Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

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.     

Microbial Utilization of Pinus radiata Bark

A screening program using suspensions of ground bark in mineral salts media, or extracts prepared from ground bark by treating with hot water, sulfuric acid, ammonium hydroxide, or sodium hydroxide, yielded more than 200 pure cultures of fungi, yeasts, and bacteria. Only 38 of these have good growth on liquid bark media. All were filamentous fungi, although many bacteria and yeasts were among the cultures that failed to give appreciable growth. Species of Penicillium, Scopulariopsis, Aspergillus, Trichoderma, Cladosporium, and Fusarium were among the most actively growing cultures. Cell biomass yields, as measured by cell nitrogen determination, were too low for economic production of single cell protein.     

Distinct responses of growth and respiration to growth temperatures in two mangrove species

Background and AimsMangrove plants are mostly found in tropical and sub-tropical tidal flats, and their limited distribution may be related to their responses to growth temperatures. However, the mechanisms underlying these responses have not been clarified. Here, we measured the dependencies of the growth parameters and respiration rates of leaves and roots on growth temperatures in typical mangrove species.MethodsWe grew two typical species of Indo-Pacific mangroves, Bruguiera gymnorrhiza and Rhizophora stylosa, at four different temperatures (15, 20, 25 and 30 °C) by irrigating with fresh water containing nutrients, and we measured growth parameters, chemical composition, and leaf and root O₂ respiration rates. We then estimated the construction costs of leaves and roots and the respiration rates required for maintenance and growth.Key ResultsThe relative growth rates of both species increased with growth temperature due to changes in physiological parameters such as net assimilation rate and respiration rate rather than to changes in structural parameters such as leaf area ratio. Both species required a threshold temperature for growth (12.2 °C in B. gymnorrhiza and 18.1 °C in R. stylosa). At the low growth temperature, root nitrogen uptake rate was lower in R. stylosa than in B. gymnorrhiza, leading to a slower growth rate in R. stylosa. This indicates that R. stylosa is more sensitive than B. gymnorrhiza to low temperature.ConclusionsOur results suggest that the mangrove species require a certain warm temperature to ensure respiration rates sufficient for maintenance and growth, particularly in roots. The underground temperature probably limits their growth under the low-temperature condition. The lower sensitivity of B. gymnorrhiza to low temperature shows its potential to adapt to a wider habitat temperature range than R. stylosa. These growth and respiratory features may explain the distribution patterns of the two mangrove species.     

Relationship of heartwood traits with diameter growth, implications for genetic selection in Pinus radiata

The heartwood of radiata pine (Pinus radiata D. Don) is not favoured commercially because of its darker appearance, unreliable durability and difficulty to treat chemically; consequently, light-coloured sapwood, which is more easily treated with decay-resistant chemicals, is of more value. To decrease the amount of heartwood or increase sapwood cross-sectional area by genetic selection, the genetics of sapwood cross-sectional area (SWA), sapwood area percentage (SWAP) and number of heartwood rings (HWR) is of interest. We investigated genetic variation in these three variables from increment cores from trees from two trial series, each planted at two sites, from a total of some 330 families (189 were half-sib and 142 were full-sib) with 10 to 30 individuals assessed per family. Trials were assessed between 16 and 23 years old when all trees had started to develop heartwood. The three sapwood-related traits were moderately heritable across pairs of sites ( h ^ i 2 $ {\widehat{h}}_i^2 $ = 0.2 to 0.4) indicating opportunity for selection. SWA was strongly correlated (r _a ?=?0.9) with diameter. SWAP and HWR were also strongly correlated (r _a ?=??0.9). The number of HWR was independent of tree diameter, providing the opportunity through genetic selection to reduce heartwood area whilst increasing tree growth rate.     

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.     

Environmental and nutritional responses of a Pinus radiata plantation to biosolids application

Since the mid-1990s, a Pinus radiata (D. Don) plantation growing on a sandy, low fertility soil at Rabbit Island near Nelson, New Zealand received aerobically digested liquid biosolids. An experimental research trial was established on the site to investigate the effects of biosolids applications on tree growth, nutrition, soil and ground water quality. Biosolids were applied to the trial site in 1997 and 2000, at three application rates: 0 (control), 300 (standard) and 600 kg N ha⁻¹ (high). Biosolids application significantly increased tree growth. This was mainly attributed to improved N supply, demonstrated by the enhanced N concentration in the tree foliage. Soil analysis indicated that biosolids application have not caused significant changes in concentrations of most nutrients. However, biosolids treatments significantly increased the available P (Olsen P). Of the heavy metals only total Cu concentrations in the soil increased after biosolids application. Groundwater quality, which was monitored quarterly, has not been affected by biosolids application. The concentrations of nitrate and heavy metals in groundwater were well below the maximum acceptable values in drinking water standards. Biological treatment of sewage and digestion of sewage sludge resulted in the enrichment of ¹⁵N in the biosolids (δ¹⁵N values between 5.0 and 8.7‰). Such enrichment was used as a tracer to study the fate of biosolids derived N. The elevated δ¹⁵N in biosolids treated pine foliage indicated that a considerable amount N was sourced from biosolids. Analysis of δ¹⁵N in understorey plants showed that both non-legume and legume understorey plants took up N from the biosolids, and acted as a N sink, reducing N availability for leaching. Our study showed that application of biosolids to a plantation forest can significantly improve tree nutrition and site productivity without resulting in any measurable adverse effect on the receiving environment.     

Pre-treatment of Pinus radiata substrates by basidiomycetes fungi to enhance enzymatic hydrolysis

Pre-treatment is important step prior to enzymatic hydrolysis of ligno-cellulosic biomass in order to obtain renewable carbon source ca. glucose. Pinus radiata biomass including wood blocks, wood chips and steam exploded wood (SEW) were used to investigate the effect of fungal pre-treatment on glucose yield. Comparison was made using one white-rot fungus (Trametes versicolor) and three brown-rot fungi (Coniophora puteana, Antrodia xantha and Oligoporus placenta). This is the first study where SEW was treated with basidiomycetes and subsequent enzymatic hydrolysis gave 5 g glucose/l which is an order of magnitude greater compared to control biomass (0.5 g glucose/l). This enhanced glucose yield is due to the novel pre-treatment sequence used in this study.     

Fine Structure of Parenchymatous and Differentiated Pinus radiata Callus

The fine structure of Pinus radiata D. Don callus before andafter differentiation into stem-like tissues has been examinedwith the electron microscope. In callus prior to differentiation(here called parenchymatous callus) the cells accumulate tanninsas they age and are quite distinct from the cells of differentiatedcallus. In the latter, cambium, phloem and xylem cells may beidentified by their general morphology and by their ultrastructuralfeatures. Differentiation into a true stem-like structure is,however, incomplete in that the tissues are not uniformly oriented,and parenchyma cells of the rays and phloem contain chloroplasts.The tracheids also show unusual differentiation in that borderedpits form over their entire surface and may be of two types.The reasons for these variations are discussed.     

Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration

An Agrobacterium tumefaciens-mediated transformation protocol was developed for detached cotyledons of Pinus radiata zygotic embryos resulting in up to 55% of cotyledons transiently expressing the reporter gene uidA. Transient expression of uidA was improved when detached cotyledons were pre-cultured on half strength medium containing cytokinin for 7 days, wounded by vortexing and then vacuum-infiltrated in a solution of A. tumefaciens. The transformation protocol was applied both to cotyledons and also to the apical meristematic dome which was the portion of the embryo remaining after cotyledons were detached, and from which the apical shoot and axillary shoots regenerate. Molecular analysis of putatively transformed shoots regenerated either adventitiously from cotyledons or via axillary shoots from apical domes, indicated the presence of uidA and nptII genes by PCR in some of these shoots. Biochemical analysis of putatively transformed shoots using nptII ELISA indicated that they contained the nptII enzyme. However, Southern hybridisation indicated stable integration of nptII only in one shoot which was regenerated from an apical dome. Shoots regenerated from cotyledons appeared to exhibit chimeric expression and were not stably transformed. Based on a comparison of time for regeneration, technical difficulty, molecular and biochemical analysis, apical domes may be more suitable as explants for transformation and subsequent regeneration of transclones than detached cotyledons.     

Biochemical genetics of some seed proteins of Pinus radiata

In a high-salt soluble fraction of the total protein from single seeds of Pinus radiata, up to 45 polypeptides were resolved on SDS-polyacrylamide gels. At least one-fifth of these polypeptides showed variation between seeds. In the 27,000–29,000 dalton region, two polypeptides were inherited as codominant alleles at a single locus and were shown to assort independently of another seed protein locus and three allozyme loci. A survey of 120 individuals from the five known native populations of P. radiata in California detected only the 27K and 29K alleles at the locus. In all populations, the 29K allele predominated, and the two island populations were monomorphic for the 29K allele. The 27 and 29 kdalton polypeptides were shown to have very similar amino acid sequences, and the allelic difference at this locus is most probably in the gene sequence for the polypeptide.     

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.     

Seasonal variation in foliar carbon exchange in Pinus radiata and Populus deltoides: respiration acclimates fully to changes in temperature but photosynthesis does not

We investigated seasonal variation in dark respiration and photosynthesis by measuring gas exchange characteristics on Pinus radiata and Populus deltoides under field conditions each month for 1 year. The field site in the South Island of New Zealand is characterized by large day-to-day and seasonal changes in air temperature. The rate of foliar respiration at a base temperature of 10 °C ( R ₁₀) in both pine and poplar was found to be greater during autumn and winter and displayed a strong downward adjustment in warmer months. The sensitivity of instantaneous leaf respiration to a 10 °C increase in temperature ( Q ₁₀) was also greater during the winter period. The net effect of this strong acclimation was that the long-term temperature response of respiration was essentially flat over a wide range of ambient temperatures. Seasonal changes in photosynthesis were sensitive to temperature but largely independent of leaf nitrogen concentration or stomatal conductance. Over the range of day time growth temperatures (5–32 °C), we did not observe strong evidence of photosynthetic acclimation to temperature, and the long-term responses of photosynthetic parameters to ambient temperature were similar to previously published instantaneous responses. The ratio of foliar respiration to photosynthetic capacity ( R _d/ A _sat) was significantly greater in winter than in spring/summer. This indicates that there is little likelihood that respiration would be stimulated significantly in either of these species with moderate increases in temperature – in fact net carbon uptake was favoured at moderately higher temperatures. Model calculations demonstrate that failing to account for strong thermal acclimation of leaf respiration influences determinations of leaf carbon exchange significantly, especially for the evergreen conifer.     

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.