DNA and protein content relationship in the cells of mature cotyledons ofPisum sativum

Summary The relationship between the DNA and reserve protein contents have been investigated in the cells of mature cotyledons ofPisum sativum. The study was made on two seeds chosen, on preliminary analysis, as having a markedly different protein content per cotyledon. The seed with the higher protein content was of theCameor variety, the other was of theAmino variety. The DNA content per cell was measured with a scanning microspectrophotometer and the protein content per cell using morphometric methods. In both seeds, nuclei of the storage parenchyma were polyploid in a range from 8 to 64 C levels, but the average DNA content per cell was higher in the Cameor specimen. The protein content also, whether expressed per unit of protoplasm or per cell, was always higher in the Cameor seed. The greater protein content of this seed was due to a higher content of protein per cell. In both seeds the level of ploidy and the reserve protein content per cell increased in the same way from the peripheral zone to the centre of the cotyledon. A high positive correlation was found for the two seeds between the DNA content and the protein content per cell.     

Morphology and phenology of seeds and whole fruit eaten by Milne-Edwards’ sifaka,Propithecus diadema edwardsi,in Ranomafana National Park,Madagascar

Although it is an anatomical folivore, the diet of the Milne-Edwards’ sifaka (Propithecus diadema edwardsiA. Grandidier, 1871) in Ranomafana National Park contained 35% seeds, 30% whole fruit, and 28% leaves. Plant species used as seed sources differed from those used as whole fruit sources in terms of temporal variation in consumption, taxonomic affiliation, morphology, and phenology. Although seeds were destroyed in both exploitation styles used by the sifakas—seed and whole fruit-eating—the gross morphology of species used as seed sources conformed to the complex of traits typical for fruits experiencing seed predation, while species used as whole fruit sources conformed to traits typical for fruits that do not experience predispersal predation. Many of the 19 plant species from which the seed was extracted and eaten contained a single seed with moderate testa thickness, and fruits containing this type of seed were medium-sized with dry or fibrous flesh, moderate skin thickness, and a dull color. In contrast, brightly colored, juicy fruits with minimally protected seeds were characteristic of the 38 plant species from which both pericarp and seed were eaten. Compared to transectwide measures of fruit availability or patterns restricted to whole fruit sources, fewer species of seed sources produced fruit per month and fruiting activity was more seasonal.     

Susceptibility of sunflower inbred lines and putative resistance sources to Smicronyx fulvus LeConte (Coleoptera: Curculionidae)

The red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), is a primary seed-feeding pest of cultivated sunflowers, Helianthus annuus L., in North America. Host plant resistance is one tool available to complement insecticide-based management of S. fulvus. Artificial infestations of 30 adult weevils per head were used to determine whether variation for susceptibility to S. fulvus exists in previously released inbred lines, and how a new weevil-resistant line, HA 488, compares with other putative sources of resistance. Correcting for the number of seeds per head, 13 older inbred lines showed variation in per cent seed damage from 20% to 38%, with two lines (HA 412 HO, HA 821) being more damaged than most of the tested lines. Among four putative resistance sources, HA 488 was significantly less damaged (5%) than two previously identified open-pollinated varieties (PI 170424, PI 253417, with 13%–14% seed damage), while the source of the resistance in HA 488, PI 431542, was statistically intermediate (12%). The resistance available in HA 488 is a marked improvement, potentially reducing damage per weevil by two thirds or more, but additional work on genetic markers for resistance, economic thresholds and basic weevil biology (e.g. degree-day models for adult emergence) is needed to support implementation of integrated pest management for this key sunflower pest.     

Estimates of nuclear DNA content in bryophyte sperm cells: phylogenetic considerations

Nuclear DNA contents of developing sperm were estimated for 17 species of bryophytes by cytophotometry in squash preparations of antheridia after Feulgen staining. Genome sizes are in the lower end of the range for land plants. Two homwort C-values have the lowest recorded for bryophytes at 0.17 and 0.26 pg DNA per nucleus. In liverworts, C-values range from 0.49 pg in Blasia pusilla to 4.05 pg in Pellia epiphylla, while moss genome sizes are less variable, ranging from 0.38 pg in Takakia ceratophylla to 0.92 pg in Atrichum oerstedianum. DNA content is not correlated with chromosome number in these bryophytes, but sperm cell size and cellular complexity are directly related to C-value. Structural variations in the locomotory apparatus are viewed as evolutionary modifications associated with changes in genomic complexity, with a generalized increase in complexity of the motile assemblage accompanying increases in DNA content. Nuclear DNA values are not as variable in bryophytes as they are in pteridophytes and seed plants. We suggest that in plants producing biflagellated gametes, lower DNA contents afford a selective advantage. Comparisons with plants that produce multiflagellated or pollen-dispersed sperm indicate operation of a nucleotypic effect in archegoniates with biflagellated sperm. This effect may be on sperm cell functioning, which in turn influences reproductive success.     

Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains

The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as “take-all decline.” Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40–50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.     

On Further Study of Extracellular Accumulation of DNA by Hydrocarbon-utilizing Pseudomonas Species

Extracellular accumulation of high molecular weight DNA was further studied using Pseudomonas species. More efficient production was obtained by the use of glucose-grown seed culture and by controlling the broth-pH at around 6.0 for first 24 hr and then around 8.0 during the fermentation. The maximum yield was 5 to 6 g per liter of the broth culture, which corresponded to 10-fold of that reported in the previous work.

Purified DNA (4 × 10⁶ daltons) was obtained successfully by applying an aqueous biphase system of dextran-polyethyleneglycol and dextranase.

Significant release of DNA occurred only with cell lysis of H-paraffin-grown bacteria. The primary cause of rapid lysis was explained by the exhaustion of cellular glucose pool. Relation of DNA accumulation to the effect of rhamnolipids on cell membrane was also investigated.     

FERTILIZATION DYNAMICS AND PARENTAL EFFECTS UPON FRUIT DEVELOPMENT IN RAPHANUS RAPHANISTRUM: CONSEQUENCES FOR SEED SIZE VARIATION

Seed weight varies significantly within and among fruits of wild radish (Raphanus raphanistrum). To determine sources of this variation, we studied fertilization and seed development following controlled pollinations. Within fruits, central ovules were fertilized prior to distal ovules and attained greater seed size. Ninety-seven percent of the variation in mean seed wt per fruit was explained by an analysis of variance incorporating parental effects, pollination date, and the number of seeds per fruit. We document strong maternal effects on the number of ovules per ovary, the number of fertilized ovules per ovary, the number of seeds per fruit, and mean individual seed wt per fruit. Across females, pollen donor had a slight but significant effect on seed wt; no paternal effects on fertilization rate, zygote number, or seed number per fruit were detected. Within females, with one exception, pollen donor had no significant effect on these components of seed development. Stronger maternal main effects may be due to donor x recipient interactions, cytoplasmic factors, the genetic inequity within triploid endosperm, and/or strict maternal control over resource allocation. The large maternal effects relative to paternal effects may limit the rate at which natural selection acts on paternal traits expressed prior to seed maturation.     

A Molecular and Fitness Evaluation of Commercially Available versus Locally Collected Blue Lupine Lupinus perennis L. Seeds for Use in Ecosystem Restoration Efforts

Dependence on wild seed sources is often impractical for large‐scale habitat restoration programs. Reliance on commercial seed supplies of unknown provenance and fitness is thereby warranted. Little consideration has been given, however, to how the large volumes of seed required should be sourced. We evaluated commercial and locally collected seed sources for potential use in a New York State‐based, landscape‐scale program for restoring blue lupine Lupinus perennis. Through analysis of microsatellite markers we determined that “native” lupine designations by some commercial suppliers were in fact interspecific hybrids and therefore unreliable; at least two commercial sources, however, were genetically as close to native New York populations as native New York populations were to one other. Common garden experiments revealed that seed source influenced first‐year overwintering survival and subsequent height growth of surviving plants; seed sources more closely related genetically to native New York populations survived better and produced more stems per individual in the field in the area targeted for restoration. We conclude that (1) commercial suppliers often but not always offer reliably characterized seed sources of sufficient genetic similarity to native populations to warrant their use in restoration projects and (2) genetic affinity of potential seed stock to native populations is positively related to its fitness in the environment targeted for restoration.     

The reserve proteins in the cells of mature cotyledons ofLupinus albus var.Lucky

Summary The nuclear DNA content of cotyledonary cells of two lupin seeds (L₁ and L₂) with markedly different total protein content, were investigated by scanning cytophotometry. Both seeds had polyloid nuclei with DNA levels varying between 8 C and 64 C, the majority being either 16 C or 32 C. The highest DNA levels were found in the abaxial and central cotyledonary zones of both seeds; seed L₂ had a higher ploidy level than L₁. It is shown that the volume of condensed chromatin (chromocenters) increased proportionally with the DNA content of the nucleus. A comparison was made between the distribution of protein, previously determined byLe Gal andRey (1986) and the DNA throughout the cotyledon. The L₂ seed, which has the highest total protein and the highest protein content per cell, also exhibited the greatest DNA content per cell. For both seeds, the r-value for association of DNA and protein content per cell was highly significant (0.98).     

Mitochondrial DMA variation in somatic embryogenic cultures ofLarix

outhern hybridization analysis using wheat mitochondrial gene-specific probes indicates that changes in mitochondrial genomic organization and the relative representation of certain genomic regions occur during in vitro somatic embryogenic cell culture ofLarix species. We observed differences in the mitochondrial (mt)DNA hybridization patterns between somatic embryogenic cell cultures and trees grown from seed forLarix leptolepis,L. decidua, and the reciprocal hybrids of these twoLarix species. This is the first study to describe the correlation of molecular changes in a gymnosperm mitochondrial genome with in vitro somatic embryogenic cell culture. Quantitative differences in mtDNA hybridization signals were also observed among a 4-year-old somatic embryogenic cell culture ofLarix ×eurolepis trees regenerated from this culture, and the seed source tree from which the somatic embryogenic cell cultures were initiated.     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

PROXIMATE AND ULTIMATE SOURCES OF WITHIN-INDIVIDUAL VARIATION IN SEED MASS IN PRUNELLA VULGARIS (LAMIACEAE)

Substantial variation in seed mass within individual seed parents is at odds with predictions of models for the evolution of optimum offspring size and with empirical observations of directional selection on seed mass. To elucidate the ultimate causes of this variation, I examined several proximal sources of within-individual variation in seed mass in the perennial herb Prunella vulgaris. Position of inflorescence, position of flower within an inflorescence, date of anthesis, and number of seeds produced per flower all explained some within-individual variation in seed mass. Hand pollination in the field failed to reveal any effect of pollen source (self pollen or outcross pollen) on seed mass. My results, in conjunction with those from studies of selection on seed mass in P. vulgaris, do not support hypotheses that within-individual variation in seed mass is favored by the pattern of natural selection on seed mass. Rather, the results suggest that seed parents are not capable of producing a uniform seed crop in the face of changes in resource availability in the course of a season. The inability to produce a uniform seed crop may persist because of selection for variability in traits correlated with seed mass or because of a true constraint on the evolution of uniform offspring size.     

Epiplastome variation of the number of chloroplasts in stomata guard cells of sugar beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations, which is typical of inbred plants. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage ("cell hereditary memory") and successive sporophyte generations.     

Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase‐like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events.     

EFFECT OF TEMPERATURE AND PHOTOPERIOD ON GROWTH AND DORMANCY OF BETULA PAPYRIFERA

The effects of day/night temperatures and photoperiod on the growth and dormancy of paper birch (Betula papyrifera) were studied in seedlings from different geographic origins. The response of Alaskan plants to temperature and photoperiod was distinctly different from other seed sources. Alaskan plants required very long days to prevent cessation of growth while plants from southern seed sources grew on photoperiods as short as 14 hr. Low night temperature (14 C) antagonized the promotive action of long photoperiods in Alaskan plants but had little effect in other seed sources. High day temperatures offset the inhibitory effect of the cool night to a lesser degree in Alaskan plants than in plants from other locations. Dormancy induced by short photoperiods was antagonized (relieved ?) to a lesser degree by high night temperatures in Alaskan birch than in other seed sources. Betula papyrifera var. humilis from Alaska may be an incipient species since its morphological traits are accompanied by adaptive physiological responses to its environment. These responses are as distinct as its morphological characteristics.     

<Emphasis Type="Italic">In Vitro</Emphasis> and cellular responses of jack pine embryos to three imbibition parameters

Summary Eighteen imbibition treatments with differing parameters of light conditions, temperature and duration were applied to jack pine seeds. After imbibition, embryos were excised and cultured in a liquid medium for 4 wk under continous agitation. At the end of the culture period, the embryos were classified according to five categories: nodule-forming, callus-forming, nodule plus callus-forming, white (non-responsive), and necrotic. Our results showed variation in the in vitro responsiveness of the embryos for the three parameters of imbibition and interactions among them. Three statistical models were tested to analyze the data on nodule formation, and two of them showed that the interaction between time and temperature, and between time and the combined variable temperature/light, were significant, while the light and duration variables had a significant effect only as single factors. The different morphogenic responses observed might indicate specific metabolic requirements of the embryos for the different developmental pathways. Afterwards, nine imbibition treatments were selected to evaluate the effects of the three parameters on the cell cycle and absolute DNA amount per nucleus. Following imbibition the number of cells in the G0–G1 phase decreased compared to the cells in dry seed, while the number of cells increased in the G2 phase after all the treatments except one. The percentage of cells in the different cell cycle phases varied significantly among the treatments. DNA amount fluctuated from 35.5 to 40.37 pg per nucleus. Compared to dry seeds with 40.19 pg DNA per nucleus, embryos from four imbibition treatments showed a pronounced decrease in the DNA by 5.9–11.8%. This might indicate underreplication of DNA sequences and reflect DNA plasticity with regards to imbibition and environmental factors.     

Seed bank versus seed rain in the regeneration of a tropical pioneer tree

Summary We used the tropical pioneer tree, Cecropia obtusifolia to evaluate the relative importance of different sources of seeds in the regeneration of species that depend on ephemeral sites. We studied seed production in a population established in a 5 ha plot, and dispersal, dormancy and seed predation in two recent treefall gaps (<1 year-old), two building or successional forest patches (10–15 since disturbed), and two mature forest patches (>35 years since disturbed) for a one year period at Los Tuxtlas (Mexico). Flowers and fruits were counted at monthly intervals. Annual fecundity per tree ranged from 1.4×10⁴ to 1.4×10⁷ seeds. Seeds were continuously available on the trees and on the ground. Average annual seed rain per m² (as measured by 0.5×0.5 m seed traps) varied from 184 to 1925 among the six sites. Distance to nearest seed source and patch type explained more than 60% of the seed rain variation among sites. Soil seed density, estimated by counting seeds from ten samples (78.5 cm²×10 cm deep) collected from each site in October and January, ranged among the six sites from 269 to 4485 seeds per m² in January and from 204 to 5073 in October. Soil seed viabilities were much lower (17.1% in October and 5.1% in January) than those of rain seeds (48.26%). Annual survivorships of 2.2% were estimated for seeds artificially sown on the soil surface of a gap and a mature patch, and 3.75% in a building patch. In two other experiments seed removal rates ranged from 27% to 98% in 4 days. Removal rates were significantly higher in gap and mature patches than in building patches. Ants (Paratrechina vividula) and grasshopper nymphs (Hygronemobius. sp.) were the main predators. We draw three main conclusions from our data: (1) Pathogens and predators determine low survivorship of C. obtusifolia's seeds in the soil and a rapid turnover rate (1.07 to 1.02 years) of its seed bank; (2) a continuous and copious seed production and an abundant and extensive seed rain replenish the soil seed pool in patches with different disturbance ages at least up to 86 m from nearest source; (3) more than 90% of the seeds contributing to C. obtusifolia seedling recruitment in gaps are less than one year-old. We discuss our results in the context of previous similar studies for tropical forests.     

Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR

Alu element-mediated polymerase chain reaction is a strategy for rapidly cloning and mapping human DNA markers from mixed DNA sources. A novel primer homologous to the 3′ end of the human Alu repeat element provides the basis for preferential synthesis of human DNA fragments from human/rodent somatic cell hybrid DNA template. This approach has been used to isolate a series of new markers from chromosome 10. The Alu element-mediated PCR probes were regionally assigned on chromosome 10 by hybridization to Southern blots of Alu PCR-synthesized DNA derived from somatic cell hybrid template DNA. Alu element-mediated PCR is generally applicable and makes possible the analysis of complex genomes with a speed and sensitivity that has not been previously possible.     

Epiplastome Variation of the Number of Chloroplasts in Stomata Guard Cells of Sugar Beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage (cell hereditary memory) and successive sporophyte generations.     

Genetic comparisons between seed bank and <Emphasis Type="Italic">Stipa krylovii</Emphasis> plant populations

The soil seed bank represents the potential plant population since it is the source for population replacement. The genetic structure of a Stipa kryiovii (Roshev.) plant population and its soil seed bank was investigated in the Xilinguole Steppe of Inner Mongolia using random amplified polymorphic DNA (RAPD) analyses. The population was sampled at two sites that were in close proximity to each other (0.5 km apart). Thirty plants and 18 seed bank samples were taken from each site to determine the genetic diversity between sites and between sources (plant or seed). The material was analyzed using 13 primers to produce 92 loci. Eighty-six were multi-loci, of which 23 loci (26.74%) of allele frequencies showed significant differences (P ≤ 0.05). The genetic similarity between two seed bank sites was 0.9843 while the genetic similarity between two plant sites was 0.9619. Their similarities were all greater than that between the seed bank and plant populations. An analysis of their genetic structure showed that 87.86% of total variation was derived by two-loci. Genetic structures between plant and soil seed bank populations in S. krylovii were different due to the variance of mean gametic disequilibria and mean gene diversity. AMOVA results showed that the majority of variance (88.62%) occurred within sites, 12.75% was from between-groups. Further research is needed to investigate the selective function in maintaining the genetic diversity of Stipa krylovii plant populations.