A culture method for mass propagation of brachythecium buchananii
By using capsules as explants, sterilizing and heat-shocking them to prepare spore suspensions, inoculating them into growth media, adding plant hormones, and culturing under light, the problems of low germination rate and slow growth of *Bryum multibranchii* were solved, achieving a rapid and efficient propagation method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEBEI NORMAL UNIV
- Filing Date
- 2024-09-09
- Publication Date
- 2026-06-26
AI Technical Summary
In the existing technology, the tissue culture germination rate of *Brucea multibranchii* is low, and the tissue growth after germination is slow, resulting in low propagation efficiency, and there is a lack of effective rapid propagation methods.
Using capsules as explants, spore suspensions were prepared by sterilization and crushing, and then subjected to heat shock treatment. The suspensions were inoculated into growth media, plant hormones were added, and the plants were cultured under specific light conditions to form sterile protonema.
It improved the germination rate and propagation speed of *Bryum multibranchii*, shortened the reproductive cycle, established a rapid propagation system, laid the foundation for large-scale production, and provided a technical basis for maintaining excellent traits.
Abstract
Description
Technical Field
[0001] This invention relates to the field of tissue culture technology of *Bryum multibranchii*, specifically to a method for the propagation and culture of *Bryum multibranchii*. Background Technology
[0002] *Bryum multibranchii* belongs to the family Bryaceae and the genus *Bryum*. The plant is small, growing in dense, small clumps, approximately 3–8 mm tall. The stem is erect, with a short main stem and numerous new branches. The leaves are relatively small, green to yellowish-green at the top and dark brown at the bottom; they lie flat and irregularly twisted when dry, and spread out when moist. The leaves are densely arranged, forming cushion-like clumps in the wild. The leaves are oblong-lanceolate or lanceolate, 1–2 mm long, almost entire, with narrowly rolled margins. The capsule is slender. The capsule is oblong-pear-shaped, relatively large, facilitating cultivation. The spores are spherical, densely covered with fine warts. It is monoecious with separate male and female spores on different plants. It grows on trees or in thin soil on rock surfaces.
[0003] *Bryum multibranchii* is a tree moss that grows densely, covering the entire tree trunk to form a moss carpet. It possesses a strong water retention capacity and a high water absorption rate; when fully hydrated, it can absorb enough water within 20 minutes, with a saturated water absorption capacity reaching 9.18 times its dry weight. This makes it well-suited to increasingly arid environments and a valuable restorative plant for rocky desertification. It also has significant application potential in the manufacture of water-absorbing and water-storage materials, environmental greening, and environmental monitoring.
[0004] Bryophytes are small plants with limited development and utilization, resulting in relatively little research on bryophyte tissue culture both domestically and internationally. Establishing a rapid propagation system for *Bryophytum multibranchii* through tissue culture can provide a technical basis for its propagation, preservation, protoplast preparation, transformation and recombination experiments, and subsequent gametophyte culture. It also provides seedling material for the use of bryophyte gametophytes in the production of absorbent materials, landscaping, rooftop greening, vertical greening, and indoor greening. However, current techniques for tissue culture of *Bryophytum multibranchii* result in low germination rates of ex vivo tissues and slow growth after germination, leading to low propagation efficiency. Summary of the Invention
[0005] This invention provides a cultivation method for the propagation of *Brachys macrantha*, effectively solving the technical problems of low germination rate and slow growth of germinating tissues in the existing tissue culture method for propagating *Brachys macrantha*. This invention establishes a propagation system for *Brachys macrantha*, laying the foundation for large-scale propagation and production of *Brachys macrantha* seedlings and creating favorable conditions for its widespread application. It also provides a cultivation method for the rapid propagation of *Brachys macrantha*.
[0006] This invention provides a method for the propagation and cultivation of *Bryum multibranchii*, comprising the following steps:
[0007] Sporangium disinfection;
[0008] The sterilized sporangia were crushed to prepare a spore suspension, and the spore suspension was subjected to heat shock treatment to obtain an activated spore suspension.
[0009] The activated spore suspension was inoculated into the growth medium, and 0.08–0.12 mg of plant hormone was added per liter of growth medium. The culture was carried out at 23–27°C and under a light intensity of 2000–2600 Lx to obtain sterile protonema.
[0010] In a preferred embodiment, the heat shock treatment specifically involves heat shock treatment at 25–45°C for 3–6 minutes.
[0011] In a preferred embodiment, the heat shock treatment is performed at 40°C for 4 minutes.
[0012] In a preferred embodiment, each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water.
[0013] In a preferred embodiment, the growth medium has a pH value of 5.7 to 6.2.
[0014] In a preferred embodiment, the plant hormone is IBA or 6-BA.
[0015] In a preferred embodiment, the amount of IBA or 6-BA added is 0.1 mg per liter of growth medium.
[0016] In a preferred embodiment, the concentration of the activated spore suspension is (0.8–1.2) × 10⁻⁶. 4 The concentration of spores per mL was 1–2:20, and the volume ratio of activated spore suspension to growth medium was 1–2:20.
[0017] In a preferred embodiment, the duration of illumination is 10-12 hours per day.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] (1) This invention uses capsules as explants to propagate *Brachys macrantha*, rapidly obtaining *Brachys macrantha* spores with a high germination rate, establishing a regeneration system for *Brachys macrantha*, laying the foundation for large-scale propagation and production of *Brachys macrantha*, shortening the propagation cycle of *Brachys macrantha*, and better maintaining its excellent traits; this invention establishes for the first time a method for culturing *Brachys macrantha* using spores, filling the gap in existing technology, and providing technical basis for the propagation, preservation, preparation and transformation recombination experiments of *Brachys macrantha*, as well as subsequent gametophyte culture.
[0020] (2) The cultivation method for propagating *Brachys macrantha* provided by this invention uses capsules as explants, sterilizes them to make a spore suspension and heat-shocks them, and cultivates the spores of *Brachys macrantha* with growth culture medium and plant hormones, which greatly improves the germination rate of spores, realizes the rapid propagation of *Brachys macrantha*, has a high yield, meets the requirements of rapid and large-scale propagation and mass production, and facilitates the large-scale promotion of *Brachys macrantha*. Detailed Implementation
[0021] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention is further described below with reference to specific embodiments. However, the embodiments are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.
[0022] As mentioned in the background section, bryophytes are small plants with limited development and utilization, resulting in relatively little research on bryophyte tissue culture both domestically and internationally. This invention establishes a rapid propagation system for *Bryophytum multibranchii* through tissue culture, providing a technical basis for its propagation, preservation, protoplast preparation, transformation and recombination experiments, and subsequent gametophyte culture. It also provides seed material for the use of bryophyte gametophytes in the production of absorbent materials, landscaping, rooftop greening, vertical greening, and indoor greening. However, existing techniques for tissue culture of *Bryophytum multibranchii* suffer from low germination rates and slow post-germination tissue growth, leading to low propagation efficiency. Furthermore, there are no reports on the use of capsules for propagation of *Bryophytum multibranchii* in culture bottles. Based on these technical problems, this invention provides a method for the propagation of *Bryophytum multibranchii*.
[0023] The technical solution of the present invention will be described in detail below.
[0024] This invention first provides a method for the propagation and cultivation of *Bryum multibranchii*, comprising the following steps:
[0025] Sporangium disinfection: The capsules were rinsed with water, disinfected with a 75% ethanol solution, and rinsed with water in sequence to obtain disinfected capsules;
[0026] The sterilized sporangia were crushed to prepare a spore suspension, and the spore suspension was subjected to heat shock treatment to obtain an activated spore suspension.
[0027] The activated spore suspension was inoculated into the growth medium, and 0.08–0.12 mg of plant hormone was added per liter of growth medium. The culture was carried out at 23–27°C and under a light intensity of 2000–2600 Lx to obtain sterile protonema.
[0028] Through the above technical solution, this invention achieves the formation and propagation of *Bryum multibranchii* protonemata in a short time. Reproduction using capsules results in a high spore germination rate and rapid subsequent protonema growth, significantly improving the propagation rate of *Bryum multibranchii* and achieving excellent propagation effects. This invention provides a technical foundation for the preservation of *Bryum multibranchii* protonemata, the preparation of its protoplasts, transformation and recombination experiments, and subsequent gametophyte culture. It also provides materials for environmental design and remediation using *Bryum multibranchii* gametophytes.
[0029] To activate the spores and shorten the propagation time of *Brachys macrantha*, the heat shock treatment specifically involved heat shock at 25–45°C for 3–6 minutes. Heat shock treatment at any temperature within the 25–45°C range for an appropriate duration yielded spores with good activity. The subsequent conversion rate of spores to sterile protonemata after propagation was high and the spores exhibited stable characteristics. Our research group previously conducted experiments exploring the heat shock temperature. If the temperature was below 25°C, the spore activation efficiency was low. For example, activation at 22°C for 10 minutes resulted in a low conversion rate of spores to protonemata, leading to poor propagation of *Brachys macrantha*. If the heat shock temperature was above 45°C, the active substances in the spores might be destroyed. In our experiment, activation at 48°C for 6 minutes, followed by spore inoculation into the growth medium, resulted in an extremely low conversion rate of sterile protonemata, failing to achieve the expected propagation effect. Within the aforementioned suitable temperature range, heat-shocking the spore suspension at 40°C for 4 minutes achieves the best heat-shock effect, resulting in the highest germination rate of *Brachys pubescens* spores.
[0030] To further achieve rapid and efficient propagation of *Bryum multibranchii*, each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water.
[0031] To provide a better growth environment for the growth of multibranched prostaglandins, the growth medium has a pH of 5.7–6.2.
[0032] To further improve the germination rate of *Brachys pubescens* spores, the amount of IBA or 6-BA added is 0.1 mg.
[0033] To ensure a good match between the spores and their growth environment and to provide sufficient nutrients for spore germination, the concentration of the spore-activating suspension is (0.8–1.2) × 10⁻⁶. 4 The concentration of spores per mL was 1–2:20, and the volume ratio of activated spore suspension to growth medium was 1–2:20.
[0034] Since spore germination requires a certain amount of light, this invention cultured the inoculated spore suspension under light conditions of 2000-2600 Lx for 10-12 hours per day, which maximized the germination rate of *Brachys pubescens* spores.
[0035] The composition of the MS culture medium of this invention is shown in Table 1.
[0036] Table 1. Components and content of MS medium
[0037] Trace elements mg / L μM <![CDATA[CoCl2·6H2O]]> 0.025 0.11 <![CDATA[CuSO4·5H2O]]> 0.025 0.10 FeNaEDTA 36.70 100.00 <![CDATA[H3BO3]]> 6.20 100.27 KI 0.83 5.00 <![CDATA[MnSO4·H2O]]> 16.90 100.00 <![CDATA[Na2MoO4·2H2O]]> 0.25 1.03 <![CDATA[ZnSO4·7H2O]]> 8.60 29.91 macro elements mg / L mM <![CDATA[CaCl2]]> 332.02 2.99 <![CDATA[KH2PO4]]> 170.00 1.25 <![CDATA[KNO3]]> 1900.00 18.79 <![CDATA[MgSO4]]> 180.54 1.50 <![CDATA[NH4NO3]]> 1650.00 20.61 Vitamins mg / L μM Glycine 2.00 26.64 myo-Inositol 100.00 554.94 Nicotinicacid 0.50 4.06 PyridoxineHCl 0.50 2.43 ThiamineHCl 0.10 0.30
[0038] The technical effects of the present invention will be described below with reference to embodiments and comparative examples.
[0039] Example 1
[0040] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0041] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0042] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 4 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0043] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0044] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0045] Example 2
[0046] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0047] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0048] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 25°C for 4 minutes to obtain a concentration of 0.8 × 10⁻⁶. 4 Activated spore suspension per mL;
[0049] S3: According to the volume ratio of activated spore suspension to growth medium of 2:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of 6-BA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0050] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 5.7.
[0051] Example 3
[0052] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0053] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0054] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 45°C for 3 minutes to obtain a concentration of 1.2 × 10⁻⁶. 4 Activated spore suspension per mL;
[0055] S3: The activated spore suspension of S2 was inoculated into the growth medium at a volume ratio of 1.5:20 to the activated spore suspension. 0.08 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 23°C, 2600 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0056] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.2.
[0057] Example 4
[0058] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0059] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0060] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 45°C for 3 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0061] S3. The activated spore suspension of S2 was inoculated into the growth medium at a volume ratio of 1:20 to the activated spore suspension. 0.12 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 27°C, 2000 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0062] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0063] Example 5
[0064] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0065] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0066] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 30°C for 5 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0067] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.08 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0068] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0069] Example 6
[0070] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0071] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0072] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 35°C for 3 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0073] S3: The activated spore suspension of S2 was inoculated into the growth medium at a volume ratio of 1.5:20 to the activated spore suspension. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 26°C, 2400 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0074] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0075] Example 7
[0076] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0077] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0078] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 38°C for 5 minutes to obtain a concentration of 1.2 × 10⁻⁶. 4 Activated spore suspension per mL;
[0079] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.08 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0080] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0081] Example 8
[0082] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0083] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0084] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 42°C for 3 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0085] S3: According to the volume ratio of activated spore suspension to growth medium of 2:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.12 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 23°C, 2400 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0086] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0087] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:
[0088] Comparative Example 1
[0089] The difference compared to Example 1 is that the spore suspension was heat-shocked at 25°C for 1 minute.
[0090] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0091] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0092] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 25°C for 1 min to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0093] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0094] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0095] Comparative Example 2
[0096] The difference compared to Comparative Example 1 is that the heat shock treatment was changed from 1 min to 2 min.
[0097] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0098] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0099] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 25°C for 2 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0100] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0101] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0102] Comparative Example 3
[0103] Compared with Comparative Example 1, the difference is that the heat shock treatment was changed from 1 min to 10 min.
[0104] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0105] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0106] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 25°C for 10 min to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0107] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0108] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0109] Comparative Example 4
[0110] The difference compared to Example 1 is that the spore suspension was heat-shocked at 40°C for 1 minute.
[0111] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0112] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0113] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 1 min to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0114] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0115] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0116] Comparative Example 5
[0117] Compared with Comparative Example 4, the heat shock treatment was adjusted from 1 min to 2 min.
[0118] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0119] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0120] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 2 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0121] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0122] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0123] Comparative Example 6
[0124] Compared with Comparative Example 4, the heat shock treatment was adjusted from 1 min to 10 min.
[0125] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0126] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0127] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 10 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0128] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0129] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0130] Comparative Example 7
[0131] The difference compared to Example 1 is that the spore suspension was heat-shocked at 60°C for 1 minute.
[0132] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0133] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0134] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 60°C for 1 min to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0135] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0136] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0137] Comparative Example 8
[0138] Compared with Comparative Example 7, the heat shock treatment was adjusted from 1 min to 2 min.
[0139] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0140] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0141] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 60°C for 2 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0142] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0143] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0144] Comparative Example 9
[0145] Compared with Comparative Example 7, the heat shock treatment was adjusted from 1 min to 4 min.
[0146] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0147] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0148] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 60°C for 4 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0149] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0150] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0151] Comparative Example 10
[0152] Compared with Comparative Example 7, the heat shock treatment was adjusted from 1 min to 10 min.
[0153] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0154] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0155] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 60°C for 10 min to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0156] S3: According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 2500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0157] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0158] Comparative Example 11
[0159] The difference compared to Example 1 is that no light is applied.
[0160] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0161] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0162] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 4 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0163] S3. According to the volume ratio of activated spore suspension to growth medium of 1:20, the activated spore suspension of S2 was inoculated into the growth medium. 0.1 mg of IBA was added to the growth medium per liter of growth medium, and the medium was cultured at 25°C for 21 days to obtain sterile protonema.
[0164] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0165] Comparative Example 12
[0166] The difference from Example 1 is that the light culture was carried out at 25°C, 500 Lx light intensity, and 12 h / d light frequency.
[0167] A method for propagating and cultivating *Bryum multibranchii* includes the following steps:
[0168] S1, Sporangium disinfection: Rinse the sporangium with sterile water, then disinfect with 75% ethanol (by volume), and then rinse with sterile water to obtain disinfected sporangium;
[0169] S2, the sterilized capsules from S1 are broken to prepare a spore suspension. The spore suspension is then heat-shocked at 40°C for 4 minutes to obtain a concentration of 1.0 × 10⁻⁶. 4 Activated spore suspension per mL;
[0170] S3. The activated spore suspension of S2 was inoculated into the growth medium at a volume ratio of 1:20 to the activated spore suspension. 0.1 mg of IBA was added to the growth medium per liter of growth medium. The culture was carried out at 25°C, 500 Lx light intensity, and 12 h / d light frequency for 21 days to obtain sterile protonema.
[0171] Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; the pH of the growth medium is 6.0.
[0172] The *Brachys macrantha* propagation culture method provided in Examples 1-8 and Comparative Examples 1-12 of this invention was used to propagate *Brachys macrantha*. The synergistic effect of the growth culture medium, spore suspension heat shock temperature and time, and light intensity used in this invention on achieving the propagation effect of *Brachys macrantha* is explained in detail below:
[0173] When the spores were inoculated onto the growth medium and cultured for 3 days, germination was observed in all treatment groups of Examples 1-8, except for Comparative Examples 9 and 10, which did not germinate. After 21 days of culture, Examples 5-8 improved the germination of *Brachys macrantha* spores to varying degrees, with Example 1 showing the highest germination rate at 67%, an increase of 27.2% compared to Example 3. Examples 9-10 significantly inhibited the germination of *Brachys macrantha* spores; after 21 days, the germination rate of Example 10 was only 5%, a decrease of 50.2% compared to Example 1. Therefore, heat shock pretreatment of the spore suspension, specifically treatment at 40°C for 4 minutes in Example 1, resulted in the highest germination rate of *Brachys macrantha* spores, reaching 67%.
[0174] In Comparative Example 11, a small number of spores showed swelling, but no spore germination was observed. However, when the inoculated spores in Comparative Example 11 were transferred to a light intensity of 2500 lx, they began to germinate within 48 hours, with a germination rate reaching 26% of the normal germination rate. There was no significant difference in the spore germination rate between Comparative Example 12 and Example 1, both reaching 58% by day 7. However, the protonema growth rate of Example 1 was faster than that of Comparative Example 12. By day 14, the number of primary branches of the protonema in Example 1 exceeded that of Comparative Example 12 by 0.5 times, and by day 21, it reached twice that of Comparative Example 12. Furthermore, the protonema growth of Comparative Example 12 was weak, while the length of the protonema in Example 1 reached 5871 μm by day 21, which was 12 times the length of the protonema in Comparative Example 12. Therefore, light is a major environmental factor affecting the germination of *Brachys fasciculata* spores, and a light intensity of 2500 lx is more suitable for the growth and development of *Brachys fasciculata* protonemata.
[0175] In summary, this invention utilizes capsules for in vitro propagation of *Brachys macrantha*, establishing for the first time a method for culturing *Brachys macrantha* using spores. It successfully establishes an in vitro regeneration system for *Brachys macrantha*, laying the foundation for large-scale propagation and production of *Brachys macrantha* and creating favorable conditions for its widespread application.
[0176] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A method for propagating *Bryum multibranchii*, characterized in that, Includes the following steps: Sporangium disinfection; The sterilized sporangia were crushed to prepare a spore suspension, and the spore suspension was subjected to heat shock treatment to obtain an activated spore suspension. The heat shock treatment specifically involves heat shock treatment at 25~45℃ for 3~6 minutes; The activated spore suspension was inoculated into the growth medium, and 0.08-0.12 mg of plant hormone was added per liter of growth medium. The mixture was then cultured at 23-27°C under a light intensity of 2000-2600 Lx to obtain sterile protonema. Each liter of the growth medium consists of the following components: 4.7 g MS medium powder, 5 g sucrose, 10 g agar, and the remainder is water; The plant hormone is IBA or 6-BA.
2. The cultivation method for propagating *Bryum multibranchii* according to claim 1, characterized in that, The heat shock treatment is performed at 40°C for 4 minutes.
3. The cultivation method for propagating *Bryum multibranchii* according to claim 1, characterized in that, The growth medium has a pH value of 5.7 to 6.
2.
4. The cultivation method for propagating *Bryum multibranchii* according to claim 1, characterized in that, The amount of IBA or 6-BA added is 0.1 mg per liter of growth medium.
5. The cultivation method for propagating *Bryum multiflorum* according to claim 1, characterized in that, The concentration of the activated spore suspension is (0.8~1.2)×10⁻⁶. 4 The spores / mL spore suspension was prepared at a volume ratio of 1-2:20 to the growth medium.
6. The cultivation method for propagating *Bryum multibranchii* according to claim 1, characterized in that, The duration of the illumination is 10-12 hours per day.