Tissue culture method of hypnum plumaeforme
By using prosthetic filament of the giant ash moss and specific induction medium for tissue culture, the problems of slow growth and lack of tissue culture technology of the giant ash moss were solved, rapid regeneration of the prosthetic filament and increase biomass were achieved, and the promotion and application of the giant ash moss was promoted.
Patent Information
- Application Number
- CN202510031987.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-03-04
AI Technical Summary
Research on tissue culture technology that grows slowly and lacks systematic influences its promotion and application.
The prosthesis of the giant ash moss was used as the explant, and the induction culture medium was used for induction culture. The composition of the induction medium included A solution, B solution, C solution, CaCl2, KNO3, FeSO4 and agar.
A large number of protofilament was induced in a short time, significantly increasing the diameter of protofilament, achieving rapid regeneration and biomass increase of protofilament in the large gray moss, and at the same time inducing the generation of gametobody.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of moss tissue culture, and in particular relates to a tissue culture method of moss. Background Art
[0002] The plant body of Hypnumplumaeforme is relatively large, mostly green or yellow-green in color. Its stem grows prostrate and can be attached to a substrate, such as the base of a tree, a rock, or the surface of the soil. Hypnumplumaeforme is evergreen all year round, has a good covering effect, forms a lawn-like shape, and is resistant to extensive management. When Hypnumplumaeforme grows densely, it can effectively reduce the erosion of soil by rainwater and prevent soil erosion. At the same time, Hypnumplumaeforme is sensitive to environmental changes, especially changes in air quality and humidity. In areas with more serious pollution, the growth of Hypnumplumaeforme may be inhibited, so it can be used as an indicator plant of environmental quality to monitor changes in the ecological environment. In addition, Hypnumplumaeforme also has medicinal effects and can be used to treat burns, epistaxis, hemoptysis, hematemesis, and metrorrhagia.
[0003] Large gray moss has good landscape effects and application prospects, but its slow growth and lack of systematic tissue culture technology research have a certain impact on its promotion and application. Summary of the invention
[0004] In view of this, the object of the present invention is to provide a tissue culture method of Glechoma longituba to promote the rapid growth of protonema, promote the formation of gametophyte and increase its biomass.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] A tissue culture method of Glechoma sphaerocephala, which uses Glechoma sphaerocephala protonema as explants and uses Glechoma sphaerocephala induction medium for induction culture;
[0007] The composition of the induction medium of the large gray moss includes: 10 mL / L of solution A, 10 mL / L of solution B, 1 mL / L of solution C, 10 mL / L of 1 M CaCl2·2H2O, 1.01 g / L of KNO3, 0.0125 g / L of FeSO4·7H2O and 9 g / L of agar;
[0008] The composition of solution A is: MgSO4·7H2O 250 g / L; the composition of solution B is: KH2PO4 250 g / L; the composition of solution C is: CuSO4·5H2O 55 mg / L, H3BO3 614 mg / L, CoCl2·6H2O 55 mg / L, Na2MoO4·2H2O 25 mg / L, ZnSO4·7H2O 55 mg / L, MnCl2·4H2O 389 mg / L, KI 28 mg / L.
[0009] Preferably, the explant preparation method comprises: collecting spores of the moss, disinfecting them with 10% sodium hypochlorite for 12 to 15 minutes, and then disinfecting them with 75% ethanol for 2 minutes, preparing a spore suspension, inoculating it into a moss induction medium, and culturing to obtain a protonema mass, namely the moss explant.
[0010] Preferably, the diameter of the protofilament mass is 0.1 to 1 cm.
[0011] Preferably, the culture conditions after the spore suspension is inoculated into the induction medium of the moss spores are: the culture temperature is 20-25°C, the illumination time is greater than 12 hours / day, and the illumination intensity is 30-100 μmolphotons m -2 s -1 Preferably, the culture time is 7 to 40 days.
[0012] Preferably, the pH of the induction medium of the Glechoma longituba is 5-7.
[0013] Preferably, the culture conditions of the induction culture are: culture temperature of 20-25°C, illumination time of more than 12 hours / day, and illumination intensity of 30-100 μmolphotons m -2 s -1 .
[0014] Another object of the present invention is to provide application of the tissue culture method in increasing the diameter of the protonema of Glechoma longituba.
[0015] Another object of the present invention is to provide an application of the tissue culture method in increasing the number of newly added gametophytes of Mossus maximus.
[0016] Another object of the present invention is to provide application of the tissue culture method in maintaining excellent traits of Glechoma longituba.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] The present invention provides a tissue culture method of a large gray moss. The present invention uses a large gray moss protonema as an explant and a large gray moss induction medium as an induction medium, can induce a large number of protonema production in a short time, and significantly increase the diameter of the protonema, realize the rapid regeneration of the large gray moss protonema and increase the biomass, and can induce the generation of gametophytes on this culture medium at the same time. The present invention provides theoretical support for the large-scale production and application of large gray moss, and is of great significance to the wide promotion of large gray moss and the industrial development of large gray moss. Therefore, studying how to establish a tissue culture system of large gray moss, promote the rapid regeneration of protonema, cultivate gametophytes, and increase its biological yield is of great significance for realizing large-scale artificial planting of large gray moss. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The average diameter of each protonema of the moss was changed when the protonema grew on different culture media for 40 days. Different letters indicate significant differences at the 0.05 level.
[0020] Figure 2 The length of the gametophyte regenerated from each protonema of the moss grosbeckia was changed when the protonema grew on different culture media for 40 days. Different letters indicate significant differences at the 0.05 level.
[0021] Figure 3 The effect of different explants on the number of regenerated gametophytes, asterisks indicate significant differences, where **** corresponds to p < 0.0001;
[0022] Figure 4 The effect of different pH values of the induction medium of G. grisea on the diameter of the protonema, where different letters indicate significant differences at the 0.05 level. DETAILED DESCRIPTION
[0023] The invention provides a tissue culture method of a large gray moss. The large gray moss protonema is used as an explant and an induction medium of the large gray moss is used for induction culture. The composition of the induction medium of the large gray moss comprises: 10 mL / L of A solution, 10 mL / L of B solution, 1 mL / L of C solution, 10 mL / L of 1M CaCl2·2H2O, 1.01 g / L of KNO3, 0.0125 g / L of FeSO4·7H2O and 9 g / L of agar; the component of the A solution is: 250 g / L of MgSO4·7H2O; the component of the B solution is: 250 g / L of KH2PO4; the component of the C solution is: 55 mg / L of CuSO4·5H2O, 614 mg / L of H3BO3, 55 mg / L of CoCl2·6H2O, 25 mg / L of Na2MoO4·2H2O, 55 mg / L of ZnSO4·7H2O, and 9 g / L of agar. 389mg / L, KI 28mg / L. The method provided by the invention can significantly increase the diameter of the protonema, induce the production and growth of the gametophyte, realize the rapid regeneration of the protonema and gametophyte of the moss of Glechoma longituba and increase the biomass.
[0024] In the present invention, the preparation method of the explant comprises: collecting spores of the moss, disinfecting them with 10% sodium hypochlorite for 12-15 minutes, and then disinfecting them with 75% ethanol for 2 minutes, preparing a spore suspension, inoculating it into a moss induction culture medium, and culturing to obtain a protonema mass, which is the moss explant.
[0025] In the present invention, the 10% sodium hypochlorite disinfection is 1 mL of 10% sodium hypochlorite disinfection / spore for disinfection; the 75% ethanol disinfection is 1 mL of 75% ethanol / spore for disinfection; after the disinfection, the spores are washed with sterile water, and the washing is repeated six times, and the washing is 1 mL of sterile water / spore for washing; after the washing is completed, one spore is placed in 1 mL of sterile water, and the spore is punctured to prepare a spore suspension.
[0026] In the present invention, the culture conditions after the spore suspension is inoculated into the induction medium of the moss are: the culture temperature is 20-25°C, the illumination time is greater than 12 hours / day, and the illumination intensity is 30-100 μmolphotons m -2 s -1 The preferred culture temperature is 22-23°C, the preferred photoperiod is 16 h light + 8 h dark, and the preferred light intensity is 60-80 μmolphotons m -2 s -1 . The culture time is 7 to 40 days. After the culture is completed, a protonema mass is obtained as an explant for induction culture of P. gigantea. The diameter of the protonema mass is 0.1 to 1 cm, preferably 0.11 to 0.5 cm, and more preferably 0.14 to 0.2 cm. After the protonema material provided by the present invention is inoculated into the P. gigantea induction culture medium, rapid regeneration of the protonema of P. gigantea can be achieved, and the diameter of the protonema and gametophyte regeneration can be significantly increased.
[0027] In the present invention, the pH of the induction medium of the moss is 5 to 7. The present invention uses the induction medium of the moss as a medium for spore germination to form protonema, and a medium for protonema induction culture, which can induce a large number of protonema in a short time and obtain a larger number of gametophytes. In the present invention, the culture conditions of the induction culture are: the culture temperature is 20 to 25°C, the illumination time is greater than 12 hours / day, and the illumination intensity is 30 to 100 μmolphotons m -2 s -1 The preferred culture temperature is 22-23°C, the preferred photoperiod is 16h light + 8h dark, and the preferred light intensity is 60-80μmolphotonsm -2 s -1 .
[0028] The invention also provides application of the tissue culture method in increasing the diameter of the protonema of the moss.
[0029] The present invention also provides application of the tissue culture method in increasing the number of newly added gametophytes of Glechoma longituba.
[0030] The invention also provides application of the tissue culture method in maintaining excellent traits of Glechoma longituba.
[0031] In the specific examples of the present invention, the experimental data were plotted using Graphpadprism 8.0, the data were analyzed using SPSS, and the data were expressed as Mean ± SEM as data analyzed using Student's t-test.
[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0033] Example 1
[0034] A tissue culture method of Glechoma longituba.
[0035] The composition of the induction medium of the large gray moss in this embodiment is: A solution 10mL / L, B solution 10mL / L, C solution 1mL / L, 1M CaCl2·2H2O 10mL / L, KNO3 1.01g / L, FeSO4·7H2O 0.0125g / L and agar 9g / L; the composition of A solution is: MgSO4·7H2O 250g / L; the composition of B solution is: KH2PO4 250g / L; the composition of C solution is: CuSO4·5H2O 55mg / L, H3BO3 614 mg / L, CoCl2·6H2O 55mg / L, Na2MoO4·2H2O 25mg / L, ZnSO4·7H2O 55mg / L, MnCl2·4H2O 389mg / L, KI 28mg / L. The pH of the induction medium of the large gray moss is 5.4.
[0036] The method for obtaining the protonema mass is as follows: spores of the moss are collected, one spore is placed in 1 mL of 10% sodium hypochlorite solution for disinfection for 15 minutes, and then the spore is placed in 1 mL of 75% ethanol for disinfection for 2 minutes. After disinfection, the spore is placed in 1 mL of sterile water for washing, and the washing is repeated 6 times. After washing, the spore is placed in 1 mL of sterile water, the spore is punctured, and a spore suspension is obtained, which is inoculated into a moss induction medium for culture. The culture temperature is 22°C, the photoperiod is 16 h light + 8 h dark, and the light intensity is 70 μmol photons m -2 s -1 Under the above conditions, after 30 days of culture, a mass of protonema with a diameter of about 1 cm was obtained.
[0037] A protonema mass with a diameter of 0.15 cm was cut from the protonema mass obtained above and inoculated into a protonema induction medium for culture. The culture conditions were as follows: culture temperature 22°C, photoperiod 16 h light + 8 h dark, light intensity 70 μmolphotons m -2 s -1 .
[0038] Example 2
[0039] A tissue culture method of Glechoma longituba, the difference between this embodiment and embodiment 1 is that the culture conditions are: the culture temperature is 20°C, the photoperiod is 16h light + 8h dark, and the light intensity is 75μmolphotons m -2 s -1 .
[0040] Example 3
[0041] A tissue culture method of Glechoma longituba, the difference between this embodiment and embodiment 1 is that the culture conditions are: the culture temperature is 25°C, the photoperiod is 16h light + 8h dark, and the light intensity is 80μmolphotons m -2 s -1 .
[0042] Example 4
[0043] A tissue culture method for Glechoma longituba. The difference between this embodiment and embodiment 1 is that the pH value of the induction culture medium of Glechoma longituba is 5.
[0044] Example 5
[0045] A tissue culture method for Glechoma longituba. The difference between this embodiment and embodiment 1 is that the pH value of the induction culture medium of Glechoma longituba is 6.
[0046] Example 6
[0047] A tissue culture method for Glechoma longituba. The difference between this embodiment and embodiment 1 is that the pH value of the induction culture medium of Glechoma longituba is 7.
[0048] Comparative Example 1
[0049] A tissue culture method for Glechoma sphaerocephala. The difference between this comparative example and Example 1 is that: Glechoma sphaerocephala gametophyte is used as explant, inoculated into Glechoma sphaerocephala induction medium and then cultured, and the Glechoma sphaerocephala gametophyte is 0.7 cm in length.
[0050] Comparative Example 2
[0051] A tissue culture method for Glechoma longituba. The difference between this comparative example and Example 1 is that: the components of the Glechoma longituba induction culture medium are as shown in Table 1, wherein the components of solution A are: MgSO4·7H2O 250 g / L; the components of solution B are: KH2PO4 250 g / L; the components of solution C are: CuSO4·5H2O 55 mg / L, H3BO3 614 mg / L, CoCl2·6H2O 55 mg / L, Na2MoO4·2H2O 25 mg / L, ZnSO4·7H2O 55 mg / L, MnCl2·4H2O 389 mg / L, KI 28 mg / L.
[0052] Table 1. Induction medium of Glechoma longituba (pH 5.8)
[0053] Element Addition amount (per liter) A solution 10mL Solution B 10mL Solution C 1mL <![CDATA[1MCaCl2·2H2O]]> 10mL sucrose 10g <![CDATA[KNO3]]> 1.01g <![CDATA[FeSO4·7H2O]]> 0.0125g Agar 9g
[0054] Comparative Example 3
[0055] A tissue culture method for Glechoma longituba. The difference between this comparative example and Example 1 is that the components of the Glechoma longituba induction culture medium are as shown in Table 2.
[0056] Table 2. Induction medium for Glechoma longituba (pH 6.2)
[0057] Element Addition amount (per liter) <![CDATA[KNO3]]> 0.25g <![CDATA[MgSO4·7H2O]]> 0.25g <![CDATA[KH2PO4]]> 0.25g <![CDATA[Ca(NO3)2·4H2O]]> 1g <![CDATA[ZnSO4·7H2O]]> 0.003g Agar 12g
[0058] Comparative Example 4
[0059] A tissue culture method for Glechoma longituba. The difference between this comparative example and Example 1 is that the components of the Glechoma longituba culture medium are as shown in Table 3.
[0060] Table 3 Large gray moss induction medium (pH 6.2)
[0061] Element Addition amount (per liter) KCl 0.25g <![CDATA[MgSO4·7H2O]]> 0.25g <![CDATA[KH2PO4]]> 0.25g <![CDATA[Ca(NO3)2·4H2O]]> 1g <![CDATA[FeSO4·7H2O]]> 0.0125g Agar 12g
[0062] Example 7
[0063] Effect of induction medium types on tissue culture of Glechoma longituba.
[0064] Treatment 1: The method of Example 1 was adopted to induce and culture the protonema of Mossus grisea, which was marked as A.
[0065] Treatment 2: The method of Comparative Example 2 was adopted to induce and culture the protonema of Glechoma longituba, which was recorded as B.
[0066] Treatment 3: The method of Comparative Example 3 was adopted to induce and culture the protonema of Mossus grisea, which was recorded as C.
[0067] Treatment 4: The method of Comparative Example 4 was adopted to induce and culture the protonema of Mossus grisea, which was recorded as D.
[0068] Treatments 1 to 4 were used to induce the culture of protonema of M. grisea. Three culture dishes were inoculated in each treatment, with 9 protonema clusters in each dish. The culture dishes were placed in an incubator and cultured for 40 days. The experiment was repeated 3 times.
[0069] The average diameter of the protonema in each treatment was counted on the 40th day of culture. The results are shown in Table 4 and Figure 1 As shown, the four culture media used in Example 1 of the present invention and Comparative Examples 2 to 4 can all be used for the regeneration of protonema of G. giganteus, and the optimal culture medium is the G. giganteus induction culture medium provided in Example 1 of the present invention. The present invention can significantly increase the protonema diameter.
[0070] Table 4 Diameter of protofilament
[0071]
[0072]
[0073] Note: In this table, 3 dishes of materials for each condition, 9 large gray mosses in each dish, a total of 27 large gray mosses were recorded as one group, repeated 3 times, and the average and standard error were calculated.
[0074] On the 40th day of culture, the average length of the gametophytes regenerated from the protonema of each treatment was counted. The results are shown in Table 5 and Figure 2 As shown, the four culture media used in Example 1 of the present invention and Comparative Examples 2 to 4 can all induce the formation of gametophytes. The induction culture medium for the giant moss used in the method provided in Example 1 of the present invention can significantly increase the length of the regenerated gametophytes, achieve rapid regeneration of the giant moss gametophytes and increase biomass.
[0075] Table 5 Gametophyte length
[0076]
[0077] Note: In this table, 3 dishes of materials for each condition, 9 large gray mosses in each dish, a total of 27 large gray mosses were recorded as one group, repeated 3 times, and the average and standard error were calculated.
[0078] Example 8
[0079] Effects of explants on tissue culture of Glechoma longituba.
[0080] Treatment 1: The method of Example 1 was adopted to induce and culture the protonema of P. grisea, which was recorded as protonema.
[0081] Treatment 2: The method of Comparative Example 1 was adopted to induce and culture the gametophyte of Glechoma gigas, recorded as gametophyte.
[0082] The method of treatment 1 was adopted to induce and culture the protonema of G. grisea. Three culture dishes were inoculated in each treatment, with 9 protonema clusters in each dish. The culture dishes were placed in an incubator for culture.
[0083] The method of treatment 2 was adopted to induce and culture the gametophytes of G. grisea. Three culture dishes were inoculated in each treatment, with 9 gametophytes in each dish. The culture dishes were placed in an incubator for culture.
[0084] After 30 days of culture, the total number of regenerated gametes in the three culture dishes of each treatment was counted. The results are shown in Table 6 and Figure 3 As shown, the present invention significantly increases the number of regenerated gametophytes obtained and achieves rapid regeneration of P. grisea.
[0085] Table 6 Number of gametophytes
[0086]
[0087] Note: In this table, 3 dishes of materials per treatment, 9 large gray mosses in each dish, a total of 27 large gray mosses were recorded as one group, repeated 3 times, and the average and standard error were calculated.
[0088] Example 9
[0089] Effect of pH of induction medium on tissue culture of Glechoma longituba.
[0090] Treatment 1: The method of Example 4 was adopted to induce and culture the protonema of Glechoma gigas, and the pH was recorded as PH5.
[0091] Treatment 2: The method of Example 5 was adopted to induce and culture the protonema of Glechoma gigas, and the pH was recorded as 6.
[0092] Treatment 3: The method of Example 6 was adopted to induce and culture the protonema of P. grisea, and the pH was recorded as PH7.
[0093] Treatments 1 to 3 were used to induce the culture of protonema of M. grisea. Three culture dishes were inoculated in each treatment, with 9 protonema clusters in each dish. The culture dishes were placed in an incubator and cultured for 28 days. The experiment was repeated 3 times.
[0094] The average diameter of the protonema in each treatment was counted on the 28th day of culture. The results are shown in Table 8 and Figure 4 As shown, the culture medium provided by the present invention can be used to obtain the protonema of the giant moss with a diameter greater than 0.7 cm after culturing for 28 days. When the pH of the giant moss induction culture medium is 7, the diameter of the obtained protonema reaches 1.06 cm. The present invention can promote the regeneration of the protonema of the giant moss.
[0095] Table 7 Diameter of protofilament
[0096]
[0097] Note: This table uses 3 dishes of materials for each condition, 9 large gray mosses in each dish, a total of 27 large gray mosses as one group, repeated 3 times, and calculated the mean and standard error.
[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A tissue culture method for Glechoma longituba, characterized in that: The protonema of the moss of the genus Psoralea was used as explant and induced culture was carried out using the induction medium of the moss of the genus Psoralea. The composition of the induction medium of the large gray moss includes: 10 mL / L of solution A, 10 mL / L of solution B, 1 mL / L of solution C, 10 mL / L of 1 M CaCl2·2H2O, 1.01 g / L of KNO3, 0.0125 g / L of FeSO4·7H2O and 9 g / L of agar; The composition of solution A is: MgSO4·7H2O 250 g / L; the composition of solution B is: KH2PO4 250 g / L; the composition of solution C is: CuSO4·5H2O 55 mg / L, H3BO3 614 mg / L, CoCl2·6H2O 55 mg / L, Na2MoO4·2H2O 25 mg / L, ZnSO4·7H2O 55 mg / L, MnCl2·4H2O 389 mg / L, KI 28 mg / L.
2. The tissue culture method according to claim 1, characterized in that The explant preparation method comprises: collecting spores of the moss, disinfecting them with 10% sodium hypochlorite for 12-15 minutes, and then disinfecting them with 75% ethanol for 2 minutes, preparing a spore suspension, inoculating it into a moss induction culture medium, and culturing to obtain a protonema mass, namely the moss explant.
3. The tissue culture method according to claim 2, characterized in that The diameter of the protofilament mass is 0.1 to 1 cm.
4. The tissue culture method according to claim 2, characterized in that The culture conditions after the spore suspension was inoculated into the induction medium of C. grisea were as follows: the culture temperature was 20-25°C, the light duration was greater than 12 h / day, and the light intensity was 30-100 μmolphotons m -2 s -1 .
5. The tissue culture method according to claim 4, characterized in that The cultivation time is 7 to 40 days.
6. The tissue culture method according to claim 1 or 2, characterized in that: The pH of the induction medium of the Glechoma longituba is 5-7.
7. The tissue culture method according to claim 1, characterized in that The culture conditions of the induction culture are: the culture temperature is 20-25°C, the illumination time is greater than 12 hours / day, and the illumination intensity is 30-100 μmol photons m -2 s -1 .
8. Use of the tissue culture method according to any one of claims 1 to 7 in increasing the diameter of protonema of Mossus maximus.
9. Use of the tissue culture method according to any one of claims 1 to 7 in increasing the number of newly added gametophytes of Mossus maximus.
10. Use of the tissue culture method according to any one of claims 1 to 7 in maintaining excellent traits of Glechoma longituba.