Cultivation method of hypnum molpadioides

Abstract

The invention belongs to the technical field of bryophyte cultivation, and particularly discloses a cultivation method of hypnum melioides, which comprises the following steps of: constructing three layers of substrates (the bottom layer is planted with golden stones of 3-5mm, the middle layer is peat soil-Kanuma soil of 1: 0.8-1.2, and the upper layer is plasma treated felt cloth); the broken stems are pretreated with an activating solution containing naphthylacetic acid and chitosan; after inoculation, culturing in a full-artificial light environment (800-1200lux, the ratio of blue light to red light is 1: (2-3)), and intermittently spraying to maintain the humidity to be 85-90%; a nutrient solution containing nitrogen, phosphorus, potassium and trace elements is sprayed every week; and the COO is enriched to 800-1000 ppm in the seedling stage. According to the method, within 21 days, the coverage degree is larger than 80%, the mildew rate is smaller than 3%, the biomass increment reaches 48.7 mg / cm, the water utilization efficiency is improved by 223%, and the method is suitable for large-scale production of vertical greening walls and ecological restoration projects.

Description

Technical Field

[0001] This invention belongs to the field of bryophyte cultivation technology, specifically relating to an efficient cultivation method for *Bryophytum comosum*. Background Technology

[0002] *Eurohypnum leptothallum*, a plant belonging to the genus *Eurohypnum* in the family Bryophyceae, is drought-tolerant and heat-resistant. It can absorb vehicle exhaust and dust, effectively improving urban air quality. It also plays a role in regulating soil and water during short-term heavy rainfall. In rocky desertification remediation, *Eurohypnum leptothallum* has demonstrated strong erosion resistance, effectively reducing soil loss and increasing enzyme activity in the cultivated soil, resulting in high water and soil conservation benefits. Furthermore, *Eurohypnum leptothallum* requires no pruning, resulting in lower maintenance costs and making it suitable for improving the urban environment. Unlike vascular plants, *Eurohypnum leptothallum* does not rely on soil; it absorbs water and nutrients directly from the environment through its leaves. Traditional agricultural cultivation techniques such as field tillage, soil fertilization, and mechanical irrigation are not suitable for *Eurohypnum leptothallum*.

[0003] Currently, large-scale moss cultivation needs to meet three core requirements simultaneously: high survival rate of propagules, rapid greening capacity within weeks, and adaptability to the substrate environment. Although existing technologies use dense substrates such as sponge cloth to improve water retention, excessive water retention can easily lead to gametophyte rot due to lack of oxygen. Even if a breathable substrate such as pumice is used to improve aeration, insufficient water retention can cause drying and death. Furthermore, natural light cultivation is affected by seasonal fluctuations, with light intensity deviations exceeding 40%, resulting in unstable chlorophyll synthesis. While stem cutting propagation is superior to mass planting, the germination time of unactivated stem cuttings can be as long as 3 weeks or more, making them prone to mold and affecting the health of the plants.

[0004] Therefore, there is an urgent need to develop a cultivation method for *Bryum micranthum* with low mold rate and rapid seedling growth. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention proposes a cultivation method for *Bryophytum comosum*.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem: A method for cultivating *Moss sphagnum* includes the following steps: (1) A three-layer composite matrix structure is adopted. The bottom layer is plant stone with a particle size of 3-5mm, the middle layer is a water-retaining layer of peat soil and Kanuma soil mixed in a mass ratio of 1:(0.8-1.2), and the top layer is a felt carrier sterilized by steam at 100℃ or above. (2) Take gametophytes of *Bryum fasciatus* and cut them into stems with a length of 0.5-1.0 cm. Soak them in an activation solution containing 0.1-0.3 mg / L naphthaleneacetic acid and 0.05-0.1% chitosan for 30-120 minutes. (3) Spread the activated stem fragments evenly on the surface of the felt carrier at a density of 200-300 fragments / cm², then cover with a black light-blocking film, maintain a temperature of 18-22℃ and a humidity of 85%-90%, spray intermittently for 5-10 minutes every 1-2 hours, and cultivate under a light intensity of 800-1200 lux, with 10-12 hours of light per day; (4) Spray nutrient solution once a week. When the coverage reaches 80%, remove the shade net and reduce the humidity to 75%-80%. Continue to cultivate until the community thickness reaches more than 5mm.

[0007] Specifically, in step (4) during the seedling stage, the CO2 concentration is maintained at 800-1000 ppm.

[0008] Furthermore, in the above-mentioned cultivation method of *Moss sphagnum*, in step (1), the thickness ratio of the three-layer composite substrate is bottom layer: middle layer: top layer = (1.5-2.0):(0.8-1.0):(0.05-0.1).

[0009] Furthermore, in the above-mentioned cultivation method of *Moss sphagnum*, in step (1), the felt carrier is plasma-treated, with a surface contact angle ≤30° and a water absorption rate ≥400%.

[0010] Furthermore, in the above-mentioned cultivation method of *Bryum micranthum*, in step (2), the pH value of the activation solution is 5.5-6.0, and the conductivity is ≤100μS / cm.

[0011] Furthermore, in the above-mentioned cultivation method of *Moss sphagnum*, in step (3), the intermittent spraying uses a micron-level atomizing nozzle with a droplet diameter of 20-50 μm and a water pressure of 0.2-0.3 MPa.

[0012] Furthermore, in the above-mentioned cultivation method of *Moss sphagnum*, in step (3), the light source used for illumination is a blue-red composite LED, with the intensity ratio of blue light wavelength 450nm to red light wavelength 660nm being 1:(2-3), and the spectral range being limited to the 400-700nm photosynthetically active radiation (PAR) range.

[0013] Furthermore, in the above-mentioned cultivation method of *Moss sphagnum*, in step (4), the nutrient solution consists of: ammonium nitrate 10-50 mg / L, potassium dihydrogen phosphate 10-30 mg / L, magnesium sulfate 5-10 mg / L, chelated iron 2-5 mg / L, vitamin B1 0.1-0.3 mg / L, selenomethionine 0.01-0.03 mg / L, and cobalamin 0.005-0.01 mg / L; the solvent is deionized water.

[0014] This invention also discloses the application of the above method in vertical green walls, moss bonsai, or ecological restoration projects.

[0015] Compared with the prior art, the present invention has the following outstanding advantages: This invention utilizes a composite matrix and an activation solution containing naphthaleneacetic acid and chitosan to pre-treat stubble of *Moss hygroscopicus*, reducing the traditional 75-day seedling growth cycle to 21 days with a coverage rate exceeding 85%, effectively improving the germination rate of the cut stems. Furthermore, the method employs a blue-red light ratio to enhance chlorophyll stability, and combined with chelated iron nutrient solution, significantly improves drought tolerance, resulting in a survival rate of up to 91% for *Moss hygroscopicus*. This method is suitable for ecological restoration in arid regions. Additionally, the *Moss hygroscopicus* cultivated using this method exhibits high peel strength, allowing for direct installation on 90° vertical walls, thus reducing the cost of green plant maintenance.

[0016] The composite matrix of this invention uses recycled cotton textile waste to prepare a felt carrier, which is green and environmentally friendly, and its three-layer structure effectively reduces water consumption. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include 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 features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0019] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0020] The raw materials involved in the embodiments of the present invention are shown in Table 1 below: Table 1 Raw Material Parameter Table Example 1 An efficient cultivation method for *Moss sphagnum* includes the following steps: 1. The matrix consists of three layers, as shown below: Bottom layer: 3mm diameter gold-bearing stone, 15mm thickness.

[0021] Middle layer: Peat and Kanuma soil are mixed at a mass ratio of 1:0.8, with a thickness of 8mm.

[0022] Top layer: Plasma-treated felt (contact angle 28°, water absorption rate 420%), thickness 0.05mm.

[0023] 2. Stem pretreatment: The gametophytes of *Bryophytum comosum* were cut into 0.5 cm sections and soaked in an activation solution (pH 5.5, conductivity 95 μS / cm) for 30 minutes. The activation solution contained 0.1 mg / L naphthaleneacetic acid and 0.05% chitosan.

[0024] 3. Inoculation and cultivation: Stem segments were scattered at a density of 200 segments / cm², covered with a black shading film with a shading rate >99%, and placed in an environment with a temperature of 18℃±1℃ and a humidity of 85%±3%. Intermittent spraying for 5 minutes every 2 hours (droplet diameter 20μm, water pressure 0.2MPa) was applied. Cultivation was carried out under a light intensity of 800lux±5%, with 10 hours of light per day. Light source: Blue-red LEDs (450nm:660nm=1:2).

[0025] 4. Nutritional supplements: Spray nutrient solution weekly (the nutrient solution includes ammonium nitrate 50mg / L, potassium dihydrogen phosphate 30mg / L, magnesium sulfate 10mg / L, chelated iron 5mg / L, and vitamin B1 0.1mg / L).

[0026] 5. Seedling Management: On day 21, when the film coverage reached 82%, the humidity was removed, the CO2 concentration was maintained at 800 ppm, and the film was cultured to a thickness of 5.2 mm.

[0027] Example 2 An efficient cultivation method for *Moss sphagnum* includes the following steps: 1. The matrix consists of three layers, as shown below: Bottom layer: 4mm diameter gold-bearing stone, 18mm thickness.

[0028] Middle layer: Peat and Kanuma soil are mixed in a 1:1 mass ratio, with a thickness of 9mm.

[0029] Top layer: Plasma-treated felt (contact angle 25°, water absorption rate 450%), thickness 0.08mm.

[0030] 2. Stem pretreatment: The stem was cut to a length of 0.8 cm, and the activation solution contained 0.15 mg / L naphthaleneacetic acid + 0.08% chitosan (pH 5.8, conductivity 85 μS / cm).

[0031] 3. Inoculation and cultivation: Stem segments were scattered at a density of 250 segments / cm², covered with a black shading film with a shading rate >99%, and placed in an environment with a temperature of 20℃±1℃ and a humidity of 88%±2%. Intermittent spraying for 5 minutes every 2 hours (droplet diameter 35μm, water pressure 0.25Mpa) was applied. Cultivation was carried out under a light intensity of 1000lux±8%, with 11 hours of light per day. Light source: blue-red light intensity ratio 1:2.5.

[0032] 4. Nutritional supplements: Spray nutrient solution weekly (the nutrient solution includes ammonium nitrate 50mg / L, potassium dihydrogen phosphate 30mg / L, magnesium sulfate 10mg / L, chelated iron 5mg / L, vitamin B1 0.1mg / L, selenomethionine 0.01mg / L, and cobalamin 0.005mg / L).

[0033] 5. Seedling Management: On day 20, when the film coverage reached 85%, the humidity was removed, the CO2 concentration was maintained at 900 ppm, and the film was cultured to a thickness of 5.2 mm.

[0034] Example 3 An efficient cultivation method for *Moss sphagnum* includes the following steps: 1. Matrix construction: Bottom layer: 5mm diameter gold-bearing stone, 20mm thickness.

[0035] Middle layer: Peat and Kanuma soil are mixed in a mass ratio of 1:1.2, with a thickness of 10 mm.

[0036] Top layer: Plasma-treated felt (contact angle 22°, water absorption rate 480%), thickness 0.1mm.

[0037] 2. Stem pretreatment: The stem was cut to a length of 1.0 cm, and the activation solution contained 0.2 mg / L naphthaleneacetic acid + 0.1% chitosan (pH 6.0, conductivity 75 μS / cm).

[0038] 3. Inoculation and cultivation: Stem fragments were scattered at a density of 300 segments / cm², covered with a black shading film with a shading rate >99%, and placed in an environment with a temperature of 22℃±1℃ and a humidity of 90%±2%. Intermittent spraying for 5 minutes every 2 hours (droplet diameter 50μm, water pressure 0.3Mpa) was applied. Cultivation was carried out under a light intensity of 1200lux±10% and 12 hours of light per day. Light source: blue-red light intensity ratio 1:3.

[0039] 4. Nutritional supplements: The nutrient solution composition is the same as in Example 2. 5. Seedling Management: On day 18, when the film coverage reached 88%, the humidity was removed, the CO2 concentration was maintained at 1000 ppm, and the film was cultured to a thickness of 5.2 mm.

[0040] Comparative Example 1 A method for cultivating *Moss sphagnum* differs from Example 2 in that: Omit the three-layer substrate: use only a single felt carrier (0.5 mm thick), and the remaining steps are the same as in Example 2.

[0041] (Results: Coverage was 46% on day 21, and mold rate was 18%).

[0042] Comparative Example 2 A method for cultivating *Moss sphagnum* differs from Example 2 in that: Stem activation omitted: The stems were inoculated directly without being treated with activation solution, and the remaining steps were the same as in Example 2.

[0043] (Result: New greening appeared delayed until day 25, and the coverage was 52% on day 35).

[0044] Comparative Example 3 A method for cultivating *Moss sphagnum* differs from Example 2 in that: Natural light cultivation: Remove the shading film and artificial light source, and use natural light (daily average light intensity fluctuation of 42%).

[0045] The remaining steps are the same as in Example 2. (Results: On day 21, the canopy cover was 61%, and the leaf chlorosis rate was 38%).

[0046] Comparative Example 4 A method for cultivating *Moss sphagnum* differs from Example 2 in that: Replace the substrate composition: Use a 1:1 mixture of vermiculite and peat moss for the middle layer. The remaining steps are the same as in Example 2. (Result: Coverage was 67% on day 21, with localized areas showing water accumulation and mold growth).

[0047] Comparative Example 5 A method for cultivating *Moss sphagnum* differs from Example 2 in that: Nutrient solution deficiency: No special nutrient solution was added throughout the entire process. The remaining steps are the same as in Example 2. (Result: Coverage was 58% on day 21, and gametes were pale yellow).

[0048] Test Example 1 A comparative experiment on the germination rate of severed stems was conducted to verify the synergistic germination-promoting effect of the composite substrate and the activation of severed stems. Experimental methods: Group processing: Examples 1-3 Control group: CK1: Unactivated broken stem + single felt cloth (Comparative Example 1).

[0049] CK2: Activated stem fragments + natural light cultivation (Comparative Example 3).

[0050] Experimental indicators: 1. Record the number of new green spots daily (≥1mm² is considered valid germination). 2. Germination rate formula: Germination rate V = (Number of germinations N in t days) t (÷Number of observation days t) ×100%.

[0051] 3. Chlorophyll a content was measured on day 7 (acetone extraction method, UV detection at 663nm).

[0052] The results are shown in Table 2 below.

[0053] Table 2 Results of the stem germination rate experiment (*P<0.01 vs CK1) Table 2 shows that the germination peak in the example group was advanced by 3.8-8.2 days (compared to CK1), and the activation solution increased the expression level of cytokinin receptors in the severed stem by 2.1 times. The three-layer matrix ensured a low water permeability (12.3±0.8 mm / h) and a low oxygen diffusion coefficient (2.7×10⁻⁶). -6 m 2 The chlorophyll synthesis efficiency was 2.6 times that of CK1, achieving a balance of / s).

[0054] Test Example 2 Drought stress resistance experiments were conducted to evaluate the mitigating effects of light environment and nutrient solution on water stress.

[0055] Experimental methods: Stress treatment: Stop water supply for 48 hours during the seedling stage (80% coverage).

[0056] Grouping: Example 2 vs. Comparative Example 5 (without nutrient solution).

[0057] Testing indicators: 1. Malondialdehyde (MDA) content (thiobarbituric acid method).

[0058] 2. Superoxide dismutase (SOD) activity (NBT photochemical reduction method).

[0059] 3. Survival rate statistics 24 hours after rehydration.

[0060] Table 3 Results of drought stress resistance experiment (*P<0.01 vs Comparative Example 5) As shown in Table 3, blue-red LED (660nm) promoted a 47% increase in proline synthesis, enhanced water retention capacity, activated SOD enzyme activity with chelated iron in the nutrient solution, and reduced MDA damage by 64.5%.

[0061] Test Example 3 Experiments were conducted on the substrate water use efficiency of different cultivation methods to analyze the water-saving effect of the three-layer substrate.

[0062] Experimental methods: Moisture monitoring: A soil moisture sensor (Decagon EC-5) was implanted to record the volumetric water content (VWC) of the middle layer matrix in real time.

[0063] Calculate water use efficiency: Water use efficiency (WUE) = (Biomass increment ΔBiomass) ÷ (Water input).

[0064] ΔBiomass represents the dry weight increment of biomass (mg / cm²); Water Input represents the total amount of water input received by the system or plant, in mL / cm².

[0065] Control group: Example 2 (three-layer matrix) vs. Comparative Example 4 (vermiculite-peat soil mixed matrix).

[0066] Table 4. Results of Water Use Efficiency (*P<0.01 vs Comparative Example 4) Table 4 shows that the bottom layer of the planting stone shortened the water retention time to 15±2 min (Comparative Example 4: 52±5 min), reducing the risk of anaerobic conditions. The pore distribution in the middle layer of Kanuma soil-peat soil was optimized (78% of the pores were 10-100 μm in diameter), resulting in a 223% increase in water retention efficiency (WUE).

[0067] Test Example 4 The quality of seedlings of *Moss sphagnum* cultivated using different cultivation methods was tested.

[0068] Detection method: Testing standards: Homogeneity: ImageJ analysis of coverage coefficient of variation (CV = standard deviation / mean) Color Measurement and Color Saturation Calculation Color determination: The Lab value of the sample is measured using a colorimeter.

[0069] L: Indicates brightness (level of darkness) a: Represents the chromaticity of the red-green axis (positive values ​​lean towards red, negative values ​​lean towards green). b: Indicates the yellow-blue axis chromaticity (positive values ​​lean towards yellow, negative values ​​lean towards blue). Color saturation The purity or vividness of the color (the higher the value, the more saturated the color).

[0070] Thickness: Scanned by a laser displacement sensor (Keyence LK-G5000).

[0071] Comparative group: Example 2 vs. commercially available moss cloth (three-dimensional moss fiber cloth).

[0072] Table 5 Seedling quality test results (*P<0.01) As shown in Table 5, plasma treatment of felt increased the gametophyte attachment density by 1.8 times, significantly reduced the CV value, enriched CO2 to promote rhizoid development, and achieved a thickness of 196% of the commercial standard.

[0073] Test Example 5 Vertical greening application test Test method: Construction scene: Example 2: Laying seedlings (5mm thick) on concrete wall (90° angle).

[0074] Control group: Traditional patch planting of *Sphagnum moss* (substrate: peat moss).

[0075] Monitoring indicators: Adhesion strength on day 30 (peel strength measured with a tensile tester, N / cm) 2 ).

[0076] Survival rate of the area that has not been irrigated for 60 consecutive days.

[0077] Table 6. Results of Vertical Greening Performance Test (*P<0.01 vs. traditional panel printing) As shown in Table 6, compared with traditional patch planting, Example 2 has higher peel strength and drought survival rate. This is because stem cutting propagation forms an interwoven network structure, which increases peel strength by 220%. In addition, the water-holding capacity of the felt carrier reduces the irrigation frequency to once a week (compared to once every 2 days in the traditional group).

[0078] In summary, this invention provides a highly efficient cultivation method for *Bryum tigrinum*. First, a three-layer substrate is constructed (bottom layer: 3-5mm of pumice; middle layer: peat moss-Kanuma soil 1:0.8-1.2; top layer: plasma-treated felt). Then, *Bryum tigrinum* stem fragments pretreated with an activation solution containing naphthaleneacetic acid and chitosan are inoculated. The mixture is then intermittently sprayed under artificial light (800-1200 lux, blue-red light ratio 1:2-3). The *Bryum tigrinum* cultivated using this method not only exhibits a high germination rate from the stem fragments but also good stress resistance, achieving a coverage rate >80% and a mold rate <3% within 21 days, with a biomass increase of 48.7 mg / cm² and a 223% improvement in water use efficiency. This method is suitable for large-scale production in vertical green walls and ecological restoration projects.

[0079] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for cultivating *Moss simonii*, characterized in that, Includes the following steps: (1) A three-layer composite matrix structure is adopted. The bottom layer is plant stone with a particle size of 3-5mm, the middle layer is a water-retaining layer of peat soil and Kanuma soil mixed in a mass ratio of 1:(0.8-1.2), and the top layer is a felt carrier sterilized by steam at 100℃ or above. (2) Take gametophytes of *Bryum fasciatus* and cut them into stems with a length of 0.5-1.0 cm. Soak them in an activation solution containing 0.1-0.3 mg / L naphthaleneacetic acid and 0.05-0.1% chitosan for 30-120 minutes. (3) Spread the activated stem fragments evenly on the surface of the felt carrier at a density of 200-300 fragments / cm², then cover with a black light-blocking film, maintain a temperature of 18-22℃ and a humidity of 85%-90%, spray intermittently for 5-10 minutes every 1-2 hours, and cultivate under a light intensity of 800-1200 lux, with 10-12 hours of light per day; (4) Spray nutrient solution once a week. When the coverage reaches 80%, remove the shade net and reduce the humidity to 75%-80%. Continue to cultivate until the community thickness reaches more than 5mm.

2. The method according to claim 1, characterized in that, In step (1), the thickness ratio of the three-layer composite matrix is ​​bottom layer: middle layer: top layer = (1.5-2.0):(0.8-1.0):(0.05-0.1).

3. The method according to claim 1, characterized in that, In step (1), the felt carrier is plasma treated, with a surface contact angle ≤30° and a water absorption rate ≥400%.

4. The method according to claim 1, characterized in that, In step (2), the pH value of the activation solution is 5.5-6.0 and the conductivity is ≤100μS / cm.

5. The method according to claim 1, characterized in that, In step (3), the intermittent spraying uses a micron-level atomizing nozzle with a droplet diameter of 20-50 μm and a water pressure of 0.2-0.3 MPa.

6. The method according to claim 1, characterized in that, In step (3), the light source used for illumination is a blue-red composite LED, with the intensity ratio of blue light wavelength 450nm to red light wavelength 660nm being 1:(2-3), and the spectral range being limited to the 400-700nm photosynthetically active radiation (PAR) range.

7. The method according to claim 1, characterized in that, In step (4), the nutrient solution consists of: ammonium nitrate 10-50 mg / L, potassium dihydrogen phosphate 10-30 mg / L, magnesium sulfate 5-10 mg / L, chelated iron 2-5 mg / L, vitamin B1 0.1-0.3 mg / L, selenomethionine 0.01-0.03 mg / L, and cobalamin 0.005-0.01 mg / L; the solvent is deionized water.

8. The application of the method as described in any one of claims 1-7 in vertical green walls, moss bonsai, or ecological restoration projects.

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