Petroleum flotation technique upgrades the germinability of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> seed lots

The germination of Casuarina equisetifolia Forst seed lots is low (40–50%) even when freshly collected. This study aimed at upgrading the germination performance by removing non-germinable seeds using petroleum flotation technique. Seeds collected from 19 individual trees in seedling seed orchards in India were assessed using X-ray to determine the proportion of different seed lot fractions. The feasibility of petroleum ether as a separation medium was tested. Morphological characters of floating and sunken seeds were further studied using image analysis technique to examine their effects on the separation efficiency. The X-ray analysis revealed a large quantity of shrivelled, empty and insect-damaged seeds, altogether accounting 50%, which were the causes of low percentage germination of un-graded seed lots (48%). Petroleum flotation resulted in 90% germination in the sunken fraction and 4% in the floating fraction. The separation was distinct for most seed lots, except few seed lots that had relatively low germination in the sunken fractions and more than 10% germination in the floating fractions. As a whole, petroleum flotation appears to be a feasible technique to upgrade the germination of C. equisetifolia seed lots, and its efficacy is influenced by wing surface area, seed density and wing quotient of filled and empty seeds.     

Effects of seed quality and seed location on the removal of acorns and beechnuts

We examine whether different guilds of foragers remove seeds differentially according to seed quality (seed size and insect infestation) and seed location (habitat and microhabitat) in a mixed oak-beech forest. Video recordings indicated that the wood mouse (Apodemus sylvaticus) was first to encounter seeds. Foragers preferred acorns to beechnuts, large to small size and sound to infested. Nevertheless, infested seeds were removed by rodents even when sound seeds were present. Seeds that were not preferred by scatter-hoarding rodents remained longer on the ground and were more vulnerable to predation and desiccation (4% moisture loss per day). However, seeds that were removed by scatter-hoarders were moved away from their mother trees (96%) and cached individually (32%), increasing their moisture content (3% per day). Buried seeds, simulating scatter-hoarding behavior, experienced only a 17% removal after 4 months. Seed removal differences among habitats were not due to habitat attributes but to the spatial distribution of rodent-preferred microhabitats. Thus, a significant lower seed removal was observed under the tree canopy with no shrubs. However, seed removal in forest gaps with deadwood cover was not significantly different from the preferred microhabitat (under shrub cover). In pure beech forests, seed removal by rodents only occurred under Ilex aquifolium (the only perennial cover) and under woody debris. This study concludes that seed quality and seed location determine the contribution of different removers (predators vs. dispersers), their seed selection and their removal speed, leading to different seed fates which will eventually affect tree regeneration.     

Effects of seed pre-soaking on the emergence and early growth of containerized Norway spruce seedlings

To study the effects of seed soaking on seedling emergence and development in Norway spruce [Picea abies (L.) Karst.] two commercial seed lots were soaked for 15 h in aerated water. After soaking, floating and sunken seeds were separated and analyzed prior to being sown in peat-filled containers. Seedling development was followed for one growing season. All sunken seeds were viable according to radiography whereas the floating seeds contained a considerable number of larvae filled and empty seeds. Mean time of emergence of the sunken seeds was approximately 1.5 days shorter than control (i.e. dry) seeds and floating seeds. Soaking had no effect on the time taken for most germinants to emerge in both seed lots, thus failing to synchronize seedling emergence. The later a seedling would emerge, the more likely it was for it to die during the growing season or to be culled at the end of the growing season. Soaking had an inconsistent effect on stem diameter at the end of the growing season and no effect on stem or root biomass. Seedling size uniformity was equal in all treatments. This study suggests that soaking and seed sorting does not guarantee a more uniform seedling crop, but this technique may be useful in upgrading poor quality seed lots of Norway spruce containing a high number of empty or larva infested seeds.     

Simultaneous detection of filled,empty and insect-infested seeds of three Larix species with single seed near-infrared transmittance spectroscopy

Near infrared spectroscopy was used for simultaneous detection of filled, empty and insect-infested seeds of Larix decidua Mill., Larix gmelinii Rupr. and Larix sukaczewii Dyl. Near-infrared spectra, expressed in the form of log (1/T), were collected from single seeds with 1225 Infratec analyzer from 850–1048 nm at 2 nm interval. Discriminant models were developed with partial least squares (PLS) regression using the normalized digital spectra as a regressor and a y-vector of artificial values (1 for filled and –1 for empty and infested seeds) as a regressand. The result showed a 100% recognition of infested and empty seeds in the test set by 3-factor PLS models computed based on calibration sets of each species and combined spectra of all species. The recognition rate of filled seeds was 90–100%; the highest being for L. sukaczewii, followed by L. decidua and L. gmelinii respectively. A similar recognition rate for empty and infested seeds was achieved by discriminant models derived from selected absorption bands, while the recognition rate of filled seeds differed slightly. Difference spectra and PLS weights indicated that the origin of spectral differences between filled versus empty and insect-infested seeds were attributed to differences in lipids and proteins.     

Physical attributes as indicator of seed maturity and germination enhancement in Himalayan Wild Cherry (<Emphasis Type="Italic">Prunus cerasoides</Emphasis> D. Don.)

Prunus cerasoides has poor regeneration in its natural habitat due to its hard seed coat. The information about maturity time of fruit/seed is scanty and studies on seed germination enhancement lacking. In the present study, the main focus has been given to the physical fruits/seeds attributes which are reliable indicators of maturity and seed pretreatments which help to enhance germination. The fruits/seeds were collected from low and high elevational sites covering the altitudinal range of the species during the period of fruit/seed development (last week of February–mid April) for two consecutive years (2003–2004). The change in fruit colour from dark green to pale red or red was a useful indicator of seed maturity in P. cerasoides. Fruit moisture content between 46.57 ± 0.45% and 56.81 ± 1.14% and seed moisture content between 29.8 ± 1.35% and 34.13 ± 1.50% coincided with maximum germination (41.11 ± 13.96% and 59.99 ± 12.05%) across sites. The removal of seed coat (mechanical treatment) enhanced the germination of seeds to 70 ± 0% at low elevation and 100 ± 0% at high elevation in comparison of control or untreated seeds (germination range between 5.83 ± 0.83% and 31.68 ± 25.02%). Positive correlation existed between seed size and germination (r = 0.280; P < 0.01).     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

Variation in cone and seed characters in clonal seed orchards of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Cone and seed characters were observed on top, middle and bottom portions of tree crown in 3 ramets/clone in three Turkish Pinus sylvestris seed orchards. Broad sense heritability (clonal repeatability), and correlations among characters were estimated. Around one quarter of the seed production occurred in the top portion, half in the middle and one quarter in the bottom portion of the crown for all orchards. The percentage of filled seeds varied little with the crown position, indicating more or less similar levels of selfing in the bottom of the crown as in the top. The seed weight was typically 11 mg. Differences were found for studied cone and seed characters among orchards and crown positions. Variation among grafts within clone was higher than among clones for most characters. The heritability was on average below 0.5 (e.g., cone diameter, number of filled seed per cone) and rarely rose above that (e.g., cone form, length/diameter; percentage of filled seed) for any individual characters. The coefficient of variation within clones was often higher than among clones. Thus, non-genetic factors seem often more important for the variation in performance of grafts than their genetic constitution. Cone form (length/diameter) was the character where the clone influence was the strongest. Cone number and cone dry weight showed significant correlations with seed characters (numbers of total and filled seed, percentage and weight of filled seeds). Significant correlation was found between seed characters.     

Longevity of <Emphasis Type="Italic">Juniperus procera</Emphasis> seed lots under different storage conditions: implications for ex situ conservation in seed banks

Juniperus procera Endl. is economically important timber species, but its populations are extremely small and fragmented in its natural habitat, thus, calling for immediate ex situ conservation. Here we examined the effects of seed sources and storage temperature on the longevity of Juniperus procera seed lots through collection and preservation of seeds in seed banks. Seeds were collected from nine sites across the species natural distribution in Ethiopia and stored in four warehouses: modern cold room (5℃), mud house (15℃), concrete block house (17℃ or corrugated iron house (20℃) for 42 months. Every three months, a random sample of stored seeds were drawn and tested for germination. A highly significant variation (p < 0.01) in germination of stored seeds was observed among different storage environments, seed lots, and duration of storage. Over the storage period, seeds stored in the cold room had the highest mean percentage germination, followed by the mud house, corrugated house and blocket house. The cold room (41%) and the mud house (38%) maintained the same level of germination as the intitial germination of the seedlotds (42%). The variation in longevity of stored seeds was significnatly correlated with the initial germination of seed lots (r > 0.80; p < 0.01). Cold storage also resulted in enhancement of germination through its stratification effect that terminated the non-deepphysiological dormancy of juniper seeds. In conclusion, seed lots with good initial germination can be effectively stored in cold room (5℃) up to four years. In the absence of modern cold stores, mud houses can be used as a good alternative to store seeds at local level.     

Cone traits of jack pine and black spruce in young seedling seed orchards

The number of fertile and infertile scales, filled and empty seeds, cone volume, seed efficiency and the incidence of insect and disease damage to seed were evaluated for seven jack pine (Pinus banksiana Lamb) and six black spruce (Picea mariana [Mill.] B.S.P.) seedling seed orchards in northern Ontario, Canada. On average, the seed potential of jack pine and black spruce cones was 50 and 82 seeds, respectively. Cone volume and the number of fertile scales were under strong genetic control and well correlated with one another for both species. Seed efficiency values were high for jack pine (60%) but poor for black spruce (24%). The incidence of seed insect damage was less than 2.5% for both species and nil for seed diseases.     

Effects of seed water content and storage temperature on the germination parameters of white spruce, black spruce and lodgepole pine seed

The effect of seed water content (WC) (2–3, 5–6 and 22–25%, on a fresh weight basis), storage temperature (+4, −20, −80 and −196°C) and storage duration (6, 12, 24, 48 and 60 months) on the germination of white spruce (Picea glauca (Moench) Voss), black spruce (Picea mariana (Mill.) B.S.P.) and lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) seed was investigated. Germination of white spruce control (untreated) seeds and seeds adjusted to 2–3% and 5–6% WC declined after 48 months of storage at −80 and −196°C, with a further decline at 60 months at −20, −80, −196°C. Germination remained high when control white spruce seeds and seeds with 2–3, 5–6% WC were stored at +4°C, over all storage durations. Generally, black spruce and lodgepole pine exhibited high germination at all storage temperatures at 2–3% and 5–6% WC as well as the control (untreated) seed, for up to 60 months in storage. Germination declined for all three species when seed was conditioned to 22–25% WC. This loss in germination was partially recovered in white spruce seed stored at +4, −20 and −80°C after storage durations of 24, 12 and 48 months, respectively, and in black spruce seeds stored at −20 and −196°C after storage durations of 24 months. Mean germination time (MGT) was relatively constant for all species, under all conditions, except for seed conditioned to 22–25% WC, where MGT increased for white spruce seed stored 48 months at −80 and −196°C, and for black spruce seed stored 24 months at +4 and −80°C and 60 months at −196°C. These results show that the optimal storage temperatures are 4°C for white spruce, and 4, −20, −80, and −196°C for black spruce and lodgepole pine, and 2–6% water content is optimal for all 3 species at these temperatures.     

Applying the IDS method to remove seeds infested with the seed chalcid,Megastigmus spermotrophus Wachtl,in Douglas-fir,Pseudotsuga menziesii (Mirb.) Franco

Douglas-fir seedlots were treated with the Incubation Drying Separation (IDS) method to test whether seeds infested with the seed chalcid, Megastigmus spermotrophus Wachtl, could be separated from non-infested seeds. Seeds were soaked in distilled water for 24 h, drained, placed in plastic bags and incubated at 15 °C for 3 days. The seeds were dried for either 0.5, 1, or 2 h at 25 °C, and then separated into floaters and sinkers in a water column. An average of 97% of the infested seed floated. The drying period did not affect the separation of infested seeds but significantly fewer sound seeds floated in the seedlots dried for 0.5 h than those dried for 1 h. Germination capacity of IDS-treated seeds did not differ from that of untreated seeds but the germination rate was significantly faster for IDS-treated seeds when all lots were stratified.     

Towards a DNA marker assisted seed source identification: a pilot study in European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The widespread European forest tree Fagus sylvatica L. is of great importance for forest management. However, information about seed dispersal is still very rare, though important for harvesting strategies and later on seed source identification. We refined a DNA fingerprinting method for beech nut shells in order to directly assign dispersed seeds to their mother trees. A pilot study was conducted in two beech stands in Germany where leaves of the adult trees and the exocarp of dispersed seeds were fingerprinted at six nSSR loci. While one stand was randomly analysed for adults and dispersed seeds the other was systematically investigated following common harvesting procedures. Imitating the typical net harvesting strategy, seeds were collected beneath 19 adult trees. Exocarp genotyping revealed that on average three different mother trees contributed to a sample of five or six seeds collected beneath a single adult tree. Of the identified mother trees most were located within a radius of 15 m from the sampling point. The repeated pattern of seed dispersal within a short distance constitutes the basis for a straightforward strategy for the assignment of seed lots to a seed source stand. This strategy is based on the matching of individual genotypes without the necessity for a full inventory of the putative source stand. Additionally, we provide allelic ladders of five nSSR loci for standardization among laboratories.     

Pre-sowing treatment for breaking dormancy in Acer velutinum Boiss. seed lots

Acer velutinum Boiss is a valuable tree species native to Iran, and its seeds possess physiological dormancy that hampers seedling production in the nursery for large-scale reforestation efforts. The aim of this study was to determine the optimal dormancy breaking treatments for A. velutinum seeds. We conducted a factorial experiment involving six seed lots collected along an elevation gradient from 300 to 1800 m at 300 m interval and four cold-moist stratification periods (0, 4, 8 and 16 weeks) at 4℃ and 70% relative humidity. The result shows that the germination of cold-moist stratified seeds was significantly (p < 0.0001) higher than the control for all seed lots. The highest germination capacity was recorded after 16 weeks of cold-moist stratification for all seed lots (68% 88% depending on the seed lot) except those collected from mid altitude sites (600 and 900 m) that germinated equally well (≥ 75%) after 4and 8-week of clod-moist stratification compared to the other seed lots. The mean germination time was significantly shorter (12 to 19 days, depending on the seed lot) for seeds stratified for 16 weeks than for untreated seeds. It can be concluded that: (1) cold-moist stratification for 16 weeks is the best pre-sowing treatment for breaking dormancy in A. velutinum seeds; and (2) seeds should be collected from mid altitude sites (600 and 900 m) to get more than 80% germination within 15 days, and these seed lots even required shorter cold-moist stratification period(eight weeks) than other seed lots.     

Some physical conditions for removal of mechanically damaged pinus sylvestris L. seeds by using the PREVAC method

If low absolute pressure is first applied to and then released from seeds of Pinus sylvestris L. lying in water, the seeds with mechanical damage, i.e. with seed coat cracked, with part of seed coat missing or with part of seed coat and gametophyte missing, quickly lose their buoyancy and sink and can thus be separated from the floating undamaged seeds (PREVAC method). The most crucial of the tested physical properties was the level of pressure. The time of treatment (1–20 min) was of less importance, and the water temperature (5–40°C) was unimportant. Treatment at approximately 97 kPa below atmospheric pressure (3–5 kPa absolute pressure) for 1 up to 20 min resulted in removal of almost all mechanically damaged seeds in the four studied seed lots (containing up to 26% damaged seeds). In total, less than 5% undamaged seeds of the whole seed lots were removed. The germination percentage of the fractions remaining after separation increased with increasing number of damaged seeds in the seed lots and vice versa for the removed fractions.     

Mating patterns in an indoor miniature <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> seed orchard as revealed by microsatellite markers

To evaluate the practical application of an indoor seed orchard, we compared the seed production, final germination rates and mating patterns of potted Cryptomeria japonica D. Don clones in two miniature seed orchards: one in a glasshouse and the other outdoors. There was no statistically significant difference in total seed production between the orchards (P = 0.275, ANOVA). However, the final germination rate of seeds produced in the indoor orchard was significantly lower than that of seeds produced in the outdoor orchard (P < 0.01, Wilcoxon test). The average self-fertilization rate was higher in the indoor orchard (27.2%) than in the outdoor orchard (5.6%), and this parameter was strongly associated with the reduced final germination rates of the seeds harvested in the glasshouse. Pollen contamination was considerably lower in the indoor seed orchard (48.9% in the outdoor seed orchard and 4.4% in the indoor seed orchard). χ² tests indicated that the paternal contributions of each constituent clone differed significantly in both orchards (P < 0.001 in both cases). The strong variations in paternal contributions among indoor orchard clones was significantly correlated with their respective pollen production capacities (Spearman ρ = 0.693, P < 0.001). To reduce the high self-fertilization rates, we recommend the use of orchard clones with similar pollen production capacity in indoor seed orchards.     

Seed rain,soil seed bank,seed loss and regeneration of Castanopsis fargesii (Fagaceae) in a subtropical evergreen broad-leaved forest

Understanding the seed rain and seed loss dynamics in the natural condition has important significance for revealing the natural regeneration mechanisms. We conducted a 3-year field observation on seed rain, seed loss and natural regeneration of Castanopsis fargesii Franch., a dominant tree species in evergreen broad-leaved forests in Dujiangyan, southwestern China. The results showed that: (1) there were marked differences in (mature) seed production between mast (733,700 seeds in 2001) and regular (51,200 and 195,600 seeds in 2002 and 2003, respectively) years for C. fargesii. (2) Most seeds were dispersed in leaf litter, humus and 0–2 cm depth soil in seed bank. (3) Frequency distributions of both DBH and height indicated that C. fargesii had a relatively stable population. (4) Seed rain, seed ground density, seed loss, and leaf fall were highly dynamic and certain quantity of seeds were preserved on the ground for a prolonged time due to predator satiation in both the mast and regular years so that the continuous presence of seed bank and seedling recruitments in situ became possible. Both longer time observations and manipulative experiments should be carried out to better understand the roles of seed dispersal and regeneration process in the ecosystem performance.     

Genetic diversity analysis in a seed orchard of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

India is the largest grower of Eucalyptus with an area of 3.943 million hectares under plantations and E. tereticornis is the predominant species in the plains of southern India with an average productivity of 12–25 m³ ha⁻¹ year⁻¹. With the aim to establish seedling seed orchards of the species, seed lots of fifteen provenances were imported from Australia and a trial was laid. In the present study the genetic diversity existing in the seed orchard was estimated using ISSR–PCR. Seven ISSR primers amplified 663 amplicons in the size ranging from 255 to 2,711 bp. The total number of polymorphic bands varied from 59 to 123 with 100% polymorphic banding profiles. The average gene diversity (Hj) of all the provenances ranged from 0.0589 to 0.1109 and the total gene diversity estimated was low (H _T = 0.130) when compared to the earlier reports from other eucalypts species. Analysis of Molecular Variance partitioned the ISSR variation into inter and intra population components. The inter population component accounted for 55.2% of the variation and the intra population component accounted for 46.3% (P < 0.001). A neighbour-joining analysis was done using the dissimilarity matrix to determine the aggregation of the individuals into clusters. Existence of population structure among the populations was revealed in STRUCTURE analysis but geographical region based clustering was not observed. The assessment of intra and inter genetic variation documented in the present study suggests that, along with the phenotypic traits, knowledge about genetic diversity measured at the DNA level in individuals of seed orchards provide an objective guide for selective culling of trees for maintaining optimal diversity for enhanced genetic gains.     

Development and evaluation of a real‐time PCR seed lot screening method for Fusarium circinatum,causal agent of pitch canker disease

Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi‐selective medium and identifying it using morphological traits. This method is time‐consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real‐time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real‐time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10^?5 ng of F. circinatum DNA per PCR. The culture‐based method detected Fusarium spp. in four of six of the same seed lots. The real‐time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture‐based method requires a minimum of 1–2 weeks. This new real‐time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.     

Effects of tallowtree seed coat on seed germination

We measured physiological parameters including water uptake, in-vitro embryo germination ratio, and seed coat structure observed by scanning electron microscopy (SEM) to explore the influence of seed coat on the germination of seeds of tallow tree (Sapium sebiferum (Linn) Roxb.). Tallow tree seeds had good water permeability. We found that germination of cabbage seeds was inhibited when cabbage seeds were soaked in extracted solutions from tallow tree seed coat. Seed coat structure at the side of the radicle appeared to be a barrier to seed germination. We tested methods to break tallow tree seed dormancy. Dormancy of tallow tree seeds was overcome by soaking the seeds in 500 mg·L-1 or 1000 mg·L-1 GA3, followed by 100 days of cold stratification.     

Effect of different pre-sowing seed treatments on the germination of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> (Lam.) and <Emphasis Type="Italic">Acacia farnesiana</Emphasis> (L.)

Leucaena leucocephala and Acacia farnesiana are tree species used for several agricultural purposes in the Mediterranean region. The seeds of these species exhibit dormancy, causing delayed germination. Two experiments were conducted to investigate the effect of pre-sowing treatments (scarification, hot water, or soaking) on seed germination of L. leucocephala and A. farnesiana. In one experiment, seeds were exposed to three pre-sowing treatments: control, sandpaper scarification, or soaking in 70°C water for 4, 8, 12, 16, 20, or 24 min. In another experiment, seeds were soaked in 70°C water for 20 min, and then soaked in water at room temperature for an additional 24, 48, or 72 h or blade scarified. In general, soaking the seeds of the two species in hot water was more effective in breaking seed dormancy than scarification. Sandpaper scarification was not effective for either species. Blade scarification increased A. farnesiana seed germination to 56%, indicating that seed dormancy was mainly a consequence of hardseededness. L. leucocephala seeds collected from Jordan University of Science and Technology (JUST) site and soaked in 70°C water for 20 min and then soaked for 24, 48, or 72 h had germination rates above 97%. Our results suggest that blade scarification of A. farnesiana seeds and soaking of L. leucocephala seeds in 70°C water for 20 min are effective treatments to break seed dormancy and enhance seed germination of these vital species.