A method for cultivating a spiny amaranth-like plant

Abstract

The application discloses a kind of cultivation methods of Sargassum siliquastrum. It is related to the field of seaweed factory seedling. The cultivation method of Sargassum siliquastrum includes the following steps: S1, Sargassum siliquastrum is taken, disinfected and crushed to obtain Sargassum siliquastrum granules; S2, the Sargassum siliquastrum granules are scattered on the attachment base; S3, the attachment base is placed in oligotrophic water and incubated until the Sargassum siliquastrum granules grow stolons and rhizoids and firmly attach to the attachment base; S4, the attachment base with Sargassum siliquastrum is transferred to seawater environment for cultivation. By crushing into granules and optimizing the culture conditions, the seedling period of Sargassum siliquastrum is shortened to about 40 days from scattering to complete attachment, which is more than 33% shorter than the traditional tissue culture method (about 60 days). The bottleneck of long seedling period and low efficiency in traditional technology is solved, and the time requirement of factory batch seedling can be met.

Description

Technical Field

[0001] This invention relates to the field of industrialized seaweed seedling cultivation technology, and in particular to a method for cultivating *Gnaphalium affine*. Background Technology

[0002] *Pterocladiella capillacea*, a representative species of the family Pterocladiaceae in the order Gynostemales of the phylum Rhodophyta, occupies a unique position in marine ecosystems due to its biological characteristics and ecological adaptability. This species typically exhibits a slender, branching structure; the thallus is purplish-red or light brown, reaching several centimeters to over ten centimeters in height. Its dense and elastic branches allow it to maintain stable attachment to the intertidal zone under the impact of waves. As a typical representative of subtropical seaweed, *Pterocladiella capillacea* has specific requirements for its growth environment, preferring shallow sea areas with water temperatures of 15–25℃ and exhibiting a certain tolerance to salinity variations. This has led to its dominance in the intertidal reef areas of Fujian, Guangdong, and Guangxi provinces along the Chinese coast. Its life cycle includes multiple stages, such as sporophyte, gametophyte, and carposporophyte, each with different requirements for light, nutrient concentration, and water flow velocity. This complex life history presents a natural challenge for artificial cultivation.

[0003] Agar, as the main polysaccharide component of the cell wall of *Gnaphalium affine*, possesses unique gelling properties that make it irreplaceable in the biomedical, food, and cosmetic fields. In the biomedical field, agar, due to its excellent biocompatibility and biodegradability, is often used as a matrix for cell culture scaffolds, drug sustained-release carriers, and tissue engineering materials. In the food industry, it is widely used as a natural thickener, stabilizer, and coagulant in the production of products such as jellies, yogurt, and meat products. In the cosmetics industry, agar's moisturizing properties and colloidal stability make it an important ingredient in high-end skincare products. Compared with other seaweed-derived gels, *Gnaphalium affine* agar's advantages are not only reflected in its gel strength and temperature stability, but its low ash content and high transparency also significantly improve the quality of end products, making *Gnaphalium affine* a favored raw material across all stages of the industry chain.

[0004] The agar industry is currently facing a profound contradiction between resource supply and market demand. With the rapid expansion of the biomedical materials market and the rise of health food consumption trends, global demand for agar is growing at an average annual rate of double digits, while the supply of raw materials is constrained by the limited availability of wild seaweed resources.

[0005] Breakthroughs in artificial aquaculture technology are considered a key path to overcoming this predicament, and the seedling stage, as the starting point of the aquaculture industry chain, directly determines the feasibility of industrialization based on its technological maturity. Currently, seedling technology for *Gnaphalium affine* mainly falls into two categories: spore cultivation and tissue culture cultivation, both of which face insurmountable technical bottlenecks. Spore cultivation technology is based on natural reproduction patterns, inducing algae to release spores, which then attach and germinate on artificial substrates, ultimately cultivating seedlings. However, spore release from *Gnaphalium affine* is regulated by multiple environmental factors, including photoperiod, temperature fluctuations, and nutrient saline solution. While spore release can be achieved under laboratory conditions, it is difficult to stably control the release efficiency in large-scale production, leading to significant batch-to-batch variations. Furthermore, the spore germination to seedling stage takes 3-4 months, requiring the maintenance of highly precise water quality parameters and incurring high management costs. Tissue culture cultivation technology achieves rapid reproduction through in vitro tissue culture, shortening the seedling cycle to about 2 months, but its core challenge lies in controlling contamination from other algae. In open cultivation systems, diatoms, green algae, and other miscellaneous algae compete with the seedlings of *Gnaphalium affine* for survival resources. Once a contamination outbreak occurs, the entire batch of seedlings may be wiped out. This high-risk characteristic makes companies cautious about large-scale application.

[0006] Therefore, there is an urgent need to develop a factory-scale artificial cultivation method for *Gnaphalium affine* that features rapid propagation, open cultivation, and low pollution risk, in order to solve the problems existing in current artificial breeding. Summary of the Invention

[0007] The purpose of this invention is to develop a method for the industrialized artificial cultivation of *Gnaphalium affine* that features rapid propagation, open cultivation, and low pollution risk.

[0008] The first aspect of the present invention is:

[0009] A method for cultivating *Gnaphalium affine* is provided.

[0010] The second aspect of the present invention is:

[0011] Application of the cultivation method of the aforementioned *Gnaphalium affine*.

[0012] Specifically, the technical solution adopted according to the first aspect of the present invention is as follows:

[0013] A method for cultivating *Gnaphalium affine* includes the following steps:

[0014] S1 Take *Pteris vittata*, and after disinfection and pulverization, obtain *Pteris vittata* algal particles;

[0015] S2 Spreads the *Pseudo-chicken feather thallus* particles onto the substrate;

[0016] S3 The attachment substrate is placed in oligotrophic water and cultured statically until the *Pseudo-chicken feather thallus* granules grow stolons and rhizoids and firmly attach to the attachment substrate.

[0017] S4. The attachment substrate with the attached *Pseudo-chicken feather algae* is transferred to a seawater environment for cultivation.

[0018] The oligotrophic water body was prepared by inoculating *Gracilaria zhangensis* in an indoor cement tank at a density of 0.8-1.2 kg / m³. 3 After culturing for 12-18 days, the oligotrophic water body was obtained.

[0019] According to embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0020] This invention shortens the seedling growth cycle of *Hymenochloa chinensis* from sowing to full attachment to about 40 days by crushing it into granules and optimizing the cultivation conditions. This is more than 33% shorter than the traditional tissue culture seedling method (about 60 days). It solves the bottleneck of "long seedling cycle and low efficiency" in traditional technology and can meet the time requirements of industrialized mass seedling production.

[0021] This invention utilizes a pre-absorbed water nutrient environment (with significantly lower concentrations of nitrogen, phosphorus, and other nutrients than ordinary seawater) created by Gracilaria zhangi, which specifically inhibits the growth of other algae. Meanwhile, the *Gnaphalium affine* granules, due to their tolerance to low nutrient levels, can still develop normally. In actual cultivation, the biomass of other algae is far lower than in traditional eutrophic environments, thus improving the purity of *Gnaphalium affine* seedlings and solving the core problem of "competition among other algae leading to seedling contamination" in large-scale seedling production.

[0022] In this invention, the *Pseudo-featherwort* thallus, after being pulverized, produces algal particles that can fit the gaps in the attachment substrate. During static cultivation, it rapidly grows stolons and rhizoids, thus firmly attaching to the substrate. This significantly improves the effectiveness compared to traditional spore-based seedling cultivation, ultimately ensuring a high survival rate of seedlings from indoor cultivation to outdoor aquaculture. After indoor cultivation, the attachment substrate containing the *Pseudo-featherwort* thallus can be transferred to any seawater environment for cultivation.

[0023] According to one embodiment of the present invention, the disinfectant used for disinfection is trichloroisocyanuric acid, preferably, the concentration of the trichloroisocyanuric acid is 0.8-1.2 g / L, and the disinfection time is 0.5-1.5 min.

[0024] According to one embodiment of the present invention, the disinfection involves immersing the *Gnaphalium affine* in the disinfectant. Preferably, the immersion time is 0.5-1.5 minutes. The present invention defines highly efficient and low-toxicity disinfection parameters, avoiding damage to the algae while killing attached microorganisms. Immersion in 0.8-1.2 g / L trichloroisocyanuric acid for 0.5-1.5 minutes is effective for disinfection, and the short treatment time reduces the impact on the activity of algal cells.

[0025] According to one embodiment of the present invention, the disinfection further includes the following steps: soaking the disinfected *Gnaphalium affine* in seawater and stirring, repeating the seawater soaking 4-5 times. Preferably, the seawater soaking time is 20-40 seconds for the first two soakings and 1.5-3 minutes for the next 2-3 soakings. The present invention, by controlling the seawater soaking time in stages, thoroughly removes residual disinfectant and avoids continued toxicity to the algae.

[0026] According to one embodiment of the present invention, the device used for pulverizing is a pulverizer.

[0027] According to one embodiment of the present invention, the specific operation of the pulverization process is as follows: *Pteris vittata* is placed in a pulverizer, and seawater is added to the pulverizer just enough to cover the *Pteris vittata*. The pulverizer is stopped for 0.5-0.6 minutes every 1-1.2 minutes of operation, and this cycle is repeated 2-4 times before filtration. The present invention avoids damaging the algae's activity by controlling the temperature rise of the pulverization process.

[0028] According to one embodiment of the present invention, the filtration process in the pulverization process is performed using a filter screen with a pore size of 0.3-0.7 cm. The present invention limits the pore size of the filter screen to limit the size of the algal particles, thereby adapting to the gaps in the substrate and improving the adhesion efficiency.

[0029] According to one embodiment of the present invention, the filter screen has a pore size of 0.5 cm. The particles obtained by the 0.5 cm pore size filter screen can be trapped in the gaps of the seedling curtain ropes, preventing them from falling off.

[0030] According to one embodiment of the present invention, the attachment substrate is a fiber-woven seedling curtain. The present invention uses a seawater-resistant blended material to ensure the stability and reusability of the attachment substrate.

[0031] According to one embodiment of the present invention, the fiber includes at least one of vinylon and polyethylene.

[0032] According to one embodiment of the present invention, the size of the seedling curtain is 20-25cm × 50-55cm. A suitable size for the seedling curtain allows it to be adapted to factory-produced cement ponds.

[0033] According to one embodiment of the present invention, in step S2, the distribution density of the *Pseudo-Gynostemma pentaphyllum* thallus particles on the substrate is 100-300 particles / dm². Overlapping of the *Pseudo-Gynostemma pentaphyllum* thallus particles should be avoided, otherwise it will affect the adhesion to the substrate.

[0034] According to one embodiment of the present invention, the temperature for static cultivation is 20-30°C; and / or the light intensity is 2500-3300 Lux. Insufficient light is detrimental to algal development, while excessive light easily leads to the growth of unwanted algae. Appropriate light intensity is beneficial to algal development and inhibits unwanted algae.

[0035] According to one embodiment of the present invention, the temperature for static culture is preferably 25-26°C, and the light intensity is preferably 2500-2800 Lux.

[0036] According to one embodiment of the present invention, the static culture time is 35-40 days.

[0037] According to one embodiment of the present invention, the water level fluctuation during the static culture period does not exceed ±2 cm. The water level fluctuation refers to the range of water level changes in the aquaculture environment.

[0038] According to one embodiment of the present invention, the substrate is fixed in the indoor cement tank by a bracket, the bracket being 5-10 cm below the water surface. Preferably, the bracket is constructed of PVC pipe. The bracket's height being 5-10 cm below the water surface ensures the substrate is completely submerged, and the appropriate water depth places the algal particles in the upper layer of the water, allowing for more sufficient light and dissolved oxygen.

[0039] According to one embodiment of the present invention, the timing for transferring the thallus to a seawater environment for cultivation is when any *Pterocarya pseudobulb* thallus particle grows three or more stolons with a length of 0.8-1.2 cm.

[0040] Specifically, the technical solution adopted according to the second aspect of the present invention is as follows:

[0041] An attachment substrate for cultivating *Gnaphalium affine* is woven from vinylon-polyethylene blended rope and measures 20-25cm × 50-55cm.

[0042] Another aspect of the present invention relates to an industrialized seedling system using the described *Gnaphalium affine* cultivation method, comprising an indoor cement tank, a *Gracilaria zhangense* inoculation unit, an attachment substrate support, and an environmental control unit, wherein the *Gracilaria zhangense* inoculation unit is used to inoculate *Gracilaria zhangense* in the indoor cement tank, and the environmental control unit is used to regulate temperature and light.

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the discovery. Attached Figure Description

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0045] Figure 1 This is a photograph of the seedling curtain in Example 1, which was sown with *Gnaphalium affine* thallus particles.

[0046] Figure 2 This is a photograph of the *Gnaphalium affine* cultivated in an oligotrophic environment as described in Example 1.

[0047] Figure 3 This is a photograph of *Gnaphalium affine* grown in a nutrient-rich environment, used in the comparative study. Detailed Implementation

[0048] The terms "preferred," "more preferred," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0049] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0050] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0051] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0052] Example 1

[0053] A method for cultivating *Gnaphalium affine* includes the following steps:

[0054] S1. Rinse the *Syngonium spp.* clean. Immerse the cleaned *Syngonium spp.* in a 1 g / L solution of trichloroisocyanuric acid in seawater for 1 minute to disinfect it. After disinfection, remove the *Syngonium spp.* and soak and stir it in seawater. Repeat this process 4 times with fresh seawater to remove any residual disinfectant from the algae. The first and second rinses should not exceed 30 seconds, and the third and fourth rinses should be 2 minutes each. Then, place the disinfected *Syngonium spp.* into a pulverizer and add seawater just enough to cover the algae. Stop the pulverizer for 0.5 minutes every 1 minute of operation to avoid overheating and damaging the algae. After three cycles of operation, filter the algae through a 0.5 cm pore size filter to obtain *Syngonium spp.* algae particles.

[0055] S2. The *Pterocarya pseudobulb* thallus particles are evenly spread on the seedling tray (a 20cm x 50cm seedling board woven from vinylon-polyethylene blended rope) as evenly as possible, ensuring the thallus particles do not overlap. A picture of the seedling tray with the *Pterocarya pseudobulb* thallus particles is shown below. Figure 1 As shown; Gracilaria zhangi cultured in seawater was added to an indoor cement tank, with a density of 1 kg / m³. 3 After about 15 days of cultivation, an oligotrophic water body is obtained; a rectangular support frame is built using PVC pipes with a diameter of 3.2cm, which is 8cm lower than the water surface of the cement pond. The seedling curtain is tied to the support frame with cable ties to fix the seedling curtain.

[0056] S3 seedling curtains inoculated with *Pseudobulbus simulans* thallus particles were placed in oligotrophic water and cultured statically for 40 days. Indoor culture conditions were 25℃ and 2800 Lux light, until most of the *Pseudobulbus simulans* thallus particles had grown multiple stolons about 1 cm long, which firmly attached themselves to the seedling ropes of the seedling curtain via rhizoids, resulting in... Figure 2 The image shows *Gnaphalium affine* cultivated in an oligotrophic environment.

[0057] S4 transferred seedlings with *Gnaphalium affine* attached to the curtains to a marine environment for cultivation.

[0058] from Figure 2 It can be seen that in the oligotrophic environment, only *Gnaphalium affine* firmly adheres to the seedling tray, without any other miscellaneous algae. This indicates that the oligotrophic environment obtained under specific culture conditions can specifically inhibit the growth of miscellaneous algae, while *Gnaphalium affine* particles can still develop normally due to their tolerance to low nutrient salts. This improves the purity of *Gnaphalium affine* seedlings and solves the core problem of "competition among miscellaneous algae leading to seedling contamination" in large-scale factory seedling production.

[0059] Example 2

[0060] A method for cultivating *Gnaphalium affine* includes the following steps:

[0061] S1. Rinse the *Syngonium spp.* clean. Immerse the cleaned *Syngonium spp.* in a seawater solution of 1.2 g / L trichloroisocyanuric acid for 0.5 min for disinfection. After disinfection, remove the *Syngonium spp.* and soak and stir it in seawater. Repeat this process 4 times with fresh seawater to remove any residual disinfectant from the algae. The first and second rinses should not exceed 30 s, and the third and fourth rinses should be 1.5 min. Then, place the disinfected *Syngonium spp.* into a pulverizer and add seawater just enough to cover the algae. Stop the pulverizer for 0.5 min every 1 min to avoid overheating and damaging the algae. After three cycles, filter the algae through a 0.3 cm pore size filter to obtain *Syngonium spp.* algae particles.

[0062] S2. The *Gnaphalium affine* thallus particles are evenly spread on the seedling tray (a 20cm x 50cm seedling board woven from vinylon-polyethylene blended rope), ensuring minimal overlap between the thallus particles. *Gracilaria zhangense* is cultured in seawater in an indoor cement tank at a density of 0.8 kg / m³. 3 After about 18 days of cultivation, an oligotrophic water body is obtained; a rectangular support frame 6cm lower than the water surface of the cement pond is built using PVC pipes with a diameter of 3.2cm; the seedling curtain is tied to the support frame with cable ties to fix the seedling curtain.

[0063] S3 placed the seedling curtains sown with *Pterocarya pseudobulb* thallus particles in a low-nutrient water body and cultured them statically for 40 days. The indoor culture conditions were a temperature of 25℃ and a light intensity of 2800 Lux, until most of the *Pterocarya pseudobulb* thallus particles grew multiple stolons of about 1 cm and were firmly attached to the seedling ropes of the seedling curtain through rhizoids.

[0064] S4 transferred seedlings with *Gnaphalium affine* attached to the curtains to a marine environment for cultivation.

[0065] Comparative Example

[0066] The difference between the comparative example and Example 1 is that in the comparative example, *Gnaphalium affine* was cultured in nutrient-rich seawater.

[0067] Specifically:

[0068] A method for cultivating *Gnaphalium affine* includes the following steps:

[0069] S1. Rinse the *Syngonium spp.* clean. Immerse the cleaned *Syngonium spp.* in a 1 g / L solution of trichloroisocyanuric acid in seawater for 1 minute to disinfect it. After disinfection, remove the *Syngonium spp.* and soak and stir it in seawater. Repeat this process 4 times with fresh seawater to remove any residual disinfectant from the algae. The first and second rinses should not exceed 30 seconds, and the third and fourth rinses should be 2 minutes each. Then, place the disinfected *Syngonium spp.* into a pulverizer and add seawater just enough to cover the algae. Stop the pulverizer for 0.5 minutes every 1 minute of operation to avoid overheating and damaging the algae. After three cycles of operation, filter the algae through a 0.5 cm pore size filter to obtain *Syngonium spp.* algae particles.

[0070] S2. Spread the *Pteris vittata* algae particles as evenly as possible on the seedling curtain (the seedling curtain is a 20cm*50cm seedling board woven from vinylon-polyethylene blended rope), ensuring that the algae particles do not overlap as much as possible; add seawater to the indoor cement tank and let it stand for 15 days to obtain eutrophic water; use 3.2cm diameter PVC pipes to build a rectangular support 8cm below the water surface of the cement tank, and tie the seedling curtain to the support with cable ties to fix the seedling curtain;

[0071] S3 seedlings seeded with *Pterocarya pseudobulb* thallus particles were placed in eutrophic water and cultured statically for 40 days. The indoor culture conditions were a temperature of 25℃ and a light intensity of 2800 Lux, yielding the following results: Figure 3 The image shows *Gnaphalium affine* cultivated in a nutrient-rich environment.

[0072] S4 transferred seedlings with *Gnaphalium affine* attached to the curtains to a marine environment for cultivation.

[0073] from Figure 3 It can be seen that in the eutrophic environment, in addition to *Gnaphalium affine*, there are also a large number of other seaweeds in the seedling trays. The seedling trays are seriously polluted, which is not conducive to mass seedling cultivation.

[0074] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for cultivating *Phyllostachys edulis*, characterized in that: Includes the following steps: S1 Take *Pteris vittata*, and after disinfection and pulverization, obtain *Pteris vittata* algal particles; S2 Spreads the *Pseudo-chicken feather thallus* particles onto the substrate; S3 The attachment substrate is placed in oligotrophic water and cultured statically until the *Pseudo-chicken feather thallus* granules grow stolons and rhizoids and firmly attach to the attachment substrate. S4. The attachment substrate with the attached *Pseudo-chicken feather algae* is transferred to a seawater environment for cultivation. The oligotrophic water body is prepared by the following method: seawater is added to an indoor cement tank and Gracilaria zhangi is inoculated at a density of 0.8-1.2 kg / m³. After culturing for 12-18 days, the oligotrophic water body is obtained.

2. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The disinfectant used for disinfection is trichloroisocyanuric acid.

3. The method for cultivating *Phyllostachys edulis* according to claim 2, characterized in that: The concentration of the trichloroisocyanuric acid is 0.8-1.2 g / L, and the disinfection time is 0.5-1.5 min.

4. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The specific operation of the crushing process is as follows: place the *Pteris vittata* in a crusher, add seawater to the crusher just enough to cover the *Pteris vittata*, stop the crusher for 0.5-0.6 minutes every 1-1.2 minutes of operation, and repeat the cycle 2-4 times before filtering.

5. The method for cultivating *Phyllostachys edulis* according to claim 4, characterized in that: The filtration process in the pulverization process is performed by using a filter screen with a pore size of 0.3-0.7 cm.

6. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The substrate is a seedling curtain woven from fibers.

7. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The static culture temperature is 20-30℃; and / or the light intensity is 2500-3300 Lux.

8. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The static incubation period is 35-40 days.

9. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The attachment substrate is fixed in the indoor cement pool by a bracket, the bracket being 5-10 cm below the water surface.

10. The method for cultivating *Phyllostachys edulis* according to claim 1, characterized in that: The timing for transferring the thallus to a marine environment is when any *Pterocarya pseudobulb* thallus particle grows three or more stolons with a length of 0.8-1.2 cm.

11. An industrialized seedling system for cultivating *Gnaphalium affine* according to any one of claims 1-9, characterized in that: It includes an indoor cement tank, a Gracilaria zhangi inoculation unit, an attachment substrate support, and an environmental control unit. The Gracilaria zhangi inoculation unit is used to inoculate Gracilaria zhangi in the indoor cement tank, and the environmental control unit is used to regulate temperature and light.

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