A new method of efficient propagation of the star fern by green globular bodies (GGBs)

Abstract

The application provides a new method for efficiently breeding star fern through green globular bodies GGBs, and belongs to the technical field of biotechnology.The new method comprises the following steps: obtaining explants, sterilizing spores, aseptically germinating spores into prothallus, proliferating prothallus, inducing and growing young sporophytes, inducing and proliferating green globular bodies GGBs, regenerating plants, and transplanting seedlings.The method can omit all culture processes of the gametophyte generation and directly propagate in the sporophyte generation, greatly shortens the production cycle of star fern seedlings, creates a complete artificial rapid propagation technology system of star fern, simplifies the tissue culture process, greatly improves the artificial in vitro propagation efficiency, and has a much higher proliferation coefficient than other species in the same family and genus reported.The star fern high-proliferation-coefficient green globular body GGBs seedling method has the characteristics of low cost, short cycle, high quality and high survival rate, and can cultivate seedlings with stable heredity and high quality to improve the artificial planting efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel method for the efficient propagation of star fern using green spherical bodies (GGBs). Background Technology

[0002] Star fern [Microsorum punctatum (L.) Copel.] belongs to the genus *Microsorum* of the family Polypodiaceae. It is mainly distributed in Gansu, Taiwan, Hainan, Guizhou, and Yunnan provinces of China, primarily growing on tree trunks or walls in shady areas of plains. Many plants in the Polypodiaceae family have high ornamental value due to their diverse plant and leaf shapes, making them suitable for indoor hanging planting or garden landscaping. Furthermore, studies have indicated that star fern contains vitamin C, vitamin E, and polyphenolic compounds, making it a potential natural source of antioxidants and anti-inflammatory compounds.

[0003] Among endangered plant conservation efforts, ferns, which emerged 300 million years ago, are the most important group and a valuable source of genetic material. The International Union for Conservation of Nature (IUCN) estimates that of the 12,000 extant fern species, overexploitation, improper agricultural practices, urbanization, and pollution threaten more than half of them with extinction. Star fern, with its low spore reproduction rate, narrow habitat, and destroyed natural habitats, has very rare wild populations, necessitating urgent research into artificial breeding.

[0004] Like all vascular plants, ferns alternate between two distinct life stages or generations: the diploid sporophyte generation and the haploid gametophyte generation. The former is primarily vegetative, later transitioning to reproductive growth, producing haploid spores through meiosis; the latter mainly performs sexual reproduction, producing male and female gametes, which are then fertilized to form a new generation of sporophytes. In ferns and other more primitive plants, the gametophyte stage, from spore germination to fertilization, is completely independent of the diploid sporophyte. Gametophyte development begins with spore germination, followed by a transition from filamentous to plate-like structures, differentiating into a slightly asymmetrical heart-shaped prothallus. Mature gametophytes produce reproductive organs such as antheridium and archegonia, which, through fertilization, produce the sporophyte. After development on the gametophyte, the gametophyte gradually yellows and dies. The sporophyte is the relatively dominant life stage in most fern groups, having evolved many anatomical and physiological features that further facilitate the regulation of nutrient transport. Generally, ferns are easier to propagate and cultivate than other plant groups due to their long-term adaptation to various environmental conditions. However, in artificial propagation, the spore germination and gametophyte-sporophyte transformation stages of ferns are relatively difficult and represent a research hotspot for this group. Environmental factors play a crucial role in spore germination and gametophyte-sporophyte transformation; changes in environmental conditions can affect intracellular and extracellular chemical signal transduction and metabolic activities, thus impacting biological development. Furthermore, completing this process requires a long growth cycle, and asexual reproduction is inefficient.

[0005] Some scholars have studied the proliferation of fern gametophyte generations, finding that some proliferation difficulties can be resolved through homogenized gametophyte culture. However, problems remain, such as difficulty in inducing sporophyte formation in vitro, lack of sporophyte proliferation, or low proliferation coefficients. In particular, the most effective induction of green globular bodies (GGBs) is still rare. Currently, *Adiantum capillus-veneris* germplasm resources are mainly wild-type. Due to the long gametophyte generation, sporophyte populations are difficult to recover once destroyed. Biological research on *Adiantum capillus-veneris* is almost nonexistent, and there are no reports on artificial propagation. Therefore, urgently seeking a new, low-cost, short-time, high-quality, and high-survival-rate artificial propagation method has become an inevitable choice for the sustainable utilization and large-scale propagation of *Adiantum capillus-veneris*. Summary of the Invention

[0006] The purpose of this invention is to provide a new method for the efficient propagation of green spherical corpuscles (GGBs) of Star Fern, in order to solve the current shortage of artificial propagation methods for Star Fern, and to lay a technical foundation for the protection of wild resources and the development of artificial cultivation. It can also provide a certain reference for the in vitro rapid propagation research of other ferns.

[0007] This invention is achieved through the following technical solution:

[0008] A novel method for the efficient propagation of star fern using green globular bodies (GGBs) is described. The steps are as follows: explant acquisition, spore sterilization, aseptic germination of spores into prothalliums, prothallium proliferation, induction and growth of young sporophytes, induction and proliferation of green globular bodies (GGBs), plant regeneration, and hardening-off transplanting.

[0009] Furthermore, the novel method for the efficient propagation of star fern using green globular granules (GGBs) includes the following steps:

[0010] (1) Explant acquisition: Select healthy fern sporophyte plants with good growth and no pests or diseases, and take their fresh reproductive leaves;

[0011] (2) Disinfection treatment: Use a clean blade to scrape off the mature spores on the back of the reproductive leaf and disinfect them;

[0012] (3) Spore germination: The spores that have been sterilized in step (2) are inoculated into culture medium A, which includes the following raw materials:

[0013] 1 / 2MS basic culture medium

[0014] sucrose

[0015] Agar powder

[0016] By controlling the light intensity, temperature, and light duration during cultivation, heart-shaped prothalliums can be obtained.

[0017] (4) Prothallus proliferation: After dividing the prothallus mass from step (3) into appropriate sizes, it is inoculated into culture medium B, which includes the following raw materials:

[0018] MS basic culture medium

[0019] 6-Benzylaminopurine

[0020] α-Naphthaleneacetic acid

[0021] kinetin

[0022] sucrose

[0023] Agar powder

[0024] Prothallus proliferation culture was carried out by controlling light intensity, temperature, and light duration;

[0025] (5) Induction of young sporophytes: When the protophyllum has multiplied to a sufficient size to conduct various experiments on inducing young sporophytes, it is divided into 1.0 cm sections. 2 The sample is transferred into culture medium C, which comprises the following ingredients:

[0026] 1 / 4MS basic culture medium

[0027] sucrose

[0028] Agar powder

[0029] Controlling light intensity, temperature, and light exposure time, and adding sterile water periodically to help male and female gametes fuse, thereby inducing the emergence of young sporophytes;

[0030] (6) Induction and proliferation culture of green globular bodies (GGBs): Young sporophytes were transferred into fresh culture medium D, which included the following raw materials:

[0031] MS basic culture medium

[0032] 6-Benzylaminopurine

[0033] α-Naphthaleneacetic acid

[0034] sucrose

[0035] Agar powder

[0036] Green spherical bodies were induced and proliferated by controlling light intensity, temperature, and light duration.

[0037] (7) Plant regeneration: The induced green spherical GGBs aggregates are divided into 2-3 clusters and transferred into culture medium A to obtain complete sporophyte plants.

[0038] (8) Hardening and transplanting: After hardening the rooted sporophyte plants from step (7) at room temperature for 3 days, take the seedlings out of the culture medium, wash the culture medium from the roots with clean water, soak them in carbendazim solution, and then transplant them into pine bark that has been sterilized at high temperature for warm and moist culture to obtain transplanted seedlings.

[0039] Furthermore, the disinfection method described in step (2) involves dispensing the scraped spores into 3 mL sterile centrifuge tubes and disinfecting them in a 0.1% mercury solution for 8-10 minutes, shaking continuously to ensure thorough mixing. Then, the mixture is poured into an empty bottle with autoclaved filter paper at the bottle opening for filtration and rinsed 4-6 times with sterile water. The filter paper is then divided into 4 equal parts using sterilized scissors, and each part of the filter paper is gently touched to the culture medium A in reverse direction to ensure that the spores on the filter paper adhere to the surface of the culture medium as much as possible.

[0040] Furthermore, the culture medium A comprises the following raw materials:

[0041] 1 / 2MS basic culture medium

[0042] 30000mg / L of sucrose

[0043] Agar powder 4700 mg / L;

[0044] The culture medium B comprises the following raw materials:

[0045] MS basic culture medium

[0046]

[0047] The culture medium C comprises the following raw materials:

[0048] 1 / 4MS basic culture medium

[0049] 30000mg / L of sucrose

[0050] Agar powder 4700 mg / L;

[0051] The culture medium D comprises the following raw materials:

[0052] 1 / 2MS basic culture medium

[0053]

[0054] The pH values ​​of the culture media A, B, C, and D are 5.4-5.8.

[0055] Furthermore, in step (8), the concentration of carbendazim is 0.1-0.5%, the size of the pine bark chips is 1.5×1.5cm, the disinfection method is boiling water for 2-3 hours, and the heat and moisture conditions are a culture temperature of 20-25℃ and a humidity of 70%.

[0056] The present invention has at least the following beneficial effects:

[0057] (1) The present invention uses tissue culture technology to achieve year-round production in the culture room, which saves land resources and improves economic efficiency, and overcomes the difficulty of year-round production by traditional propagation methods;

[0058] (2) After the prothallus stage of the haploid generation is completed and the young sporophyte is induced to green spherical bodies GGBs, the entire culture process can be simplified to two culture media, D and A, thus omitting all the long culture stages of the haploid generation and directly completing the entire breeding process in the sporophyte generation; therefore, through this invention, a large number of seedlings can be obtained in a short time, with low cost, short cycle, easy standardization and industrial operation, effectively improving the quality of seedlings, and providing high-quality seedlings with unified standards for large-scale planting;

[0059] (3) This invention overcomes the slow growth of ferns and has the characteristics of high reproductive efficiency. A culture cycle can be completed in 120 days and the proliferation coefficient can reach more than 32.0.

[0060] (4) The test-tube seedlings of this invention are derived from green globular bodies (GGBs), which are similar to the somatic embryos of angiosperms and have bipolar growth characteristics. The regenerated seedlings are strong and have thick roots, resulting in high survival rates after hardening and transplanting. Attached Figure Description

[0061] Figure 1 This is a diagram of the aseptic germination of *Adiantum capillus-veneris* spores.

[0062] Figure 2 Diagram of the proliferation of the prothallus of *Asteris spp.*

[0063] Figure 3 Diagrams illustrating the formation of male and female gametophytes, fertilization of male and female gametophytes, and induction of sporophytes in *Dryopteris spp.*

[0064] Figure 4 Diagram showing the induced development of green globular corpuscles (GGBs) in star fern;

[0065] Figure 5 Diagram showing the proliferation, regeneration, and plant domestication of GGBs (green spherical granules) of Star Fern;

[0066] Figure 6 This is a diagram of the extracorporeal circulation regeneration of *Stellaria media*.

[0067] Figure 1 In Chinese: AD scale = 2cm, E and H scales = 50μm, F and G scales = 100μm; Figure 1 - Diagrams showing the germination process of spores in sterile bottles A, B, C, and D; Figure 1 -E indicates that spores begin to germinate 20 days after inoculation, producing primary rhizoids and primitive cells. Figure 1 -F shows the primitive cells after 30 days of culture, which have developed into cylindrical shapes with thin walls, bulging sidewalls, and no branches, representing the filamentous stage. Figure 1 -G, After about 45 days of culture, the cells will be arranged in a single row at the base, and a wedge-shaped group of apical cells will appear at the top, and rhizoids will begin to appear, which is the plate-like stage. Figure 1 -H is a diagram of a mature prothallus that has grown into a heart shape after 70 days of cultivation, with symmetrical broad wings and multiple rhizoids appearing on both wings and at the base of the petiole.

[0068] Figure 2 In Chinese: AD scale = 2cm; Figure 2 -A shows the size of the fragmented prothallus clusters after 15 days of culture, which increased significantly in volume and the newly formed prothallus were bright green. Figure 2 -B shows the prothallus mass continuing to enlarge after 30 days of cultivation, with a denser texture and larger terminal leaflets; Figure 2 -C shows the protophyllum clusters after 45 days of cultivation, which further increased in size. Due to their excessive density, yellowing and wilting began to occur at the edges and base. Figure 2 -D indicates that after 60 days, the prothallus mass occupies the entire surface of the culture bottle, the yellowing phenomenon intensifies, some prothallus die and turn black, and rhizoids appear, the texture becomes loose, and the proliferation coefficient can reach more than 9.5 (Figure).

[0069] Figure 3In Chinese: A scale = 500 μm, B and D scales = 40 μm, C scale = 200 μm, EH scale = 2 cm; Figure 3 -A shows the prothallus after 30 days of cultivation. It is heart-shaped, symmetrical, and bright green. The indentation at the top is the growth point. Figure 3 -B shows multiple protruding or spherical stalked anthers near the rhizoids, and the iconic cap cell layer structure can be seen. Figure 3 -C and D show 7-9 rows of archegonia produced below the growth point of the prothallus. Each archegonia is spindle-shaped, narrow at the top and swollen in the middle and lower part, with two distinct rows of neck groove cells. Figure 3 -E indicates that after culturing in sterile water for 20 days, single-leaf sporophytes can be seen growing on the surface of the prothallus mass, and the prothallus remains emerald green. Figure 3 -F represents the early sporophytes that, after 60 days of culture, mostly have 2-3 leaves, and new sporophytes continue to appear, while the prothallus still shows signs of continued proliferation. Figure 3 -G indicates that after 90 days of culture, a large number of young sporophytes appear on the prothallus, the prothallus basically stops proliferating, and many rhizoids appear on the surface, turning yellowish-green. At this time, there are as many as 50-55 young spores per bottle, with an average occurrence coefficient of about 10.0 (Figure 1). Figure 3 -H represents the rooting culture of young sporophytes or the direct induction of green globular bodies (GGBs).

[0070] Figure 4 In Chinese: A and E scales = 1 cm, BD and H scales = 1 mm, F scale = 500 μm, G scale = 5 mm; Figure 4 -A shows the transfer of spores induced from the prothallus after 60 days into medium D. Figure 4 -B and C are images showing small, green, spherical clusters that are difficult to separate from the tender epidermis of the rhizome at the base of the young sporophyte after 30 days of culture. Figure 4 -D and E show the differentiation of green globular bodies (GGBs) aggregates after being peeled off and transferred to fresh culture medium and cultured for 30 days. The proliferation coefficient at this time can reach about 32.0. Figure 4 -F is a diagram of green spherical bodies with single leaves growing on their surface; Figure 4 -G and H are separate individual green spherical bodies. GGB continues to be cultured and gradually differentiates into young sporophytes.

[0071] Figure 5 In Chinese: AD scale = 1cm, E scale = 5cm, FH scale = 3cm; Figure 5 -A and B are diagrams showing the proliferation of green spherical bodies (GGBs); Figure 5 -C shows the growth of numerous bifoliate sporophytes after a single green spherical GGB body was peeled off and cultured for 20 days. Figure 5Figure D shows the sporophyte further enlarging and leaves gradually increasing after 30 days of cultivation, indicating excellent growth. Figures E, F, G, and H show the spore acclimatization process, with a survival rate of over 90% after 60 days of acclimatization. Detailed Implementation

[0072] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the relevant technical field, and all reagents, raw materials and equipment used are commercially available.

[0073] Example 1

[0074] A novel method for the efficient propagation of star fern using green globular granules (GGBs) includes the following steps:

[0075] (1) Obtaining explants: Select healthy star fern plants with good growth and no pests or diseases, and take their tender leaves;

[0076] (2) Disinfection treatment: Use a clean blade to scrape off the mature spores from the back of the reproductive leaf in step (1) and divide them into 3 mL sterile centrifuge tubes. Disinfect them in a 0.1% mercuric chloride solution for 8 min, shaking constantly to mix thoroughly. Then pour the solution into an empty bottle with autoclaved filter paper at the mouth for filtration and wash it 4 times with sterile water. Use sterile scissors to divide the filter paper into 4 equal parts and gently touch each part of the filter paper to the culture medium A in reverse so that the spores on the filter paper can adhere to the surface of the culture medium as much as possible.

[0077] (3) Spore germination: The spores that have been sterilized in step (2) are inoculated into the following culture medium A for aseptic germination:

[0078] 1 / 2MS basic culture medium

[0079] 30000mg / L of sucrose

[0080] Agar powder 4700mg / L

[0081] The pH of culture medium A is 5.4.

[0082] Culture conditions: The spores were cultured under the following conditions: light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 20 days of inoculation, the spores began to germinate, producing primary rhizoids and primitive cells. After 30 days of culture, the primitive cells developed into cylindrical shapes with thin walls and outward bulging lateral walls, without branches, which is the filamentous stage. After about 45 days of culture, the cells arranged in a single row at the base, and a wedge-shaped apical cell group appeared at the apex, and rhizoids began to appear, which is the plate-like stage. After 70 days of culture, the cells elongated into a heart shape with symmetrical broad wings, and multiple rhizoids appeared on both wings and at the base of the petiole, which is the mature prothallus stage.

[0083] (4) Prothallus proliferation: The prothallus mass from step (3) is divided into 1.0 cm sections. 2 After reaching the desired size, inoculate into the following culture medium B:

[0084] MS basic culture medium

[0085]

[0086] The pH of culture medium B is 5.4.

[0087] Culture conditions: Cultured under conditions of 1500-2000 lx light intensity, 10 h / d light duration, and 23±2℃ temperature. After 15 days of culture, the volume of the prothallus clusters increased significantly, and the newly formed prothallus were bright green. After 30 days of culture, the prothallus clusters continued to increase in size, with a denser texture and larger terminal leaves. After 45 days of culture, the prothallus clusters increased further in size, and due to their excessive density, yellowing and wilting began to occur at the edges and base. After 60 days of culture, the prothallus clusters occupied the entire surface of the culture bottle, the yellowing intensified, some prothallus died and turned black, and rhizoids appeared, making the texture looser. At this time, the prothallus proliferation coefficient reached 9.77.

[0088] (5) Induction of young sporophytes: When the protophyllum has multiplied to a sufficient size to conduct various experiments on inducing young sporophytes, it is divided into 1.0 cm sections. 2 Transfer the sample to the following culture medium C:

[0089] 1 / 4MS basic culture medium

[0090] 30000mg / L of sucrose

[0091] Agar powder 4700mg / L

[0092] The pH of culture medium C is 5.4.

[0093] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature controlled at 23±2℃. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, and bright green, with the growth point at the upper notch. Multiple protruding or spherical stalked anthers appear near the rhizoids, and the characteristic cap cell layer structure is visible. Below the growth point of the prothallus, 7-9 rows of archegonia are produced. Each archegonia is spindle-shaped, narrow at the top and swollen in the middle and lower parts, with two distinct rows of neck groove cells. After 10 days of culture, [the process begins...]. Sterile water was injected into the culture vessel every 15 days. After 20 days of culture with sterile water, single-leaf sporophytes were observed growing on the surface of the prothallus mass, while the prothallus remained bright green. After 60 days of culture, the sporophytes that were produced earlier mostly had 2-3 leaves, and sporophytes continued to appear, while the prothallus still showed signs of continued proliferation. After 90 days of culture, a large number of sporophytes appeared on the prothallus, and the prothallus basically stopped proliferating. Many rhizoids appeared on the surface and turned yellowish-green. At this time, there were as many as 50-55 sporophytes per bottle, with an average occurrence coefficient of about 10.25. At this point, the sporophytes were acclimatized and transplanted.

[0094] (6) Induction of green globular bodies (GGBs): Transplant individual young sporophytes into the following culture medium D:

[0095] MS basic culture medium

[0096]

[0097] The pH of culture medium D is 5.4.

[0098] Culture conditions: Cultured under the conditions of 1500-2000 lx light intensity, 10 h / d light duration, and 23±2℃ temperature, the induced spores from the prothallus cultured for 60 days were cultured for 30 days, and small green spherical bodies that were not easy to separate appeared on the tender epidermis of the rhizome at the base. At this time, the green spherical bodies were peeled off and transferred to fresh culture medium for 30 days, after which they differentiated into aggregates of green spherical bodies GGBs, with a proliferation coefficient of 32.15. The separate individual green spherical bodies GGBs continued to be cultured and gradually differentiated into spore-bearing bodies.

[0099] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 clusters and transferred into fresh culture medium A. After culturing for 30 days under the conditions of light intensity of 1500-2000 lx, light duration of 10 h / d and temperature of 23±2℃, complete sporophyte plants could be obtained.

[0100] (8) Hardening and transplanting: After hardening the rooted plants from step (7) at room temperature for 3 days, take the seedlings out of the culture medium, wash the culture medium from the roots with clean water, soak them in a 0.1% carbendazim solution, and then transplant them into 1.5×1.5cm pieces of pine bark that have been boiled in boiling water for 2.5 hours. Keep them warm and moist for 60 days at 20℃ and 70% humidity to obtain transplanted seedlings.

[0101] Example 2

[0102] A novel method for the efficient propagation of star fern using green globular granules (GGBs) includes the following steps:

[0103] (1) Obtaining explants: Select healthy plants with good growth and no pests or diseases, and take their tender leaves.

[0104] (2) Disinfection treatment: Using a clean blade, scrape off the mature spores from the back of the reproductive leaves from step (1) and aliquot them into 3mL sterile centrifuge tubes. Disinfect the tubes in a 0.1% mercuric chloride solution for 9 minutes, shaking constantly to ensure thorough mixing. Then pour the solution into an empty bottle with autoclaved filter paper at the top for filtration. Rinse the bottle 4-6 times with sterile water. Divide the filter paper into 4 equal portions using sterile scissors. Gently touch each portion of filter paper to the culture medium A from the opposite direction to ensure that the spores on the filter paper adhere to the surface of the culture medium as much as possible.

[0105] (3) Spore germination: The spores that have been sterilized in step (2) are inoculated into the following culture medium A for aseptic germination:

[0106] 1 / 2MS basic culture medium

[0107] 30000mg / L of sucrose

[0108] Agar powder 4700mg / L.

[0109] The pH of culture medium A is 5.6.

[0110] Culture conditions: The cells were cultured under the following conditions: light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. Spores began to germinate 20 days after inoculation, producing primary rhizoids and primitive cells. After 30 days of culture, the primitive cells developed into cylindrical shapes with thin walls, bulging lateral walls, and no branches, which is the filamentous stage. After about 45 days of culture, the cells arranged in a single row at the base, and a wedge-shaped apical cell group appeared at the apex, and rhizoids began to appear, which is the plate-like stage. After 70 days of culture, the cells elongated into a heart shape with symmetrical broad wings, and multiple rhizoids appeared on both wings and at the base of the petiole, which is the mature prothallus stage.

[0111] (4) Prothallus proliferation: The prothallus mass from step (3) is divided into 1.0 cm sections. 2 After reaching the desired size, inoculate into the following culture medium B:

[0112] MS basic culture medium

[0113]

[0114] The pH of culture medium B is 5.6.

[0115] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 15 days of culture, the volume of the prothallus clusters increased significantly, and the newly formed prothallus were bright green. After 30 days of culture, the prothallus clusters continued to increase in size, with a denser texture and larger terminal leaves. After 45 days of culture, the prothallus clusters increased further in size, and due to their excessive density, yellowing and wilting began to occur at the edges and base. After 60 days of culture, the prothallus clusters occupied the entire surface of the culture bottle, the yellowing intensified, some prothallus died and turned black, and rhizoids appeared, making the texture looser. At this time, the prothallus proliferation coefficient reached 9.75.

[0116] (5) Induction of young sporophytes: When the protophyllum has multiplied to a sufficient size to conduct various experiments on inducing young sporophytes, it is divided into 1.0 cm sections. 2 Transfer the sample to the following culture medium C:

[0117] 1 / 4MS basic culture medium

[0118] 30000mg / L of sucrose

[0119] Agar powder 4700mg / L

[0120] The pH of culture medium C is 5.6.

[0121] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, and bright green, with the growth point at the upper notch. Multiple protruding or spherical stalked anthers appear near the rhizoids, and the characteristic cap cell layer structure is visible. Below the growth point of the prothallus, 7-9 rows of archegonia are produced. Each archegonia is spindle-shaped, narrow at the top and swollen in the middle and lower parts, with two distinct rows of neck groove cells. After 10 days of culture, a 1 Sterile water is injected every 5 days. After 20 days of culture with sterile water, single-leaf sporophytes can be seen growing on the surface of the prothallus mass, while the prothallus remains bright green. After 60 days of culture, the sporophytes that are produced first mostly have 2-3 leaves, and sporophytes continue to appear, while the prothallus still shows signs of continued proliferation. After 90 days of culture, a large number of sporophytes appear on the prothallus, the prothallus basically stops proliferating, and more rhizoids appear on the surface, turning yellowish-green. At this time, there are as many as 51-57 sporophytes per bottle, with an average occurrence coefficient of about 10.45. At this point, the sporophytes can be directly acclimatized and transplanted.

[0122] (6) Induction of green globular bodies (GGBs): Transplant individual young sporophytes into the following culture medium D:

[0123] MS basic culture medium

[0124]

[0125] The pH of culture medium D is 5.6.

[0126] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃, the immature spores induced from the prothallus after 60 days were cultured for 30 days. At this time, small, green, spherical bodies that were not easy to separate appeared on the tender epidermis of the rhizome at the base of the prothallus. After the green spherical bodies were peeled off and transferred to fresh culture medium, they differentiated into aggregates of green spherical bodies (GGBs) after 30 days of culture. The proliferation coefficient at this time could reach 32.10. The separate individual green spherical bodies (GGBs) continued to be cultured and gradually differentiated into immature sporophytes.

[0127] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 clusters and transferred into fresh culture medium A. After culturing for 30 days under the conditions of light intensity of 1500-2000 lx, light duration of 10 h / d and temperature of 23±2℃, complete sporophyte plants could be obtained.

[0128] (8) Hardening and transplanting: After hardening the rooted plants from step (7) at room temperature for 3 days, take the seedlings out of the culture medium, wash the culture medium from the roots with clean water, soak them in a 0.3% carbendazim solution, and then transplant them into 1.5×1.5cm pieces of pine bark that have been boiled in boiling water for 2.5 hours. Keep them warm and moist for 60 days at a temperature of 22℃ and a humidity of 70% to obtain transplanted seedlings.

[0129] Example 3

[0130] A novel method for the efficient propagation of star fern using green globular granules (GGBs) includes the following steps:

[0131] (1) Obtaining explants: Select healthy plants with good growth and no pests or diseases, and take their tender leaves.

[0132] (2) Disinfection treatment: Using a clean blade, scrape off the mature spores from the back of the reproductive leaves from step (1) and aliquot them into 3mL sterile centrifuge tubes. Disinfect the tubes in a 0.1% mercuric chloride solution for 10 minutes, shaking constantly to ensure thorough mixing. Then pour the solution into an empty bottle with autoclaved filter paper at the top for filtration. Rinse the bottle 4-6 times with sterile water. Divide the filter paper into 4 equal portions using sterilized scissors. Gently touch each portion of filter paper to the culture medium A from the opposite direction to ensure that the spores on the filter paper adhere to the surface of the culture medium as much as possible.

[0133] (3) Spore germination: The spores that have been sterilized in step (2) are inoculated into the following culture medium A for aseptic germination:

[0134] 1 / 2MS basic culture medium

[0135] 30000mg / L of sucrose

[0136] Agar powder 4700mg / L

[0137] The pH of culture medium A is 5.8.

[0138] Culture conditions: The cells were cultured under the following conditions: light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. Spores began to germinate 20 days after inoculation, producing primary rhizoids and primitive cells. After 30 days of culture, the primitive cells developed into cylindrical shapes with thin walls, bulging lateral walls, and no branches, which is the filamentous stage. After about 45 days of culture, the cells arranged in a single row at the base, and a wedge-shaped apical cell group appeared at the apex, and rhizoids began to appear, which is the plate-like stage. After 70 days of culture, the cells elongated into a heart shape with symmetrical broad wings, and multiple rhizoids appeared on both wings and at the base of the petiole, which is the mature prothallus stage.

[0139] (4) Prothallus proliferation: The prothallus mass from step (3) is divided into 1.0 cm sections. 2 After reaching the desired size, inoculate into the following culture medium B:

[0140] MS basic culture medium

[0141]

[0142] The pH of culture medium B is 5.8.

[0143] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 15 days of culture, the volume of the prothallus clusters increased significantly, and the newly formed prothallus were bright green. After 30 days of culture, the prothallus clusters continued to increase in size, with a denser texture and larger terminal leaves. After 45 days of culture, the prothallus clusters increased further, and due to their excessive density, yellowing and wilting began to occur at the edges and base. After 60 days of culture, the prothallus clusters occupied the entire surface of the culture bottle, the yellowing intensified, some prothallus died and turned black, and rhizoids appeared, making the texture looser. At this time, the prothallus proliferation coefficient reached 9.55.

[0144] (5) Induction of young sporophytes: When the protophyllum has multiplied to a sufficient size to conduct various experiments on inducing young sporophytes, it is divided into 1.0 cm sections. 2 Transfer the sample to the following culture medium C:

[0145] 1 / 4MS basic culture medium

[0146] 30000mg / L of sucrose

[0147] Agar powder 4700mg / L

[0148] The pH of culture medium C is 5.8.

[0149] Culture conditions: Cultured under light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 30 days of culture, the prothallus is heart-shaped, bilaterally symmetrical, and bright green, with the growth point located at the upper notch. Multiple protruding or spherical anthers with stalks appear near the rhizoids, and the characteristic cap cell layer structure is visible. Below the growth point of the prothallus, 7-9 rows of archegonia are produced. Each archegonia is spindle-shaped, narrow at the top and swollen in the middle and lower parts, with two distinct rows of neck groove cells. After 10 days of culture, spermatogenesis begins... Sterile water is injected every 15 days. After 20 days of culture with sterile water, single-leaf sporophytes can be seen growing on the surface of the prothallus mass, while the prothallus remains bright green. After 60 days of culture, the sporophytes that are produced first mostly have 2-3 leaves, and sporophytes continue to appear, while the prothallus still shows signs of continued proliferation. After 90 days of culture, a large number of sporophytes appear on the prothallus, the prothallus basically stops proliferating, and many rhizoids appear on the surface, turning yellowish-green. At this time, there are as many as 50-54 sporophytes per bottle, with an average occurrence coefficient of about 10.25. At this point, the sporophytes can be directly acclimatized and transplanted.

[0150] (6) Induction of green globular bodies (GGBs): Transplant individual young sporophytes into the following culture medium D:

[0151] MS basic culture medium

[0152]

[0153] The pH of culture medium D is 5.8.

[0154] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000 lx, light duration of 10 h / d, and temperature of 23±2℃. After 60 days of culture, the immature spores induced from the prothallus were cultured for 30 days. At this time, small green spherical bodies that were not easy to separate appeared on the tender epidermis of the rhizome at the base. After the green spherical bodies were peeled off and transferred to fresh culture medium, they were cultured for 30 days and differentiated into aggregates of green spherical bodies GGBs. The proliferation coefficient at this time could reach 32.15. The separate individual green spherical bodies GGBs continued to be cultured and gradually differentiated into immature sporophytes.

[0155] (7) Plant regeneration: The induced green spherical GGBs aggregates were divided into 2-3 clusters and transferred into fresh culture medium A. After culturing for 30 days under the conditions of 1500-2000 lx light intensity, 10 h / d light duration and 23±2℃ temperature, complete sporophyte plants can be obtained.

[0156] (8) Hardening off and transplanting: Take the rooted plants from step (7) and place them in the bottle at room temperature for hardening off for 3 days. Take the seedlings out of the culture medium, wash the culture medium on the roots with clean water, soak them in a 0.5% carbendazim solution, and then transplant them into 1.5×1.5cm pieces of pine bark that have been boiled in boiling water for 2.5 hours. Keep them warm and moist for 60 days at a temperature of 25℃ and a humidity of 70% to obtain transplanted seedlings.

[0157] Technical principle of the invention:

[0158] 1. In the artificial propagation of ferns, although mature spores are a commonly used source of explants, the series of steps from spore germination to prothallium formation and proliferation, and the transformation into sporophyte after fertilization, are quite complex, with each step requiring different culture conditions. Furthermore, in the fern life cycle, reproductive cells germinate through spores to form oocytes (female gametophytes) and spermatids (male gametophytes); this life cycle characteristic makes callus induction less convenient than in seed plants. Therefore, it is quite difficult to generate regenerated plants from spores. In addition, the lack of ovules and vegetative tissues in the sporophyte of ferns also limits callus formation and culture. This invention solves the above problems, successfully completing spore germination, prothallium formation, and proliferation, and achieving a satisfactory proliferation coefficient for the prothallium.

[0159] 2. This invention solves the key problem of fertilization of male and female gametophytes in star fern bottles by adding sterile water at regular intervals, which can induce a large number of young sporophytes on the prothallus in the bottle. This is a key node connecting the previous and subsequent processes. The appearance of green spherical bodies (GGBs) is the basis for producing high-yield reproduction lines.

[0160] 3. This invention employs two approaches ( Figure 6 To achieve sporophyte regeneration, two methods are employed: first, the prothallus mass is treated as "callus tissue" in angiosperms, providing appropriate culture conditions to efficiently transform it into young sporophytes, which are then considered "clustered buds"; second, the sporophyte is used as material to induce the production of green globular bodies (GGBs), thereby differentiating into sporophytes. In the production application of *Adiantum capillus-veneris*, besides the initial culture of the gametophyte generation (spore germination, etc.), once the young sporophyte appears, the green globular bodies (GGBs) differentiate from the tender epidermis of the rhizome. The appearance of GGBs significantly increases the proliferation coefficient, completely eliminating the need for gametophyte generation culture and achieving the propagation goal of repeatedly inducing regenerated sporophyte plants through GGBs in the sporophyte generation. Thus, the artificial rapid propagation cycle of *Adiantum capillus-veneris* is greatly shortened, and it features low variability, low cost, and high efficiency.

[0161] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various adjustments and improvements without departing from the concept of the technical solution of this application, and these all fall within the scope of protection of this application.

Claims

1. A method for the efficient propagation of star fern using green globular bodies (GGBs), characterized in that, The method includes the following steps: (1) Obtaining explants: Select healthy sporophyte plants with good growth and no pests or diseases, and take their fresh reproductive leaves; (2) Disinfection treatment: Use a clean blade to scrape off the mature spores on the back of the reproductive leaf and disinfect them; (3) Spore germination: The spores that have been disinfected in step (2) are inoculated into culture medium A for culture to obtain heart-shaped prothallus. Culture medium A is composed of the following raw materials: 1 / 2MS basic culture medium, 30000 mg / L sucrose and 4700 mg / L agar powder. (4) Prothallus proliferation: After dividing the prothallus mass in step (3) into appropriate sizes, it is inoculated into culture medium B for prothallus proliferation culture. The culture medium B is composed of the following raw materials: MS basic culture medium, 6-benzylaminopurine 0.3-0.5 mg / L, α-naphthaleneacetic acid 1.0-1.5 mg / L, sucrose 30000 mg / L, agar powder 4700 mg / L; (5) Induction of young sporophytes: When the protophyllum has multiplied to a sufficient size to conduct various experiments on inducing young sporophytes, it is divided into 1.0 cm sections. 2 The cells were transferred to culture medium C, which consisted of the following ingredients: 1 / 4 MS basal medium, 30,000 mg / L sucrose, and 4,700 mg / L agar powder. The light intensity, temperature, and light exposure time were controlled, and sterile water was added periodically to help the male and female gametes fuse, thereby inducing the emergence of young sporophytes. (6) Induction and proliferation culture of green globular bodies (GGBs): The young sporophytes were transferred into fresh culture medium D for induction and proliferation culture of green globular bodies. The culture medium D was composed of the following raw materials: MS basic culture medium, 1.5-2.0 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of α-naphthaleneacetic acid, 30000 mg / L of sucrose, and 4700 mg / L of agar powder; (7) Plant regeneration: The induced green spherical GGBs aggregates are divided into 2-3 clusters and transferred into culture medium A to obtain complete sporophyte plants; (8) Hardening and transplanting: Take the rooted sporophyte plants from step (7) and harden them at room temperature for 3 days. Then, take the seedlings out of the culture medium, wash the culture medium from the roots with clean water, soak them in carbendazim solution, and then transplant them into pine bark that has been sterilized at high temperature for warm and moist culture to obtain transplanted seedlings. The cultivation conditions in steps (3), (4), (5), and (6) are: light intensity 1500–2000 lx, light duration 10 h / d, and temperature 23±2℃.

2. The method according to claim 1, characterized in that, The disinfection method described in step (2) is as follows: the scraped spores are aliquoted into 3 mL sterile centrifuge tubes and disinfected in 0.1% mercuric chloride solution for 8-10 min, shaking continuously to mix thoroughly; then poured into an empty bottle with autoclaved filter paper at the bottle mouth for filtration, and washed with sterile water 4-6 times; the filter paper is divided into 4 equal parts with sterile scissors, and each part of the filter paper is gently touched to culture medium A in reverse so that the spores on the filter paper can adhere to the surface of the culture medium as much as possible.

3. The method according to claim 1, characterized in that, The pH values ​​of culture media A, B, C, and D are all 5.4 to 5.

8.

4. The method according to claim 1, characterized in that, The concentration of the carbendazim solution in step (8) is 0.1-0.5%, the size of the pine bark pieces is 1.5×1.5 cm, and the disinfection method is boiling water for 2-3 hours.

5. The method according to claim 1, characterized in that, The heat preservation and humidity conditions mentioned in step (8) are a culture temperature of 20-25℃ and a humidity of 70%.

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