Aquatic Ecosystem Health & Management

ISSN: 1463-4988 (Print) 1539-4077 (Online) Journal homepage: https://www.tandfonline.com/loi/uaem20

A low-cost and effective seeding technique using

protective core for restoration of Zostera marina
habitats

Kun-Xiu Xie, Zheng Li, Chao Li, Yan-Shan Liu, Wen-Tao Li & Pei-Dong Zhang

To cite this article: Kun-Xiu Xie, Zheng Li, Chao Li, Yan-Shan Liu, Wen-Tao Li & Pei-Dong
Zhang (2020) A low-cost and effective seeding technique using protective core for restoration
of Zostera�marina habitats, Aquatic Ecosystem Health & Management, 23:3, 341-349, DOI:
10.1080/14634988.2020.1831360

To link to this article: https://doi.org/10.1080/14634988.2020.1831360

Published online: 17 Dec 2020.

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 A low-cost and effective seeding technique using
protective core for restoration of Zostera
marina habitats
Kun-Xiu Xie#, Zheng Li#, Chao Li, Yan-Shan Liu, Wen-Tao Li,
and Pei-Dong Zhang

Key Laboratory of Mariculture (Ocean University of China), Ministry of Education,
Qingdao, People’s Republic of China

Corresponding author: zhangpdsg@ouc.edu.cn

In this study, a protective seed core method for planting seeds of the common eelgrass Zostera
marina was developed and evaluated through laboratory and field experiments. We studied the suit-
able desiccation period of the protective seed cores, and their effect on seed germination and seedling
establishment in a laboratory experiment. New patches established by seeds planted with the protect-
ive seed cores were evaluated through an 11-month-seed-sowing field experiment. Observations of the
stability of the protective seed cores and the change in viability of Z. marina seeds placed in the pro-
tective seed cores demonstrated that the optimum desiccation period was 2 days. Seed germination
and seedling establishment of Z. marina were promoted by the protective seed cores during the 90-
day laboratory experiment. The field experiment showed that the seedling establishment of Z. marina
seeds was significantly enhanced by the protective cores, ranging from 12% to 64%, with an average
of 30%, 6 months after seeding. New patches were successfully established by protective seed cores
with a maximum shoot density of 367 shoots m2 11 months after planting. The average planting cost
per Z. marina seed was $0.03; thus, $14,820 would be incurred for planting 1 ha of Z. marina beds
using this method. These results indicate that the protective seed core method is an efficient and cost-
effective restoration technique with a potential in facilitating large-scale restoration projects.

Keywords: Seagrass meadows, protective seed core, seed germination, seedling establishment,
new patch, cost

Introduction health of the coastal areas (Cullen-Unsworth and

Unsworth, 2013; Duarte et al., 2013). However,
As vital components in coastal ecosystems, the species diversity and coverage of seagrasses
seagrass meadows have high ecological value declined sharply worldwide due to natural and
and serve numerous functions that relate to the anthropogenic causes (Orth et al., 2006a;

#
The authors contributed equally to this work.

341

Aquatic Ecosystem Health & Management, 23(3):341–349, 2020. Copyright # 2020 AEHMS. ISSN: 1463-4988 print / 1539-4077
online DOI: 10.1080/14634988.2020.1831360
342 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
Waycott et al., 2009). Seed planting methods can
reduce the damage to donor beds and maintain
the genetic diversity and population structure of
restored seagrass beds (Orth et al., 2000; Greve
et al., 2005), and therefore have been suggested
to be more effective than vegetative transplant-
ation alone for seagrass restoration in
recent years.
The common eelgrass Zostera marina
Linnaeus has been the focus of most seagrass res-
toration projects along the temperate coasts of
Europe, North America, and the northwest
Pacific (Zhang et al., 2015b). Seed planting meth-
ods focusing on Z. marina have been developed
by applying buoy-deployed planting techniques
(Pickerell et al., 2005; Marion and Orth, 2010),
hand-broadcast techniques (Golden et al., 2010),
mechanical seed planters (Traber et al., 2003;
Orth et al., 2009), and seed protection techniques
(Harwell and Orth, 1999). However, the rate of
seedling establishment in most seeding restor-
ation projects is usually less than 10% of the total
seeds used (Orth et al., 2009). Low seedling
establishment rates have led to low efficiency of
restoration and high consumption of donor seeds,
and hence remain a bottleneck for seed-based eel-
grass restoration projects.
Previous studies have demonstrated that the
lack of seedling establishment may be mostly
attributed to seed losses resulting in part from
deep burial, predation, or the lateral transport of
seeds by deep-water bottom currents and erosion
events (Harwell and Orth, 1999, Orth et al.,
2003). Several seed planting trials using seed pro-
tection techniques have achieved considerable
success. For example, Zhang et al. (2015a) con-
ducted a Z. marina seed planting trial using a
protective bag method over a three-year period
and demonstrated the successful re-establishment
of Z. marina with shoot densities of 156 to 657
shoots m2. However, the protective bag method
is relatively costly, which may limit the general
application of this method. Subsequently, Zhao
et al. (2016) developed a small protective bag
method for planting seeds of Z. marina and found
that new patches from the planted seeds were
fully developed 11 months after planting with a
maximum shoot density of 350 shoots m2. This
method was labor-consuming and could not be
applied using mechanical seed planting
technology, which may limit its potential use in
large scale restoration projects.
In the present study, a cost-effective protective
core method of planting Z. marina seeds was
developed for potential use in large-scale restor-
ation projects. The objectives of this study were:
(1) to determine the key conditions required by
the seeds of Z. marina to germinate, (2) to assess
the effectiveness of this method to successfully
establishment of new patches by the seeds
through a seed-sowing field experiment over 11
months, and (3) to determine the cost of this
method associated with seed collection, process-
ing, storage, and dispersal.
Methodology
Experimental site
The experiment was conducted in Swan Lake
(37 210 N, 122 340 E) of Rongcheng, Shandong
Province, China. The lake is characterized by a
tidal lagoon located on the eastern end of
Shandong peninsula with an area of 4.94 km2,
and is separated from the open sea (i.e.
Rongcheng Bay and the Yellow Sea) by a
2.5 km-long sand spit, lying to the east of the
lagoon. The growing season of Z. marina in
Swan Lake is from April to September, and the
detailed information on environmental conditions
and seagrass vegetation traits can be found in
Zhang et al. (2015a).
Seed collection and storage
In order to reduce the damage to the existing
beds of Z. marina during seed collection, vegeta-
tive shoots and underground parts were avoided,
and reproductive shoots only, with mature and
nearly mature seeds were collected by hand in
July 2013 at Lidao Bay (37 120 N, 122 360 E) in
the Shandong Peninsula. The reproductive shoots
were kept in nylon mesh bags (mesh size
< 1 mm). The bags were placed in the donor site
and submerged in ambient water until the shoots
degenerated and the seeds were released (up to 5
weeks after collection). The seeds were then
sieved and placed in 2-litre polythene cups with
natural seawater for transport to the laboratory. In
the laboratory, the mature seeds with rigid seed
 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
coats were selected and stored inside mesh bags,
contained in tanks with flowing seawater at air
temperature until the seeds were prepared for
experiments.
The experiment included five processes from
the production of protective seed cores (PSCs) to
cost estimation (Fig. S1).
Protective seed cores
The PSCs were made of high-silted sediments
collected from the beach outside the experimental
site. Fine sand (sediment particle size between 63
and 250 lm) and silt and clay (sediment particle
size < 63 lm) were obtained using a standard
series of sieves. The mixtures of 25% fine sand
and 75% silt and clay by weight of sediments
were used for PSCs. Mud-pies were produced by
adding water into the high-silted sediments (about
1:4 in weight).
Core tubes (PVC; 7 cm diameter  3 cm) were
partially filled with mud-pies, pressed firmly with
a stainless-steel spoon. Fifty seeds were placed at
depths between 1 and 2 cm in the core, and then
filled to the top of the core tube with the remain-
der of the mud-pie (Fig. S2). The viability of the
seeds was determined using the “squeeze test”
described by Marion and Orth (2010) prior to
placement of the seeds inside the cores. Seed
depth was selected based on observations of seed
germination and seedling emergence of Z. marina
subjected to different seed burial depths in previ-
ous laboratory and field studies (Jarvis and
Moore, 2015). The core tubes were removed 24 h
after seed planting.
Air exposure of protective seed cores
To determine the suitable desiccation period
of PSCs, the stability of the PSCs was observed,
and the viability of Z. marina seeds placed in the
PSCs was detected. A total of 40 PSCs were pre-
pared as described above. These PSCs were kept
in a laboratory at air temperature (average tem-
perature: 18.3  C; average relative humidity:
80%) for 4 days. On days 1, 2, 3, and 4 after air
exposure began, ten PSCs were randomly
selected. Firstly, the PSCs were placed into sea-
water for 30 min in a 30 L tank and the structure
of the PSCs was observed and recorded. Then the
343
PSCs were wet sieved (710-micrometer mesh) to
collect all seeds in each PSC. The seeds were
collected daily, and along with a sub-sample of
250 seeds selected directly from the stored seeds
(0 days of air exposure) were tested for viability
using tetrazolium chloride as described by Jarvis
et al. (2014).
Effect of protective seed cores on seed
germination and seedling establishment
To determine whether PSCs enhanced seed
germination and seedling establishment, a labora-
tory experiment was conducted under controlled
conditions. The seeds were planted for 90 days
using five different methods as follows: (1) the
seeds were directly sown in seawater (seed þ sea-
water); (2) seeds were sown on the surface of
fine sand (seed þ sand); (3) seeds were buried 2-
cm deep in fine sand (seed þ sand burial); (4)
seeds were sown with the PSCs which were dir-
ectly placed on the surface of fine sand (cor-
e þ surface); and (5) seeds were sown with the
PSCs which were buried at a depth of 2 cm in
fine sand (core þ sand burial). A total of 30 PSCs
were prepared as described above and exposed to
air at 18.5  C for 2 days prior to use.
Fifteen replicates were prepared for each of
the planting treatments. A total of 3 750 seeds
were randomly selected from the stored seeds
and divided into 75 groups with 50 seeds each.
500-milliliter beakers were used as planting con-
tainers for each group. The 15 beakers for each
treatment were placed at random in a
50  40  20 cm glass tank with an effective
water volume of 30 L (Fig. S3). Temperature,
irradiance, salinity, and photoperiod of the water
in the tank were maintained at 18 ± 0.5  C, 130
mmol m2 s1, 32 ± 1, and 12 L:12 D, respect-
ively. Each tank was continuously aerated using
an air stone, and the dissolved oxygen (DO) was
maintained above 6.0 mg L1. The water in the
tank was renewed every two days with filtered
seawater. On days 15, 30, 45, 60, 75, and 90 after
planting, germinated seeds and emerged seedlings
in each group were carefully counted to calculate
the seed germination rate and seedling establish-
ment rate.
344 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
Assessment of the establishment success
of new patches
Two seeding ways to bury the PSCs were
tested in a field experiment. One way was to
bury the PSCs flush with the sediment surface
(PSCs þ 0 cm burial), in which a 3-centimeter-
height sediment plug was extracted and replaced
with one PSC (Fig. S4-A and B). The other way
was that the PSCs were buried 2 cm below the
sediment surface (PSCs þ 2 cm burial), in which
a 5-centimeter-height sediment plug was
extracted, replaced with one PSC and then buried
at a depth of 2 cm with natural sediments (Fig.
S4-C and D). Two 2 m  2 m experimental plots
with a water depth of 0.5 m at neap tides were
established in a 9 m  12 m unvegetated subtidal
zone of Swan Lake. Two permanent buoys were
fixed to the seabed near the plot using steel
stakes as markers. A total of 200 PSCs were pre-
pared as described above and exposed at air tem-
perature 2 days prior to use on 22 October 2013.
One hundred PSCs were randomly selected from
these PSCs and planted according to the descrip-
tion for the PSCs þ 0 cm burial treatment, fol-
lowing a randomized block design in an
established plot. The remainder of the PSCs were
planted according to the description for the PSCs
þ 2 cm burial treatment in the other established
plot. The PSCs were arranged 20 cm apart from
one another within each plot.
Five PSCs were randomly selected and
extracted from the sediments at each of the two
experimental plots using SCUBA in March 2014.
The PSCs were wet sieved (710-micrometer
mesh) to separate all seeds. Germinated seeds
(cotyledon extended through the seed coat, still
attached to the seed), intact seeds (intact seed
coat with embryo), and seed coats (seed coat split
open with missing embryo) were sorted and
counted. Retention of seeds was determined as
the percentage of the planted seeds that were con-
sidered to have germinated and to be still intact.
Five PSCs were randomly selected at each of
the experimental plots using SCUBA in April
2014. Germinated seeds with cotyledons emerg-
ing from the sediment surface were counted to
estimate the seed germination rate (i.e. the per-
centage of initial planted seeds that were consid-
ered to have germinated). Seedlings with leaves
from the sediment were counted to estimate the
seedling establishment rate (i.e. the proportion of
the planted seeds that had developed into seed-
lings). After the initial survey, the five PSCs
sampled from each plot were extracted from the
sediments and wet sieved (710-micrometer mesh)
to separate all seedlings. Seedlings in each core
were rinsed thoroughly in seawater to remove
sediments and then placed in sealed polythene
bags, stored on ice, transported to the laboratory,
and stored at 4  C before sorting for analysis.
Each seedling was cleaned with gauze, and mor-
phometrics were analyzed by measuring the shoot
height, leaf length, and root length (cm).
To assess the successful establishment of new
patches after seedling emergence, five
0.25  0.25 m quadrats were placed haphazardly
within the experimental plot in every month
between May and September 2014, and the num-
ber of shoots was counted using SCUBA. These
measurements were expressed as shoots per unit
area (shoots m2). One quadrat was randomly
selected from the five sampled, and all intact
shoots in this quadrat were collected carefully by
hand to avoid damage to belowground structures.
The samples were rinsed thoroughly in seawater
to remove the sediments and other debris. These
shoots were placed in sealed polythene bags,
stored on ice, transported to the laboratory, and
stored at 4  C before sorting for analysis. In the
laboratory, shoots in the quadrat were counted to
correct the potential error in the density estimate
obtained by SCUBA observation. The samples
were sorted, gently cleaned with gauze to remove
epiphytes, and washed with tap water. Plant mor-
phometrics were analyzed by measuring shoot
height, leaf length, and internode length (cm).
Subsequently, the samples were rinsed in deion-
ized water, and dried at 60  C for 48 h to deter-
mine their biomass (g dry wt m2).
Costs
The total costs associated with seed collection,
processing, storage and dispersal, such as staff
labor, travel and boat support, and expendable
supplies, were assessed. Single purchases of
equipment, utilities, pre- and post-project moni-
toring and other costs were not included to aid
comparison with a previous study by Busch et al.
(2010). In the present study, approximately 56
000 seeds were manually collected by four
 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
Figure 1. Rates of seed germination (open bars) and seedling
establishment (solid squares) of Zostera marina seeds planted
in the PSCs þ 0 cm burial and PSCs þ 2 cm burial treatments
in April 2014. Data are expressed as means ± S.D.
persons over 3 days in mid-July 2013, when at
least 60% of the seeds within reproductive shoot
spadices were already mature enough to ensure
harvesting during the peak of seed production.
The seeds were stored for 2 months prior to dis-
persal in a 120-litre tank recirculating system,
and viability was determined by counting repli-
cate 5-milliliter samples of seed material using
tetrazolium chloride. A total of 28 500 viable Z.
marina seeds were obtained on 15 October 2013
after the processing and storage procedures. The
dispersal cost was calculated by adding the costs
of making and placing the PSCs in the sediment.
Statistical analysis
Data were tested for normality and homogen-
eity of variance to determine the assumptions of
parametric statistical analysis, before conducting
one-way analyses of variance (ANOVAs) or
independent sample t-tests. Duncan’s multiple
range test was used to establish the significant
differences among the samples when the
ANOVA result was significant at p ¼ 0.05.
Statistical analyses were performed using SPSS
Windows Program (Version 18.0, SPSS, Inc.).
Results
Air exposure of protective seed cores
Observations showed that PSCs were not firm
enough to hold structures when they were placed
into seawater after 1 day of air exposure (Fig.
345
S5). Viability of Z. marina seeds in the PSCs
decreased significantly from initial seed viabil-
ities of 95% when days after air exposure
exceeded 2 (p < 0.05; Fig. S6). These results sug-
gest that the optimum desiccation period of the
PSCs is 2 days after air exposure.
Effect of protective seed cores on seed
germination and seedling establishment
The germination and seedling establishment of
all Z. marina seeds planted with the methods of
seed þ seawater, seed þ sand, and seed þ sand
burial were lower than 4% during the experimen-
tal period, but those of Z. marina seeds planted
with PSCs displayed a rapid increase with aver-
ages of 25.2% and 19.8% at 90 days after planting
(Fig. S7 and S8). A one-way ANOVA indicated
that the germination rate and seedling establish-
ment rate of Z. marina seeds planted with the
method of core þ surface were significantly higher
(p < 0.05) than those of Z. marina seeds planted
with other methods at each sampling time point.
Assessment of the successful
establishment of new patches
Retention of Z. marina seeds planted using the
PSCs þ 0 cm burial method ranged from 28 to
74% with an average of 58% 5 months after
planting, which was similar to that of Z. marina
seeds planted using the PSCs þ 2 cm burial
method, where the range was 14 to 76%, and the
average was 54% (Fig. S9).
Seeds planted in cores partially developed into
seedlings with leaves in April 2014 (Fig. S10). No
significant differences were found in seed germin-
ation rate and seedling establishment rate between
Z. marina seeds planted using PSCs þ 0 cm burial
and PSCs þ 2 cm burial methods in April 2014; the
averages were 49% and 30%, respectively (Fig. 1).
No marked differences were found in seedling mor-
phometrics between the two planting treatments,
with averages of 5.4 cm for shoot height, 3.0 cm for
leaf length, and 1.9 cm for root length.
Random samples obtained each month showed
that shoot density increased steadily from May to
September, with a maximum value of 377 shoots
m2 for plants grown in the PSCs þ 0 cm burial
346 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
Figure 2. Shoot density of Zostera marina plants grown in the
PSCs þ 0 cm burial and PSCs þ 2 cm burial treatments from
May to September 2014. Data are expressed as means ± S.D.

above error bar indicates significant difference between plant-
ing treatments in the same sampling month (p < 0.05).
treatment and 357 shoots m 2 for plants grown in
the PSCs þ 2 cm burial treatment (Fig. 2 and Fig.
S11). No significant differences were found in shoot
density between the two planting treatments in each
sampling month, except May and July 2014.
The biomass of Z. marina plants increased
rapidly from May to July, after which it leveled
off, and a significant decrease was observed from
August to September 2014 (Fig. 3). The max-
imum values of 81.5 g dry wt m2 for plants
grown in the PSCs þ 0 cm burial treatment and
82.2 g dry wt m2 for plants grown in the PSCs
þ 2 cm burial treatment were recorded in August
2014. No significant differences were found in
biomass between the two planting treatments in
each sampling month, except July 2014.
The shoot height and leaf length of Z. marina
plants exhibited similar trends to the biomass,
with significant increases from May to August,
and decreases between August and September
2014. Internode length displayed a rapid increase
from May to September 2014, with a maximum
value of 1.6 cm. No significant differences were
found in shoot height, leaf length, and internode
length between the two planting treatments in
each sampling month, except June and July 2014
for leaf length (Fig. S12).
Costs
The total cost of collection, processing and
storage for 28 500 viable Z. marina seeds was
Figure 3. Biomass of Zostera marina plants grown in the
PSCs þ 0 cm burial and PSCs þ 2 cm burial treatments from
May to September 2014. Data are expressed as means ± S.D.

above error bar indicates significant difference between plant-
ing treatments in the same sampling month (p < 0.05).
$570, or $0.02 per seed, while the cost of making
and filling each PSC was $0.22. At a rate of 250
PSCs per day per person, the cost of burying
each PSC was $0.25, resulting in a total dispersal
cost of $0.47 per PSC. The cost per seed
depended on the number of seeds enclosed in
each PSC. For the seed density used in this study
(50 seeds per PSC), the cost was $0.03. The cost
per ha depended on the number of seeds dis-
persed per ha. Based on the seeding density (494
000 seeds ha1) reported by Busch et al. (2010),
the cost of seeding 1 ha of suitable sea bed was
$14 820 ha1.
Discussion
In seagrass systems, the period between seed
germination and seedling establishment is one of
the most vulnerable phases for successful repro-
duction via seed (Alagna et al., 2013; Jarvis and
Moore, 2015). The protective cores used in the
present study not only prevented seeds and seed-
lings from settling in an unsuitable habitat but
also prevented deep burial, predation, and losses,
and resulted in nearly 60% retention of seeds
from preparation to initial sampling 5 months
later. Moreover, the sediment with 75% silt and
clay content proved to be the most suitable sub-
strate for the growth and establishment of Z. mar-
ina transplants (Zhang et al., 2015b), thereby
promoting the growth of plants in association
 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
with an increase in the availability of nutrients
(Halun et al., 2002).
Although seeds of some Potamogeton species
are capable of surviving desiccation (Ailstock
et al., 2010), our results demonstrated that
Z. marina seeds can only tolerate a short expos-
ure period of desiccation (<3 days). Seagrass
seeds are usually recalcitrant seeds which are
sensitive to dryness, as they have a moisture con-
tent of >30%; and even a short exposure period
induces cell-membrane dysfunction and accumu-
lation of harmful substances such as intracellular
toxins, resulting in the loss of seed viability (Park
et al., 2014).
Several studies have found that seed burial can
greatly improve seed germination and seedling
establishment of Z. marina with burial depths
ranging from 1 to 1.5 cm in laboratory experi-
ments (Jarvis and Moore, 2015), and from 2 to
3 cm in field experiments (Marion and Orth, 2010,
2012). For example, Marion and Orth (2012) con-
ducted a manipulative field experiment in
Chesapeake Bay and found that the seedling estab-
lishment rate of Z. marina seeds buried at 2 to
3 cm (average 22.5%) was significantly higher
than that of seeds planted at the sediment surface
(average 2.6%) 6 months after seeding, and that
this was largely due to seed and seedling loss. The
seedling establishment rate obtained 6 months
after seeding in the present study (30%) confirms
that seed burial is beneficial to establishment of
Z. marina seedlings at a suitable burial depth.
The germination rate of Z. marina seeds
planted in the protective cores ranged from 26%
to 70%, with an average of 49% six months after
planting. This germination rate was considerably
higher than that achieved in most previous seed-
sowing field experiments, when generally less
than 40% of the seeds germinated (Orth et al.,
2003, 2009). Furthermore, the seedling establish-
ment rate of Z. marina in the present study
ranged from 12% to 64%, with an average of
30% after 6 months, which was also higher than
that achieved in previous seed-sowing field
experiments; 0.1% to 6.9% was observed for the
buoy-deployed planting method (Pickerell et al.,
2005; Marion and Orth, 2010), 1.2% to 10.1%
for the mechanical seed planter method (Orth
et al., 2009), and zero to 5.5% for the broadcast
method (Golden et al., 2010; Marion and
Orth, 2010).
347
The present results indicate that, after seedling
emergence, the plants fully developed into new
patches with an average shoot density of 367
shoots m2 in September 2014. The shoot den-
sities were approximately equal to the natural bed
densities of an undisturbed area of the experi-
mental site, where an average of 398 shoots m2
was observed in September 2013 (Qin et al.,
2016). The results also exceeded the shoot den-
sities in the burlap bag protection method (361.0
shoots m2 11 months after planting) reported by
Zhang et al. (2015a) and the small gauze bag pro-
tection method (350.0 shoots m 2 11 months
after planting) reported by Zhao et al. (2016).
The results of the present study indicated that the
seed core method for the dispersal of Z. marina
seeds is an efficient technique for growing
Z. marina from seeds.
To compare the financial investments made
using different restoration methods, the total costs
were divided by the total number of viable seeds
or shoots planted. Several studies have shown
that the costs of seed planting methods were sig-
nificantly lower than those of plant transplant-
ation methods (Table S1). The cost per shoot in
previous shoot transplant trials ranged from $0.29
to $0.39, whereas the cost per seed in previous
seed dispersal trials ranged from $0.10 to $0.22.
Further, the cost per seed for the seed core
method used here was only $0.03.
The total cost of large-scale Z. marina restor-
ation depends on plant density and restoration
area, and it can be calculated by multiplying res-
toration area by seed costs per ha. The cost of
seeds per ha is equal to the number of seeds
needed multiplied by the cost per seed, which
includes materials, labor, and travel expenses.
The cost per ha varies greatly from approxi-
mately $14 000 to more than $110 000 per ha
due to the manpower and material resources
involved in different restoration methods.
However, direct comparison of the costs of proj-
ects is difficult because of regional differences in
the costs of materials, and especially, labor in
this study.
Conclusions
Seed planting methods have the potential to
facilitate large-scale seagrass restoration projects,
however, the low seedling establishment rates
348 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
remain a bottleneck. In the present study, we
developed a protective core method for Z. marina
seed dispersal. New patches formed by the seeds
were fully developed 11 months after planting
with a maximum shoot density of 367 shoots
m2. Economic analysis revealed that the cost
per seed was significantly lower than the costs
reported for previous Z. marina shoot transplant
and seed dispersal trials. The results indicated
that the seed core method is an efficient and cost-
effective restoration technique for growing
Z. marina from seeds. In particular, we developed
a mechanical seed planter to make the protective
core and successfully applied it to the large-scale
seagrass restoration projects in Shandong
Province. Therefore, the seed core method has
valuable potential to improve the efficiency of
large-scale restoration projects. In general, restor-
ation success through planting seeds is highly site
specific, further studies may be necessary for
repeated tests at more potential sites.
Acknowledgments
We thank Qian Zhang, Qiang Xu, and
Lezheng Qin for their assistance during sample
collection and calculations.
Funding
This study was supported by funds from the
National Key R&D Program of China
(2019YFD0902101), Fundamental Research
Funds for the Central Universities (201822021)
and National Natural Science Foundation of
China (41576112).
References
Ailstock, M.S., Shafer, D.J., and Magoun, A.D., 2010.
Protocols for use of Potamogeton perfoliatus and Ruppia
maritima seeds in large-scale restoration. Restoration
Ecology 18, 560–573. doi:10.1111/j.1526-100X.2010.
00696.x
Alagna, A., T.V. Fernandez, A. Terlizzi, and F. Badalamenti.
2013. Influence of microhabitat on seedling survival and
growth of the mediterranean seagrass Posidonia oceanica
(l.) Delile. Estuarine Coastal and Shelf Science 119,
119–125. doi:10.1016/j.ecss.2013.01.009
Busch, K.E., Golden, R.R., Parham, T.A., Karrh, L.P.,
Lewandowski, M.J., and Naylor, M.D., 2010. Large-scale
Zostera marina (eelgrass) restoration in Chesapeake Bay,
Maryland, USA. Part I, a comparison of techniques and
associated costs. Restoration Ecology 18, 490–500. doi:
10.1111/j.1526-100X.2010.00690.x
Cullen-Unsworth, L., and Unsworth, R., 2013. Seagrass
meadows, ecosystem services, and sustainability.
Environment: Science and Policy for Sustainable
Development 55 (3), 14–28. doi:10.1080/00139157.2013.
785864
Duarte, C.M., Losada, I.J., Hendriks, I.E., Mazarrasa, I., and
Marba, N., 2013. The role of coastal plant communities
for climate change mitigation and adaptation. Nature
Climate Change 3, 961–968. doi:10.1038/nclimate1970
Golden, R.R., Busch, K.E., Karrh, L.P., Parham, T.A.,
Lewandowski, M.J., and Naylor, M.D., 2010. Large-scale
Zostera marina (eelgrass) restoration in Chesapeake Bay,
Maryland, USA. Part II: a comparison of restoration meth-
ods in the Patuxent and Potomac Rivers. Restoration
Ecology 18, 501–513. doi:10.1111/j.1526-100X.2010.
00691.x
Greve, T.M., Krause-Jensen, D., Rasmussen, M.B., and
Christensen, P.B., 2005. Means of rapid eelgrass (Zostera
marina L.) recolonization in former dieback areas.
Aquatic Botany 82, 143–156. doi:10.1016/j.aquabot.2005.
03.004
Halun, Z., Terrados, J., Borum, J., Kamp-Nielsen, L., Duarte,
C.M., and Fortes, M.D., 2002. Experimental evaluation of
the effects of siltation-derived changes in sediment condi-
tions on the Philippine seagrass Cymodocea rotundata.
Journal of Experimental Marine Biology and Ecology
279, 73–87. doi:10.1016/S0022-0981(02)00366-0
Harwell, M.C., and Orth, R.J., 1999. Eelgrass (Zostera marina
L.) seed protection for field experiments and implications
for large-scale restoration. Aquatic Botany 64, 51–61. doi:
10.1016/S0304-3770(99)00008-X
Jarvis, J.C., and Moore, K.A., 2015. Effects of seed source,
sediment type, and burial depth on mixed-annual and per-
ennial Zostera marina L. seed germination and seedling
establishment. Estuaries and Coasts 38, 964–978. doi:10.
1007/s12237-014-9869-3
Jarvis, J.C., Moore, K.A., and Kenworthy, W.J., 2014.
Persistence of Zostera marina L. (eelgrass) seeds in the
sediment seed bank. Journal of Experimental Marine
Biology and Ecology 459, 126–136. doi:10.1016/j.jembe.
2014.05.024
Marion, S.R., and Orth, R.J., 2010. Innovative techniques for
large-scale seagrass restoration using Zostera marina (eel-
grass) seeds. Restoration Ecology 18, 514–526. doi:10.
1111/j.1526-100X.2010.00692.x
Marion, S.R., and Orth, R.J., 2012. Seedling establishment in
eelgrass: seed burial effects on winter losses of developing
seedlings. Marine Ecology Progress Series 448, 197–207.
doi:10.3354/meps09612
Orth, R.J., Harwell, M.C., Bailey, E.M., Bartholomew, A.,
Jawad, J.T., Lombana, A.V., Moore, K.A., Rhode, J.M.,
and Woods, H.E., 2000. A review of issues in seagrass
seed dormancy and germination: implications for conser-
vation and restoration. Marine Ecology Progress Series
200, 277–288. doi:10.3354/meps200277
 Xie et al. / Aquatic Ecosystem Health and Management 23 (2020) 341–349
Orth, R.J., Fishman, J.R., Harwell, M.C., and Marion, S.R.,
2003. Seed-density effects on germination and initial seed-
ling establishment in eelgrass Zostera marina in the
Chesapeake Bay region. Marine Ecology Progress Series
250, 71–79. doi:10.3354/meps250071
Orth, R.J., Carruthers, T.J.B., Dennison, W.C., Duarte, C.M.,
Fourqurean, J.W., Heck, K.L., Hughes, A.R., Kendrick,
G.A., Kenworthy, W.J., Olyarnik, S., Short, F.T.,
Waycott, M., and Williams, S.L., 2006a. A global crisis
for seagrass ecosystems. Bioscience 56, 987–996. doi:10.
1641/0006-3568(2006)56[987:AGCFSE]2.0.CO;2
Orth, R.J., Marion, S.R., Granger, S., and Traber, M.S., 2009.
Evaluation of a mechanical seed planter for transplanting
Zostera marina (eelgrass) seeds. Aquatic Botany 90,
204–208. doi:10.1016/j.aquabot.2008.07.004
Park, J.I., Lee, K.S., and Son, M.H., 2014. Germination rate
of Phyllospadix japonicus seeds relative to storage meth-
ods and periods. Ocean Science Journal 49, 67–72. doi:10.
1007/s12601-014-0007-8
Pickerell, C.H., Schott, S., and Wyllie-Echeverria, S., 2005. Buoy-
deployed seeding: demonstration of a new eelgrass (Zostera
marina L.) planting method. Ecological Engineering 25,
127–136. doi:10.1016/j.ecoleng.2005.03.005
Qin, L.Z., Li, W.T., Zhang, X.M., Zhang, P.D., and Qiao,
W.M., 2016. Recovery of the eelgrass Zostera marina fol-
lowing intense Manila clam Ruditapes philippinarum har-
vesting disturbance in China: the role and fate of
seedlings. Aquatic Botany 130, 27–36. doi:10.1016/j.aqua-
bot.2016.01.002
349
Traber, M.S., Granger, S., and Nixon, S., 2003. Mechanical
seeder provides alternative method for restoring eelgrass
habitat (Rhode Island). Ecological Restoration 21,
213–214.
Waycott, M., Duarte, C.M., Carruthers, T.J.B., Orth, R.J.,
Dennison, W.C., Olyarnik, S., Calladine, A., Fourqurean,
J.W., Heck, K.L., Hughes, A.R., Kendrick, G.A.,
Kenworthy, W.J., Short, F.T., and Williams, S.L., 2009.
Accelerating loss of seagrasses across the globe threatens
coastal ecosystems. Proceedings of the National Academy
of Sciences of the United States of America 106 (30),
12377–12381. doi:10.1073/pnas.0905620106
Zhang, P.D., Fang, C., Liu, J., Xu, Q., Li, W.T., and Liu,
Y.S., 2015a. An effective seed protection method for
planting Zostera marina (eelgrass) seeds: implications for
their large-scale restoration. Marine Pollution Bulletin 95,
89–99. doi:10.1016/j.marpolbul.2015.04.036
Zhang, Q., Liu, J., Zhang, P.D., Liu, Y.S., and Xu, Q., 2015b.
Effect of silt and clay percentage in sediment on the sur-
vival and growth of eelgrass Zostera marina: transplant-
ation experiment in Swan Lake on the eastern coast of
Shandong Peninsula, China. Aquatic Botany 122, 15–19.
doi:10.1016/j.aquabot.2015.01.001
Zhao, J.S., Y.S. Liu, Zhang, P.D., Li, W.T., and Fang C.,
2016. Assessment of the establishment success of eelgrass
Zostera marina (Alismatales: Zosteraceae) from seeds in a
cost-effective seed protection method: implications for
large-scale restoration. Botanica Marina 59(4), 259–2 doi:
10.1515/bot-2016-0028