Abstract
Trembling aspen (Populus tremuloides Michx. [Salicaceae]) is an important early successional reclamation species with small, wind-dispersed seeds that have short viability once released. Seeds can be placed into freezers to maintain viability, but information regarding the viability of aspen seeds in long-term storage is limited. Furthermore, seed ripeness at the time of collection may also impact the germination and longevity of seeds in storage. In this study, we collected aspen seeds in 2012, 2013, 2015, and 2016 within the Peace Region of Alberta. Seedlots were categorized into 3 ripeness categories, based on color, and tested for germination. Seeds were subsequently stored at –20 °C (–4 °F) at a moisture content of 6% on average. Germination tests were repeated on seedlots over several years, with the most recent tests occurring in 2024. Germination was significantly higher in the ripest seeds (85 ± 4.8%) relative to less ripe seeds (61.5 ± 8.9%). Seed age did not significantly affect germination, demonstrating that aspen seeds can be stored for more than a decade without losing viability. We recommend collecting ripe seeds with a brown seedcoat as they maintained the highest germination. The ability to maintain aspen seedlots for 10+ y will be beneficial for ecological restoration, particularly in regions where local populations are fragmented by industrial activity.
NOMENCLATURE
USDA NRCS (2024)
Deciduous forest in northern Alberta dominated by aspen (Populus tremuloides).
Trembling aspen (Populus tremuloides Michx.) belongs to the Salicaceae family and is widely distributed throughout North America (Perala 1990). Aspen is desirable for use in reclamation as it is an early successional species that can rapidly colonize disturbed soils and produces leaf litter high in nitrogen, calcium, and phosphorous (Fung and Hamel 1993). This leaf litter quickly decomposes, potentially providing a source of nutrients and organic matter to reclamation soils that are often lacking (Fung and Hamel 1993). Over the past few decades, extensive research has been performed to increase our knowledge of aspen ecology and reproduction, which has produced best practices for reforesting disturbed sites (Landhäusser and others 2019). For instance, site preparation and microsite creation can improve the success of nursery-grown aspen stock in the field and is often more successful than direct seeding (Landhäusser and others 2019). Despite this extensive research, however, there are still knowledge gaps surrounding aspen seed storage longevity.
Aspen is a prolific seed producer with relatively small seeds (Wyckoff and Zasada 2008) that have a very short period of viability (2–4 wk) and require a narrow range of moisture requirements to successfully establish (Moss 1938). As such, asexual reproduction through root suckering is often the more successful colonization method, especially when regenerating from disturbance (Brinkman and Roe 1975). For reforestation practices, seedling production is more cost-effective especially for large-scale operations, relative to direct seeding (Fung and Hamel 1993; Landhäusser and others 2019). Direct seeding is challenging as the seeds are small and have low rates of survival when broadcast (Landhäusser and others 2019). However, seeds can be used to produce seedlings that can help introduce greater genetic diversity to an ecosystem (Smreciu and others 2013).
Historically, the short viability of aspen seed was reported as a general constraint to storing and utilizing seed (Moss 1938); however, more recent studies have revealed practices associated with seed collection, handling, and storage that have maintained viability over longer periods of storage (Benson and Harder 1972; Simpson and others 2004). Aspen seeds are considered as orthodox seeds (Baah-Acheamfour and Sobze 2022), which can tolerate desiccation below 12% and freezing down to –20 °C (–4 °F) (CPC 2019). Seed should be collected when a portion of the catkins begins to open and the pappus is exposed (Fung and Hamel 1993; Smreciu and others 2013). It is crucial to collect seeds immediately after capsules have begun to open in order to maintain high viability (Daigle and Simpson 2009; Smreciu and others 2013). However, as seed production varies by region, tree age, and weather conditions, the optimal collection time can vary by year, so trees should be closely monitored in the weeks prior to collection (Fung and Hamel 1993). For seeds of many northern tree species, high viability can be maintained in storage if the seed is dried to a moisture content between 5% and 8% and stored promptly in an airtight container below –20 °C (–4 °F) (Simpson and others 2004). To date, quantitative guidance is limited with respect to maximum storage length for aspen.
Flattened petioles of Populus tremuloides leaves.
Small seeds of Populus tremuloides after collection.
We expect seed ripeness at the time of collection may also impact the viability of seeds in storage. Aspen seed may be collected either by felling trees and collecting the catkins or by pruning branches (Smreciu and others 2013). Branches may be soaked in water in a warm location to promote the ripening and maturation of seeds (Moench 1999). This increases the window of opportunity for collection as the seed can be collected in earlier stages of ripening. With branch collections, there may be circumstances in which the time to allow seeds to ripen is insufficient, and the seed remains in an underripe or marginally ripe state. Underripe seed will not have completed the phases of seed development (Ellis 2019) and may have reduced germination as a result. To date, little is known about the differential performance of aspen seed of various ripeness over time. Seeds are generally considered underripe when green or white (Brown 1989), and ripe when straw colored (Faust 1936; Brown 1989; Wyckoff and Zasada 2008).
The purpose of this study was to assess the effects of seed ripeness at the time of collection on seed germination for seed stored up to 12 y. We hypothesized that unripe seeds would have lower germination rates over time as unripe seeds often do not have developed embryos. Furthermore, we expected seed germination to decline as the seeds aged, with unripe seeds experiencing seed germination declines earlier than ripe seeds, which is supported by the results found in Simpson and others (2004).
METHODOLOGY
Collection and Extraction
Aspen seed was collected May 2012, 2013, 2015, and 2016 from the Peace River region of Alberta, Canada (Appendix Table S1–S2). Seeds were collected utilizing 2 methods: 1) the aspen branches were pruned and placed in water, once the capsules began to burst, individual catkins were harvested; or 2) in other cases, the aspen trees were felled and catkins were collected off the branches. A seedlot consisted of seeds collected from at least 10 trees spaced a minimum of 500 m (1640 ft) apart within a 5 km (16,404 ft) radius (Smreciu and Sobze 2011).
Catkins were placed on screened trays to allow capsules to burst over a 2–4 d period. We used an ash vacuum (Snow Joe, New Jersey, USA) with the filter removed to separate seed from pappus. Following this step, seed moisture content was tested with a water activity and temperature HC2-AW-USB probe (Rotronic, Bassersdorf, Switzerland) to determine Equivalent Relative Humidity (ERH). Seeds with ERH of 15–25% at 20 to 30 °C (68–86 °F), which is equivalent to 4–8% moisture, were immediately placed in cold storage at –20 °C (–4 °F) (Baah-Acheamfour and Sobze 2022). In cases when moisture content was greater than 8%, it was further air-dried on the bench for 24 to 48 h, and the moisture content was re-tested. While seed moisture content was being completed, seeds were stored in a cooler at 4 °C (39.2 °F) to reduce deterioration.
Seed Ripeness Assessment
Color is important in determining ripeness, and aspen seed is considered under-ripe when translucent and ripe when brown to pink (Moench 1999). We determined seed color with the Munsell Colour system using a grouping of 50 seeds per lot. This system has been used in prior studies to quantify seed color (Thompson and others 1996). Colors were broadly grouped into 3 ripeness categories: 1) less ripe, 2) medium ripe, and 3) most ripe (Table S3; Figure 1), which was determined by the authors.
Germination Testing
We performed germination tests on 4 replicate Petri plates per seedlot. Fifty aspen seeds were randomly selected and distributed evenly across each plate, and a total of 200 seeds per seedlot were used each test year. Seeds were placed into a germination chamber (BioChambers, Manitoba, Canada), which was set to 25 °C (77 °F) and 75% relative humidity. Fluorescent lights along the sides of the chamber provided an estimated 110 umol/s/m² (9.58 umol/s/ft²) PPFD for 12 h each day. During this time, germination was monitored daily for 8 d, and the sand was saturated with deionized water daily. We classified seeds as germinated once cotyledons had expanded. Seedlings were left in the Petri dish and recounted each day along with new germinants. The average of the 4 replicate dishes was calculated for each seedlot. These seedlots were re-tested on a regular basis between 2012 and 2024 (Table S4). For re-testing, we removed the seeds from the freezer and re-tested with the methods described above.
Aspen (Populus tremuloides [Salicaceae]) seeds were categorized for ripeness at the time of collection based on a Munsell Colour chart. They were categorized as less ripe, medium ripe, and most ripe.
Coefficient terms for the generalized linear mixed model (GLMM) with an ordbeta distribution predicting germination rates of aspen (Populus tremuloides) seed.
Statistical Analysis
A generalized linear mixed model (GLMM) was used to assess the effects of seed age and seed ripeness on germination with seedlot as a random factor in RStudio (R Core Team 2024). The interaction term was not tested as replication among treatment groups was not balanced. A GLMM with an ordbeta family was specified using the ‘glmmTMB’ package (Brooks and others 2017). Model assumptions were verified by plotting residuals as well as calculation of a dispersion statistic using the ‘DHARMa package’ (Hartig and others 2024). Post hoc testing was performed using ‘emmeans’ package and Tukey tests were run (version 1.1, Lenth and others 2021).
RESULTS AND DISCUSSION
Seed ripeness had a marginally significant effect on germination (χ² = 5.7, df = 2, P = 0.057; Tables S5–S7). The ripest seeds (85 ± 4.8%) had significantly greater germination than did the less ripe seeds (61.5 ± 8.9%) (Figure 2). Germination in medium ripe seeds (74.0% ± 4.4%) did not significantly differ from either the most or less ripe categories (Figure 2). There was greater spread of germination values in less ripe seeds (SD = 30.7 %, range = 91.0 %) than in medium (SD = 20.2%, range = 89.5%) and more 108 ripe seeds (SD = 12.4%, range = 56.5%) (Figure 2). Findings from this study show that seed quality in long-term storage can be impacted by ripeness stage at collection. These results suggest that fully ripe seed is the best candidate for maintaining viability in long-term storage, as germination rates for the most ripe seeds were the least variable. Marginally ripe seed may also have favorable long-term storage, although the effects are more variable. Any seeds that are considered less ripe are expected to have more variable and lower germination rates. The grouping of colors into ripeness groups can be subjective; in particular, the grouping of colors in the medium and less ripe groups can be debatable. However, the dark brown ripe seeds have greater germination than both other groupings (Figure 2). The colors in Figure 1 can help guide collection activities.
Seed age did not significantly impact germination (χ² = 0.2, df = 1, P = 0.634; Tables S5–S7), and germination for the seeds that were 10 (58.8 % ± 9.6%), 11 (78.9% ± 4.1%), and 12 (63.8% ± 14.1%) y old were relatively high (Figure 3). These results contrast those of Simpson and others (2004), in which declines occurred within the first 12 y of storage. The variability observed within aspen seedlots across seed ages was likely a result of the inherent variability of seed quality within each lot (Figure S1–S2). Furthermore, within-seedlot variation may help explain why there is sometimes higher germination in older seeds. Our results demonstrate that aspen seeds can be stored more than a decade without major impacts on germination success. It was once commonly thought that aspen seeds were short-lived in storage (Worrell and others 1999), but our study has shown that this is incorrect for the Peace River region of Alberta. In addition, Fechner and others (1981) found that aspen (Populus tremuloides) had 93.6% germination after 2 y of storage at –18 °C (–1 °F). Unpublished results from other studies by Palamarek have indicated a decline over 12 y for seeds stored at –20 °C (–4 °F) (Smreciu and Gould 2017). Young and Young (1992) have suggested that mature aspen seeds can last between 2 and 6 y, and Robb (2019) indicated that private industry-owned aspen seeds often retain viability for 3 to 4 y. This study also illustrates that prompt cold storage can maintain viability, even a decade later, which is in agreement with the results found by Simpson and others (2004).
Future studies should examine if our results hold true for aspen seeds collected in other regions. In particular, if seeds from warmer southern climates will show different responses under long-term storage. This information is very important as Alberta is divided into various seed zones, and reclamation guidelines require that seedlings are produced from propagules from the same seed zone. Understanding the responses of aspen seeds in different regions will help nurseries plan collection dates.
The maintenance of seed viability over time is a positive indication that the seedlots were collected and stored appropriately. Seed quality in storage can be significantly impacted by various factors including improper collection, storage, and handling. Aspen seed that has been collected too late or left at ambient conditions for long periods prior to extraction, cleaning, and storage will often show a loss of viability (Moss 1938; Baah-Acheamfour and Sobze 2022). Similarly, high moisture content or storage temperatures above freezing will result in loss of seed viability, especially as the storage period increases (Simpson and others 2004). Declines in seed viability can be associated with an increase in reactive oxygen species that cause metabolic damage in orthodox seeds, which can result from changes in temperature, moisture, and oxygen concentration in storage (Corbineau 2024).
Average proportion of seeds germinated ± SE for aspen seeds classified as less ripe, medium ripe, and most ripe. Seeds were collected in the Peace Region of Alberta in 2012, 2013, 2015, and 2016 and tested for germination. Seeds were subsequently stored, and the seeds were re-tested regularly. The initial seed was categorized for ripeness at the time of collection based on a Munsell Colour chart. A GLMM with seed age and seed ripeness as fixed factors and seedlot as a random factor was used to model germination. Subsequent Tukey tests were conducted on the 3 ripeness groups and different letters indicate a significant difference.
Proportion of seeds germinated ± SE for aspen seeds stored for different number of years. Seeds were collected in the Peace Region of Alberta in 2012, 2013, 2015, and 2016 and tested for germination. Seeds were subsequently stored, and the seeds were re-tested on a regular basis. A GLMM with seed age and seed ripeness as fixed factors and seedlot as a random factor was used to model germination. The regression line (black) with confidence bands (gray) for seed age is presented along with the observed germination values (black dots). The effect of seed age is averaged over different maturity groups.
CONCLUSIONS
Focusing harvest on fully ripe seeds (brown, reddish brown, light reddish brown) can increase the quality of seedlots and nursery stock through improved germination. Collection efforts should focus on gathering fully ripe seed, especially in lots slated for long-term storage. When stored properly, these seeds can remain viable for well over a decade with minimal losses in viability. In turn, these seedlots can provide a seedbank for restoration activities, offering a selection of diverse seeds that may help to enhance both ecosystem resilience and resistance.
ACKNOWLEDGMENTS
The authors thank all the staff who have assisted with seed germination testing, including Ryan O’Neill, Erica Iwabuchi-Althoff, and Shae-Lynn Stewart. The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this research through Technology Access Centre (TAC) and Innovation Enhancement (IE) grants. Competing Interest Statement: The authors declare that there are no conflicts of interest. Data Availability: Data are available upon request. Author Contribution: JM and AS conceptualized the project design and research questions. Data were collected by staff, including PH. Data were analyzed by RSK and AS. The manuscript draft was written by RSK and PH. JM and AS provided editorial and scientific feedback to the manuscript.
SUPPLEMENTARY APPENDIX INFORMATION
Aspen (Populus tremuloides) seeds were collected in the Peace Region of Alberta in 2012, 2013, 2015, and 2016. The initial seed was categorized for ripeness at the time of collection based on a Munsell Colour chart, and it was categorized as either less ripe, medium ripe, or most ripe.
Aspen (Populus tremuloides) seedlot collection sites.
Categorization of aspen (Populus tremuloides) seeds into 3 different ripeness categories by color description. The color of aspen seed at the time of collection was determined using a Munsell Colour chart.
Aspen (Populus tremuloides) seeds were collected in the Peace Region of Alberta and subsequently placed into long-term storage. Seedlots were tested over time for germination, and an X indicates that the seedlot was assessed for germination that year while an — indicates no test was run. No seeds were tested in 2014, 2020, and 2021.
Mean germination and standard errors (SE) for the seedlots classified as “less ripe” at the time of collection. Seeds were stored at -20 °C (-4 °F) after collection and re-tested regularly.
Mean germination and standard errors (SE) for the seedlots classified as “medium ripe” at the time of collection. Seeds were stored at -20 °C (-4 °F) after collection and re-tested regularly.
Mean germination and standard errors (SE) for the seedlots classified as “most ripe” at the time of collection. Seeds were stored at -20 °C (-4 °F) after collection and re-tested regularly.
The mean germination values of aspen (Populus tremuloides) seeds of varying age were predicted from the generalized linear mixed model (GLMM) for the seeds categorized as A) most ripe, B) medium ripe, and C) less ripe. The confidence interval for predicted values was plotted and represented by the shading around each line. The observed germination of the seedlots are plotted alongside the regression lines as points. Seeds were collected in the Peace Region of Alberta in 2012, 2013, 2015, and 2016 and tested for germination. Seeds were subsequently stored, and the seeds were re-tested regularly. The initial seed was categorized for ripeness at the time of collection based on a Munsell Colour chart.
The observed germination of aspen (Populus tremuloides) seedlots that were categorized as (A) less ripe, (B) medium ripe, or (C) most ripe. The seedlots, represented by a different color in each panel, were stored and tested for germination on a regular basis. The initial seed was categorized for ripeness at collection based on a Munsell Colour chart.
This open access article is distributed under the terms of the CC-BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/) and is freely available online at: https://npj.uwpress.org.
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