Method for establishing a suspension system of adiantum reniforme
By establishing a GGB suspension culture system for Adiantum repens, the problems of resource scarcity and low propagation efficiency have been solved, achieving efficient plant regeneration and secondary metabolite production, with the advantages of high propagation coefficient and low pollution rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 三峡植物园管理处(宜昌市林业科学研究所 宜昌市国有金银岗试验林场管理处)
- Filing Date
- 2024-01-19
- Publication Date
- 2026-06-19
AI Technical Summary
Existing technologies have failed to establish a stable and efficient GGB suspension culture system for Adiantum repens, resulting in resource scarcity, insufficient propagation materials, and a low propagation coefficient, which cannot meet the needs of scientific research.
By establishing sterile green spherical bodies, using specific culture media and suspension culture methods, we induced and cultured Adiantum repens GGB to form a stable suspension system, and then transplanted it into the substrate to differentiate into plants.
This study achieved stable and efficient propagation of Adiantum repens suspension culture, shortened the experimental cycle, improved propagation efficiency, reduced labor costs, and can be used for plant regeneration and secondary metabolite production.
Smart Images
Figure CN117958139B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, and in particular to a method for establishing a suspension system of Adiantum capillus-veneris inflorescence. Background Technology
[0002] Adiantum nelumboides, belonging to the genus Adiantum in the family Pteridaceae, is the only single-leaved fern in this genus distributed in Asia. Also known as moneywort or lotus-leaved moneywort, it is a Quaternary relict plant with an elegant appearance, suitable for garden potted plants. The whole plant can be used medicinally, possessing the effects of clearing heat and detoxifying, and promoting diuresis.
[0003] Existing technologies document the propagation of *Adiantum repens* using its mature spores through plant tissue culture or sowing, as well as division propagation. For example, Chinese patent application CN201510087219.4 uses naturally dehiscent spores as explants, with the culture process as follows: spore germination—prothallus proliferation—GGB induction—GGB differentiation into seedlings. The entire process is conducted on solid culture media, requiring hormone induction and a sterile environment for seedling formation. To date, no research has been found on establishing a stable and efficient suspension propagation system for *Adiantum repens* GGB (green spherical bodies). Plant cell suspension culture offers advantages such as rapid growth, high propagation coefficient, easily controllable nutritional and environmental requirements, and lack of geographical limitations. Furthermore, it may generate new compounds suitable for pharmaceutical research from suspension systems. Therefore, researching a stable and efficient suspension culture system for *Adiantum repens* is of great significance for the artificial propagation and population recovery of *Adiantum repens*, as well as for plant biodiversity and development. Summary of the Invention
[0004] The purpose of this invention is to provide a method for establishing a suspension system of *Adiantum repens* (lotus leaf maidenhair fern). A stable suspension system is established using sterile green spherical bodies. The spherical bodies obtained through cultivation are directly removed from the sterile environment for sowing, thus completing plant regeneration. This addresses the gap in suspension culture technology for *Adiantum repens*, as well as the current problems of resource scarcity, insufficient propagation materials, and low propagation coefficient.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for establishing a suspension system of *Adiantum capillus-veneris* (lotus leaf fern), comprising the following steps:
[0007] (1) Inducing the prothallus of Adiantum repens into primary green spherical bodies;
[0008] (2) The primary green spherical bodies were subjected to suspension culture to obtain suspended proliferating green spherical bodies of Adiantum repens;
[0009] (3) The green spherical bodies of the Adiantum repens are cultured in a suspension culture medium to obtain a stable Adiantum repens suspension system. The suspension system is then transplanted into a substrate to differentiate into plants.
[0010] Preferably, the induction medium in step (1) is a solid medium, the solid medium having the following components: MS + 0.4-0.6 mg / L 6-BA + 0.08-0.12 mg / L NAA + 1%-3% sucrose + 0.4-0.6% agar + 0.02-0.04% activated carbon.
[0011] Preferably, the induction time in step (1) is 30 to 90 days.
[0012] Preferably, the culture medium for suspension culture in step (2) consists of 1 / 2 MS + 0.4-0.6 mg / L 6-BA + 0-0.1 mg / L NAA + 2.5-3.5% sucrose or MS + 0.4-0.6 mg / L 6-BA + 0-0.1 mg / L NAA + 2.5-3.5% sucrose.
[0013] Preferably, the suspension culture in step (2) is a shaking culture. During the shaking culture, the rotation speed is 100-120 r / min, the temperature is 23-27℃, the light intensity is 900-1100 Lx, the light time is 14-16 h / d, and the culture cycle is 16-20 days.
[0014] Preferably, the suspension culture medium in step (3) consists of MS + 0.4-0.6 mg / L 6-BA + 2.5-3.5% sucrose.
[0015] Preferably, the green spherical bodies of the Adiantum repens described in step (3) need to pass through a 50-70 mesh cell sieve, and the cell clusters on the sieve are taken and cultured in the suspension culture medium.
[0016] Preferably, the culture in step (3) is a shaking culture. During the shaking culture, the rotation speed is 100-120 r / min, the temperature is 23-27℃, the light intensity is 900-1100 Lx, the light time is 14-16 h / d, and the culture period is 16-20 days.
[0017] Preferably, the matrix in step (3) is perlite or peat moss.
[0018] The method for establishing a suspension system of *Adiantum repens* provided by this invention induces *Adiantum repens* GGB from sterile *Adiantum repens* prothallus, eliminating the need for explant sterilization and solving the problem of scarce *Adiantum repens* resources and the inability to continuously provide raw materials for scientific research. A stable *Adiantum repens* GGB suspension system is established through cell suspension culture, exhibiting good dispersibility, high propagation efficiency, a large proliferation coefficient, and a short cycle. The established GGB suspension system can achieve an average biomass increase of 3 times in two weeks, significantly shortening the experimental cycle and improving the propagation efficiency of the rare plant *Adiantum repens*. Furthermore, the nutritional and environmental requirements during the propagation process are easily controlled. It can achieve year-round growth, unaffected by geographical factors; therefore, the advantages of the GGB suspension system of Adiantum repens are: First, it can be propagated efficiently through generations; Second, it can be directly used for sowing to complete the plant regeneration process, with high survival rate and good uniformity of regenerated plants; In this invention, the GGB morphology is removed from the sterile environment and transferred to seedling trays for processing. Compared with removing the seedlings from the sterile environment after they have matured, the GGB stage is removed from the sterile environment, which simplifies cleaning, reduces the contamination rate, increases the synchronization of seedling emergence, and increases the seedling emergence rate, while greatly reducing time and labor costs; Third, it can be used for the production and utilization of secondary metabolites, genetic transformation research, and large-scale production in bioreactors. Attached Figure Description
[0019] Figure 1 The prothallus of the lotus-leaved maidenhair fern used in Example 1;
[0020] Figure 2 The primary GGB obtained after induction in Example 1;
[0021] Figure 3 The green, non-browning Adiantum repens GGB, which was propagated in suspension in Example 1;
[0022] Figure 4 The stable *Adiantum repens* suspension system obtained in Example 1;
[0023] Figure 5 The GGB prepared for sowing after rinsing with clean water in Example 1;
[0024] Figure 6 This is a seeding diagram of the lotus leaf maidenhair fern suspension system from Example 1;
[0025] Figure 7 This refers to the regenerated plant of *Adiantum repens* from Example 1;
[0026] Figure 8 This is a *Adiantum capillus-veneris* after its first transplant in Example 1;
[0027] Figure 9 This refers to the Adiantum capillus-veneris after its second transplant in Example 1;
[0028] Figure 10 The browned Adiantum capillus-veneris GGB is from Example 2. Detailed Implementation
[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0030] Example 1
[0031] A method for establishing a suspension system of *Adiantum repens* (lotus leaf fern) comprises the following steps:
[0032] (1) GGB induction: Aseptically cultured Adiantum repens prothallus ( Figure 1 The cells were transferred to GGB induction medium for culture. The GGB induction medium consisted of MS medium containing 0.5 mg / L 6-BA, 0.1 mg / L NAA, 3% sucrose, 0.5% agar, and 0.03% activated carbon. After 60 days of culture, primary green spheroids (GGB) were obtained. Figure 2 It is dark green and granular.
[0033] (2) GGB suspension proliferation: The primary green spherical bodies obtained in step (1) were transferred to GGB suspension culture medium for suspension culture. The suspension culture medium formula was MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose. Shaking culture was used under the following conditions: rotation speed 110 r / min, culture temperature 25℃, light intensity 1000 Lx, light duration 15 h / d, and culture period 18 days. Suspension proliferation of *Adiantum repens* GGB was obtained. The proliferated GGBs were green spherical or nearly spherical particles of uniform size. Figure 3 The suspension was clear and showed no browning.
[0034] (3) Establishment of the *Adiantum repens* suspension system: The green spherical cells of *Adiantum repens* obtained in step (2) were passed through a 60-mesh cell sieve. After sieving, the liquid was allowed to stand for 1 hour, and the supernatant was discarded. An equal amount of fresh suspension culture medium was added, and the cell clusters on the sieve were inoculated into the fresh suspension culture medium for further culture. The suspension culture medium formula was MS + 0.5 mg / L 6-BA + 3% sucrose, and shaking culture was used. The conditions were: rotation speed 110 r / min, culture temperature 25℃, light intensity 1000 Lx, and light duration 15 h / d. The culture period was 18 days, and a stable *Adiantum repens* suspension system was obtained. After subculturing with suspended GGB, the proliferation rate was fast, the size was uniform, and the suspension was clear without browning. Figure 4 ).
[0035] (4) Plant regeneration: The stable Adiantum repens suspension obtained in step (3) was passed through a cell sieve and rinsed three times with water. Figure 5 Spread evenly on a moist peat moss substrate. Figure 6 ), retaining water and moisture, to obtain regenerated plants of Adiantum capillus-veneris (lotus leaf maidenhair fern). Figure 7The germination rate of regenerated plants reached 90%, with uniform emergence and no deformities observed. They grew normally after transplanting. Figure 8 , 9 ).
[0036] MS basal medium: can be prepared according to Table 6 on page 485 of the literature (Murashige T, Skoog FA revised medium for rapid grouth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15:485).
[0037] Example 2
[0038] A method for establishing a suspension system of *Adiantum repens* (lotus leaf fern) includes the following steps:
[0039] (1) GGB induction: The prothallus of Adiantum repens obtained by sterile culture was transferred to GGB induction medium for culture. The induction medium for primary green spheroids was MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 2% sucrose + 0.5% agar + 0.03% activated carbon. A small amount of GGB was induced after 60 days of culture.
[0040] (2) GGB suspension proliferation: The GGB obtained in step (1) was transferred to a suspension culture medium for suspension culture. The suspension culture medium formula was MS. The culture was shaken and cultured under the following conditions: rotation speed 110 r / min, culture temperature 25℃, light intensity 1000 Lx, and light duration 15 h / d. The culture period was 18 days to obtain suspended proliferated Adiantum repens GGB. The proliferated GGB was green spherical or nearly spherical particles with uniform size, and the suspension was slightly browned.
[0041] (3) Establishment of the lotus leaf maidenhair fern suspension system: The green spherical bodies of lotus leaf maidenhair fern obtained in step (2) were passed through a 60-mesh cell sieve. After sieving, the liquid was allowed to stand for 1 hour, and the supernatant was discarded. An equal amount of fresh suspension culture medium was added, and the cell clusters on the sieve were inoculated into the fresh suspension culture medium for further culture. The suspension culture medium formula was MS + 0.5 mg / L 6-BA + 3% sucrose. The shaking culture conditions were 110 r / min, 25℃, 1000 Lx light intensity, and 15 h / d light duration. The culture period was 18 days. The lotus leaf maidenhair fern suspension system was brown spherical. Figure 10 The suspension turned brown and could not be reused for further propagation.
[0042] (4) Plant regeneration: The Adiantum lancifolium GGB obtained in steps 2) and 3) is passed through a cell sieve, rinsed several times with clean water, and then spread evenly on a moist perlite substrate to retain moisture and obtain regenerated Adiantum lancifolium plants. The germination rate of the regenerated plants is 40%, no deformities are observed, and they can grow normally after transplanting.
[0043] Example 3
[0044] A method for establishing a suspension system of *Adiantum repens* (lotus leaf fern) comprises the following steps:
[0045] (1) GGB induction: Sterile lotus leaf maidenhair fern prothallus was transferred to GGB induction medium for culture. The induction medium was MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.5% agar + 0.03% activated carbon. GGB was obtained after 60 days of culture and appeared as dark green granules.
[0046] (2) GGB suspension proliferation: The GGB obtained in step (1) was transferred to GGB suspension culture medium for suspension culture. The suspension culture medium was MS + 0.5 mg / L 6-BA + 3% sucrose. The culture was shaken at a speed of 110 r / min, a temperature of 25℃, a light intensity of 1000 Lx, and a light duration of 15 h / d. The subculture cycle was 18 days to obtain suspended proliferated Adiantum repens GGB. The proliferated GGB was green spherical or nearly spherical particles of uniform size, and the suspension was clear without browning.
[0047] (3) Establishment of the *Adiantum repens* suspension system: The green spherical *Adiantum repens* cells obtained in step (2) were passed through a 60-mesh cell sieve. After sieving, the liquid was allowed to stand for 1 hour, and the supernatant was discarded. An equal amount of fresh suspension culture medium was added, and the cell clusters on the sieve were inoculated into the fresh suspension culture medium for further culture. The suspension culture medium was MS + 0.5 mg / L 6-BA + 3% sucrose. The cells were cultured under shaking conditions of 110 r / min, 25℃, 1000 Lx light intensity, and 15 h / d light duration for 18 days to obtain a stable *Adiantum repens* suspension system. The suspended GGB cells proliferated rapidly, were uniform in size, and the suspension was clear without browning after subculturing.
[0048] (4) Plant regeneration: The suspension system of Adiantum lancifolium obtained in step (3) was passed through a cell sieve, rinsed with water 3 times, and then spread on a moist 1:1 perlite + peat moss substrate to retain water and moisture, and Adiantum lancifolium regenerated plants were obtained. The germination rate of the regenerated plants was 50%, no deformities were observed, and they could grow normally after transplanting.
[0049] Example 4
[0050] A method for establishing a suspension system of *Adiantum repens* (lotus leaf fern) includes the following steps:
[0051] (1) GGB induction: Sterile Adiantum repens prothallus was transferred to GGB induction medium for culture. The induction medium for primary green spheroids was MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 1% sucrose + 0.5% agar + 0.03% activated carbon. A small amount of GGB was obtained after 60 days of culture, which was dark green granular.
[0052] (2) GGB suspension proliferation: The primary green spherical bodies obtained in step (1) were transferred to GGB suspension culture medium for suspension culture. The suspension proliferation culture medium was 1 / 2 MS. The culture was shaken under the following conditions: rotation speed 110 r / min, culture temperature 25℃, light intensity 1000 Lx, and light duration 15 h / d. The culture period was 18 days. The proliferation of GGB obtained from the Adiantum capillus-veneris was not obvious, the GGB was brown, and the suspension was browned.
[0053] (3) Establishment of the *Adiantum repens* suspension system: The proliferation product obtained in step (2) was passed through a 60-mesh cell sieve. After sieving, the liquid was allowed to stand for 1 hour, and the supernatant was discarded. An equal amount of fresh suspension culture medium was added, and the cell clusters on the sieve were inoculated into the fresh suspension culture medium for further culture. The suspension culture medium was MS + 0.5 mg / L 6-BA + 3% sucrose. The culture was carried out under the following conditions: rotation speed 110 r / min, culture temperature 25℃, light intensity 1000 Lx, and light duration 15 h / d. The culture period was 18 days to obtain the *Adiantum repens* GGB suspension system. The suspended GGB did not proliferate after subculturing, and the suspension showed severe browning.
[0054] (4) Plant regeneration: The Adiantum fusiforme GGB obtained in steps 2) and 3) is passed through a cell sieve, rinsed several times with clean water, and then spread evenly on a moist peat moss substrate to retain moisture and obtain regenerated Adiantum fusiforme plants. The germination rate of the regenerated plants is 60%, no deformities are observed, and they can grow normally after transplanting.
[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for establishing a suspension system of Adiantum raddianum, characterized in that, Includes the following steps: (1) Inducing the prothallus of *Adiantum repens* into primary green spherical bodies; the induction medium is a solid medium, the composition of which is MS + 0.4~0.6 mg / L 6-BA + 0.08~0.12 mg / L NAA + 1%~3% sucrose + 0.4~0.6% agar + 0.02~0.04% activated carbon; (2) The primary green spherical bodies are subjected to suspension culture to obtain suspended proliferating green spherical bodies of Adiantum repens; the culture medium for suspension culture consists of MS + 0.4~0.6 mg / L 6-BA + 0~0.1 mg / L NAA + 2.5~3.5% sucrose; (3) The green spherical bodies of the Adiantum repens are cultured in a suspension culture medium to obtain a stable Adiantum repens suspension system, and the suspension system is transplanted into a substrate to differentiate into plants; The suspension culture described in step (2) is a shaking culture. During the shaking culture, the rotation speed is 100~120 r / min, the temperature is 23~27℃, the light intensity is 900~1100Lx, the light duration is 14~16h / d, and the culture period is 16~20 days. The suspension culture medium in step (3) consists of MS + 0.4~0.6 mg / L 6-BA + 2.5~3.5% sucrose; The culture described in step (3) is a shaking culture. During the shaking culture, the rotation speed is 100~120 r / min, the temperature is 23~27℃, the light intensity is 900~1100Lx, the light time is 14~16h / d, and the culture cycle is 16~20 days.
2. The establishment method of claim 1, wherein, The induction time in step (1) is 30~90 days.
3. The establishment method of claim 1, wherein, In step (3), the green spherical bodies of the Adiantum capillus-veneris described above need to be passed through a 50-70 mesh cell sieve, and the cell clusters on the sieve should be taken and cultured in the suspension culture medium.
4. The establishment method of claim 1, wherein, The substrate in step (3) is perlite or peat moss.