem (PS) II photochemical efficiency of Scots pine (Pinus sylvestris L.) during the winter. In contrast, the in vivo PSI photochemistry is less inhibited during winter as shown by in vivo measurements of deltaA820/A820 (P700+). There was also an enhanced cyclic electron transfer around PSI in winter-stressed needles as indicated by 4-fold faster reduction kinetics of P700+. The differential functional stability of PSII and PSI was accompanied by a 3.7-fold higher intersystem electron pool size, and a 5-fold increase in the stromal electron pool available for P700+ reduction. There was also a strong reduction of the QB band in the thermoluminescence glow curve and markedly slower Q-A re-oxidation in needles of winter pine, indicating an inhibition of electron transfer between QA and QB. The data presented indicate that the plastoquinone pool is largely reduced in winter pine, and that this reduced state is likely to be of metabolic rather than photochemical origin. The retention of PSI photochemistry, and the suggested metabolic reduction of the plastoquinone pool in winter stressed needles of Scots pine are discussed in terms of the need for enhanced photoprotection of the needles during the winter and the role of metabolically supplied energy for the recovery of photosynthesis from winter stress in evergreens.     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

The effects of heat treatment on some technological properties of Scots pine (Pinus sylvestris L.) wood

Heat treatment is often applied to wood species to improve their dimensional stability. This study examined the effect of heat treatment on certain mechanical properties of Scots pine (Pinus sylvestris L.), which has industrially high usage potential and large plantations in Turkey. Wood specimens obtained from Bolu, Turkey, were subjected to heat treatment under atmospheric pressure at varying temperatures (120, 150 and 180 degrees C) for varying durations (2, 6 and 10h). The test results of heat-treated Scots pine and control samples showed that technological properties including compression strength, bending strength, modulus of elasticity in bending, janka-hardness, impact bending strength and tension strength perpendicular to grain suffered with heat treatment, and increase in temperature and duration further diminished technological strength values of the wood specimens.     

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     

Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings

Summary Ultraviolet-B light (UV-B) and ultraviolet-A light (UV-A) at higher doses exert a strong inhibitory (toxic) effect on axis growth in Scots pine (Pinus sylvestris L.) seedlings. This effect is unrelated to control of growth rate by phytochrome. Rather, after a toxic UV dose growth of the pine seedling no longer responded to phytochrome. Both, the effect of UV-B as well as the inhibiting effect of UV-A could be photoreactivated by blue light (B). The action of UV-A was 2 fold: (i) it exerted a toxic effect which could be photoreactivated by B, and (ii) applied after UV-B it photoreactivated to some extent the toxic UV-B effect. Obviously, the UV-A range causes a toxic effect, and at the same time is capable of photoreactivating the toxic UV effect. At higher doses the toxic effect prevails.     

High-throughput genotyping and mapping of single nucleotide polymorphisms in loblolly pine (Pinus taeda L.)

The development and application of genomic tools to loblolly pine (Pinus taeda L.) offer promising insights into the organization and structure of conifer genomes. The application of a high-throughput genotyping assay across diverse forest tree species, however, is currently limited taxonomically. This is despite the ongoing development of genome-scale projects aiming at the construction of expressed sequence tag (EST) libraries and the resequencing of EST-derived unigenes for a diverse array of forest tree species. In this paper, we report on the application of Illumina’s high-throughput GoldenGate™ SNP genotyping assay to a loblolly pine mapping population. Single nucleotide polymorphisms (SNPs) were identified through resequencing of previously identified wood quality, drought tolerance, and disease resistance candidate genes prior to genotyping. From that effort, a 384 multiplexed SNP assay was developed for high-throughput genotyping. Approximately 67% of the 384 SNPs queried converted into high-quality genotypes for the 48 progeny samples. Of those 257 successfully genotyped SNPs, 70 were segregating within the mapping population. A total of 27 candidate genes were subsequently mapped onto the existing loblolly pine consensus map, which consists of 12 linkage groups spanning a total map distance of 1,227.6 cM. The ability of SNPs to be mapped to the same position as fragment-based markers previously developed within the same candidate genes, as well as the pivotal role that SNPs currently play in the dissection of complex phenotypic traits, illustrate the usefulness of high-throughput SNP genotyping technologies to the continued development of pine genomics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.