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.     

Optimized conditions for rapd analysis inPinus radiata

Summary Pinus radiata is the most important softwood plantation species in Australia and New Zealand. The improtance of this species in forestry has led to an increasing demand to improve the efficiency of selection time of the production population, which currently takes 13 yr by traditional methods. With the application of molecular biology techniques such as random amplified polymorphic DNA (RAPD) the selection period can be reduced to 6 yr. In this study, the conditions for RAPD were optimized and the feasibility of this marker system was investigated with different families ofPinus radiata from Tasmania and South Australia. Best concentrations of Taq-polymerase (1 U), magnesium chloride (2 mM), and template DNA (20 ng) were selected to test different polymerase chain reaction (PCR) thermocycler profiles. Devey's et al. (1996) program was the most effective for production of clear RAPD bands. Best conditions were investigated to screen 10–12 bp arbitrary Breasatec and Operon primers. Both types were found useful at detecting genetic variation between families. Seventy percent and thirty percent of the selected Bresatec and Operon primers, respectively, produced polymorphic bands.     

Stem deformity inPinus radiata plantations in south-eastern Australia:

Visual symptoms of stem deformity similar to those of Cu deficiency are common inP. radiata established on fertile sites previously used for agricultural production in south-eastern Australia. In this study, Cu fertiliser was applied at rates of 0, 2, 5, 10, 20 and 50 kg ha⁻¹ to deformedP. radiata at ages 3 and 6 years. Available soil Cu and contents of Cu in the foliage increased significantly only in the younger plantation. Cu fertiliser did not affect growth nor did it improve stem form. Levels of N, Cu and Cu/N ratios in foliage of straight and deformed trees were similar. However, contents of Cu in apical buds were significantly lower in deformed trees. It was concluded that stem deformity inP. radiata as observed on these fertile pasture sites, cannot be corrected by application of Cu fertiliser. Differences in Cu levels in apical buds of straight and deformed trees suggest that Cu may still be involved in this syndrome. There was also no indication of other nutrient deficiencies that could be associated with the deformity.     

Interpretation of nutrient concentrations inPinus radiata foliage at Belanglo state forest

Foliage from six youngPinus radiata trees on a phosphorus limited site were chemically analysed annually for ten years. Nutrient differences were found between trees and between years. The pre-sampling period rainfall (last month of summer plus all autumn) accounted for 68% of variance of annual phosphorus concentrations and pre-sampling rainfall and age together accounted for 83%. The figures for calcium and nitrogen were 65% and 56% respectively. Testing the relationships in older stands of the same forest indicated age was only critical in stands which were less than about 16 years of age. When the information was used in conjunction with a site survey which showed Site Index to be related to phosphorus status, it was shown that the required concentrations of phosphorus needed to be modified according to rainfall. These relationships are discussed in relation to the use of foliage analysis for determining fertilizer requirements.     

Molecular control of early cone development inPinus radiata

Summary A family of genes expressed during early stages of shoot development were isolated fromPinus radiata. A homologue of theLEAFY/FLORICAULA flower meristem-identity genes,NEEDLY (NLY), and three MADS-box genes,PrMADS1, PrMADS2 andPrMADS3 (Pinus radiata MADS-box genes), were expressed at early stages of initiation and differentiation of reproductive (male and female) cone buds, as well as vegetative buds. Expression ofNLY in transgenicArabidopsis thaliana promoted floral fate, demonstrating that it encodes a functional ortholog of theFLORICAUL A/LEAFY genes of angiosperms.Abbreviations DSB dwarf shoot bud - LSTB long-shoot terminal bud - PCB pollen cone bud - SCB seed cone bud - LD long day - SD short day     

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.     

The 5S RNA genes inPinus radiata and the spacer region as a probe for relationships betweenPinus species

The 5S RNA genes inPinus radiata occur in two size classes of about 850 and 525 bp in length. Representatives from both the long and short size classes were cloned and sequenced. The primary difference in the two size classes was shown to be a 330 bp insertion in the spacer region of the long units. Within an individual breeding clone ofP. radiata there was some sequence heterogeneity between representatives of the short class. The 5S RNA genes in thirty pine species were characterised using either a clone of the short size class or a subclone of the 330 bp insertion characterizing the long size class. Eleven species of subg.Strobus were examined and all lacked the long type of unit of radiata pine. The New World species of subg.Pinus all had both short and long units whereas the Old World species had long units. The implications of these results for the evolution of the 5S DNA sequences and the phylogenetic relationships withinPinus are discussed.     

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.     

Copper deficiency inPinus radiata planted in a podzol in Victoria,Australia

Summary Copper deficiency inPinus radiata in Gippsland, Victoria was found to be associated with acid, organic-rich sandy podzols. A trial designed to find a Cu supplement for P and NPK fertilisation on these soils showed significant growth in stem-length following Cu-solution treatment in the nursery, and Cu fertiliser applied in the field up to at least 13.5 kg Cu ha^–1.The Steenbjerg effect was evident in foliar concentrations of nutrients following fertilisation; this makes diagnosis from foliar analysis difficult. Foliar CuN ratios proved a more sensitive indicator of Cu fertiliser treatment than either Cu or N alone.Two phases of seasonal drainage and podzol development were identified in the experiment. Trees growing on the better-drained, more acid soil with a high content of organic matter responded better to Cu fertiliser.     

Cytological and molecular characterization of oat x maize partial hybrids

In cereals, interspecific and intergeneric hybridizations (wide crosses) which yield karyotypically stable hybrid plants have been used as starting points to widen the genetic base of a crop and to construct stocks for genetic analysis. Also, uniparental genome elimination in karyotypically unstable hybrids has been utilized for cereal haploid production. We have crossed hexaploid oat (2n=6x=42, Avena sativa L.) and maize (2n=2x=20, Zea mays L.) and recovered 90 progenies through embryo rescue. Fifty-two plants (58%) produced from oatxmaize hybridization were oat haploids (2n=3x=21) following maize chromosome elimination. Twenty-eight plants (31%) were found to be stable partial hybrids with 1–4 maize chromosomes in addition to a haploid set of 21 oat chromosomes (2n=21+1 to 2n=21+4). Ten of the ninety plants produced were found to be apparent chromosomal chimeras, where some tissues in a given plant contained maize chromosomes while other tissues did not, or else different tissues contained a different number of maize chromosomes. DNA restriction fragment length polymorphisms (RFLPs) were used to identify the maize chromosome(s) present in the various oat-maize progenies. Maize chromosomes 2, 3, 4, 5, 6, 7, 8, and 9 were detected in partial hybrids and chromosomal chimeras. Maize chromosomes 1 and 10 were not detected in the plants analyzed to-date. Furthermore, partial self-fertility, which is common in oat haploids, was also observed in some oat-maize hybrids. Upon selfing, partial hybrids with one or two maize chromosomes showed nearly complete transmission of the maize chromosome to give self-fertile maize-chromosome-addition oat plants. Fertile lines were recovered that contained an added maize chromosome or chromosome pair representing six of the ten maize chromosomes. Four independently derived disomic maize chromosome addition lines contained chromosome 4, one line carried chromosome 7, two lines had chromosome 9, one had chromosome 2, and one had chromosome 3. One maize chromosome-8 monosomic addition line was also identified. We also identified a double disomic addition line containing both maize chromosomes 4 and 7. This constitutes the first report of the production of karyotypically stable partial hybrids involving highly unrelated species from two subfamilies of the Gramineae (Pooideae — oat, and Panicoideae — maize) and the subsequent recovery of fertile oat-maize chromosome addition lines. These represent novel material for gene/ marker mapping, maize chromosome manipulation, the study of maize gene expression in oat, and the transfer of maize DNA, genes, or active transposons to oat.Joint contribution of the Minnesota Agricultural Experiment Station and USDA-ARS. Scientific journal series paper No. 21 859 of the Minnesota Agricultural Experiment Station. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS or the University of Minnesota and does not imply approval over other products that also may be suitable     

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.     

Characterization of diploid,tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA₃ (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x=17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.     

Chromosomal sex determination and heterochromatin structure in date palm

In the date palm, a dioecious mode (separate male and female individuals) and the late initial reproductive age (5–10 years) are major practical constraints for genetic improvement. Early selection on young seedlings could enhance breeding programmes and generate experimental male and female genetic stocks, but no cytogenetic protocol exists for sex determination in an immature date palm. Here we describe a cytological method based on chromomycin staining which demonstrates the occurrence of sexual chromosomes carrying distinctive nucleolar heterochromatin and thus offers, for the first time, the possibility of identifying male and female individuals by simple analysis of root meristems. This observation has been extended by in situ rDNA hybridization, confocal microscopy and dual-label flow cytometry of nuclei.     

Cytogenetic characterization of six species of flatfishes with comments to karyotype differentiation patterns in Pleuronectiformes (Teleostei)

The karyotypes and cytogenetic characteristics of flatfishes species Paralichthys orbignyanus , Paralichthys patagonicus , Citarichthys spilopterus and Etropus crossotus (Paralichthyidae), Bothus ocellatus (Bothidae) and Symphurus tessellatus (Cynoglossidae) were investigated by conventional [Giemsa staining, C-banding, Ag- and chromomycin (CMA₃)-stainings] and molecular [ in situ hybridization (ISH)] cytogenetic techniques. The results showed 2n = 46 and FN = 48 (2msm + 46sta) in P. orbignyanus , 2n = 46 and FN = 46 (46sta) in P. patagonicus , 2n = 26 and FN = 44 (18msm + 8sta) in C. spilopterus , 2n = 38 and FN = 64 (26msm + 12sta) in E. crossotus , 2n = 32 and FN = 50 (18msm + 14sta) in B. ocellatus , and 2n = 46 and FN = 62 (46msm + 62sta) in S. tessellatus . All species exhibited weak C-band positive segments in terminal and centromeric positions of some chromosome pairs. Silver staining of the nucleolus organizer regions (Ag-NOR) technique showed a single Ag-NOR-bearing chromosome pair in all species except E. crossotus . All these sites were CMA₃ positive and showed clear ISH signals after probing with a 18S rRNA probe. Etropus crossotus presented until seven chromosomes with Ag-NORs and CMA₃ positively stained segments in five chromosome pairs. Conversely only one chromosome pair was identified with the ISH experiments in this species. The available results show that the fishes of the order Pleuronectiformes experienced a marked chromosome evolution that included reduction in diploid number, mainly due to Robertsonian rearrangements, and several chromosome inversions.     

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.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

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     

Seed source variation in 32P uptake in inus taeda L. seedlings: A possible regulation by efflux

Short-term ³²P uptake experiments were conducted with intact seedlings of loblolly pine (Pinus taeda L.) to examine possible seed source variation in net accumulation of ³²P in roots and shoots, and in rates of unidirectional influx. Seed source had a highly significant effect on biomass and P concentrations of shoots and roots. Seedlings from two seed sources representing fast-growing populations (a broadly-adapted and wet-site seed source) accumulated over 60% more total seedling P than smaller seedlings from a drought-hardy seed source, reflecting higher biomass and root P concentrations. Rates of unidirectional ³²P influx in seedlings from the drought-hardy seed source were more than twice the rates of the seedlings from the broadly-adapted seed source. However, after 24 h in labeled uptake solution, net accumulation of ³²P was similar, suggesting that rates of unidirectional efflux from roots of the drought-hardy seed source were also high. Although there were no significant differences in biomass and tissue P concentrations between the two fast-growing seed sources, rates of unidirectional influx in seedlings from the broadly-adapted seed source were 42% lower than rates in seedlings from the wet-site source. Yet, after 24 h in labeled uptake solution, net accumulation of ³²P in seedlings from the broadly-adapted seed source was 50% higher. Unidirectional efflux out of the root may regulate net uptake of P as much, if not more, than influx in loblolly pine seedlings-at least under high-P growth conditions. The results in this study do not support previous studies with herbaceous plants suggesting that fast-growing species typically exhibit higher rates of nutrient uptake than slow-growing species.     

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.