Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Heritability of juvenile characters of white spruce (Picea glauca (Moench.) Voss.) in central Newfoundland,Canada

Summary The paper presents results of a growth chamber progeny test of selected phenotypically superior trees from two white spruce (Picea glauca (Moench.) Voss.) populations in central Newfoundland, Canada. On the basis of heritability of 11 juvenile characters the superior trees have been demonstrated to be suitable as a base population for continued advanced generation breeding. Family selection would produce good genetic gains in the first generation but these can be enhanced considerably by selection of best individuals within the best families for clonal propagation in the second generation or propagation by seed in the second and subsequent generations.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

The effectiveness of various nitrogen sources in white spruce [Picea glauca (Moench) Voss] somatic embryogenesis

The effects of glutamine-based dipeptides, glutamine and casein hydrolysate, as well as the deletion of organic nitrogen, were investigated during white spruce [Picea glauca (Moench) Voss] somatic embryogenesis. There were no differences in the fresh weight increase of the tissue masses grown on initiation medium with different combinations of organic nitrogen. This was also the case for subsequent growth on kinetin medium, except that glutamine alone produced a significantly lower fresh weight increase than the other organic nitrogen combinations. Without organic (i.e. with only inorganic) nitrogen in the medium, the fresh weight increase was significantly less than with organic nitrogen on both initiation and kinetin medium. No differences were found between the dry/fresh weight ratios obtained with the various nitrogen treatments. The number of mature embryos produced per gram fresh weight when cultured in the absence of organic nitrogen was significantly higher than that obtained in its presence. There were no differences in the total number of mature embryos produced in cultures grown with various organic nitrogen combinations or without organic nitrogen. There were large clone differences with respect to the number of mature somatic embryos per gram tissue and the total number of somatic embryos produced. Hence, nitrogen type influences culture growth rate but not the number of mature somatic embryos produced. The latter was clone dependent.Abbreviations BA 6-benzylaminopurine - CH casein hydrolysate     

Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds

The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca²⁺, Mg²⁺ and Mn²⁺; only the nucleases active at neutral pH were inhibited by Zn²⁺. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.     

Ascorbic acid improves conversion of white spruce somatic embryos

Summary The effects of exogenous applications of ascorbic acid on white spruce somatic embryogenesis were examined. Increasing concentrations of ascorbate (1 μM to 100 μM) in the germination medium enhanced somatic embryo conversion in a linear fashion. At the optimal ascorbate level (100 μM) the number of embryos able to undergo normal conversion, i.e., emergence of both root and shoot, increased from 34% (control) to 58%. The effect of ascorbate had a more pronounced effect on shoot growth than on root emergence; and at 100 μM ascorbate, the percentage of embryos able to produce new leaf primordia increased from 47% (control) to 79%. Root emergence increased slightly from 64% in the control embryos to 74% in the presence of ascorbic acid. The ascorbate-treated embryos were characterized by an enlarged apical region, presumably due to a larger number of leaf primordia produced, and by dark green leaves. When allowed to grow further, these embryos were able to develop into normal plantlets.     

The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

A comparison of BA,zeatin and thidiazuron for adventitious bud formation from Picea glauca embryos and epicotyl explants

Picea glauca (white spruce) zygotic embryos and one-week-old-seedling epicotyl explants were placed on either Woody Plant Medium (WPM) or half-strength Schenk & Hildebrandt (1/2S&H) medium supplemented with varying levels of benzyladenine (BA) (0.1, 1.0, 10, 50, 100 M), zeatin (10, 50, 100 M) or thidiazuron (TDZ) (0.01, 0.1 M). In addition to differences in the number of buds induced at three months on the two media, buds induced on WPM were visually more uniform, less vitrified and elongated faster. On 1/2S&H supplemented with BA, maximum bud induction from embryos occurred on 1.0 M BA with 0.01 M TDZ with higher BA concentrations inhibitory to bud induction. In contrast, on WPM there was little difference in the number of buds induced from embryos placed on 10, 50 and 100 M BA with or without TDZ. One-week-old-seedling epicotyl explants required higher BA levels on 1/2S&H, as bud induction at three months was greatest at 10 M BA. On WPM, as with the embryos, there were only minor differences in the number of buds induced from epicotyl explants on the various BA levels. Zeatin was more effective at inducing buds than BA with both media. From embryos, bud induction was greatest on 50 or 100 M zeatin without TDZ and 50 or 100 M zeatin with or without TDZ on 1/2S&H and WPM respectively. From epicotyl explants on 1/2S&H, there was little difference in the number of buds induced with the zeatin concentrations used, while with WPM, 50 and 100 M zeatin induced the greatest number of buds. Interestingly, with BA, the epicotyl explants needed a higher level than the embryos for maximal response, while with zeatin, the level was the same for both embryos and epicotyl explants. Long-term (six month) survival was higher on WPM than with 1/2S&H. Additionally, embryos had a higher percentage of genotypes surviving at six-months when compared with epicotyl explants. For overall survival and development of the buds, 50 M zeatin with 0.01 M TDZ was the best treatment tested.Abbreviations BA benzyladenine, 1/2S&H-half-strength Schenk & Hildebrandt medium - TDZ thidiazuron - WPM woody plant medium     

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

Summary Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.Abbreviations ABA ± abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - CTAB cetyldimethylethyl-ammonium bromide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid disodium salt - IBA indole-3-butyric acid - LN liquid nitrogen - PEG polyethylene glycol - SLS N-lauroyl sarcosine - Tris tris[hydroxymethylamino] methane - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cell wall changes in white spruce (Picea glauca) needles subjected to repeated drought stress

White spruce [ Picea glauca (Moench) Voss] seedlings were preconditioned by subjecting them to 3 cycles of a mild drought stress. After 1 week of stress relief their water status, soluble carbohydrate content and cell wall composition in newly formed needles were examined and compared with those in control seedlings. Both preconditioned and control seedlings were subsequently subjected to a severe drought stress and again analyzed. Preconditioning treatment both before and during subsequent stress exposure lowered osmotic potentials at full hydration, and after the loss of turgor, decreased lignin content and increased hemicellulose content of the cell walls. Severe drought had similar but more drastic effects on seedling water relations, sugar accumulation and cell wall hemicellulose content; it also decreased cell wall pectin levels. The decrease in pectin levels was accompanied by a loss of galactose and glucose from pectic substances. Little change in cellulose content was observed as a result of preconditioning and severe drought.     

Production of vigorous,desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor

Summary This report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20–50 M abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.Plant Research Centre contribution No. 1523     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Cytochrome and alternative pathway respiration in white spruce (Picea glauca) roots. Effects of growth and measurement temperature

Interactions between growth temperature and measurement temperature were examined for their effects on white spruce [ Picea glauca (Moench) Voss] root respiration. Total dark respiration rates increased with measurement temperature and were unaffected by growth temperature. Partitioning of respiratory electron flow between the cytochrome and alternative pathways was also unaffected by growth temperature. The proportion of respiration mediated by the alternative pathway was constant at measurement temperatures between 4°C and 18°C, but was increased at higher temperatures. Changes in alternative pathway activity were paralleled by changes in capacity, and the alternative pathway was almost fully engaged at all temperatures. Roots grown at low temperature displayed higher carbohydrate levels than roots grown at higher temperatures, but respiration rate was unaffected. Spruce root respiration did not appear to acclimate to growth temperature, and the alternative pathway was not preferentially engaged at low temperature.     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.