Proteome analysis of early somatic embryogenesis in Picea glauca

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.     

Seasonal variation in the tissue water relations of Picea glauca

Seasonal variation in water relations of 3-yearold white spruce (Picea glauca (Moench) Voss) shoots, monitored with pressure-volume curves over 28 months, was closely related to shoot phenology and was sensitive to environmental fluctuations during both summer growth and winter dormancy. Turgor maintenance capacity was lowest during rapid shoot elongation from late May to early July; this was indicated by the lowest total turgor pressures, the highest (least negative) osmotic potentials at full turgor and the turgor loss point, the smallest differences between osmotic potentials at full turgor and the turgor loss point, the highest relative water contents at turgor loss and a linear decline in cell elasticity with decreasing turgor pressure. This suggests that the high susceptibility of white spruce seedlings to growth check after transplanting is largely attributable to the poor turgor maintenance capacity of this species in early summer.     

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     

Some aspects of differentiation in cells of Picea glauca cultivated in vitro

Microscopic observation of cells of Picea glauca, grown in tissue culture under a variety of cultural conditions, reveals a series of phenomena which are believed to be associated with the development of specific types of cells recognizable in intact plants. Characteristically patterned deposits of resin drops, and strands, furrows, and channels in the cytoplasm appear to be capable of serving as templates around which cellulose and lignin may later be deposited. The cytoplasm is extruded into characteristic papillae, and cell content may be extruded slowly or ejected with force through the cell walls, through multiple pores of characteristic distribution. Material ejected from such cells condenses in characteristic extracellular patterns. All of these processes may contribute to the differentiation of recognizable distinct cell types.     

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.     

Natural Variation in the Strength and Direction of Male Mating Preferences for Female Pheromones in Drosophila melanogaster

Many animal species communicate using chemical signals. In Drosophila, cuticular hydrocarbons (CHCs) are involved in species and sexual identification, and have long been thought to act as stimulatory pheromones as well. However, a previous study reported that D. melanogaster males were more attracted to females that were lacking CHCs. This surprising result is consistent with several evolutionary hypotheses but is at odds with other work demonstrating that female CHCs are attractive to males. Here, we investigated natural variation in male preferences for female pheromones using transgenic flies that cannot produce CHCs. By perfuming females with CHCs and performing mate choice tests, we found that some male genotypes prefer females with pheromones, some have no apparent preference, and at least one male genotype prefers females without pheromones. This variation provides an excellent opportunity to further investigate the mechanistic causes and evolutionary implications of divergent pheromone preferences in D. melanogaster males.     

Within and Between Species Variation in Male Mating Behaviors in the Mexican Sailfin Mollies Poecilia velifera and P. petenensis

Polymorphism in male morphology is often correlated with the expression of alternate behavioral tactics. This relationship between behavioral and morphological polymorphisms, however, is less well understood. We characterized male mating behaviors and morphological variation within and between Poecilia velifera and P. petenensis to understand mating signal evolution in the sailfin molly lineage. In addition, we examined whether differences between these species in the size range of mature males and the strength of allometry between dorsal fin size and body length could explain the variation observed in their expression of different mating behaviors. We determined each male's mating behavior profile by observing the behavior of a single male in the presence of a receptive female. We found that P. velifera showed evidence of an alternate male mating strategy, with small males generally performing only gonopodial thrusts (forced insemination attempts) towards receptive females, while large males performed courtship displays as well as gonopodial thrusts. Males of P. petenensis performed similar rates of courtship displays and gonopodial thrusts regardless of body length. Little variation existed among populations of P. velifera in mating behaviors, while males from different populations of P. petenensis showed population‐specific average rates of each mating behavior. Population differences in P. petenensis may be driven, in part, by its occurrence in more variable habitats than those occupied by P. velifera. Variation among individuals in the mating repertoire of P. velifera, but not P. petenensis, suggests that the greater range of variation in male size at maturity, as well as considerably stronger allometry between dorsal fin size and body length, may explain why males of P. velifera show the greatest degree of expression of alternate male mating behaviors when compared to other sailfin species. These results also suggest an important role of morphological polymorphisms in predicting the expression of alternate male mating behaviors.     

Postglacial range expansion and population growth of Picea mariana, Picea glauca and Pinus banksiana in the western interior of Canada

The fossil pollen records from the sediments of thirteen lakes are used to reconstruct the postglacial spread and population expansions of Picea mariana (Mill.) B.S.P., Picea glauca (Moench) Voss and Pinus banksiana Lamb. across the western interior of Canada. The objective of this study was to examine temporal and spatial patterns in the growth of the populations and to determine if intraspecific and interspecific variations exist across a range of latitudes and elevations. Pollen accumulation rates (PARs) are estimated from pollen grain counts and sedimentation rates throughout the early to mid-Holocene and are used to represent the population level at the time of pollen deposition. Exponential equations are used to calculate population growth rates. Three-dimensional diagrams (time, space, abundance) are constructed to illustrate range expansion and population growth.The rates of population growth varied from south to north for all three species. Pinus banksiana , on average, had slower population growth rates, and its expansion across the region began over 4000 years later and lasted approximately 1000 years longer than the two Picea species. All three tree taxa experienced reduced rates of population growth at northern high elevation sites. These variations are examined in light of the physical and biological environmental conditions which existed during postglacial range expansion and population growth.     

Mating and Residence Patterns of Male Patas Monkeys

A longterm study of a population of patas monkeys (Erythrocebus patas) in Kenya indicated fluid male residence patterns. While a single adult male was present in each of the two main study groups for most of each year, several males were present and mating in groups during some conception periods. Tenures of males in heterosexual groups were generally short; some males were resident in both groups and some had more than one tenure in the same group. When not in heterosexual groups, males lived as solitaries or in all-male associations.     

The influence of cambial age on breeding for wood properties in Picea glauca

We investigated the influence of cambial age on correlations between different wood traits and the possibility of early selection in order to help decision-making for the improvement of juvenile wood in white spruce (Picea glauca (Moench) Voss). Increment cores were analysed from 375 trees covering 25 open-pollinated families from a 30-year-old provenance–progeny trial in Quebec, Canada. Genetic and phenotypic correlations between different mechanical and fibre anatomy-related wood traits were found to vary with cambial age. Most correlations became stronger in magnitude in rings closer to the bark. An exception is the correlation between microfibril angle (MFA) and the modulus of elasticity where correlations were strongly negative from the pith to the bark. Age–age correlations for different wood traits were found to be high and possible gains from early selection were estimated to be good in ring 8 and older for most traits. MFA was the trait with the strongest potential for selection as early as ring 4, but a detrimental correlation with wood density may represent a drawback of such a juvenile selection approach. Estimates showed that selection concentrated on a few easily measurable traits such as wood density and core length holds promise to obtain superior genetic gains for mechanical properties, but negative impacts would be expected on fibre anatomy traits related to pulp quality. These findings show the need for more carefully planned breeding and selection strategies if one wishes to improve several traits for different end uses.     

Male Remating in Drosophila ananassae: Evidence for Interstrain Variation in Remating Time and Shorter Duration of Copulation during Second Mating

In Drosophila ananassae, male remating was studied using ten mass culture stocks which were initiated from flies collected from different geographic localities. Male remating occurs at a high frequency and varies within narrow limits (84-96 percent) in different strains. Interestingly, male remating time (in min) varies from 7.41 (Bhutan) to 21.59 (PAT) in different strains and the variation is highly significant. Further, the results also show that males copulate for shorter duration during second mating. This is the first report in the genus Drosophila which provides evidence for interstrain variations for male remating time as well as for shorter duration of copulation during second mating as compared to first mating in D. ananassae.     

Male Dominance Rank,Female Mate Choice and Male Mating and Reproductive Success in Captive Chimpanzees

Mating and consequently reproductive success in male vertebrates are predominantly determined by intermale competition and female mate choice. Their relative importance however, is still poorly understood. We investigated the interrelationship between male dominance rank — a formal indicator of male competitive ability — female mate choice, and male mating success in a multimale-multifemale group of captive chimpanzees. In addition, we examined the relationship between male dominance rank and reproductive success determined by genetic paternity analysis over a 13-yr period in the same captive population. We related the frequencies of sociosexual behaviors to the female anogenital swelling stage and female fertile phase as determined by urinary and fecal progestogen analysis. Rates of behaviors in both sexes increased with increasing intensity of female swelling, but they were not influenced by the timing of the fertile phase. Male mating success was clearly related to male dominance rank, with high-ranking males performing the overwhelming majority of copulations. This was mainly due to both rank-related rates of male soliciting behavior and intermale aggressiveness during the period of well-developed female anogenital swelling. Although females solicited copulations mainly from the high-ranking males and thus expressed a mate choice based on rank, their overall contribution in initiating copulations and thus influencing male mating success was low. The data on paternity from the population, which always contained 4 adult males, revealed that -males sired the majority (65%) of offspring. We conclude, that male dominance rank is an important determinant of male mating and reproductive success in captive (and presumably wild) chimpanzees and that female mate choice is of minor importance in modulating male reproductive outcome.     

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.     

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     

Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.)

Interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.) cotyledonary somatic embryos were encapsulated in sodium alginate. Somatic embryo viability was retained, but germination occurred at a reduced frequency compared with the equivalent zygotic embryos. The addition of 0.5% (w/v) activated charcoal to the alginate capsule significantly enhanced root development and germination for somatic embryos but not for zygotic embryos. The possibility of developing an artiflcal endosperm was also investigated, by addition of Litvay (Litvay et al. 1981) nutrients with or without 90 mM sucrose to the alginate-charcoal capsule. This treatment significantly enhanced root development for all embryo categories with the exception of black spruce somatic embryos. Encapsulated and non-encapsulated somatic embryos survived one month cold storage at 4 °C without reduction in germination frequency.NRCC No. 35895     

Male Lekking duration and Mating Benefits in a Lek-Forming Hawaiian Drosophila

Variation in lekking duration of males of a Hawaiian Drosophila, D. grimshawi, was examined in laboratory enclosures. The relationship between variation in male lekking activity and number of eggs laid by females, proportion of eggs hatching, and total offspring production was investigated. Females mating highly active males laid fewer eggs and thus had lower offspring production than females mating less active males, even though other studies have shown that D. grimshawi females prefer more active males as mates. These results are discussed in terms of conflict between males and females over mating benefits and in trade-offs faced by males between lekking duration and offspring production per mating. Testis mass was not related to lekking activity levels, but males with heavier testes produced more offspring per mating, suggesting that males may vary in other ways that correlate with possible direct fitness gains for females.     

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.