Application of silicon dioxide nanoparticles in promoting growth of moss leaf incision stem cells and method of silicon dioxide nanoparticles in promoting growth of moss leaf incision stem cells

Abstract

The invention provides application of silicon dioxide nanoparticles in promoting moss leaf incision stem cell development and a method, and relates to the technical field of plant regeneration. Depending on the totipotency of the plant stem cells, the silicon dioxide nanoparticles are used for promoting the development of the stem cells of the in-vitro moss leaves subjected to mechanical damage, promoting the rapid growth of leaf incisions, generating more new growth points, remarkably increasing the number of the epiphytic points of the newly born protonema of the leaves, shortening the cultivation time and improving the cultivation efficiency; and an effective nanometer strategy is provided for rapid propagation of moss.

Description

Technical Field

[0001] This invention belongs to the field of plant regeneration technology, specifically relating to the application and method of silica nanoparticles in promoting the development of stem cells in moss leaf incisions. Background Technology

[0002] Bryophytes play a vital role not only in environmental protection, ecological conservation, and horticulture, but also in economic value, such as medicinal and ornamental value. As one of the earliest colonized higher plants, bryophytes differ significantly from angiosperms in many aspects. They have simple structures, lack vascular tissue, and their leaf-like structures are typically composed of a single layer of cells. After germination, bryophyte spores form filamentous tissues called protonemata, and each protonemata contains a apical stem cell, known as the apical cell. Generally, bryophytes exhibit strong recovery capabilities after mechanical damage; the leaf cells at the site of injury can respond to damage signals, inducing surrounding cells to differentiate into stem cells and develop into complete plants. However, damaged bryophytes grow relatively slowly. Both wild and artificially cultivated bryophytes have inherent drawbacks of slow growth and low yield, restricting their industrial application. Therefore, to overcome the bottlenecks in the application of bryophytes and improve their reproductive capacity, it is urgent to provide a method to promote the development of stem cells at the cut surfaces of bryophyte leaves.

[0003] In recent years, nanomaterials have played a vital role in agriculture, offering advantages such as low cost, high stability, and durability. They also reduce environmental pollution and energy waste caused by traditional chemical fertilizers and pesticides, contributing to the construction of an environmentally friendly society. The application of nano-fertilizers and nano-pesticides has provided innovative solutions to practical challenges in agricultural production, achieving remarkable progress. However, the impact of nanoparticles on mosses remains poorly understood, and their influence on moss growth and development has not been fully explored. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of silica nanoparticles in promoting the development of stem cells in moss leaf cuts. This invention induces stem cell growth and development at the cut site by first soaking the moss leaf cut with silica nanoparticles, thereby promoting the rapid generation of new growth points, shortening the cultivation time, and improving cultivation efficiency.

[0005] A second objective of this invention is to provide an agent that promotes the development of stem cells from cut surfaces on moss leaves.

[0006] A third objective of this invention is to provide a culture method for promoting the development of stem cells from cut surfaces on moss leaves.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: This invention provides the application of silica nanoparticles in promoting the development of stem cells in moss leaf incisions.

[0008] In one embodiment, the concentration of the silica nanoparticles is 1~100 mg / L.

[0009] In one embodiment, the silica nanoparticles include VMSN@Mn nanoparticles or VMSN nanoparticles.

[0010] In one embodiment, the moss includes *Moss styracifolium*.

[0011] The present invention also provides an agent for promoting the development of stem cells in cut scalp moss leaves, the agent comprising silica nanoparticles.

[0012] In one embodiment, the silica nanoparticles include VMSN@Mn nanoparticles or VMSN nanoparticles, and the concentration of the silica nanoparticles is 1~100 mg / L.

[0013] This invention also provides a method for promoting the development of stem cells from moss leaf incisions, wherein the above-described preparation is used to culture moss leaf incisions.

[0014] One implementation method includes the following steps: the cut surface of moss leaf is soaked in a dispersion of 1-100 mg / L silica nanoparticles for 3-10 h, and then cultured in a basal culture medium.

[0015] In one implementation, the basal culture medium is BCDAT medium.

[0016] As one implementation method, the cultivation conditions are 20~28℃, humidity 35%~45%, photoperiod 16 h / 8 h, and light intensity 60~80 μmol•photons•m. -2 •s -1 .

[0017] The advantages of this invention compared to existing technologies are as follows: The silica nanoparticles of this invention promote the development of stem cells in moss leaf incisions. After the incision is immersed in VMSN@Mn nanoparticle dispersion or VMSN nanoparticle dispersion, it can promote the rapid growth of leaf incisions. After 6 days of culture, the number of moss incision stem cells and the number of new protonema attachment points on the leaves are significantly increased, thereby shortening the moss cultivation time and improving the cultivation efficiency. This provides an effective nano-strategy for the rapid propagation of moss. Attached Figure Description

[0018] Figure 1The images show representative pictures of detached leaves that have suffered mechanical damage, after being soaked in sterile double-distilled water for 4 hours and cultured for 6 days. The arrows in the images indicate the attachment points of newly formed protonema. Figure 2 A bar chart showing the number of new protonema attachment points at leaf cuts 6 days after treatment with different concentrations of VMSN@Mn nanoparticles. Figure 3 A bar chart showing the number of new protonema attachment points on moss leaves 6 days after mechanical damage, treated with different concentrations of VMSN@Mn nanoparticles. Figure 4 A bar graph showing the number of new protonema attachment points at leaf cuts 6 days after treatment with different concentrations of VMSN nanoparticles. Figure 5 A bar chart showing the number of new protonema attachment points on moss leaves 6 days after mechanical damage, treated with different concentrations of VMSN nanoparticles. Detailed Implementation

[0019] This invention constructs detached moss leaves subjected to mechanical damage. Experimental results show that the silica nanoparticle dispersion can promote the development of stem cells in mechanically damaged moss leaves and can be used for the cultivation of stem cells from moss leaf incisions.

[0020] In this invention, the silica nanoparticle dispersion is prepared by dissolving silica nanoparticles in water, preferably in double-distilled water. The concentration of the silica nanoparticle dispersion is 1~100 mg / L, preferably 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, or 100 mg / L. The silica nanoparticles include VMSN@Mn nanoparticles or VMSN nanoparticles, wherein the VMSN nanoparticles are virus-like mesoporous silica nanoparticles synthesized by a two-phase diffusion system, with a hydrated particle size of 160-200 nm. In this invention, as an optional embodiment, the VMSN nanoparticles are prepared by mixing a hexadecyltrimethylammonium bromide solution and a mixed solution at a volume ratio of 1:(1-3) and reacting at 55-65°C, followed by filtration and purification. The mixed solution includes tetraethyl orthosilicate and cyclohexane at a volume ratio of 1:(3-5). The VMSN@Mn nanoparticles are nanoparticles coated with a layer of manganese dioxide on the surface of VMSN, and their hydrated particle size is identical to that of the VMSN nanoparticles. In this invention, as an optional embodiment, the VMSN@Mn nanoparticles are prepared by ultrasonically dispersing VMSN in a 0.1-0.2 mM MES solution, then slowly adding a 4-6 mM potassium permanganate solution dropwise, followed by ultrasonic dispersion and centrifugation. The ultrasonic dispersion is continued until uniform dispersion is achieved. In this invention, soaking mechanically damaged moss leaves in silica nanoparticles significantly increases the number of moss cuts and the number of new protonema attachment points. As an optional embodiment, the moss includes *Sphaerocera minor*. *Sphaerocera minor* plays an important role in ecological environment, horticultural landscape, and medicinal applications. Furthermore, *Sphaerocera minor* is a non-vascular plant with a simple structure; its pseudo-leaf consists of a single layer of cells, allowing for asexual reproduction, which is beneficial for research on its growth and development.

[0021] This invention also provides a formulation for promoting the development of stem cells in moss leaf incisions, the formulation comprising silica nanoparticles. In this invention, the silica nanoparticles comprise VMSN@Mn nanoparticles or VMSN nanoparticles, and the concentration of the silica nanoparticles is 1~100 mg / L, preferably 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, or 100 mg / L.

[0022] This invention also provides a method for culturing moss leaf incisions to promote stem cell development, wherein the moss leaf incisions are cultured in the culture medium described above. As an optional embodiment, the culture method includes the following steps: the moss leaf incisions are soaked in a 1-100 mg / L silica nanoparticle dispersion for 4-10 h, and then cultured in a basal culture medium. In this invention, after soaking the moss in the silica nanoparticle dispersion of the stated concentration for 4 h, the silica nanoparticles can enter the moss and promote stem cell development at the incision site. To shorten the moss culture time, the soaking time is preferably 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, or 10 h. After soaking, the detached moss leaves are washed with double-distilled water, preferably once with sterile double-distilled water. In this invention, all the above operations are performed aseptically in a laminar flow hood.

[0023] After washing, the detached moss leaves are placed in a basal culture medium for cultivation, preferably BCDAT medium. In this invention, the BCDAT medium can be prepared according to the following formula. The BCDAT medium was formulated as follows: 1 mM MgSO4•7H2O, 0.0184 mM KH2PO4 (pH=6.5), 10 mM KNO3, 0.045 mM FeSO4•7H2O, 0.00022 mM CuSO4•5H2O, 0.010 mM H3BO3, 0.00023 mM CoCl2•6H2O, 0.001 mM Na2MoO4•2H2O, 0.00019 mM ZnSO4•7H2O, 0.002 mM MnCl2•4H2O, 0.00017 mM KI, 5 mM ammonium tartrate, 1~10 mM CaCl2·2H2O, and 9 g / L Agar. As an optional implementation, the concentration of CaCl2·2H2O is preferably 1 mM, 3 mM, 5 mM, 7 mM, or 9 mM. As another implementation, the culture temperature is 20–28°C, preferably 23°C, 25°C, or 27°C; the culture humidity is 35%–45%, preferably 38%, 40%, or 43%; the photoperiod is 16 h / 8 h; and the light intensity is 60–80 μmol•photons•m. -2 •s -1 Preferably, it is 65 μmol•photons•m -2 •s -1 70 μmol•photons•m -2 •s -1 Or 75 μmol•photons•m -2 •s-1 The culture time is 6-7 days. This invention significantly increases the number of moss incision stem cells and the number of attachment points for new leaf protonema after 6 days of culture, thus shortening the moss cultivation time.

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0026] The formula for BCDAT medium is: 1 mM MgSO4•7H2O, 0.0184 mM KH2PO4 (pH=6.5), 10 mM KNO3, 0.045 mM FeSO4•7H2O, 0.00022 mM CuSO4•5H2O, 0.010 mM H3BO3, 0.00023 mM CoCl2•6H2O, 0.001 mM Na2MoO4•2H2O, 0.00019 mM ZnSO4•7H2O, 0.002 mM MnCl2•4H2O, 0.00017 mM KI, 5 mM ammonium tartrate, 1 mM CaCl2·2H2O, and 9 g / L Agar.

[0027] Preparation method of VMSN nanoparticles: 2 g of hexadecyltrimethylammonium bromide (CTAB) and 1.6 mL of NaOH (0.1 M) were added to 100 mL of water, and stirred in a round-bottom flask at 60 ℃ and 400 rpm for 2 h. After the CTAB was completely dissolved, 200 mL of a mixture of tetraethyl orthosilicate (TEOS) and cyclohexane (volume ratio 1:4) was added to the solution, and the mixture was reacted in an oil bath at 60 ℃ for 72 h. Then, the supernatant was removed by centrifugation, and the mixture was washed several times with water and ethanol to obtain a mixture of spiky mesoporous silica nanoparticles containing CTAB. 200 mL of acetone was added to the mixture, and the mixture was refluxed in an oil bath at 60 ℃ with cooling water for 48 h to remove CTAB. The supernatant was then removed by centrifugation to obtain the filtered solid, which was washed 6 times with ethanol and 3 times with water to obtain spiky mesoporous silica nanoparticles (VMSN) stored in the aqueous phase.

[0028] Preparation method of VMSN@Mn nanoparticles: VMSN is obtained by coating a layer of manganese dioxide onto the surface of stably synthesized VMSN. Specifically, 5 mg of stably synthesized VMSN was dispersed in 0.25 mL of 0.1 mM morpholine ethanesulfonic acid monohydrate (MES) solution (pH 6.0) and ultrasonically dispersed. After uniform dispersion, 0.2 mL of 5 mM potassium permanganate (KMnO4) solution was slowly added dropwise and ultrasonically dispersed. After uniform dispersion, the mixture was ultrasonicated for 30 min, then centrifuged to remove the supernatant. The resulting solid was washed with water, and this process was repeated three times to obtain manganese-coated, spike-like mesoporous silica nanoparticles (VMSN@Mn) stored in the aqueous phase.

[0029] Example 1 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerocarpa*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN@Mn nanoparticles were dissolved in double-distilled water to prepare a 1 mg / L VMSN@Mn nanoparticle dispersion. The detached moss leaves were immersed in this 1 mg / L VMSN@Mn nanoparticle dispersion for 4 h. After 4 h of immersion, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at a temperature of 25℃, humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 60 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0030] Example 2 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerocarpa*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN@Mn nanoparticles were dissolved in double-distilled water to prepare a 10 mg / L VMSN@Mn nanoparticle dispersion. The detached moss leaves were immersed in this 10 mg / L VMSN@Mn nanoparticle dispersion for 4 h. After 4 h, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at a temperature of 25℃, humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 80 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0031] Example 3 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerocarpa*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN@Mn nanoparticles were dissolved in double-distilled water to prepare a 50 mg / L VMSN@Mn nanoparticle dispersion. The detached moss leaves were immersed in this dispersion for 4 h. After 4 h, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at 25°C, 40% humidity, a photoperiod of 16 h / 8 h, and a light intensity of 70 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0032] Example 4 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerophyte*, grown for one month, were cut off with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN@Mn nanoparticles were dissolved in double-distilled water to prepare a 100 mg / L VMSN@Mn nanoparticle dispersion. The detached moss leaves were immersed in this 100 mg / L VMSN@Mn nanoparticle dispersion for 4 hours. After 4 hours of immersion, the detached moss leaves were washed once with sterile double-distilled water. Then, the detached moss leaves were carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at a temperature of 25°C, a humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 65 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0033] Example 5 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerophyte*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN nanoparticles were dissolved in double-distilled water to prepare a 1 mg / L VMSN nanoparticle dispersion. The detached moss leaves were immersed in this dispersion for 4 h. After 4 h, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at 25°C, 40% humidity, a photoperiod of 16 h / 8 h, and a light intensity of 75 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0034] Example 6 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerophyte*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN nanoparticles were dissolved in double-distilled water to prepare a 10 mg / L VMSN nanoparticle dispersion. The detached moss leaves were immersed in this 10 mg / L VMSN nanoparticle dispersion for 4 h. After 4 h of immersion, the detached moss leaves were washed once with sterile double-distilled water. Then, the detached moss leaves were carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at a temperature of 25℃, humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 60 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0035] Example 7 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerophyte*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN nanoparticles were dissolved in double-distilled water to prepare a 50 mg / L VMSN nanoparticle dispersion. The detached moss leaves were immersed in this dispersion for 4 h. After 4 h, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at 25°C, 40% humidity, a photoperiod of 16 h / 8 h, and a light intensity of 80 μmol•photons•m. -2 •s-1 Cultured in a light incubator for 6 days.

[0036] Example 8 A method for promoting the development of stem cells from cut surfaces of moss leaves includes the following steps: Under a desktop magnifying glass, 1-3 mm leaflets of the gametophyte *Moss sphaerophyte*, grown for one month, were cut with a sterile scalpel to serve as detached moss leaves subjected to mechanical damage. VMSN nanoparticles were dissolved in double-distilled water to prepare a 100 mg / L VMSN nanoparticle dispersion. The detached moss leaves were immersed in this 100 mg / L VMSN nanoparticle dispersion for 4 h. After 4 h of immersion, the detached moss leaves were washed once with sterile double-distilled water, and then carefully placed into BCDAT medium using tweezers. All of the above operations were performed in a clean bench. The medium was then placed at a temperature of 25℃, humidity of 40%, a photoperiod of 16 h / 8 h, and a light intensity of 68 μmol•photons•m. -2 •s -1 Cultured in a light incubator for 6 days.

[0037] Comparative Example 1 Compared with Example 1, the VMSN@Mn nanoparticle dispersion was replaced with double-distilled water, and the detached moss leaves were soaked in double-distilled water for 4 hours, while the other steps were the same.

[0038] Experimental Example 1 In a sterile environment, 100 detached leaves of *Moss sphaerocephala* were cut and divided into 5 groups of 20 leaves each. After culturing for 6 days according to the methods of Examples 1-4 and Comparative Example 1, photographs were taken for observation and data analysis. Representative images of mechanically damaged detached leaves after soaking in sterile double-distilled water for 4 hours and culturing for 6 days are shown below. Figure 1 As shown in the figure, the arrows indicate the attachment points of newly formed protonema. The number of attachment points of newly formed protonema in mechanically damaged detached leaves was statistically analyzed using GraphPad Prism 8.0 software. Data are expressed as mean ± standard error, and t-tests were used to analyze differences. * indicates... p <0.05, ** indicates p <0.01, ns indicates no significant difference.

[0039] The results of the analysis of the number of new protonema attachment points at the leaf cut and the total number of new protonema on the leaves 6 days after mechanical damage in each of the groups of Examples 1-4 with different concentrations of VMSN@Mn nanoparticles are shown in Table 1 and 2. Figures 2-3 As shown in Table 1, the number of new protonema attachment points at the cut and the total number of new protonema on the leaves after mechanical damage 6 days after treatment with different concentrations of VMSN@Mn nanoparticles in Examples 1-4 were greater than those in Comparative Example 1 (Mock).

[0040] Table 1. Number of new protonema attachment points at leaf cuts and total number of leaves treated with VMSN@Mn nanoparticles 6 days after mechanical damage. Depend on Figure 2 It can be seen that the number of new protofilament attachment points generated at the cut site after treatment with 10 mg / L and 50 mg / L VMSN@Mn nanoparticles is significantly different compared with Comparative Example 1. Figure 3 It can be seen that the number of new protonema attachment points generated in the leaves after treatment with 1 mg / L VMSN@Mn nanoparticles is significantly different from that in Comparative Example 1.

[0041] Experimental Example 2 In a sterile environment, 100 detached leaves of *Moss sphaerocephala* were cut and divided into 5 groups of 20 leaves each. After culturing for 6 days according to the methods described in Examples 5-8 and Comparative Example 1, photographs were taken for observation and data analysis. The number of new protonema attachment points in mechanically damaged detached leaves was statistically analyzed using GraphPad Prism 8.0 software. Data are expressed as mean ± standard error, and t-tests were used to analyze differences. * indicates... p <0.05, ** indicates p <0.01, ns indicates no significant difference.

[0042] The results of the analysis of the number of new protonema attachment points at the leaf cut and the total number of new protonema on the leaves 6 days after mechanical damage in Examples 5-8 were obtained by treating moss with different concentrations of VMSN nanoparticles. The results are shown in Table 2 and... Figures 4-5 As shown in Table 2, the number of new protonema attachment points at the cut surface of the leaves treated with different concentrations of VMSN nanoparticles in Examples 5-7 was greater than that of Comparative Example 1 (Mock) 6 days after mechanical damage. The number of new protonema attachment points of the leaves treated with different concentrations of VMSN nanoparticles in Examples 5-8 was also greater than that of Comparative Example 1 (Mock) 6 days after mechanical damage.

[0043] Table 2. Number of new protonema attachment points at leaf cuts and total number of leaves 6 days after mechanical damage treated with different concentrations of VMSN nanoparticles.

[0044] Depend on Figure 4 It can be seen that the number of new protonema attachment points at the leaf cut surface 6 days after mechanical damage treated with different concentrations of VMSN nanoparticles is significantly different from that of Comparative Example 1 (Mock). Figure 5It can be seen that the number of new protonema attachment points generated in the leaves when treated with 50 mg / LVMSN nanoparticles in Example 7 is significantly different from that in Comparative Example 1.

[0045] In summary, this invention leverages the totipotency of plant stem cells and utilizes silica nanoparticles to promote the development of stem cells in mechanically damaged detached moss leaves. This increases the number of stem cells at the cut surfaces of the moss leaves, i.e., the number of new protonema attachment points at the cut surfaces, thereby generating more new growth points, shortening the cultivation time, and improving cultivation efficiency. Compared to untreated mechanically damaged detached moss leaves, the number of new protonema attachment points at the cut surfaces and the total number of new protonema attachment points on the treated leaves are significantly increased, showing a clear difference, especially after treatment with VMSN@Mn nanoparticles. Therefore, the technical solution provided by this invention can promote the rapid growth of mechanically damaged detached leaves, compensating for the inherent slow growth of bryophytes and achieving rapid propagation.

[0046] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Application of silica nanoparticles in promoting the development of stem cells in moss leaf incisions.

2. The application according to claim 1, characterized in that, The concentration of the silica nanoparticles is 1~100 mg / L.

3. The application according to claim 1, characterized in that, The silica nanoparticles include VMSN@Mn nanoparticles or VMSN nanoparticles.

4. The application according to claim 1, characterized in that, The moss includes *Moss styracifolium*.

5. A preparation for promoting the development of stem cells in cut surfaces of moss leaves, characterized in that, The formulation comprises silica nanoparticles.

6. The formulation according to claim 5, characterized in that, The silica nanoparticles include VMSN@Mn nanoparticles or VMSN nanoparticles, and the concentration of the silica nanoparticles is 1~100 mg / L.

7. A method for culturing stem cells to promote the development of moss leaf incisions, characterized in that, The incision of moss leaf is cultured using the formulation described in any one of claims 5 to 6.

8. The cultivation method according to claim 7, characterized in that, Includes the following steps: Moss leaf cuts were soaked in a 1-100 mg / L silica nanoparticle dispersion for 4-10 h, and then cultured in a basal culture medium.

9. The cultivation method according to claim 8, characterized in that, The basal culture medium is BCDAT medium.

10. The cultivation method according to claim 8, characterized in that, The cultivation conditions were 20–28°C, 35%–45% humidity, a photoperiod of 16 h / 8 h, and a light intensity of 60–80 μmol•photons•m. -2 •s -1 .

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