Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

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.     

Biochemical genetics of a 7S globulin-like protein from Pinus pinaster seed

The megagametophytes of seeds of Pinus pinaster Ait. contain two types of oligomeric globulins of approximately 175 and 190 kDa that are comprised of 47-kDa and 27- and 22-kDa, monomers, respectively, joined by weak interactions. The 27- and 22-kDa components were purified and their N-terminal sequences determined. Both polypeptides were inherited as if they were coded by a single unit of recombination. The results obtained suggest that these two polypeptides originate from a single protein that undergoes proteolytic processing. The characteristics of this P. pinaster globulin indicate that it is a member of the 7S globulin family of seed storage proteins.     

Gene silencing studies in the gymnosperm species Pinus radiata

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.     

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.     

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.     

Effect of inbreeding on production of filled seed in Pinus radiata — experimental results and a model of gene action

Summary Pinus radiata D. Don was inbred to different degrees, commencing with a founder population of 8 clones. Yield of filled seed was determined for each mating type. Mean yields (%), relative to outcrossing, were: half-sib 102; full-sib 97; S₁ 43; S₂ 42. An epistatic model was developed to predict the empty seed yield following selfing, assuming that homozygosity for several recessive co-lethals is necessary to kill an embryo. Calculations were then extended to predict the yields following different degrees of inbreeding. The proposed model gave a better fit to present results, and to other published results of similar investigations, than did an alternative based on action of independent recessive lethals. Implications for breeding and seed production strategy 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.     

Lime-induced chlorosis inPinus radiata

Summary Addition of marl (CaCO₃) and/or manure to an acid soil in pots caused lime-induced chlorosis inPinus radiata seedlings, especially after excessive irrigation for 15 days. Chlorotic symptoms and their intensity were found to be related more to soil moisture and to the HCO₃ ^– concentrations, than to percentage of free CaCO₃, in the soil mixtures.Comparative chemical analysis showed lower total Fe and Mn concentrations and higher concentrations of cations and organic anions in the needles of seedlings with chlorotic symptoms than in the needles of healthy ones.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

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     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variations in response to induced stem bending in seedlings of Pinus radiata

Summary The occurrence of stem deformation in Pinus radiata plantations has been examined previously in terms of the factors leading to the onset of bending symptoms. In this study the influence of seedling family and nitrogen availability on the ability of seedlings to recover from deformation was investigated under glasshouse conditions. Seedlings from four families ranging from resistant to susceptible to stem deformation were used. Stem deformation was induced by loading stems with a known weight and comparing bending moments and rates of recovery. Significant differences between families were found with more susceptible families exhibiting greater initial magnitudes of stem lean. Similarly these families also exhibited greater rates of apical elongation following bending despite the larger stem leans. This resulted in more exaggerated stem bends as a consequence of the recovery of the lower stem pushing the upper stem away from the vertical. The strength characteristics of the stems could not explain differences between families. Susceptibility to stem deformation was explained more by stem slenderness expressed as stem height to root collar diameter.     

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.     

Alterations in endogenous levels of cytokinins following grafting of Pinus radiata support ratio of cytokinins as an index of ageing and vigour

Markers for ageing or maturation in woody plants provides the possibility of identifying genotypes with a prolonged juvenile phase, or to evaluate reinvigoration procedures, such as grafting of adult material. This study focuses specifically on cytokinins (Cks) as markers, due to their role in the transition process from juvenile to adult states. Previous reports from our group disclosed that the ratio of isopentenyladenine-type (iP-type) to zeatin-type (Z-type) Cks decreases during tree maturation, a maturation and ageing index being likely in Pinus radiata. Based on these results, Cks were tested in terminal buds of adult and grafted adult P. radiata material to corroborate the importance of the aforementioned balance as an index in the reinvigoration process of adult trees associated with grafting procedures. Results revealed the reversion of this index pattern parallel to the reactivation of some juvenile traits, namely, increasing values of the ratio throughout the reinvigoration process. According to previous results, however, the opposite was the case during the reverse process. This fact allowed us to validate the iP-type/Z-type Ck ratio as an ageing and vigour index.     

Effects of aluminium on growth and cation uptake in seedlings of Eucalyptus mannifera and Pinus radiata

This experiment was designed to examine the effects of aluminium (Al) on the growth of Pinus radiata (D. Don) and Eucalyptus mannifera subsp. mannifera (Mudie) seedlings in culture solutions in a glasshouse to help explain the failure of radiata pine trees on some acid, low fertility soils in Australia on which the native eucalypts flourish. Aluminium (Al) in culture solution increased the growth of roots and shoots of seedlings of both species but while growth of the eucalypt continued to increase with increases in Al to 2.222 μM, growth of the pine was largest at 370 μM Al. In addition to total root length, specific root length (length per unit dry weight), a measure of fineness of the root, increased in the eucalypt seedlings as the substrate Al increased. Growth of the shoots and roots of the pine in the absence of any added Al was extremely poor suggesting that Al, in low concentrations, may be an essential element or ameliorate some other factors in solution culture at low pH. Root and shoot concentrations of K increased with increasing Al, whilst Ca and Mg Concentrations decreased and Mn concentrations were unaffected in both species. Tissue Ca and Mg concentrations were 2 to 3 times higher in the eucalypt seedlings than the pine at all levels of added Al due to greater uptake of these elements by the eucalypt. In contrast, at the highest concentration of Al in the medium, shoot Al concentrations were lower in the cucalypt than in the pine due to a greater proportion of Al being retained in the eucalypt roots. These differences between the seedlings in terms of root growth and tissue cation concentrations may help explain the ability of eucalypt species to maintain vigorous growth on acid soils high in Al and low in Ca and P, where growth of the pines failed.     

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.     

Biodegradation of Pinus radiata softwood by white- and brown-rot fungi

The weight and component losses of Pinus radiata wood after decay by six species of white-rot and two species of brown-rot fungi for periods varying from 30 to 360 days were evaluated. Three groups of decayed wood samples were identified based on the principal component analysis (PCA) of the data on their weight and component losses. Selective lignin degradation was produced by Ceriporiopsis subvermispora and Punctularia atropurpurascens within different periods, the longest one lasting 90 days, and also by Merulius tremellosus after 90 days of biodegradation. Comparing the data on biodegradation of P. radiata by Trametes versicolor with the ones reported for biodegradation of Eucalyptus globulus and E. grandis indicated that P. radiata is as susceptible to wood decay by this white-rot fungus as the two types of hardwood.     

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.