Background

Seeds are an innovation of Spermatophyta (seed plants). They are specialized dispersal units that safely package the plant embryo to allow for dispersal in space and time. Seeds are critical for successful propagation of the individual and maintenance of populations within a species but also for terrestrial ecosystems including forest ecosystems. Seeds can vary drastically in their characteristics, such as size, mass, color, or durability [1]. The smallest orchid seeds, for example, weigh less than 1 μg, while the large seeds of the double coconut palm (Lodoicea maldivica) can reach up to 25 kg in weight [2–4]. Poplar seeds lose viability within a few days or weeks after release, while seeds of a date palm from an archeological site were reported to have germinated after 2,000 years [5,6]. This means that seed collection, handling, processing, and storage need to be tailored carefully to the species or taxonomic group of interest to ensure seed viability and successful plant growth from the seed. Here, we focus on the seeds of temperate forest trees of the genus Populus.

Despite their importance for healthy forests, much remains to be learned about seed traits and biology in forest trees, ranging from the molecular mechanisms of seed production or seed performance under climate change to applications in forest genetic resources preservation and long-term forest management [7–11].

Trees in the genus Populus are among the most widespread trees in North America, including some of the largest and fastest growing hardwoods in this large geographic area [5,12,13]. This taxonomically complex genus comprises poplars, cottonwoods, and aspens. They are early successional in natural plant communities but are also used widely in intensive culture for biomass, pulp, and paper production, for lumber, as shelterbelts or urban trees, or in land reclamation [5,13]. Populus species are dioecious, reach sexual maturity at 10–15 years of age, and produce seeds every year once matured. The number of seeds produced is remarkable: 28–54 million seeds have been reported for individual trees of Eastern cottonwood (P. deltoides) and European aspen (P. tremula) [5,14,15]. The seeds themselves are small and covered with a significant amount of hair (cotton or white fluff) that allows for easy wind dispersal. Seeds do not show dormancy, germinate quickly under suitable conditions, and can lose viability rapidly under natural conditions [5,16]. These characteristics impose challenges on seed collection, cleaning, and storage. Seed collection, therefore, needs to be timed precisely with the seasons and seed ripening on the trees. Moreover, extraction of seeds from the difficult-to-separate seed hair requires careful handling to prevent damage to the thin seed coat. Finally, seeds need to be stored properly to ensure longer lasting viability.

Agroforestry equipment such as seed macerators or grinders and fanning mills can be used to extract large numbers of Populus seeds [5,17]; however, seed viability declines strongly within two days, possibly due to damage of the delicate seed coat. Similarly, brushing against a coarse screen released seeds from the white cotton, but the extraction efficiency was low (20%), and seeds remained viable only for a few days [18]. Smaller quantities can be collected using a vacuum cleaner, and separation from cotton can be done in a nested set of soil sieves and applying a stream of air [19–21]. Based on this rationale, we established a method for seed extraction that leaves the thin seed coat intact and that yields very clean seeds, suitable for molecular analyses. The method is fast, simple, and effective. It uses household items that can be obtained easily, that are used in every lab, and importantly, that can be cleaned and sterilized easily. Storage of the extracted seeds was tested under different conditions. Germination tests confirmed high viability of seeds directly after extraction as well as after extended storage time. Growth in tissue culture without contamination furthermore confirmed purity of seeds.

Due to the high quality, intactness, and purity of seeds extracted with this protocol, these lend themselves well to analyses of seed responses under controlled conditions in tissue culture, also germinating well on soil and other substrates. The protocol was specifically developed for Populus to separate seeds from the attached tufts of hair but could equally be adjusted for the extraction of small, delicate seeds in other species that need separation from attached structures or impurities.