Eelgrass seed after-ripening method

Abstract

The invention belongs to the technical field of sea grass culture, and particularly relates to an eelgrass seed after-ripening method. The invention provides an eelgrass seed after-ripening method which comprises the following steps: collecting eelgrass reproductive branches, and removing main branches of the eelgrass reproductive branches to obtain eelgrass reproductive branch branches with Buddhist bracts; hanging the eelgrass reproduction branch branches in seawater for after-ripening to obtain eelgrass seeds. According to the method, the main branches of the eelgrass reproduction branches are removed, nutrient resource consumption of redundant tissues to the branches is reduced, more favorable conditions are created for development of the Buddha bracts and the seeds, the perfectness ratio and the seed maturity of the Buddha bracts in the after-ripening period are increased by 30% or above, the seed recovery rate is increased to 90% or above, the seed activity reaches 100%, the cost is reduced by 30% compared with that of a conventional method, and the method is suitable for large-scale popularization and application. After sowing, the seedling establishment rate exceeds 50%, the overall effect far exceeds that of a conventional collection and after-ripening mode, and the method is easy to operate, low in cost and more suitable for large-scale repair and reconstruction of the seaweed bed.

Description

A method for post-ripening eelgrass seeds Technical Field

[0001] This invention belongs to the field of seaweed cultivation technology, specifically relating to a method for the post-ripening of eelgrass seeds. Background Technology

[0002] Seagrass is a type of monocotyledonous herbaceous plant widely distributed in tropical and temperate seas. Seagrass beds, formed by large-scale aggregations of seagrass, play a vital ecological role. The average carbon sequestration rate of seagrass bed ecosystems is as high as 83 g C / m³. 2 / a, the total carbon sequestration is equivalent to 10% to 15% of the total global ocean carbon sequestration. Although the global seagrass bed area is only about 0.15% of the ocean area, its average annual net primary productivity can be as high as 10¹² g DW / m². 2 Seagrass beds provide habitats, nurseries, and shelters for a wide range of marine life. In addition, most seagrasses have well-developed root systems and strong rhizomes that form a crisscrossing network of roots underground, which helps to stabilize sediments and bottom mud.

[0003] However, under the multiple pressures of natural environmental changes and human activities, seagrass beds are showing a serious trend of degradation. Simply relying on the natural growth and propagation of seagrass is insufficient to meet the needs for rapid recovery; therefore, artificial restoration of seagrass beds is extremely necessary. Artificial restoration methods for seagrass beds can be divided into two main categories: plant transplantation and seed sowing. Seed sowing has advantages such as small seed size, convenient transportation, and minimal damage to the donor seagrass bed, and has become a hot topic in research on large-scale seagrass bed restoration methods. However, seagrass seeds are only produced in specific seasons and need to be collected at the appropriate time and undergo post-ripening to obtain high-quality seeds.

[0004] Eelgrass (Zostera marina) is a dominant seagrass species widely distributed in temperate waters of my country and a commonly used core species for seagrass bed ecological restoration. Currently, the main methods for collecting and processing eelgrass reproductive branches and for seed after-ripening involve: determining the timing of manual collection based on the appearance and development of the reproductive branches; after collection, multiple processes are required to obtain spathes containing only seeds; and then the spathes are directly placed in seawater for seed after-ripening. However, the existing process is prone to a series of problems: approximately 20% of seeds are empty, about 18% are rotten, and seed viability decreases during direct after-ripening in the sea, resulting in a low yield. Furthermore, the process of manually collecting eelgrass reproductive branches and processing them to obtain spathes is cumbersome, time-consuming, and labor-intensive, significantly increasing labor and time costs. Therefore, there is an urgent need to provide a method for eelgrass seed after-ripening that can increase seed yield and reduce costs. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the post-ripening of eelgrass seeds, which can reduce costs and obtain eelgrass seeds with high maturity and high recovery rate.

[0006] This invention provides a method for the after-ripening of eelgrass seeds, comprising the following steps: collecting eelgrass reproductive branches, removing the main branches of the eelgrass reproductive branches to obtain eelgrass reproductive branch branches with spathes; suspending the eelgrass reproductive branch branches in seawater for after-ripening to obtain eelgrass seeds.

[0007] As a preferred embodiment, the seawater surface temperature is 23~25℃ when collecting the reproductive branches of Eelgrass.

[0008] As a preferred embodiment, the conditions for collecting *Erigeron brevis* reproductive branches include collecting them from areas with dense *Erigeron brevis* reproductive branches; the density of reproductive branches in the dense *Erigeron brevis* reproductive branch area is ≥300 plants / m². 2 .

[0009] As a preferred embodiment, the criterion for the end of post-ripening is a spathe splitting rate of ≥90%.

[0010] As a preferred embodiment, the post-ripening time is 1.5 to 2 months.

[0011] As a preferred embodiment, the suspension in seawater includes suspension in the eelgrass habitat marine environment.

[0012] As a preferred embodiment, the marine environment of the eel grassland is located at a distance of 0.5 to 1.0 meters from the seabed.

[0013] As a preferred embodiment, the suspension includes: placing the eelgrass reproductive branches into a net bag and suspending the net bag to a fixing device.

[0014] As a preferred embodiment, the mesh bag has an aperture of 0.5~1.0 mm.

[0015] As a preferred embodiment, the fixing device includes a buoyancy float 4, a rope 5, a net bag 6, an anchor rope 7, and a fixing anchor 8.

[0016] Beneficial Effects: This invention provides a method for the after-ripening of seagrass seeds, comprising the following steps: collecting seagrass reproductive branches, removing the main branches of the seagrass reproductive branches to obtain branches of the seagrass reproductive branches with spathes; suspending the branches of the seagrass reproductive branches in seawater for after-ripening to obtain seagrass seeds. This invention removes the main branches of the seagrass reproductive branches, reducing the consumption of nutrient resources by redundant tissues on the branches, creating more favorable conditions for the development of spathes and seeds, and increasing the spathe integrity rate and seed maturity by more than 30% during after-ripening. Example results show that using the method described in this invention, the maturity of seagrass seeds is increased by 35%, the seed recovery rate is increased to over 90%, seed vigor reaches 100%, the cost is reduced by 30% compared to conventional methods, and the seedling establishment rate after sowing exceeds 50%. The overall effect far exceeds that of conventional collection and after-ripening methods. Furthermore, this method is simple to operate, low in cost, and more suitable for the large-scale restoration and reconstruction of seagrass beds. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 is a schematic diagram of the reproductive branches of *Erigeron annuus*; where 1 is the main branch of the reproductive branch; 2 is a branch of the reproductive branch; and 3 is the spathe. Figure 2 is a comparison of the collection areas and seed after-ripening time of different reproductive branches. Figure 3 is a schematic diagram of the suspension and fixing device for the reproductive branches of *Erigeron annuus*; where 2 is a branch of the reproductive branch; 4 is a buoyancy float; 5 is a rope; 6 is a net bag; 7 is an anchor rope; and 8 is a fixing anchor. Figure 4 shows the seed yield and dry weight per 100 seeds under different collection periods and after-ripening methods; where A is the seed yield and B is the dry weight per 100 seeds. Figure 5 shows the seedling establishment rate and plant height at different collection periods for different after-ripening methods; where A is the seedling establishment rate and B is the plant height. Figure 6 shows the seedling yield and principal component analysis comprehensive score at different collection periods; where A is the seedling yield and B is the factor score for each collection period. Detailed Implementation

[0019] This invention provides a method for the after-ripening of eelgrass seeds, comprising the following steps: collecting eelgrass reproductive branches, removing the main branches of the eelgrass reproductive branches to obtain eelgrass reproductive branch branches with spathes; suspending the eelgrass reproductive branch branches in seawater for after-ripening to obtain eelgrass seeds.

[0020] This invention involves collecting reproductive branches of *Erigeron annuus* to obtain reproductive branches. The main branch of the *Erigeron annuus* reproductive branch of this invention bears reproductive branches, and each reproductive branch bears several spathes. These reproductive branches include lateral branches and terminal branches. The main branch of the reproductive branch is the "stem axis" of the reproductive branch, or it can also be referred to as the "main axis" of the reproductive branch. A schematic diagram of the *Erigeron annuus* reproductive branch is shown in Figure 1, where 1 is the main branch of the *Erigeron annuus* reproductive branch, i.e., the stem axis or main axis of the reproductive branch; 2 is a reproductive branch with a spathe; and 3 is the spathe of *Erigeron annuus*. The reproductive branches and spathes shown in Figure 1 (2 and 3) are for illustrative purposes only and are not fully labeled. As one implementation method, the conditions for collecting *Erigeron annuus* reproductive branches include collection in areas with dense *Erigeron annuus* reproductive branches; the density of reproductive branches in the dense *Erigeron annuus* reproductive branch area is ≥300 plants / m². 2 In this invention, a reproductive branch containing one main branch (i.e., one stem axis) is defined as one plant, and the density of the reproductive branches is 1m². 2 The number of internal reproductive branches. This invention selects a reproductive branch density ≥300 branches / m². 2 Collecting eelgrass reproductive branches in designated areas ensures that seagrass beds maintain their genetic diversity and population continuity through sexual reproduction, preventing irreversible damage to the natural recovery process caused by low-density collection. On the other hand, this density standard also takes into account seed collection efficiency, satisfying the practicality of manual collection while providing sufficient seed sources for seagrass bed restoration.

[0021] In one implementation method, the sea surface temperature is 23-25°C when collecting *Erigeron annuus* reproductive branches. The sea surface temperature referred to in this invention refers to the seawater temperature within a range of no more than 10 meters below the sea surface. The flowering and seed-setting process of *Erigeron annuus* reproductive branches is significantly affected by seawater temperature, and the seed maturation process is accompanied by changes in seawater temperature. Therefore, this invention comprehensively considers core factors such as dynamic changes in environmental temperature, seed maturity, and expected harvest yield to define the suitable time for collecting reproductive branches. Through coupling key environmental factors, key seed indicators, and seedling establishment effects, this embodiment of the invention found that *Erigeron annuus* seeds begin to appear in early June, and the sea surface temperature is approximately 23-25°C, which is suitable for collecting reproductive branches for after-ripening. The sea surface temperature of 23-25°C mentioned in this invention corresponds to the period from early to mid-July to early August, when *Erigeron annuus* seeds are mature. Collecting reproductive branches at this stage can reduce the empty shell rate and increase the seed yield.

[0022] After collecting the reproductive branches of *Erigeron annuus*, this invention removes the main branches of the reproductive branches to obtain branches of the reproductive branches with spathes. In one embodiment, the branches of the *Erigeron annuus* reproductive branches have intact, unopened spathes. Removing the main branches of the reproductive branches reduces the consumption of nutrient resources by redundant tissues on the branches, creating more favorable conditions for the development of spathes and seeds. This results in an increase of over 30% in spathe integrity and seed maturity during after-ripening, and a reduction in seed decay rate.

[0023] After obtaining the reproductive branches of *Erigeron brevis*, this invention suspends these branches in seawater for after-ripening to obtain *Erigeron brevis* seeds. As one embodiment, suspending them in seawater includes suspending them in the native marine environment of the *Erigeron brevis*. This invention utilizes the natural adaptive ecological conditions of the in-situ habitat, such as water temperature, salinity, and photoperiod, to reduce the adverse effects of environmental stress on the seeds and effectively ensure the seed after-ripening process.

[0024] In one embodiment, the suspension includes: placing the eelgrass reproductive branches into a net bag and suspending the net bag to a fixing device. In another embodiment, the mesh size of the net bag is 0.5~1.0 mm; this invention limits the mesh size to be smaller than the minor diameter of the eelgrass seed to prevent seed loss. In yet another embodiment, the eelgrass reproductive branches occupy 50% of the net bag's volume. This proportion prevents the reproductive branches from crowding and piling up, and reduces the labor costs of deployment and retrieval in the sea area.

[0025] In one embodiment, the fixing device includes a buoyancy float 4, a rope 5, a net bag 6, an anchor rope 7, and a fixed anchor 8, as shown in Figure 3. The core feature of the fixing device of this invention is that the net bag is precisely suspended at a position 0.5-1.0 m above the seabed through the combination of an anchor rope and a buoyancy float. The anchor rope 7 is fixed at both ends by the fixed anchor 8 anchored to the seabed, keeping the anchor rope 7 close to the seabed. The anchor rope 7 is connected to the rope 5. The anchor rope 7 can connect to multiple ropes 5, preferably two ropes 5, with an interval of 1.0-1.5 m between adjacent ropes 5. One end of the rope 5 is connected to the buoyancy float 4, which floats on the sea surface. The net bag 6 is fixed to the rope 5, allowing the net bag to be submerged in seawater. In one embodiment, the rope is a nylon rope. In one embodiment, the net bag is located at a position 0.5-1.0 m above the seabed. During the direct after-ripening process of eelgrass seeds in the sea area, the seeds are prone to a decrease in viability to about 80% due to exposure to air, or to being preyed upon by crabs due to their proximity to the seabed, thus reducing the effective yield of seeds. The fixing device described in this invention uses the flexibility of nylon ropes to adapt to ocean current fluctuations, and the pull of buoyancy floats to keep the net bag stably suspended in the seawater. This avoids the risk of seeds being preyed upon or buried by benthic organisms when they are close to the seabed, and also prevents problems such as dehydration, embryo activity decline, and reduced seed viability caused by prolonged exposure to air. It simplifies the operation while improving the quality and survival rate of after-ripening seeds.

[0026] In one implementation method, the standard for the end of after-ripening is a spathe splitting rate ≥90%. In another implementation method, the after-ripening period is 1.5–2 months. The production, maturation, and shedding of *Erigeron annuus* seeds have certain seasonal and phenological characteristics. Determining seed collection time solely based on the developmental state of reproductive branches cannot fully reflect the internal maturity of the seeds, potentially missing the optimal collection time and leading to inconsistent seed quality. This invention uses a spathe splitting rate ≥90% to determine the end of the after-ripening process, which can improve seed maturity and recovery rate.

[0027] As one implementation method, after the post-ripening is completed, the process further includes: removing the reproductive branches from the net bag to obtain eelgrass seeds. This invention does not impose any special limitations on the separation method of the eelgrass seeds; conventional separation methods in the art can be used. In a specific embodiment of this invention, removing the reproductive branches from the net bag includes: transferring the contents of the net bag into a bucket of water, repeatedly separating the remaining reproductive branches and seeds in the net bag using the seawater specific gravity method, removing the reproductive branches, and obtaining eelgrass seeds.

[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening, the steps are as follows: (1) In mid-June, reproductive branches with complete spathes are artificially collected from a natural Eriocaulon buergerianum bed. The collection time is 30 min each time (Examples 1-6 and Comparative Examples 1-19 of this invention limit the collection time of Eriocaulon buergerianum reproductive branches to 30 min, mainly to eliminate the interference of collection time on experimental results, and to avoid the actual number of spathes harvested deviating from the true difference of density gradient due to the collection time of a certain group being too long or too short, so as to ensure that the experimental data are related to the target variables such as "reproductive branch density" and "after-ripening method", thereby accurately verifying the influence of core variables such as different reproductive branch densities and different after-ripening times on seed yield and germination rate); the reproductive branch density of the natural Eriocaulon buergerianum bed is 300 plants / m². 2(2) Treatment of reproductive branches: Remove the main branches from the artificially collected Eriocaulon buergerianum reproductive branches in step (1), and retain only the reproductive branches with complete spathes, that is, retain the secondary branches with lateral spathes attached. The reproductive branches with complete spathes are shown in the black box in Figure 1. (3) Seed after-ripening in the sea area: Place the treated reproductive branches in the net bag into the net bag at 50% of the net bag volume. The net bag has a mesh diameter of 1.0 mm, which is smaller than the short diameter of the Eriocaulon buergerianum seed. Subsequently The net bag was submerged to a depth of 1.0m from the seabed using an underwater fixing device (see Figure 3) for post-ripening. After 1.5 months of post-ripening, the spathe splitting rate was ≥90% and the post-ripening was completed. The anchor rope 7 of the underwater fixing device is connected to two ropes 5, and each rope 5 is connected to a net bag 6 (the same below). (4) Collection of mature seeds: After the post-ripening cycle of step (3) is completed, the contents of the net bag are transferred into a water bucket, and the residual reproductive branches and seeds in the net bag are repeatedly sieved using the seawater specific gravity method.

[0030] Example 2: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that after-ripening is performed for 2 months until the spathe splitting rate is ≥90% in step (3) after the seed has splitted.

[0031] Example 3: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 400 plants / m². 2 .

[0032] Example 4: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 3, except that after-ripening is performed for 2 months until the spathe splitting rate is ≥90% in step (3).

[0033] Example 5: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 500 plants / m². 2 .

[0034] Example 6: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 5, except that after-ripening is performed for 2 months until the spathe splitting rate is ≥90% in step (3).

[0035] Comparative Example 1: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 100 branches / m². 2 In step (3), after one month of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0036] Comparative Example 2: A method for collecting and processing reproductive branches of *Erigeron annuus* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 100 branches / m². 2 In step (3), after 1.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0037] Comparative Example 3 describes a method for collecting and processing reproductive branches of *Erigeron annuus* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 100 branches / m². 2 In step (3), after 2 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0038] Comparative Example 4 describes a method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 100 branches / m². 2 In step (3), after 2.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0039] Comparative Example 5 describes a method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 100 branches / m². 2 In step (3), after 3 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0040] The reproductive branches of *Erigeron brevis* used in Comparative Examples 1-5 were collected from the same batch.

[0041] Comparative Example 6 describes a method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 200 branches / m². 2 In step (3), after one month of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0042] Comparative Example 7: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 200 branches / m². 2 In step (3), after 1.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0043] Comparative Example 8: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 200 branches / m². 2 In step (3), after 2 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0044] Comparative Example 9: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 200 branches / m².2 In step (3), after 2.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0045] Comparative Example 10: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 200 branches / m². 2 In step (3), after 3 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0046] The reproductive branches of *Erigeron brevis* used in Comparative Examples 6-10 were collected from the same batch.

[0047] Comparative Example 11 describes a method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that after-ripening is performed for one month until the spathe splitting rate is ≥90% before the end of the ripening process.

[0048] Comparative Example 12: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that in step (3), after-ripening is performed for 2.5 months until the spathe splitting rate is ≥90% before the end of the ripening.

[0049] Comparative Example 13 describes a method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are similar to those in Example 1, except that after-ripening is performed for 3 months until the spathe splitting rate is ≥90% before the end of the ripening process.

[0050] The reproductive branches of *Erigeron brevis* used in Examples 1-2 and Comparative Examples 11-13 were collected from the same batch.

[0051] Comparative Example 14: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 400 branches / m². 2 In step (3), after one month of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0052] Comparative Example 15: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 400 branches / m². 2 In step (3), after 2.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0053] Comparative Example 16: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 400 branches / m². 2 In step (3), after 3 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0054] The reproductive branches of *Erigeron brevis* used in Examples 3-4 and Comparative Examples 14-16 were collected from the same batch.

[0055] Comparative Example 17: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 500 plants / m². 2 In step (3), after one month of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0056] Comparative Example 18: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 500 branches / m². 2 In step (3), after 2.5 months of ripening to achieve a spathe splitting rate of ≥90%, the ripening is completed.

[0057] Comparative Example 19: A method for collecting and processing reproductive branches of *Eriocheir sinensis* and for seed after-ripening. The steps are similar to those in Example 1, except that the density of reproductive branches in step (1) is 500 branches / m². 2 In step (3), after 3 months of ripening, the spathe splitting rate is ≥90% and then the ripening is completed.

[0058] The eelgrass reproductive branches used in Examples 5-6 and Comparative Examples 17-19 were collected from the same batch.

[0059] The seed yields of *Erigeron annuus* in Examples 1-6 and Comparative Examples 1-19 were statistically analyzed, and the results are shown in Table 1 and Figure 2. The data in the tables are the average values ​​from five replicate experiments. After two months of after-ripening, the reproductive branch density was 300 plants / m². 2 Mature seeds obtained from the high-density area were more abundant than those from the low-density area (100 plants / m²). 2 The seed yield was 31.1% higher. Additionally, after a post-ripening period exceeding 2.5 months, the empty seed shell rate reached 10.5%. However, during the post-ripening process, in Examples 5, 7, and 12, one net bag each was not securely fixed, resulting in seeds sticking to the bottom and being exposed to air. The seeds in the air-exposed bags had a viability of 70%, while the bags sticking to the bottom showed varying degrees of damage, leading to a higher seed loss rate. These three net bags were not included in the seed yield statistics. Therefore, the optimal density of reproductive branches for collection should not be less than 300 plants / m². 2 The seed after-ripening time is 1.5 to 2 months, and the net bag should be suspended in the water during the after-ripening process.

[0060] Table 1. Effects of different reproductive branch densities and seed after-ripening time on the yield of *Erigeron annuus* seeds (×10⁻¹⁰). 3 The influence of particles

[0061] Example 7 A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening, the steps are as follows: (1) Reproductive branch collection: From the time range from the beginning of seed production to the basic completion of seed shedding, on June 1, June 15, July 1, July 15 and August 1, respectively, at a reproductive branch density of not less than 300 plants / m². 2 (1) Collect reproductive branches with complete spathes from natural eelgrass beds. Each collection lasts for 30 minutes. (2) Treatment of reproductive branches: Remove the main branches (i.e., the main axis of the reproductive branches) from the reproductive branches collected in step (1) and retain only the branches with complete spathes, i.e., the secondary branches attached to the lateral spathes. (3) Seed after-ripening in the sea area: Place the treated reproductive branches in the net bag at 50% of the net bag volume. The net bag diameter is smaller than the short diameter of the seed, which is 1.0 mm. Then, use an underwater fixing device to immerse the net bag to 1.0 m from the seabed, so that the net bag is suspended in the water (Figure 3) and allows it to ripen in the in-situ sea area environment. After 2 months of ripening, the spathe splitting rate is ≥90% and the ripening ends. (4) Collection of mature seeds: After the ripening cycle of step (3) is completed, transfer the contents of the net bag into a water bucket and use the seawater specific gravity method to repeatedly sieve away the reproductive branches and seeds remaining in the net bag.

[0062] Comparative Example 20: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are the same as in Example 7, except that: (2) Reproductive branch processing: The main branches and branches of the reproductive branches collected in step (1) are removed, leaving only the complete spathe.

[0063] The surface water temperature and surface sunshine duration at different collection periods of *Erigeron annuus* reproductive branches in Example 7 and Comparative Example 20 were statistically analyzed, and the results are shown in Table 2. Simultaneously, the seed yield and dry weight per 100 seeds of *Erigeron annuus* in Example 7 and Comparative Example 20 were statistically analyzed, and the results are shown in Table 3 and Figure 4. (Figure 4 shows...) The uppercase and lowercase letters indicate significant differences between the two ripening methods at the same collection time, respectively, indicating significant differences between different seed collection periods for the same ripening method.

[0064] Table 2 Key Environmental Conditions at Different Collection Periods

[0065] Table 3 Seed yield and 100-seed dry weight under different collection periods and after-ripening methods

[0066] The results showed that seed availability initially increased and then decreased, while the dry weight per 100 seeds showed an increasing trend. The highest seed availability was observed in early July, with the seed availability under two after-ripening methods—artificial spathe removal and no spathe removal—being 82.5% and 77.2% higher than in early June, respectively (Figure 4). Regarding the dry weight per 100 seeds, the highest and most similar values ​​were observed in mid-July and early August. Temperature and sunshine duration were key environmental factors affecting reproductive branch growth and seed maturation, with surface water temperature being the core factor.

[0067] Example 8: Effects of Different Collection Periods and After-Ripening Methods on Seed Germination and Seedling Establishment. Mature *Eriocheir sinensis* seeds obtained in Example 7 and Comparative Example 20 were sterilized and disinfected. The sterilized seeds were then stratified for one month in 4°C water under dark conditions to obtain stratified *Eriocheir sinensis* seeds. The sowing method employed was the small-bag sowing method. A composite substrate with a mud-to-sand ratio of 3:1 was filled into cotton mesh bags (length × width = 20 cm × 20 cm, mesh diameter 0.5 mm), with a mesh diameter smaller than the seed's short diameter, filling half the bag's volume. Stratified *Eriocheir sinensis* seeds were sown into the cotton mesh bags, 200 seeds per bag. These cotton mesh bags containing *Eriocheir sinensis* seeds and the composite substrate were then sown in natural sea areas at a depth of 3–5 cm until initial *Eriocheir sinensis* seedlings were established. For each treatment, a total of 6 mesh bags were sown in natural sea areas, and the experiment was repeated 12 times.

[0068] The seed germination rate and seedling establishment rate were calculated using the formulas for seed germination rate and seedling establishment rate. The results are shown in Table 4 and Figure 5. All data represent the average of 12 replicate experiments. (Figure 5 shows...) The uppercase and lowercase letters indicate significant differences between the two ripening methods at the same collection time, respectively, indicating significant differences between different seed collection periods for the same ripening method.

[0069] Seed germination rate = (Number of germinated seeds / Number of seeds sown) × 100; Seedling establishment rate = (Number of established seedlings / Number of seeds sown) × 100.

[0070] The results showed that the seed germination rate and seedling establishment rate increased. The highest germination rate and seedling establishment rate were observed in seeds collected in early August. Significant differences were found in seedling establishment rates between artificially removing the spathe and not removing it, with the seedling establishment rate reaching 39.1% in the latter method (Figure 5). R language was used to calculate factor scores for each collection period, integrating key seed indicators and seedling establishment effects. OriginLab 2025b was used to fit the factor scores and seawater temperatures at different collection periods. The comprehensive evaluation concluded that early June, when *Erigeron annuus* seeds begin to appear and the seawater surface temperature is approximately 23–25°C, is the ideal time to collect seeds. The optimal after-ripening method is to not remove the spathe and select branches with spathes for after-ripening (Figure 6).

[0071] Table 4 Comparison of seed germination rate and seed formation rate between different seed collection strategies and after-ripening methods for Eelgrass

[0072] Example 9 A method for collecting and processing reproductive branches of Eelgrass and for seed after-ripening, comprising the following steps: (1) Reproductive branch collection: using the method described in this invention, that is, when the surface seawater temperature reaches 24℃, and the density of reproductive branches is not less than 300 plants / m 2 (1) Collect reproductive branches with complete spathes from natural eelgrass beds. Each collection lasts for 30 minutes. (2) Treatment of reproductive branches: Remove the main branches (main axis of reproductive branches) from the reproductive branches collected in step (1) and retain only the branches with complete spathes (secondary branches attached to lateral spathes). (3) Seed after-ripening in the sea area: Place the treated reproductive branches in the net bag at 50% of the net bag volume. The net bag diameter is smaller than the short diameter of the seed, which is 1.0 mm. Then, use an underwater fixing device to immerse the net bag to 1.0 m from the seabed, so that the net bag is suspended in the water (Figure 3) and allows it to ripen in the in-situ sea area environment. After 2 months of ripening, the spathe splitting rate is ≥90% and the ripening ends. (4) Collection of mature seeds: After the ripening cycle of step (3) is completed, transfer the contents of the net bag into a water bucket and use the seawater specific gravity method to repeatedly sieve away the reproductive branches and seeds remaining in the net bag.

[0073] Comparative Example 21: A method for collecting and processing reproductive branches of Eriocaulon buergerianum and for seed after-ripening. The steps are the same as in Example 9, except that: (2) Reproductive branch processing: The reproductive branches collected in step (1) are not processed in any way (the main axis, branches and spathe of the reproductive branches are retained).

[0074] Comparative Example 22: A method for collecting and processing reproductive branches of Eelgrass and for seed after-ripening. The steps are the same as in Example 9, except that: (1) Reproductive branch collection: The conventional method is used, that is, the time for artificial collection of Eelgrass reproductive branches is determined based on the appearance and development status of the reproductive branches as judged by humans, when the density of reproductive branches is not less than 300 plants / m². 2 Reproductive branches with intact spathes were artificially collected from natural eelgrass beds, with each collection session lasting 30 minutes.

[0075] Comparative Example 23: A method for collecting and processing reproductive branches of Eelgrass and for seed after-ripening. The steps are the same as those in Comparative Example 21, except that: (1) Reproductive branch collection: The conventional method is adopted, that is, the time for artificial collection of Eelgrass reproductive branches is determined based on the appearance and development status of the reproductive branches as judged by humans, when the density of reproductive branches is not less than 300 plants / m². 2 Reproductive branches with intact spathes were artificially collected from natural eelgrass beds, with each collection session lasting 30 minutes.

[0076] The seed yields of *Erigeron annuus* in Examples 9 and Comparative Examples 21-23 were statistically analyzed, and the results are shown in Table 5. The seed yield varied significantly under different collection times and after-ripening methods. Under the reproductive branch collection time conditions described in this invention, the average seed yield was 1.4 times that of manual judgment (conventional method), and the average seed yield under the after-ripening method without removing the spathe was 1.3 times that of the reproductive branches without any treatment. This further demonstrates the feasibility of the reproductive branch collection and treatment and seed after-ripening methods described in this invention.

[0077] Table 5 Seed yield under different after-ripening methods

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for the post-ripening of eelgrass seeds, characterized in that, Includes the following steps: Collect eelgrass reproductive branches, remove the main branches of the eelgrass reproductive branches to obtain eelgrass reproductive branch branches with spathes; hang the eelgrass reproductive branch branches in seawater for after-ripening to obtain eelgrass seeds.

2. The method according to claim 1, characterized in that, When collecting the reproductive branches of Eelgrass, the surface temperature of the seawater was 23~25℃.

3. The method according to claim 1, characterized in that, The conditions for collecting eelgrass reproductive branches include collecting them from areas with dense eelgrass reproductive branches; the density of reproductive branches in the dense eelgrass reproductive branch area is ≥300 plants / m². 2 .

4. The method according to claim 1, characterized in that, The standard for the end of post-ripening is that the spathe splitting rate is ≥90%.

5. The method according to claim 1 or 4, characterized in that, The post-ripening period is 1.5 to 2 months.

6. The method according to claim 1, characterized in that, The suspension in seawater includes suspension in the marine environment of the eelgrass habitat.

7. The method according to claim 6, characterized in that, The eel grassland is located in the marine environment at a distance of 0.5 to 1.0 meters from the seabed.

8. The method according to claim 1, 6, or 7, characterized in that, The suspension process includes placing the reproductive branches of the eelgrass into a net bag and suspending the net bag to a fixing device.

9. The method according to claim 8, characterized in that, The mesh size of the bag is 0.5~1.0mm.

10. The method according to claim 8, characterized in that, The fixing device includes a buoyancy float (4), a rope (5), a net bag (6), an anchor rope (7), and a fixing anchor (8).

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