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.     

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.     

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

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.     

Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos.

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by "rescuing" structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316-319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, L-galactono-gamma-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex)

Putrescine, spermidine, and spermine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified On abscisic acid supplemented growth medium putrescine and spermidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Escherichia coli arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), following affinity purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses, Immunoassays using these antibodies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce.Abbreviations ADC arginine decarboxylase - BSA bovine serum albumin - ODC ornithine decarboxylase - PBS phosphate buffered saline - PCA perchloric acid - SDS-PAGE sodium dodecyl sulfateporyacrylamide gel electrophoresis     

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss]

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N⁶-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA N⁶-benzyladenine - cDNA complementary deoxyribonucleic acid - Em early methionine-labelled - HSP heat-shock protein - LEA late embryogenesis abundant - PEG polyethylene glycol The authors thank Mr. Terry Bethune for his assistance, and Dr. Larry Pelcher, Mr. Barry Panchuk and Mr. Don Schwab for DNA sequencing and primer synthesis. This is National Research Council of Canada publication number 38929.     

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     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Understanding changes in black (Picea mariana) and white spruce (Picea glauca) foliage biomass and leaf area characteristics

Key message

Growth conditions related to inter-tree competition greatly influence black and white spruce foliage biomass and projected leaf area characteristics.

Abstract

Foliage characteristics such as biomass and area are important among other reasons because they can be related to tree growth. Despite their economic and ecologic importance, equations to characterize foliage biomass and projected area of black (Picea mariana (Miller) BSP) and white (Picea glauca (Moench) Voss) spruces are sparse. Total foliage biomass and projected leaf area, foliage biomass and leaf area density, and relative vertical distribution of black and white spruces foliage biomass and leaf area were modelled with linear and nonlinear mixed effect models. A total of 65 white spruces and 57 black spruces were destructively sampled at four different locations in Alberta, Québec, and Ontario, Canada. Our results show that for each species, total tree foliage biomass and projected leaf area is proportional to stem diameter, total height, and crown length. The addition of crown length in the equations improved the precision of the predictions of total foliage biomass for both species and diminishes greatly the site level random effect. An increase in DBH for black spruce and in the DBH to total height ratio for white spruce skewed the relative vertical foliage biomass distribution toward the base of the living crown. According to our results, growth conditions or tree development stage influence both foliage biomass and leaf area characteristics of black and white spruces. Our results emphasize the importance of inter-tree competition on foliage biomass characteristics.     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

In white spruce ( Picea glauca [Moench.] Voss.) seeds, the raffinose family oligosaccharides (RFOs) provide carbon reserves for the early stages of germination prior to radicle protrusion. Some seedlots contain seeds that are dormant, failing to complete germination under optimal conditions. Since dormancy may be imposed through a metabolic block in reserve mobilization, the goal of this project was to identify any impediment to RFO mobilization in dormant relative to nondormant seeds. Desiccated seeds contain primarily, and in order of abundance on a molar basis, sucrose and the first 3 members of the RFOs, raffinose, stachyose and verbascose. Upon radicle protrusion at 25°C, the contents of RFOs decreased to low amounts in all seed parts, regardless of prior dormancy status and sucrose was metabolized to glucose and fructose, which increased in seed parts. During moist chilling at 4°C, RFO content initially decreased before stabilizing and then increasing. In seeds that did not complete germination, the synthesis of RFOs at 4°C favored verbascose, so that at the end of 14 (nondormant) or 35 (dormant) weeks, verbascose contents in megagametophytes exceeded the amount initially present in the desiccated seed. This was also true in the embryos of the dormant seedlot. In seed parts from both seedlots after months of moist chilling, stachyose amounts exceeded raffinose amounts. Upon radicle protrusion at 4°C, RFO contents decreased to amounts most similar to those present in seeds that completed germination at 25°C. Hence, the RFOs are utilized as a source of energy, regardless of the temperature at which white spruce seeds complete germination. Based on the similarity of sugar contents in seed parts between dormant and nondormant seeds that did not complete germination, differences in sugar metabolism are probably not the basis of dormancy in white spruce seeds.     

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.