A method for direct organ regeneration of sporophyte leaves of *Adiantum capillus-veneris*

Abstract

The application discloses a method for regenerating direct organs of Cyatheasporophyte leaves, and relates to the field of in-vitro rapid propagation technology.The method induces spores from Cyatheasporophyte protoleaves, then induces green globular bodies (GGBs) from the spores, and after the GGBs germinate into new spores, the new spores are used to induce spore leaf clusters through a leaf direct organ pathway, and the regenerated spore leaf clusters are subcultured and rooted to form complete plants.The method provided in the application has a high spore propagation coefficient and a short cycle through the spore leaf direct organ pathway.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro rapid propagation, and particularly relates to a method for direct organ regeneration of Drynaria roosii sporophyte leaves. Background Art

[0002] Drynaria roosii Drynaria roosii Nakaike is the only source plant of the traditional Chinese medicine "Rhizoma Drynariae", which is rich in flavonoid active ingredients such as naringin, and has the effects of tonifying the kidney and strengthening bones, promoting blood circulation and healing injuries, etc. The demand in the fields of osteoporosis treatment and the development of orthopedic Chinese patent medicines continues to rise. However, the special epiphytic habit, slow growth rate and strict habitat requirements of Drynaria roosii lead to weak natural regeneration ability. Coupled with the superimposed effects of long-term unregulated excavation and habitat destruction, the wild population has shrunk sharply and cannot meet the market demand. In traditional introduction, due to problems such as low spore germination rate, slow seedling growth, and low transplanting survival rate, it is difficult to implement large-scale artificial cultivation. At present, the supply of Drynaria roosii medicinal materials still mainly relies on wild resources, and the contradiction between the rigid growth of market demand and ecological carrying capacity is becoming increasingly acute. Facing this crisis, how to break through the shortage of Drynaria roosii resources through artificial breeding technology has become the core problem亟待解决的核心问题 in the sustainable development of traditional Chinese medicine.

[0003] Tissue culture technology provides a new breeding strategy for plants that are difficult to reproduce by seeds or have a low traditional breeding rate. At present, in the continuous regeneration research of ferns, there is a bottleneck problem of difficulty in the transformation from the gametophyte stage to the sporophyte stage. In the existing tissue culture research of Drynaria roosii, the induction rate and proliferation efficiency of sporophytes in vitro are extremely low. Although the green globular bodies (GGBs) technology can increase a certain multiplication coefficient, its long transformation cycle and cumbersome multi-step seedling formation process still难以达到槲蕨商业化生产的效率要求. Summary of the Invention

[0004] The purpose of the present invention is to at least solve one of the technical problems existing in the prior art, and provide a method for direct organ regeneration of Drynaria roosii sporophyte leaves.

[0005] The technical solution of the present invention is as follows:

[0006] A method for direct organ regeneration of Drynaria roosii sporophyte leaves, comprising the following steps:

[0007] S1: Select fresh green, fluffy and rhizoid-free prothallus clumps, divide them into small prothallus clumps, inoculate them into a rhizome induction medium, and after culturing for 15-20 days, add sterile water to the medium and continue culturing to obtain a sporophyte with a single leaf;

[0008] The rhizome induction medium was MS medium containing 0.5–2.0 mg / L 6-BA and 0.1–1.0 mg / L NAA.

[0009] S2: Remove the sporophyte that has grown a single leaf, peel off the prothallus, and then inoculate the sporophyte into GGBs induction medium. When the GGBs aggregates differentiate significantly, divide the induced GGBs into small clusters and transfer them to GGBs differentiation medium for culture to obtain young sporophytes.

[0010] The GGBs induction medium was an MS medium containing 1.0–3.0 mg / L 6-BA, 0.1–1 mg / L NAA, and 0.01–0.5 mg / L 2,4-D, with sucrose at 30,000 mg / L and agar at 4,000 mg / L. The GGBs differentiation medium was an MS medium containing 0.05–0.5 mg / L NAA and 1,000 mg / L activated carbon, with sucrose at 30,000 mg / L and agar at 4,000 mg / L.

[0011] S3: The young sporophytes are inoculated into the sporophyte leaf direct regeneration medium for regeneration culture to obtain regenerated young sporophytes;

[0012] S4: The regenerated young sporophytes are inoculated into a sporophyte leaf direct regeneration medium with 2-3 leaves per plant for subculture. The sporophytes that have been subcultured once are then rooted in a rooting medium to form a complete plant.

[0013] In steps S3 and S4, the direct regeneration medium for sporophyte leaves is MS medium containing 1.0~3.0 mg / L 6-BA, 0.5~1.5 mg / L NAA, 0.01~0.1 mg / L 2,4-D, and 1000 mg / L activated carbon.

[0014] Preferably, in step S4, the rooting medium contains MS basic culture medium, NAA: 0.1~1.0 mg / L, activated carbon: 1000 mg / L, sucrose: 30000 mg / L, and agar powder: 4000 mg / L.

[0015] Preferably, in step S1, the sterile water level is 0.5-1.5 mm above the surface of the culture medium.

[0016] Preferably, the cultivation conditions in steps S1-S4 are as follows: temperature controlled at 22±1℃, light intensity 1800~2500 lx, and light duration 8~12 h·d. -1 .

[0017] The beneficial effects of this invention are as follows: This invention successfully developed a new strategy for regenerating sporophyte shoots via the direct organ regeneration pathway of the sporophyte leaves in *Adiantum capillus-veneris*. This method effectively avoids the generational transition in ferns, achieving direct organ regeneration of the sporophyte and significantly shortening the plant regeneration cycle. This innovation provides efficient and feasible technical support for the industrialized seedling production and sustainable resource utilization of *Adiantum capillus-veneris*, and also provides important experimental evidence for the in vitro rapid propagation research of other ferns. Attached Figure Description

[0018] Figure 1 Sporophytes were induced from prothallus; where A: heart-shaped leaf of prothallus; B: green spheroids on prothallus; C: spheroids on prothallus after 2 months of growth; D and E: rhizome precursors directly induced from prothallus; F: new sporophyte leaves that develop from small spheres after 60 days.

[0019] Figure 2 The diagram shows the induction of GGBs; where A: green spherical bodies induced from sporophytes; B: GGBs aggregates grown from green spherical bodies; C and D: young sporophytes differentiated from GGBs; E: sporophytes with roots.

[0020] Figure 3 The images show the direct organ regeneration of sporophyte leaves and the rooting status; A: direct regeneration of sporophyte leaves; B: sporophyte leaf clusters directly regenerated from leaves after 90 days; C: back of sporophyte leaf clusters directly regenerated from leaves after 90 days; D: sporophyte leaf clusters directly regenerated from leaves in a bottle after 90 days; E: sporophyte leaf clusters 30 days after the first subculture; F: plant status 30 days after rooting culture. Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0022] The technical solution of the present invention will be further illustrated by specific experiments below.

[0023] 1. Experimental Materials

[0024] In this embodiment, MS medium was used as the basal medium. Naphthaleneacetic acid (NAA), 2,4-dichlorobenzeneacetic acid (2,4-D), and 6-benzylaminopurine [N-(phenylmethyl)-9H purin-6-amine, (6-BA)] were all analytical grade and purchased from Jiangxi Xiangyu Biotechnology Co., Ltd. along with sucrose and agar. The pH of the medium was 5.4–5.8, and it was sterilized in an autoclave at 121°C for 20 min before use.

[0025] 2. Experimental Design

[0026] Comparative Example 1: Sporophyte Induction via Prothallium Pathway

[0027] Fresh, green, fluffy prothallus masses without rhizoids were selected and divided into smaller prothallus masses of approximately 1.0 cm² as experimental materials. These were inoculated into MS and rhizome induction media, respectively. The rhizome induction media was MS medium containing 6-BA (2.0 mg / L) and NAA (0.2 mg / L). Three small prothallus masses were inoculated into each bottle. After 20 days of transfer, sterile water was added to the culture bottles, ensuring that the sterile water level was approximately 1.0 mm (2-3 mL) above the surface of the medium to promote sperm motility. Sterile water was then added every 15 days to maintain the fertilization environment within the medium. The culture room temperature was controlled at 22 ± 1℃, the light intensity at 2200 lx, and the photoperiod at 10 h•d. -1 .

[0028] Comparative Example 2: GGBs-induced sporophyte formation

[0029] Step 1: Select fresh, green, fluffy prothallus masses without rhizoids on the surface, and divide them into smaller prothallus masses of approximately 1.0 cm² as experimental materials. Inoculate these smaller prothallus masses into MS medium containing 6-BA (2.0 mg / L) and NAA (0.2 mg / L). Inoculate 3 smaller prothallus masses into each bottle. After 20 days of transfer, add sterile water to the culture bottle, ensuring that the sterile water level is approximately 1.0 mm (2-3 mL) above the surface of the medium to promote sperm motility. Thereafter, add sterile water every 15 days to maintain the fertilization environment within the medium.

[0030] Step 2: Remove the sporophytes that have grown single leaves in Step 1, carefully peel off the prothallus attached to the periphery, and then inoculate the sporophytes onto GGBs induction medium. The GGBs induction medium is MS medium (sucrose: 30000 mg / L, agar powder: 4000 mg / L) containing 6-BA (2.5 mg / L), NAA (0.5 mg / L), and 2,4-D (0.01 mg / L). When the GGBs aggregates have clearly differentiated and about 1 / 4 are adhered and linked, divide the induced GGBs into small clusters (about 4-5 GGBs) and transfer them to GGBs differentiation medium. The GGBs differentiation medium is MS medium (sucrose: 30000 mg / L, agar powder: 4000 mg / L) containing NAA (0.1 mg / L) and activated carbon (1000 mg / L).

[0031] Step 3: The seedlings in the GGBs differentiation medium were separated into individual plants by the single spherical body at the bottom and transferred to the rooting medium for rooting culture. The rooting medium contained MS basic culture medium, NAA: 0.5 mg / L, activated carbon: 1000 mg / L, sucrose: 30000 mg / L, and agar powder: 4000 mg / L.

[0032] The temperature in the culture room was controlled at 22±1℃, the light intensity was 2200 lx, and the light duration was 10 h•d. -1 .

[0033] Comparative Example 3

[0034] Step 1: Select fresh, green, fluffy prothallus masses without rhizoids on the surface, and divide them into smaller prothallus masses of approximately 1.0 cm² as experimental materials. Inoculate these smaller prothallus masses into MS medium containing 6-BA (2 mg / L) and NAA (0.2 mg / L). Inoculate 3 smaller prothallus masses into each bottle. After 20 days of transfer, add sterile water to the culture bottle, ensuring that the sterile water level is approximately 1.0 mm (2-3 mL) above the surface of the medium to promote sperm motility. Thereafter, add sterile water every 15 days to maintain the fertilization environment within the medium.

[0035] Step 2: The sporophytes that had grown single leaves in Step 1 were divided into groups of 1-2 leaves each (2-3 cm tall) and inoculated into sporophyte leaf direct organ regeneration medium (MS + 3.0 mg / L 6-BA + 1.0 mg / L NAA + 0.01 mg / L 2,4-D + 1000 mg / L activated carbon) for differentiation culture. The results showed that no new sporophytes were produced.

[0036] The temperature in the culture room was controlled at 22±1℃, the light intensity was 2200 lx, and the light duration was 10 h•d. -1 .

[0037] Example 1

[0038] Step 1: Select fresh, green, fluffy prothallus masses without rhizoids on the surface, and divide them into smaller prothallus masses of approximately 1.0 cm² as experimental materials. Inoculate these smaller prothallus masses into MS medium containing 6-BA (2.0 mg / L) and NAA (0.2 mg / L). Inoculate 3 smaller prothallus masses into each bottle. After 20 days of transfer, add sterile water to the culture bottle, ensuring that the sterile water level is approximately 1.0 mm (2-3 mL) above the surface of the medium to promote sperm motility. Thereafter, add sterile water every 15 days to maintain the fertilization environment within the medium.

[0039] Step 2: Remove the sporophytes that have grown single leaves, carefully peel off the prothallus attached to the periphery, and then inoculate the sporophytes onto GGBs induction medium. The GGBs induction medium is MS medium (sucrose: 30000 mg / L, agar powder: 4000 mg / L) containing 6-BA (2.5 mg / L), NAA (0.5 mg / L), and 2,4-D (0.01 mg / L). When the GGBs aggregates have clearly differentiated and about 1 / 4 are adhered and linked, divide the induced GGBs into small clusters (about 4-5 GGBs) and transfer them to GGBs differentiation medium. The GGBs differentiation medium is MS medium (sucrose: 30000 mg / L, agar powder: 4000 mg / L) containing NAA (0.1 mg / L) and activated carbon (1000 mg / L).

[0040] Step 3: Divide the young sporophytes differentiated from GGB into 1-2 leaf-shaped plants (plant height 2-3cm) and inoculate them into sporophyte leaf direct organ regeneration medium (MS + 3.0mg / L 6-BA + 1.0mg / L NAA + 0.01mg / L 2,4-D + 1000mg / L activated carbon) for differentiation culture.

[0041] Step 4: Young sporophytes regenerated from sporophyte leaves are inoculated into sporophyte leaf direct regeneration medium at 2-3 leaf sections per plant (plant height ≤ 1 cm) for subculture. Then, the subcultured sporophytes (plant height 2-3 cm) are used for rooting. The rooting medium contains MS basal medium, NAA: 0.5 mg / L, activated charcoal: 1000 mg / L, sucrose: 30000 mg / L, and agar powder: 4000 mg / L.

[0042] For direct regeneration of sporophyte leaves, newly formed sporophytes, i.e., rhizomes before they swell, must be used as inoculation material. The culture room temperature was controlled at 22±1℃, light intensity at 2200 lx, and photoperiod at 10 h•d. -1 .

[0043] 3. Conclusion

[0044] (1) In Comparative Example 1, the mature prothallus is a heart-shaped leaf, symmetrical on both sides, and emerald green in color. The upper depression is the growth point. Figure 1 (A). In prothallus proliferation culture, it was found that prothalluses only increased in size in anhydrous medium, without any sporophytes appearing, indicating that fertilization of *Quercus acutissima* prothalluses requires a dispersal medium. After 40 days of culture in MS medium with added sterile water, single-leaf sporophytes began to form on the surface of the prothallus mass, while the prothalluses remained bright green; after 60 days, sporophytes continued to appear on the prothallus mass, and the prothalluses still showed signs of continued proliferation. Figure 1 (B and C) After 90 days, the prothallus basically stopped proliferating and gradually turned yellowish-green, with more rhizoids appearing on the surface. The sporophyte produced by fertilization of the prothallus has rhizomes and roots. The average number of sporophytes produced by the prothallus was 18±2.646, which also indicates that water can be used as a fertilization medium for oak ferns.

[0045] Prothallophyll was cultured in rhizome induction medium. After 40 days, the prothallophyll curled up, and small green round spheres grew from the ribs (see image). Figure 1 (D); After 90 days, the prothallus is basically covered with small spheres, and yellowish-brown hairs grow on the surface of the spheres, which adhere to the surface of the prothallus as a whole. Figure 1 (E). Morphologically, the pubescent globules resemble the rhizomes of *Quercus acutissima* in their natural state. When the globules adhering to the surface of the prothallus were separated into individual globules and inoculated into MS medium, new sporophytes would develop on the globules after 60 days. The globules would have roots at their base. Figure 1 (Middle F).

[0046] (2) In Comparative Example 2, the sporophytes induced from the prothallus ( Figure 1 After being transferred to GGB induction medium and cultured for 90 days, tightly packed, green, spherical clumps that were not easily separated appeared on the tender epidermis of the rhizome at the base of the sporophyte. Figure 2 (A). After isolating the green spherical bodies, they were transferred to a new GGBs induction medium for proliferation culture. After 60 days, the green spherical bodies differentiated into GGBs and single-leaf aggregates (GGBs and single-leaf aggregates). Figure 2 (Medium B), the number of aggregates was 40±7.024. Aggregates of GGBs that had not yet developed single leaves, divided into 4 to 5 small spheres, could continue to proliferate on a new GGBs induction medium. When cultured individually on MS medium, they differentiated into sporophytes after 60 days. Figure 2 (C, D); Sporophytes were inoculated as single plants onto rooting medium. After 60 days, the sporophyte rhizomes swelled, and the surface was covered with brown hairs and roots ( Figure 2 (E) During the statistical period, the sporophyte rhizomes that rooted were small and the sporophytes were weak, making them unsuitable for domestication and transplantation.

[0047] (3) In Comparative Example 3, the sporophyte induced by the prothallus in Comparative Example 1 was directly used. Figure 1In order to rapidly increase the production of fern sporophytes, direct organ regeneration was carried out in the middle F stage. However, the sporophytes in this state could not produce new sporophytes, which may be related to the weak development, maturation and differentiation ability of the sporophytes.

[0048] (4) In Example 1, the newly induced young sporophytes from Comparative Example 2 were used as materials for direct organ regeneration culture of sporophyte leaves, and it was found that the young sporophytes had a strong differentiation ability.

[0049] The sporosomes grown from GGBs ( Figure 2 (B) Inoculated into the direct organogenesis medium of sporophyte leaves and cultured, after 30 days the sporophyte leaves grew as a whole, rhizomes emerged, and sporophytes were directly regenerated on the underside of the leaves in contact with the culture medium. Figure 3 (A); After 90 days, the number of sporophytes continues to increase, forming sporophyte clusters around the prosporophyte ( Figure 3 (B and D). Simultaneously, roots grow on the small green dot-like spheres at the base of the spore bud cluster, which can be used to distinguish individual plants. Figure 3 The number of sporophyte leaves produced was 130.67 ± 3.712. Sporophytes were divided into clusters of 2-3 leaves and inoculated into a new culture medium for subculturing. After 90 days, sporophyte shoot clusters emerged, and the sporophyte leaf clusters showed relatively uniform growth, demonstrating that the leaves produced by the direct organogenesis of sporophytes can continue their direct organ regeneration ability in this culture medium. The sporophyte height doubled after one subculture, making it suitable for rooting culture. Figure 3 (E). Sporophytes were inoculated as single plants onto rooting medium. After 60 days, the small, dot-like bulbils swelled into rhizomes with brown hairs, and roots formed on the rhizomes. Figure 3 (Middle F).

[0050] Traditionally, the rapid in vitro propagation of ferns using mature spores requires a complex and cumbersome process involving spore germination, prothallium formation and proliferation, and fertilization to sporophyte formation. Directly utilizing sporophytes for regeneration culture is an effective way to shorten the in vitro propagation cycle. However, the highly differentiated mature tissues of sporophytes, the lack of active meristematic tissue, complex hormone requirements, and high sensitivity to the in vitro environment make it difficult for their cells to dedifferentiate into pluripotent callus tissue or to directly induce adventitious organs from explants. Therefore, regenerating plants from spores in vitro is quite challenging. To address this problem, this invention employs four schemes (Comparative Examples 1-3 and Example 1) to achieve sporophyte regeneration. Scheme 1 (Comparative Example 1) involves the prothallium mass transforming into sporophytes through fertilization in a suitable environment, or directly inducing the prothallium to form rhizomes. In Scheme 1, the prothallium can either produce sporophytes through fertilization in MS medium or be induced to form rhizomes in a rhizome induction medium, thereby producing sporophytes. Prothallus-induced sporophytes possess rhizomes and roots, allowing for direct domestication and transplantation. However, Scheme 1 (Comparative Example 1) has significant drawbacks: the number of sporophytes produced by fertilization is limited, and multiple water additions are required; the induction of rhizomes takes a long time, and the rhizomes are small in size, making them prone to structural damage and death during segmentation. Scheme 2 (Comparative Example 2) uses sporophytes as material to induce GGBs aggregates, and then produces sporophytes through GGBs differentiation. This method significantly increases the proliferation coefficient of induced sporophytes, but it also has drawbacks: the induction cycle is longer than Scheme 1, and it requires repeated replacement and iteration of GGBs induction and differentiation media, resulting in relatively higher time and material costs. Scheme 3 (Comparative Example 3) involves direct organ regeneration based on Scheme 1. However, the sporophyte in this state cannot produce new sporophytes. Scheme 4 (Example 1) involves direct regeneration of sporophyte shoots on sporophyte leaves. Although this scheme requires a long period of preparation, the sporophyte leaves are produced through direct organ regeneration and possess complete plant organs. After rooting and seedling strengthening, the sporophyte rhizomes are larger and have more roots, making them more suitable for domestication. Furthermore, the proliferation coefficient of Scheme 4 is significantly higher than that of Scheme 2.

[0051] It is worth noting that, from the perspective of production application, although the growth cycle of Scheme 2 (Comparative Example 2) is shorter than that of Scheme 4 (Example 1), Scheme 2 (Comparative Example 2) requires three different culture media to produce rooted seedlings, and the rooted seedlings are relatively weak. Scheme 4 (Example 1) only requires two different culture media, and its proliferation coefficient is significantly higher than that of Scheme 2 (Comparative Example 2). Therefore, Scheme 4 (Example 1) is a more economical production method.

[0052] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for direct organ regeneration of a Cyatheasporophyte leaf, characterized in that, The method comprises the following steps: S1: selecting fresh green, fluffy and root-free protocorm clumps, dividing the protocorm clumps into small protocorm clumps, inoculating the protocorm clumps into rhizome induction medium, and culturing for 15-20 days, then adding sterile water to the medium to continue culturing, and obtaining spore bodies growing single leaves; The rhizome induction medium is MS medium containing 0.5-2.0 mg / L 6-BA and 0.1-1.0 mg / L NAA; S2: taking out the spore bodies growing single leaves, peeling the protocorm, and then inoculating the spore bodies into GGBs induction medium, until the GGBs aggregates are obviously differentiated, dividing the induced GGBs into small groups, and transferring the GGBs into GGBs differentiation medium for culturing to obtain young spore bodies; The GGBs induction medium is MS medium containing 1.0-3.0 mg / L 6-BA, 0.1-1 mg / L NAA and 0.01-0.5 mg / L 2,4-D, wherein the sucrose is 30000 mg / L and the agar powder is 4000 mg / L; the GGBs differentiation medium is MS medium containing 0.05-0.5 mg / L NAA and 1000 mg / L activated carbon, wherein the sucrose is 30000 mg / L and the agar powder is 4000 mg / L; S3: inoculating the young spore bodies into spore body leaf direct regeneration medium for regeneration culturing to obtain regenerated young spore bodies; S4: inoculating the regenerated young spore bodies into spore body leaf direct regeneration medium for subculture, and subculturing the spore bodies once to perform rooting culturing in rooting medium; in steps S3 and S4, the spore body leaf direct regeneration medium is MS medium containing 1.0-3.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 0.01-0.1 mg / L 2,4-D and 1000 mg / L activated carbon.

2. The method of claim 1, wherein the method is characterized by, In step S4, the rooting medium contains MS basic medium, NAA: 0.1-1.0 mg / L, activated carbon: 1000 mg / L, sucrose: 30000 mg / L and agar powder: 4000 mg / L.

3. The method of claim 1, wherein the method is characterized by, In step S1, the sterile water is higher than the surface of the medium by 0.5-1.5 mm.

4. The method of claim 1, wherein the method is characterized by, The conditions for the culture in steps S1-S4 are as follows: temperature control at 22±1℃, light intensity of 1800-2500lx, light time of 8-12h·d -1 .

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