Timing matters: earlier planting leads to increased seedling emergence success in Zostera marina seeds

Globally seagrass restoration efforts have been increasing in recent years. Seed-based restoration is often favoured, as it provides higher genetic diversity and less impact on donor meadows. Restoration success is highly dependent on site-selection, methods and timing. To increase the success of seedling establishment for restoration programmes we carried out experiments investigating timing, planting methods and sediment type under semi-controlled conditions in a mesocosm setup. In December, February and March Zostera marina seeds were placed in hessian bags or directly into the sediment in small boxes. Both set ups contained two different types of sediment, one coming from our donor meadow (SG) and one from an unvegetated beach (noSG). Our experiments showed that the Zostera marina population from Porthdinllaen, North Wales, is a winter seedling emergence population. Seedling establishment was most successful at the lowest temperature in December when planted directly into the sediment (43.2 ± 8.1%). Planting time and method had a significant effect on seedling emergence. The use of hessian bags decreased seedling emergence to 23 ± 6% in December and down to 0.8 ± 1.4% in March. Hessian bags in March were covered in epiphytic algae. Sediment type furthermore had a significant effect on the leaf length; 6.4 ± 1.2 (SG) to 3.7 ± 0.86 (noSG) cm in December and 3.79 ± 0.64 (SG) to 3.02 ± 0.82 (noSG) cm in March. The presented results here could have important implications for success of restorations activities.

The decline of seagrass meadows remains a global issue (Dunic et al., 2021), resulting in the loss of valuable ecosystem services such as biodiversity support, water purification, carbon sequestration, and coastal protection (Campagne et al., 2015; Nordlund et al., 2016). The east part of the temperate North Atlantic lost about 69% of its seagrass meadows over the last 100 years (Dunic et al., 2021), and remaining meadows continue to face pressure from anthropogenic impacts, often occurring in unison (Jones et al., 2025). While recovery has been documented in some regions (de los Santos et al., 2019; Lefcheck et al., 2018), this extensive decline has left large areas locked in a state of feedbacks unable to recover (Maxwell et al., 2017).

In an attempt to combat this, seagrass restoration projects have increased in number and scale worldwide (van Katwijk et al., 2016; Unsworth et al., 2019; Orth et al., 2020). In temperate systems with seagrass species like Zostera, which produce high numbers of small seeds, seed-based restoration is often favoured over shoot-based transplantation, as it maintains higher genetic diversity and minimizes impacts on donor meadows (Reynolds et al., 2012; Yue et al., 2019). Although, shoot-based restoration outperformed seed-based restoration in all approaches collected by van Katwijk et al. (2016) and showed high establishing rate of 70-fold increase after 2 years (Lange et al., 2022) in comparison to low germination and seedling establishment in seed-based restoration (Unsworth et al., 2023), seed-base restoration may be favourable for large scale projects, since seeds are less expensive and logistically more feasible to use (Kettenring and Tarsa, 2020). Two of the most successful large-scale restoration projects in Chesapeake Bay, US and the Dutch Wadden Sea were seed based (Orth et al., 2020; Gräfnings et al., 2023). Both methods underly high variability and depend on species, location and life history. Seagrass life histories differ among and within species, depending on their distribution and environmental conditions (van Katwijk et al., 2000; Zhang et al., 2022). Found across the Northern Hemisphere, Zostera marina is one of the most widely distributed seagrass species globally (Green and Short, 2003; Short et al., 2007). Its seeds develop in spathes, which are released once maturity is reached, and the negatively buoyant seeds sink to the seabed. The timing of germination varies across regions and populations: on European coasts, germination mainly occurs in spring, though winter germination has also been observed (Harrison, 1993; van Lent and Verschure, 1994). In China, Zhang et al. (2022) reported contrasting strategies, with seeds from one bay germinating in autumn immediately after release, while seeds from a nearby bay germinated only the following spring, even after reciprocal transplantation.

The success of restoration projects strongly depends on timing, site selection, and planting method (Unsworth et al., 2024). Z. marina generally germinates between 5 and 20 °C (Orth and Moore, 1983; Kawasaki et al., 1986). Several studies found that the highest germination occurs at the lower end of the local temperature range (McMillan, 1983; Kawasaki et al., 1986; Orth and Moore, 1983; Abe et al., 2008). The seeds of annual seagrass populations often undergo a period of dormancy and germinate in the following spring, once environmental conditions are more favourable. The germination ecology of perennial UK populations remains poorly understood, and it is unclear whether seeds of perennial plants undergo dormancy. The success of seed-based projects strongly depend on seed storage facility. Seed storage conditions and time has an effect on seed quality. For Z. marina best seed storage conditions were found to be cold (1–5 °C) temperature and high-salinity seawater (< 40 PSU) to suppress germination and prolong viability (Pan et al., 2014; Xu et al., 2019). Nevertheless, seed viability decreases with storage duration, even under optimal conditions (Thomson, 2023).

Winter planting may be optimal, as it provides more natural conditions and reduces seed storage time. Unsworth et al. (2022), for instance, achieved successful seedling establishment after planting in December. However, winter planting is not without risk. Unsworth et al. (2019) lost a number of hessian bags planted in November to a winter storm. For this reason, spring planting is more common in practice, as it avoids the risk of winter storms. Also, in some locations, however, permission to plant may be restricted due to the presence of overwintering birds (Project Seagrass, personal communication). Seedling emergence in the field is rarely documented as wild seedling emerge during times of the year when monitoring is difficult and finding small seedling within an established meadows is challenging.

Restoration projects are also constrained by licensing, seed sourcing and storage, weather conditions, and logistical opportunities (Unsworth et al., 2023). Habitat suitability models (HSMs) are commonly used to guide site selection (Bertelli et al., 2022). These models are usually based on parameters such as bathymetry, wave energy, and light availability, which are critical for meadow persistence. However, germination and establishment may also depend on other local factors such as sediment anoxia, pore-water nutrients, and proximity to existing seagrass (Moore et al., 1993; van Katwijk et al., 2016). Indeed, restoration success is often higher near existing seagrass meadows (van Katwijk et al., 2016), potentially due to favourable environmental conditions and the presence of microbial communities that support seed germination and seedling survival. Seagrass roots and surrounding sediments host abundant microbial taxa involved in sulphate reduction, sulphur oxidation, and nitrogen fixation (Cucio et al., 2016; Fahimipour et al., 2017; Gorvel et al., 2025). These root-associated communities might support seedling establishment and subsequent growth — for example by detoxifying sulphide and supplementing nitrogen availability rather than germination itself. If seed germination might be supported by specific microbial taxa currently unknown. Because restoration often occurs in areas where seagrass has either been absent for decades, or never been documented (new habitat creation), supplementing with natural seagrass sediment or inoculating with microbial communities may help improve outcomes. Methods such as hessian seed bags or injection with carrier substrates (e.g., the Dispenser Injection Seeding, DIS) provide opportunities to add natural seagrass sediment to restoration sites (Coals et al., 2025). Both approaches have been successfully applied in restoration projects (Unsworth et al., 2019; Gräfnings et al., 2022). The effect of adding seagrass related material or microbiome is not well studied. Adding seagrass detritus to hessian bags in the field did not show any beneficial effect on seedling emergence (Unsworth et al., 2022), but Sun et al. (2024) demonstrated that the addition of plant growth-promoting rhizobacteria enhanced Z. marina growth after transplanting.

To improve reliability for subtidal Z. marina restoration and to fill knowledge gaps of the life history of our donor population from Porthdinllaen, Noth Wales, we carried out experiments under semi-controlled mesocosm conditions. We tested the effects of planting time and method on seedling emergence, using two approaches, hessian bags and direct planting into sediment (Figure 1). The studies objectives were to find the optimal planting month and method for Z. marina. We additionally compared two sediment types: sediment from a natural seagrass meadow (SG) and sediment collected from a non-seagrass site (noSG). These combinations of method and sediment testing were applied during early winter (December) into spring (February and March) to test the effect of planting time on seedling emergence.

Seed collection and storage

Z. marina seeds were harvested under permission in August 2022 from a meadow in Porthdinllaen, Wales, UK. Seeds were collected by hand diving and at low tide. During collection people tried to only pick seeds spates where seed had passed stage 4b from De Cock (1980) seed development table. After collection, reproductive shoots were kept at ambient water temperature with aeration until ripe seeds naturally released from the spathes. Seeds were cleaned of residual biomass and stored at 5 °C (Zarranz et al., 2010; Dooley et al., 2013) and 50 PSU salinity, which has been found to be optimal storage conditions (Pan et al., 2014; Xu et al., 2019), from end of August until used in the experiments. Storage temperature was below ambient water temperature of the area. Water exchanges were carried out regularly to reduce microbial growth, but seed were not treated with any detergence to reduce infections (Govers et al., 2017; Xu et al., 2019). To investigate the life history of our seeds and test the effect of planting time, seeds were planted at three dates during winter and early spring, approximately six weeks apart: 12.12.2022 and 13.12.2022, 31.01.2023 and 01.02.2023, and 15.03.2023. Seedling emergence was monitored for a period of 3 - 5 months. For each planting time, two methods (hessian bags and direct planting) and two sediment types were tested (Figure 2).

Experimental setup

Two different planting methods were used (Figure 1). For direct planting seeds were planted directly into sediment in small plastic boxes (15.0 x 11.0 x 9.5 cm). Each box was filled with approximately 1600 mL of sediment, leaving 1.5 cm of headspace. 50 seeds were planted into every box (n = 5). Seeds were positioned at a depth of 2 cm using wooden sticks to create appropriate wells (Marion and Orth, 2012; Xu et al., 2021). For the hessian bag planting ten uncoated hessian bags were filled with a sediment-seed mixture containing 50 seeds each. Bags had a mesh size of about 1 mm with a thickness of 13 x 7.5 cm once filled. In field settings, the same hessian bags used in this study have been observed to have broken down into fragments by 8 months (Unsworth et al., 2019). Approximately 100 g of sediment-seed mix was filled into each bag, so the bags had a maximal hight of 2 cm. The bags were placed in shallow trays for incubation. Two different sediment types were tested: sediment collected from within a seagrass meadow at Porthdinllaen (SG) and sediment from Pendine Beach, Wales, UK, where we have no record of current or historic seagrass occurrence (noSG). Both sediment types were sieved to < 1 mm and not sterilized before use.

Planting was executed at the 12.12.2022 (hessian bags) and 13.12.2022 (boxes) for the December trial, at the 31.01.2023 (hessian bags) and 01.02.2023 (boxes) for the February trial and at the 15.03.2023 (both hessian bags and boxes) for the March trial.

Boxes and trays were placed in ponds inside a polytunnel. Ponds were filled with filtered seawater up to a depth of 50 cm and run with a closed recirculating system. Water temperature in the nursery ponds was supposed to follow natural conditions in Porthdinllaen (Figure 2). In late December, after the December planting, system failure required temporary transfer to a closed system with artificial seawater (Tropical Marine, Germany). During this period, water temperature deviated from natural conditions. In December it was colder, but during spring the temperature increased faster than in the sea. End of March, after repairs, the system was switched back to a flow-through setup with natural seawater. Salinity in both set ups was between 28 and 30 PSU.

Temperatures at the time of each planting event were 6.6 °C (December), 7.9 °C (February), and 10.7 °C (March), compared to a mean ambient temperature in Pwllheli, North Wales (seaTemperature.org) of 10.9 °C (December), 8.5 °C (February), and 8.2 °C (March). Water temperature in our experimental ponds was therefore lower than ambient in December but already increasing by March. The lowest temperature measured was 5.9 °C shortly after December planting took place. Light levels inside the ponds were ~40% of natural ambient light, while absolute irradiance was not measured. Day length was extracted from SunCalc.org for the nursery location and the day of the planting. It was 7h 53min, 9h 6min, and 11h 50min for the December, February, and March planting times, respectively.

Seedling emergence and shoot length

Seedling emergence in box treatments and hessian bags was checked approximately once per month until end of June (Figure 2). Seedling emergence was validated only when a green shoot emerged from the sediment or the surface of a hessian bag. Monitoring of hessian bag treatments stopped earlier due to extensive epiphyte overgrowth and subsequent seedling loss. Percentage of emerged seedlings was calculated by dividing counted seedling by the number of seeds planted (50 for all treatments). For maximum emergence, the sampling point with the highest observed value was used independent of the time of occurrence. For further analysis all replicate data of this sampling point were used.

Leaf length was measured only for the box treatments. In each box the length of the longest leaf of 5 -10 plants was measured. Length of the leaf blade was measured in cm from above the sheath to the tip of the leaf blade. For the December trial, leaf length was measured 98 and 127 days after planting. For February trial, measurements were taken 64 days after planting, and for the March trial 35 days after planting. Growth was calculated as cm day -1 by dividing measured length (cm) by days after planting of the measuring day.

Data analysis

Data analysis and figure generation was carried out usings R (version 4.3; R Core Team 2023), we analysed seedling emergence as a proportion of seeds planted using a generalized linear mixed model (GLMM) with a binomial error distribution and logit link in the R package lme4 (Zuur et al., 2009). The response was the seedling emergence proportion per observation, with the number of trials specified via the weights argument. Fixed effects included the categorical predictors month, method, and sediment, with their prior interactions month × method; month × sediment; and sediment × method to test for context-dependent effects. To account for repeated measures and hierarchical structure, we included random intercepts for ID and time nested within ID ((1 | ID/time)), where ID represents the individual experimental box and time represents the repeated sampling occasions at which seedling emergence was recorded within each box. For the model runs using results from the first month and the maximal emergence no random intercept was included. The model was fitted by maximum likelihood (Laplace approximation) using the bobyqa optimizer to aid convergence (Bates et al., 2015). The GLMM was used for three different approaches, using the entire data set of all sampling times (All data), the data for the first month sampled only (First month) and the month where maximum seedling emergence occurred (Max emergence). Additionally, we applied a Type II analysis of deviance based on likelihood-ratio tests (ANOVA) to the fitted GLMM. ANOVA was also used to test the effect of the categorial predictors and their interactions on the slope from leaf length (growth cm day -1).

Seedling emergence

The effects of planting time and method, on seedling establishment were tested across three planting periods: December, February, and March. Additional two different sediment types were tested to investigate the possible effect of seagrass related sediment on germination and establishment. Seedling emergence was monitored approximately once per month (Figure 2). Because of the relatively long intervals between samplings, it was not possible to identify a clear plateau in the seedling emergence curves, nor calculate mean seedling emergence time, but clear plateaus of the graphs are visible. Seedlings from seeds planted directly into boxes in December emerged later but ultimately reached a higher percentage of establishment than seeds planted later in the season (Figure 3). A similar trend was observed in hessian bags. Seedling emergences in March were almost 0% and sampling was terminated early due to overgrowth of epiphytic algae, which appeared to inhibit seedling growth and even reduced seedling numbers over time.

To capture different patterns in the data, a GLMM was run for three scenarios: the full sampling period (all data), sampling after one month (first month), and maximal seedling emergence (max emergence) (Figure 4). For maximum emergence, the sampling point with the highest observed value was used independent of the time of occurrence (Figure 5b). ANOVAs were additionally conducted for emergence after the first month and for maximum emergence (Table 1). These analyses revealed a significant effect of planting method (<0.001) both after one month and for maximum emergence. Across all planting dates, seedling emergence was consistently higher in direct-planted boxes compared with hessian bags. Planting month significantly affected maximum emergence, while sediment type only influenced the establishment of seedlings after the first month.

After one month (first month), seedling emergence was lower in non-seagrass sediment compared with seagrass sediment (Figure 5a). Effects of sediment type on maximum seedling emergence varied with planting month and method. In boxes, non-seagrass sediment had a negative effect, particularly on later plantings. In contrast, in hessian bags, emergence was slightly higher in non-seagrass than in seagrass sediment.

The highest number of seedling emerged was 24 shoots out of 50 seeds (48%) observed in both sediment types, when planted in December, and February into seagrass sediment. The highest average emergence was 43.2 ± 8.1% standard deviation after 63 days for the December plantings in boxes with seagrass sediment. For hessian bags planted in December, seedling emergence increased until the second sampling (64 days after planting), reaching a maximum of 17 seedlings (34%) in non-seagrass sediment (average 23.0 ± 6.0% standard deviation) and declined by the third sampling (98 days after planting). February plantings had consistently lower seedling emergence than December, and the lowest success overall occurred in hessian bags in March, with only 0.8 ± 1.4% seedling emergence across both sediment treatments. These bags were not sampled further due to poor conditions and complete algal overgrowth (Figure 6).

For the GLMM, direct-planted boxes, December planting, and noSG sediment were set as reference levels (Table 2). Figure 4 shows the coefficient estimate for all predictors for the separately tested set ups. When testing seedling emergence after one month the GLMM shows that February and March have a significant positive estimate, but this was not found over the entire sampling period (model: all data) or for maximum emergence. These two model sets had a significant negative estimate for the month of March, indicating that planting in March negatively affects seedling emergence. This undermines the trend visible in Figure 3, that seedlings from seeds planted in December emerged later, but more seedlings established. This was observed for both methods, direct and hessian bag planting. Even though the hessian bag experiment was stopped earlier, seedling emergence success for seeds planted in December was more than double that of seeds planted in February, while in March hardly any emergence occurred.

The model testing “all data” and “max emergence” also showed a negative effect of the method hessian bags (HB) and all three tested models for the combined predictors hessian bags and SG sediment. SG sediment tested alone had a positive effect when applying all data and testing maximum emergence. The lowest coefficient estimate was found for all models for March trials using hessian bags.

Leaf length

Leaf length was only measured for seedlings emerging in boxes (Figure 7a). Growth rates (cm day -1) were calculated from repeated measurements (Figure 7b). Leaves grew faster in March plantings, when water temperature (10.7 °C) and day length (11h 50min) were higher, compared with December plantings (6.6 °C, 7h 53min). Growth rates were also higher in seagrass sediment than in non-seagrass sediment. The maximum growth rate was 0.10 ± 0.018 cm day -1 (average ± standard deviation) in March plantings with seagrass sediment, whereas the lowest rate was 0.034 ± 0.010 cm day -1 in December plantings with non-seagrass sediment. Both planting month (<0.001) and sediment type (<0.001) had significant effects on the slope according to ANOVA (Table 1). Seedlings generally did not exceed 7 cm in length, likely due to the constraints of the small box environment.

To improve the understanding of seedling emergence and seedling establishment of subtidal Z. marina and to improve seed-based restoration, we tested the effects of planting time, method, and carrier sediment in a semi-controlled mesocosm setup. Three different planting periods (December, February, and March), two methods (hessian bags and direct planting), and two sediment types (seagrass and non-seagrass) were compared. Our results showed that planting shortly after the seeds were ripened and went into storage (December) produced higher maximum seedling emergence than later plantings (February and March). Direct planting into sediment consistently yielded a higher seed emergence rate than planting into hessian bags, particularly when using sediment collected from a seagrass meadow.

Z. marina germinates and establishes seedling at different times depending on location, life history, and environment (Zhang et al., 2022). Our experiment suggests that seeds from the perennial, subtidal population at Porthdinllaen (North Wales) germinate in late autumn or winter and seedlings grow rapidly during spring, consistent with observations from the east coast of the Atlantic (Marion and Orth, 2012). Seeds appeared not to undergo an extended dormancy but seedling emerge shortly after release. Unfortunately, our experiment only started in mid-December, whereas seeds went into storage after most seeds have fallen out of the seed spate end of August. Due to the timing we cannot make any predictions about earlier emergence. If germination is triggered by low temperature, the threshold may already have been met in the December planting, since the lowest temperature in the nursery, compared to ambient water temperatures, was already reached in December.

Potential dormancy in Zostera spp. has been described by Orth et al. (2000) as secondary dormancy, driven by environmental conditions. In the Northern hemisphere, two different periods for germination were observed mainly depending on environmental factors. Some populations, especially in tidal areas, do not germinate before the next spring, but subtidal plants as the Porthdinllaen population more often already germinate shortly after seed were released in late summer or early autumn. Temperature is likely a key factor to introduce seedling emergence. For Z. marina it was found to be optimal between 5 and 20 °C (McMillan, 1983; Orth and Moore, 1983; Kawasaki et al., 1986). Studies often found the highest germination at the lower end of the temperature range of the seed’s origin (Kawasaki et al., 1986; Abe et al., 2008). Recent studies from Zhang et al. (2025) showed that mean time to germinate was shortest at 10 °C in comparison to 5 °C and longest at 20 °C. But they also found differences in temperature dependence for different Zostera population growing in different locations under different environmental conditions. In Porthdinllaen, minimum sea temperatures of ~7–8 °C occur between January and March (www.seaTemperature.org). In our experiment, minimum temperatures of 5.9 °C was reached already in December, shortly after the first planting occurred. After this temperature increased during February (7.9 °C) and March (10.7°). December plantings reached higher seed emergence rates, but over an extended period, while February and March reached their maximal emergence earlier, but also started declining earlier, especially when planted into noSG sediment.

Similar to most restoration projects, we stored seeds at cold temperature (<5 °C) and high salinity (50 PSU) to suppress germination and reduce pathogen growth (Zarranz et al., 2010; Dooley et al., 2013). Storage, however, affects viability. Thomson (2023) reported a 15% decline in viable seeds after 112 days at 4 °C and 50 PSU. This most likely affects autumn germinating seeds, like ours, more than seeds geminating in spring, as our setup supressed natural germination. Before the start of each planting seeds were stored for 73 (December), 122 (February) and 165 days (March). If the viability of our seeds also declined about 15% within 112 days, this is a possible explanation for the decrease in seedling emergence between December and March in direct planting, but seed viability alone cannot explain the almost zero percent seedling emergence success in hessian bags in March. Unfortunately, we did not monitor seed viability during the storage period or before seeds were used in the experiment. Monitoring seed viability over the storage period will help selecting the best time for planting for your restoration project.

Fewer seedlings developed in hessian bags compared with direct planting. Seeds placed individually at 2 cm depth in boxes had higher seedling emergence, consistent with earlier studies that identified ~2 cm as the optimal depth for Z. marina seedling establishment (Marion and Orth, 2012; Xu et al., 2021; Gräfnings et al., 2023). In contrast, seeds in hessian bags were randomly distributed, and seedlings had to grow through the bag material to access light. This barrier, together with surface algal fouling, likely contributed to reduced success in bags. The algae film reduced the light availability for early stages of seedlings and even led to a dieback of some green shoots. Hessian bags do not generally inhibit seedling establishment. Zhang et al. (2015) have found emergence success of up to 49% with an average emergence of 38% during their experiment. These bags were also not buried, but the experiment was carried out in the field. Maybe the nursery conditions induced algae growth at the surface of the hessian bags.

Sediment type influenced seedling emergence and growth. Fewer seedlings developed in non-seagrass (noSG) sediment compared with seagrass meadow (SG) sediment. The SG sediment was finer (silty sand) than the noSG sediment (sand), which likely contributed. Finer sediments generally have higher nutrient and carbon content (Koch, 2001; Duarte et al., 2005). Seagrass patches enrich sediments in organic matter and nutrients (Gacia et al., 2002; Duarte et al., 2005; Ricart et al., 2015). Tanner and Parham (2010) also reported higher germination in fine compared to coarse sediments.

In addition to physical properties, seagrass sediments host microbial communities important for sulphur and nitrogen cycling (Gorvel et al., 2025). These microbes can support plants with detoxifying sulphide (Van der Heide et al., 2012) and providing nitrogen (Nielsen et al., 2001). Community composition of these microbes differs significantly between vegetated and unvegetated sediments (Ettinger et al., 2017). SG sediment may have contained microbial taxa that promoted seedling emergence and establishment. While Unsworth et al. (2022) did not find benefits of adding seagrass detritus to hessian bags in the field, other recent work shows promise. Sun et al. (2024) demonstrated that plant growth-promoting rhizobacteria enhanced Z. marina growth after transplanting. Bacterial inoculation to support seagrass seed germination remains a new field, but results are beginning to emerge. During our experiment we observed a rather contradictory effect of sediment coming from a seagrass meadow. The sediment clearly supports emergence and especially seedling growth when seeds were planted directly into a larger amount of loose sediment, but when using the seagrass sediment within a hessian bag, it seemed to have a negative effect on seedling emergence. We hypothesise that the enclosure of the bag supported the development of unfavourable environmental conditions like hypoxia. Hypoxia was found to increase (Infantes et al., 2016) and depress (Hootsmans et al., 1987; Churchill, 1992) seed survival. Seeds were found to germinate earlier under anoxic conditions (Moore et al., 1993, Orth et al., 2000), maybe due to the high respiratory demand of seagrass seeds (Brodersen and Kuehl, 2023). However, when opening the bags at the end of the experiment to recover the remaining seeds there was no sign of hypoxia like darkening of the sediment inside the bags. Remaining seeds were recovered from the bags and boxes after the experiment ended and tested for viability using the squeeze test (Marion and Orth, 2010). Seeds were squeezed using a forceps and seeds that did not break or smashed were assumed to be still viable. The highest number of seeds still assumed to be viable was 7 out of 50 were recovered from the different treatments. The ANOVA results showed that method had a significant effect (< 0.05) on the amount of viable seeds, where less viable seeds were found inside the hessian bags than being recovered from the sediment in the boxes. The sediment had no effect on the number of viable seeds recovered from the different treatments. Still seedling emergence from hessian bags was higher when using noSG sediment. This could have been due to a more mechanical reason. Potentially the more sandy and coarse noSG sediment made it easier for the cotyledons and seedling emerged inside the already tight environment within the bag to make its way through.

Hessian bags suffered from algal overgrowth, especially in March, which suppressed seedling emergence. Epiphytic growth is also a problem in nature, particularly under eutrophic conditions (Nelson, 2017). In field studies, burying hessian bags can reduce fouling and improve stability. However, also under field conditions, hessian bags generally yield lower germination than injection methods such as DIS (Coals et al., 2025). Still, hessian bags have advantages. They are easy to deploy at scale, inexpensive, suitable for community-led projects and avoid diving when deploy in deeper waters. Furthermore, bags protect seeds from predators like crabs or burial by worms (Unsworth et al., 2024). Some projects using direct seed planting methods suffered immensely from losses due to predation and burying (Infantes et al., 2016; Kwakernaak et al., 2023). Restoration success depends highly on choosing the right method for the right area.

Our findings demonstrate that planting time has a strong effect on seedling establishment. Under nursery conditions seeds from the Porthdinllaen population seedling emerge before spring. However, knowing when natural seedling emergence occurs is not the same as identifying the optimal planting window for restoration. Planting in autumn and early winter maximizes seedling emergence potential under controlled conditions but may expose seeds and seedlings to high losses from winter storms in the UK (Coals et al., 2025). Therefore, restoration planning must balance biological timing with environmental risks. Knowledge of local seed behaviour remains critical for tailoring strategies to specific populations. Predictably, seeds have a higher seedling emergence rate when planted carefully into the right depth, but there are many areas where hessian bags are a more suitable method due to accessibility or costs. For example at sites with a high risk of seed predation or relocation.

Our experiment adds a huge value for future restoration projects, especially when using seeds from subtidal North Wales Zostera population. Future investigations should investigate the two factors timing and temperature and test germination at different temperature and times. The community would also benefit from knowing when seeds germinate in natural conditions in the field, which could also be simulated under ambient condition using seed without storage.