Competitive processes in a monoculture of Pinus radiata D. Don

Summary The changes between 9 and 40 years of age of the frequency distribution of tree diameters were studied in an experiment in a plantation of Pinus radiata D. Don in South Australia in which the spacing between trees at planting varied. Empirical functions were developed to relate tree diameter to growth rate in diameter, variance of growth rate in diameter and mortality rate. These functions were used in solving a forward diffusion equation to predict future diameter distributions. The contributions from each of the terms in the diffusion equation were examined and it was found that the drift (growth rate in diameter) term made the greatest contribution to the solution, whilst the diffusion (variance in growth rate) term had neglible effect. This implied that competition between individual trees for light was the dominant competitive process operating in this plantation, rather than below ground competition for soil resources. The ramifications of this finding to forest growth modelling are discussed.     

Inter- and intraspecific polymorphism at chloroplast SSR loci and the inheritance of plastids in Pinus radiata D. Don

DNA sequence analysis of chloroplast genomes has revealed many short nucleotide repeats analogous to nuclear microsatellites, or simple sequence repeats (SSRs). We designed PCR primers flanking five of these regions identified in the chloroplast sequence from Pinus thunbergii and tested them for amplification in Pinus radiata, P. elliotii, P. taeda, P. strobus, Pseudotsuga menziesii, Cupressus macrocarpa, four New Zealand native conifer species (Podocarpus totara, Podocarpus hallii, Podocarpus nivalis, Agathis australis), and four angiosperms (Vitex lucens, Nestegis cunninghamii, Actinidia chinensis, and Arabidopsis thaliana). A PCR product in the expected size range was amplified from all species and interspecific polymorphism was detected at all five loci. Intraspecific polymorphism was detected in P. radiata with four of the five primer pairs. One of these polymorphic chloroplast SSR (cpSSR) was then used to determine the inheritance of chloroplasts in 206 progeny from four control-pollinated, full-sibling P. radiata families. Approximately 99% of the progeny had the cpSSR variant of the pollen parent indicating that in Pinus radiata, like most other conifers, chloroplasts are typically inherited from the paternal parent. These results suggest that polymorphic chloroplast SSRs will be a valuable tool for studying chloroplast diversity, cyto-nuclear disequilibrium, and plastid inheritance in a range of species, and for the analysis of gene flow via pollen and paternity in species with paternal transmission of chloroplasts.     

Boron deficiency inPinus radiata D. Don and the effect of applied boron on height growth and nutrient uptake

Summary Severe dieback observed inP. radiata D. Don plantations during dry years in Southeastern Australia was found to be due to boron deficiency. Borax applied in spring (August 1982) at rates of 50, 100 and 150 kg ha^–1 resulted in a marked increase in foliar B and an alleviation of B deficiency symptoms. During the dry summer following the application of borax no further dieback was observed and height growth of fertilized trees was significantly improved. Correction of B deficiency enhanced the uptake of P by the trees.     

Pinus radiata D. Don in Chile. Fertilization trials with copper

Summary YoungPinus radiata D. Don were fertilized with copper oxychloride and copper sulfate on a random block basis with four replicates. Roots, needles, stems and shoot apices were analyzed separately for Cu, Fe, Mn and Zn contents at different times from fertilization. The results showed a rapid plant copper absorption, no effects being found on the Fe, Mn and Zn content. The dynamic internal distribution of copper among different plant parts is shown. The treatments applied did not cause any phenotypical alteration nor phytotoxicity in the plants, and no significant effect of the fertilizer chemicals on each other.     

Testing for linkage disequilibrium in the New Zealand radiata pine breeding population

Linkage analysis is commonly used to find marker-trait associations within the full-sib families of forest tree and other species. Study of marker-trait associations at the population level is termed linkage-disequilibrium (LD) mapping. A female-tester design comprising 200 full-sib families generated by crossing 40 pollen parents with five female parents was used to assess the relationship between the marker-allele frequency classes obtained from parental genotypes at SSR marker loci and the full-sib family performance (average predicted breeding value of two parents) in radiata pine (Pinus radiata D. Don). For alleles (at a marker locus) that showed significant association, the copy number of that allele in the parents was significantly correlated, either positively or negatively, with the full-sib family performance for various economic traits. Regression of parental breeding value on its genotype at marker loci revealed that most of the markers that showed significant association with full-sib family performance were not significantly associated with the parental breeding values. This suggests that over-representation of the female parents in our sample of 200 full-sib families could have biased the process of detecting marker-trait associations. The evidence for the existence of marker-trait LD in the population studied is rather weak and would require further testing. The exact test for genotypic disequilibrium between pairs of linked or unlinked marker loci revealed non-significant LD. Observed genotypic frequencies at several marker loci were significantly different from the expected Hardy-Weinberg equilibrium. The possibilities of utilising marker-trait associations for early selection, among-family selection and selecting parents for the next generation of breeding are also discussed.     

Ethanol production from sulphuric acid wood hydrolysate ofPinus radiata using free and immobilized cells ofPichia stipitis

Summary Ethanol was produced by a strain ofPichia stipitis adapted to an inhibitory acid wood hydrolysate ofPinus radiata. The best ethanol productivity for batch cultures was 0.21 g/l h at 0.7% ethanol. Varying culture conditions increased ethanol concentration to 0.76%, however the productivity decreased to 0.18 g/l h. A decrease in ethanol concentration in the culture fluid was noted late in the batch which suggested ethanol catabolism. Values of kinetic parameters (K _m,K _s, _max, andV _max) were evaluated for this system. The use of calcium alginate immobilized cells in a continuous-flow stirred tank reactor lead to enhanced fermentative performance, namely a maximum productivity of 0.27 g/l h and 1.13% ethanol yield. The immobilized cells in continuous flow reactors represent an attractive option for fermenting sugars released by sulphuric acid hydrolysis ofP. radiata wood.     

Growth and uptake of N,P, K and B by Pinus radiata D. Don in response to applications of borax

Leader dieback associated with B deficiency in P. radiata D. Don plantations was treated with borax applied at rates of 50, 100 and 150 kg ha⁻¹. This initially increased B in foliage from 5 to 40, 80 and 110 μg g⁻¹ respectively, and was followed by a rapid decline and stabilisation at around 25 μg g⁻¹ for the duration of the study. Annual fluctuations in foliage B levels were strongly correlated with rainfall during the preceding spring and summer. Uptake of N, P and K increased as a result of applied B and comparison of the distribution of these nutrients in crowns of fertilized and unfertilised trees six years after application indicated continued uptake of these nutrients probably as a result of improved root growth due to B. Foliage concentrations of B like N, P and K, increased in young needles towards the upper crown and this, together with a decline in needle concentrations of B as foliage aged, indicated some redistribution of B from older to new foliage. A limit of 5 μg g⁻¹ was found below which little redistribution seems to occur. Application of B prevented further leader dieback, improved apical dominance and height growth and increased volume production by 25 m³ ha⁻¹ at age 8 years. Differences between application rates of B were not significant in terms of growth.     

Photosynthesis and transpiration ofPinus radiata D. Don under natural conditions in a forest stand

Summary Gas-exchange ofPinus radiata foliage was measured with climatised cuvettes under natural light in the sun-crown of 8 m tall trees in a forest stand. Measurement began during a period of drought (_WS –8.2 bar, _We –10.5 bar) and continued after elimination of soil moisture-deficit by watering (_WS –0.5 bar, _We –5.5 bar). Soil and air moisture-deficits severely restricted gas-exchange. Watering resulted in an immediate decline in stomatal resistance (r _s ) and an increase in net photosynthesis (P _N ) of 13%. A slower progressive gas-exchange recovery occurred additionally during the 10 days after watering leading to a further decline inr _s to 3 s cm^-1 and an ultimate increase inP _N of 38% when measured under comparable conditions at 8 mb v.p.d. Radiata pine had a high photosynthetic capacity with a measured maximumP _N of 10.2 mg CO₂ dm^-2 h^-1 total needle surface (11.4 mg CO₂ g^-1 DM h^-1).Optimum temperature forP _N in March (late summer) occurred at ca. 18°C. Rate ofP _N was 95% saturated at irradiance of 900 E m^-2 s^-1 and 50% saturated at only 270 E m^-2 s^-1. Radiata pine needles responded directly to changes in atmospheric humidity by adjusting their stomatal diffusive resistance. As a result, between 8 and 18 mb v.p.d.P _N declined by 2.3% mb^-1 increase.     

Effects of micronutrients on the growth form of two families of Pinus radiata (D.Don) seedlings

Micro-element deficiencies have been implicated in the development of poor stem form in Pinus radiata plantations. Cu, Mn and B have been implicated in previous studies as influencing tree form and/or the process of lignification in plants. Therefore an experiment was initiated to examine the individual and interactive effects exhibited by these trace elements on stem form and lignification.The investigation showed that Cu deficiency reduced both lignification and stem form in seedlings. Mn, competing with Cu for uptake, enhanced the symptoms of Cu deficiency. Boron addition did not alleviate the expression of deformity or increase levels of lignification. There was a clear family influence on the development of seedling stem form in response to variations in mineral nutrition.     

Influence of urea and superphosphate fertilizers on microarthropod numbers and fungal activity during a short-term incubation of material from aPinus radiata D. Don forest floor

Urea and superphosphate dramatically increased the concentrations of exchangeable ammonium-N and water-soluble P in incubated forest floor samples. Urea also increased absolute but not relative nitrate production. Superphosphate depressed pH while urea elevated it. Microarthropod numbers and fungal activity were depressed by fertilizer addition. Mites were more sensitive to urea than were collembola, which were more sensitive to superphosphate. Both groups were more adversely effected by superphosphate than urea. In contrast, fungal activity was more retarded by urea than superphosphate. It is uncertain whether the observed changes resulted from increased osmotic potentials, pH or pH induced toxicities.     

Water potential and gas exchange did not reflect performance of Pinus radiata D. Don in an agroforestry system under conditions of soil-water deficit in a temperate environment

In order to understand how radiata pines respond to declining supply of soil-water in agroforestry systems, we monitored water potential in xylem (ψ _x ), osmotic potential (ψ) and relative water content (q) for fascicles at pre-dawn and at mid-day for 3-year-old trees that were raised from either seedlings (Seedling) or from tissue culture (TC3 and TC4), and grown either alone (Control) or over lucerne (Medicago sativa) pasture (Lucerne). Water relations at dawn were mostly similar for all the pines, except late in the season when ψ was lower, bulk turgor pressure (P), deduced as the difference between ψ _x and ψ, was higher, for TC3 than for the other two pines. At mid-day, Seedling often had higher ψ _x and ψ, but because of its poor osmotic adjustment (OA) had lower P, than either TC3 or TC4. The cell walls were more elastic in Seedling with modulus of elasticity (e) of 6.5 MPa compared with 8.1 MPa for both TC3 and TC4, while loss of turgor was estimated to occur at ψ _x of −1.45 MPa for Seedling, −1.38 MPa for TC3 and −1.35 MPa for TC4. All trees irrespective of their origin had higher ψ _x , P, CO₂ assimilation (A), and stomatal conductance (g _s ), but lower ψ, in Control than in Lucerne in which the soil profile was consistently drier. The trends in ψ _x , ψ, q and A did not reflect the known differences in dry weight of trees, P was in the order TC3 > TC4 > Seedling, consistent with previously reported tree weights. Both TC3 and TC4 had higher P, due to their larger OA, than Seedling, although the latter had higher A. Thus ψ _x and A that are routinely measured may not always adequately explain differences in growth amongst pines; it is advisable that ψ be determined to allow deductions of P be made when using water relations to analyse plant growth.     

A biolistic approach for the transfer and expression of a gusA. reporter gene in embryogenic cultures of Pinus radiata

Summary The biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10–14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.Abbreviations ccc covalently closed circular DNA - lin linearised DNA - E restriction enzyme Eco RI - Sph restriction enzyme SpH I - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

A method for extraction of total RNA fromPinus radiata and other conifers

Carbohydrates, polyphenolic compounds, terpenoids and tannins interfere with the extraction of intact, uncontaminated total RNA from conifers. A method for extraction of total RNA fromPinus radiata is described. This method uses cesium trifluoroacetate in the ultracentrifugal separation of RNA to overcome the problems of co-purification of contaminating secondary metabolites.     

Inferring relatedness and heritability using molecular markers in radiata pine

A set of eight unlinked microsatellite markers was used to estimate relatedness among 355 individuals of a Pinus radiata breeding population. The average performance of open-pollinated progeny of each individual, for wood density, was considered to represent the phenotype of all 355 individuals. Marker-based estimates of relationship were compared with the pedigree-based coefficients of relationships. The phenotypic similarity among all pairs of individuals was regressed on marker-estimated relatedness to estimate the inheritance of wood density. The marker-based estimate of heritability was compared with that obtained using classical quantitative genetic methods. Overall, a low correlation (0.13) was observed between marker-based and pedigree-based estimates of relatedness. After discarding negative estimates of relatedness, the average coefficient of relationship among known groups of maternal half-sibs, full-sibs and unrelated individuals, increased from 0.24 to 0.29 (0.25 expected), from 0.43 to 0.48 (0.50 expected) and from –0.04 to 0.15 (0 expected), respectively. Marker-based and conventional estimates of heritability of wood density were 0.79 and 0.38, respectively. However, by using only marker loci with expected Hardy–Weinberg frequencies, marker-based estimate of heritability was 0.33, which is very similar to that obtained from conventional approaches. The use of molecular markers to understand quantitative genetic variation is discussed.     

The origin and genetic diversity of Pinus radiata in Australia

Summary Despite the fact that forest trees are in early stages of domestication there has been little direct evaluation of either the origin of, or genetic diversity within the breeding material in tree improvement programs. Allozyme variation was used to compare the total genetic diversity in the breeding programs of P. radiata within Australia and the five wild populations in North America. The current breeding populations were very similar genetically and were essentially homogenous with only 1.8% of the variation among programs. The total genetic diversity in the species was 0.12, which is a low estimate compared to most conifers. Overall in the Australian material the genetic diversity was somewhat less. The comparison of allelic frequencies in the five native populations with the Australian material indicates that the Monterey and Año Nuevo populations were probably the major source of the original introductions and that a substantial portion of the genetic diversity in the two populations has been captured in current breeding programs. The three southern populations do not appear to be currently represented in the breeding programs. The implications for future breeding strategies are discussed.     

Relationships between stem structure and bending strength in Pinus radiata seedlings

Summary Seedlings from nine families of Pinus radiata were grown in a glasshouse under conditions of high and low nitrate nitrogen availability to investigate effects on anatomical and strength characteristics of stems. Families were classified into groups dependent upon their previously determined susceptibility to stem deformation prevalent in plantations established on fertile ex-pasture. Nitrogen treatments significantly affected seedling form in terms of both branch production and stem slenderness. The high N treatment resulted in shorter seedlings, a proportion of which were obviously stunted. Stem strength of seedlings, physically supported throughout the experiment, was assessed as stem lean at harvest as well as the bending strength of the fresh stem at 50% stem height. These two variables were found not to be correlated. Stem lean at harvest was greatest in families known to be susceptible to stem deformation. These families produced stems that were also more slender than families of low susceptibility. Increased stem lean was associated mostly with increased stem slenderness while elasticity was more influenced by pith diameter, stem density and wood radius.     

Strength and structure of stems from fast grown Pinus radiata

Summary Segments of living stems from 2-year-old, fast growing Pinus radiata, established on a fertile ex-pasture, were examined in terms of their bending strength parallel to the grain. Stem anatomical and structural variables were examined to identify variables that could explain variation in bending strength. Half of the seedlings were physically supported to minimise the confounding effects of compression wood production in response to environmental stresses. Differences between seedlings and cuttings were examined. Variation in microfibril angle and lignin content was sufficient to account for 81% of the variation in bending strength. A positive correlation between both of these variables and elasticity was evident. Few differences were found to be caused by the support treatment or between seedlings and cuttings. The support treatment resulted in significantly fewer branches in the top most whorl, while cuttings had less branches in this whorl than seedlings.     

Identification of an agar constituent responsible for hydric control in micropropagation of radiata pine

Tissue cultured Pinus radiata grown on media containing agar as the gelling agent display toxic symptoms and poor long-term shoot survival, however it does have the attribute of hydric control, through a mechanism which, until now, has not been elucidated. Gelrite as an alternative gelling agent is clearly non-toxic but results in hyperhydric (vitrified) tissues. In an effort to overcome these problems, the controlling mechanism found in agar was examined. Hydric control was shown to be effected by a non-gelling, cold-water soluble constituent of a commercial agar, rather than by physical properties of the gel. It could be separated from low molecular weight components of the agar responsible for the toxic symptoms by dialysis. It was identified as being an agaroid-type xylogalactan bearing pyruvate and sulphate substituents. Improved management of gelling agents in culture medium has contributed substantially to a thirty fold increase in propagation rates.     

Cytological characterization of heterochromatin and rDNA inPinus radiata andP. taeda

Fluorochrome C-banding ofPinus radiata andP. taeda metaphase chromosomes showed many pericentromeric DAPI bands and interstitial CMA bands inP. radiata, and centromeric and interstitial CMA bands inP. taeda. Giemsa C-band patterns differed between the species with centromeric bands inP. radiata but no consistent bands inP. taeda. A karyotype ofP. radiata was developed based on banding patterns that distinguished all but two of the 12 pairs of chromosomes. In situ hybridization (ISH) using probes for high-copy ribosomal DNA (rDNA) showed 10 pairs of 18S–25S sites and two pairs of 5S sites in both species. Most of the sites were interstitial or centromeric.     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.