A method for rapidly obtaining a large number of gametophytes of the desiccation-tolerant moss Erythromyces dentata

Abstract

The present invention relates to the technical field of propagation and cultivation of drought-tolerant mosses, and in particular to a method for rapidly obtaining a large number of gametophytes of the drought-tolerant moss Erythromyces serratus. The method is either method one or method two; method one comprises the following steps: soaking Erythromyces serratus in an activation solution for 2-3 hours, then washing, and sowing the gametophytes on a substrate block for cultivation; method two comprises the following steps: soaking the substrate block in a nutrient solution, sowing the gametophytes on the substrate block for cultivation. The present invention provides a method for rapidly obtaining a large number of gametophytes of the drought-tolerant moss Erythromyces serratus indoors, which has the characteristics of a simple operation method and is suitable for industrial-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of propagation and cultivation of drought-tolerant mosses, in particular to a method for rapidly obtaining a large number of drought-tolerant moss Erythromyces dentata gametophytes. Background Art

[0002] Mosses are widely distributed worldwide. They are a group of plants that transition from aquatic to terrestrial habitats, belonging to the lowest order of higher plants. They can thrive in environments where other terrestrial plants struggle to survive, making them pioneers of environmental succession. Dessication-tolerant mosses (DTs), in particular, possess the unique ability to survive drought and even regenerate from the dead. Their unique stress-resistance properties are attracting increasing attention and have become a hot topic in stress-resistance biology research.

[0003] The drought-tolerant moss Erythromyces dentata, known for its ability to adapt to rapid changes in desert water content, is a dominant species among desert biocrusts. In extreme drought conditions, Erythromyces dentata assumes a dormant state, resembling a gray-black "shell" covering the desert surface. Upon encountering precipitation, Erythromyces dentata rapidly rehydrates within 30 seconds, returning to a vibrant green color and photosynthesizing. Further research has shown that this species exhibits faster photosynthetic recovery, a shorter rehydration recovery process, and more rapid protein synthesis than the drought-tolerant model species, Erythromyces dentata. This suggests that this species may possess more robust cellular protection and repair mechanisms, making it a valuable experimental material for studying drought tolerance mechanisms.

[0004] In the wild, there are almost no sporophytes on the gametophytes of Erythromyces serrulate, and the development and maintenance of its population mainly rely on asexual reproduction, but it takes several years or even decades from colonization to the formation of moss crust. Adequate experimental materials are essential for a series of subsequent research work. However, the problem of rapid reproduction of Erythromyces serrulate indoors has not been well solved. The cultivation of drought-resistant mosses mainly includes tissue culture, sand culture, substrate cultivation, native soil culture, liquid culture, etc., depending on the substrate. Erythromyces serrulate is an extremely drought-resistant moss. Its tissue structure and living habits are quite different from those of conventional higher plants, and conventional cultivation methods are not applicable. In particular, during the tissue culture process of Erythromyces serrulate, there are a series of problems: ① The explants are not thoroughly disinfected and are easily contaminated by green algae or microorganisms; ② In solid Knop culture medium, the explants can grow callus tissue and differentiate into new plants, but the growth cycle is long, the new plants have weak vitality, and eventually turn brown and die; ③ Protonema can be produced smoothly, but in liquid culture medium, the protonema grows compactly and does not differentiate, making it difficult to expand the reproduction and culture of protonema; ④ During the sandy soil culture process, the explants grow slowly, and individuals are prone to death in the later stages of culture. Therefore, it is urgent to take measures to solve the difficult problem of "the long growth cycle of Erythromyces serrulate gametes, the weak vitality of new plants, the ultimate browning and death, and the difficulty of expanding reproduction and culture", so as to ensure the sustainable and rapid reproduction of Erythromyces serrulate indoor culture materials. Summary of the Invention

[0005] Based on the above content, the present invention provides a method for quickly obtaining a large number of drought-tolerant moss Erythromyces dentata gametophytes, which can quickly obtain stable re-propagation plant gametophytes.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for rapidly obtaining a large number of gametophytes of the drought-tolerant moss Erythromyces dentata, which is method one or method two;

[0008] The method 1 comprises the following steps:

[0009] Soak Erythromyces dentata in the activation solution for 2-3 hours, then wash it and sow it on the substrate block for cultivation;

[0010] The second method comprises the following steps:

[0011] The substrate block is soaked in nutrient solution, and Erythromyces dentata is sown on the substrate block for cultivation.

[0012] Furthermore, in the method 1, the activation solution is a glucose solution.

[0013] Furthermore, the mass concentration of glucose in the glucose solution is 4%, and the solvent is water.

[0014] Furthermore, in the method 1, the cleaning is specifically: repeatedly rinsing with distilled water until no activation solution remains on the surface of the Erythromyces costatus.

[0015] Furthermore, in the second method, the nutrient solution is one of Knop medium, 2,4-D solution, IAA solution or 6-BA solution.

[0016] Furthermore, the composition of the Knop medium includes: 1000 mg / L calcium nitrate, 250 mg / L potassium nitrate, 250 mg / L potassium dihydrogen phosphate, 250 mg / L magnesium sulfate and 3 mg / L zinc sulfate; the pH of the Knop medium is 8.0.

[0017] Furthermore, the concentration of 2,4-D in the 2,4-D solution is 0.1 mg / L, and the solvent is water.

[0018] Furthermore, the concentration of IAA in the IAA solution is 0.01 mg / L, and the solvent is water; the concentration of 6-BA in the 6-BA solution is 0.01 mg / L, and the solvent is water.

[0019] Furthermore, in the second method, the nutrient solution submerges the lower 1 / 3 of the substrate block (the amount used is about 60 mL).

[0020] Furthermore, the composition of the matrix blocks in both the method 1 and the method 2 is: coconut husk, peat, and lignin as main raw materials.

[0021] Furthermore, the culture in both the method 1 and the method 2 is: the photoperiod is 16 hours / 8 hours, the light intensity is 150 μmol / m 2 / s, day / night temperature is 25℃ / 15℃, and indoor relative humidity is 60%.

[0022] Indoor cultivation of drought-tolerant mosses is plagued by a long gamete growth cycle, weak vitality of new plants, and eventual browning and death, making expanded propagation difficult. The present invention provides a method for rapidly propagating the gametophytes of the drought-tolerant moss Erythrocostales using matrix block tissue culture. The method ultimately determines that pre-treating and soaking Erythrocostales individuals in a 4% glucose solution, followed by washing and culturing them in matrix blocks, can yield stable re-propagated gametophytes, thus filling a current technological gap. While it is a consensus among researchers that glucose solutions promote plant growth, it is important to emphasize that there are few literature reports on mosses cultivated using sugar solutions. In particular, drought-tolerant mosses become completely contaminated by glucose within approximately seven days of contact, and the addition of sugar sources has long been considered unsuitable reagents for artificial cultivation of Erythrocostales. However, the present invention breaks with conventional experimental protocols by activating drought-tolerant mosses with glucose solution for 2-3 hours, followed by washing and matrix block culture. This method demonstrates significant differences in culture performance compared to other treatments during the later stages of the culture process. The treatment method of the present invention can provide sufficient experimental materials for further indoor experimental work, and also provide technical support and a continuous supply of biological materials for the implementation of the subsequent "desert carpet" project and biological control of desertification.

[0023] The present invention discloses the following technical effects:

[0024] The invention provides a method for quickly obtaining a large number of drought-resistant moss Erythromyces dentata gametophytes indoors. The method has the characteristics of simple operation method and is suitable for industrial-scale production.

[0025] The invention soaks the Erythromyces serrulate in a 4% glucose solution for 2-3 hours for activation treatment, then cleans the treated material and places it in a substrate block filled with water, thereby achieving stronger growth and reproduction of the Erythromyces serrulate in indoor culture; promotes the stability of the rapid reproduction gametophyte regeneration system of the Erythromyces serrulate, and can obtain a large number of monoclonal strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the CK group in Example 2.

[0028] Figure 2 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata in Example 1.

[0029] Figure 3This is the culture condition of the drought-tolerant moss Erythromyces dentata in the Knop culture medium group in Example 2.

[0030] Figure 4 This is the cultivation status of the drought-tolerant moss Erythromyces dentata of the 2,4-D group in Example 2.

[0031] Figure 5 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the IAA group in Example 2.

[0032] Figure 6 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the 6-BA group in Example 2.

[0033] Figure 7 It is the weight gain of the drought-tolerant moss Erythromyces dentata in Examples 1 and 2 after 56 days of cultivation.

[0034] Figure 8 This is a photo of the comparative example 1 in which the drought-tolerant moss Erythromyces dentata was cultivated for 7 days. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Unless otherwise specified, the raw materials used in the embodiments of the present invention can be obtained through commercial channels.

[0041] Unless otherwise specified, the "%" mentioned in the present invention is based on mass percentage.

[0042] The matrix block used in the embodiment of the present invention is composed of a new type of seedling substrate pot formed by high pressure using coconut husk, peat and lignin as main raw materials, wrapped with a layer of degradable mesh-shaped non-woven fabric, and purchased from Dalian Haiming Horticulture Co., Ltd.

[0043] Example 1

[0044] Step 1, material pretreatment: moss plants collected from the Gurbantunggut Desert are placed on kraft paper and air-dried to obtain specimens. Foreign matter such as lichens, algae, vascular plant roots, dead leaves, and mud and sand are removed from the specimens. The plants are stored in a dry and cool place to obtain dried Erythromyces dentata plants.

[0045] Step 2, rehydration of the material: Place the dried Erythromyces dentata plant in a culture dish containing sand soaked in distilled water in an incubator at 25°C for 24 hours until it is fully revived;

[0046] Step 3, cleaning of materials: In order to remove impurities and sand on the surface of the Erythromyces serrulate, transfer the revived Erythromyces serrulate to a glass beaker, gently stir it with a glass rod for 3 minutes, wash off the soil, and then place it in another beaker, and repeat the washing 5 times to obtain the cleaned Erythromyces serrulate.

[0047] Step 4: Prepare the activation solution: 4% glucose solution. Prepare as follows: Accurately weigh 4g of glucose, dissolve it in an appropriate amount of distilled water, and gradually add water to the 100mL mark to obtain a 4% glucose solution. (During the actual preparation process, the amount of glucose and distilled water added can be adjusted proportionally as needed.)

[0048] Step 5: Soak the substrate block in distilled water to make it swollen; select the single plant of Erythromyces serrulate that has been cleaned in step 3, has good growth and regular morphology, and soak it in the activation solution prepared in step 4 for 2.5 hours (activation process of Erythromyces serrulate), then clean the activation solution on the surface of Erythromyces serrulate with distilled water, and sow it neatly on the above substrate block (two rows of cuttings on the side) for cultivation (the photoperiod in the incubator is 16 hours / 8 hours, and the light intensity is 150 μmol / m 2 / s, day / night temperature of 25°C / 15°C, relative humidity of 60% (indoor), and changes in plant overall morphology were observed and recorded. Three replicates were set up. Throughout the incubation process, the substrate blocks did not come into contact with the activation solution used to soak the Erythromyces serrata.

[0049] Comparative Example 1

[0050] Step 1 is the same as step 1 in Example 1.

[0051] Step 2 is the same as step 2 in Example 1.

[0052] Step 3 is the same as step 3 in Example 1.

[0053] Step 4 is the same as step 4 in Example 1.

[0054] Step 5: Soak the substrate block in distilled water until it is in an imbibition state, then place the substrate block in a tissue culture bottle filled with 30 mL of activation solution (4% glucose solution). Select individual Erythromyces dentata plants that were cleaned in step 3, have good growth, and are relatively regular in shape, and sow them neatly on the substrate block (two rows of cuttings on the side) for cultivation (the photoperiod in the incubator is 16 hours / 8 hours, and the light intensity is 150 μmol / m 2 / s, day / night temperature of 25°C / 15°C, relative humidity of 60% in the room), observe and record the changes in the overall morphology of the plants, and set three replicates.

[0055] The photos of this comparative example after 7 days of cultivation are as follows Figure 8 As shown by Figure 8 It can be seen that the plant body of Erythromyces serrulate is completely contaminated.

[0056] Example 2

[0057] Step 1 is the same as step 1 in Example 1.

[0058] Step 2 is the same as step 2 in Example 1.

[0059] Step 3 is the same as step 3 in Example 1.

[0060] Step 4, prepare various nutrient solutions: (modified) Knop medium, 0.1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.01 mg / L IAA, 0.01 mg / L 6-BA, and use distilled water as a control (CK);

[0061] Preparation method of modified Knop medium: 1000 mg / L calcium nitrate (Ca(NO3)2·4H2O), 250 mg / L potassium nitrate (KNO3), 250 mg / L potassium dihydrogen phosphate (KH2PO4), 250 mg / L magnesium sulfate (MgSO4·7H2O), and 3 mg / L zinc sulfate (ZnSO4·7H2O) are placed in a beaker and fully dissolved. Add water to 1000 mL and adjust the pH to about 8.0 to prepare 1 L of modified Knop medium.

[0062] To prepare 0.1 mg / L 2,4-D: Weigh 0.1 mg of 2,4-D and adjust the total volume to 1000 mL with distilled water. Place the resulting solution in a brown glass bottle.

[0063] To prepare 0.01 mg / L IAA: Weigh 0.01 mg of IAA and adjust the total volume to 1000 mL with distilled water. Place the resulting solution in a brown glass bottle.

[0064] Preparation method of 0.01 mg / L 6-BA: Weigh 0.01 mg of 6-BA and mix it with distilled water to 1000 mL. Place it in a glass bottle, label it with information such as the drug name, concentration, and preparation date, and store it in a refrigerator at 4°C.

[0065] Step 5: Soak the matrix blocks in distilled water until they are in an imbibition state, and then place the matrix blocks in tissue culture bottles filled with 30 mL of the above-mentioned nutrient solution. Select the single plants of Erythromyces dentata that were cleaned in step 3, have good growth and regular morphology, and sow them neatly on the above-mentioned matrix blocks (two rows of cuttings on the side) for cultivation (the photoperiod in the incubator is 16 hours / 8 hours, and the light intensity is 150 μmol / m 2 / s, day / night temperature of 25°C / 15°C, relative humidity of 60% in the room), observe and record the changes in the overall morphology of the plants, and set three replicates.

[0066] Figure 1 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the CK group in Example 2.

[0067] Figure 2 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata in Example 1.

[0068] Figure 3 This is the culture condition of the drought-tolerant moss Erythromyces dentata in the Knop culture medium group in Example 2.

[0069] Figure 4 This is the cultivation status of the drought-tolerant moss Erythromyces dentata of the 2,4-D group in Example 2.

[0070] Figure 5This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the IAA group in Example 2.

[0071] Figure 6 This is the cultivation situation of the drought-tolerant moss Erythromyces dentata of the 6-BA group in Example 2.

[0072] Compare Figure 2-Figure 6 It can be seen that the survival rate of the drought-tolerant moss Erythromyces dentata in the 6-BA group can reach 100%, but the treatment has no significant effect on the length and weight gain of the gametophyte, and the growth of Erythromyces dentata is weak over time. At 56 days, the gametophyte of the entire plant turned yellow-brown.

[0073] Figure 7 The weight gain of the dry-tolerant moss Erythromyces dentata after culturing for 56 days in Examples 1 and 2. Figure 7 It can be seen that the gametophytes of Erythrocephala dentata treated with 4% glucose solution grew the best, and the changes in gametophyte weight gain were significantly different from those of other treatments (P < 0.05).

[0074] Comparative Example 2

[0075] Step 1 is the same as step 1 in Example 2.

[0076] Step 2 is the same as step 2 in Example 2.

[0077] Step 3 is the same as step 3 in Example 2.

[0078] Step 4, same as step 4 in Example 2

[0079] Step 5: Soak the substrate block in distilled water to make it swollen; select the single plants of Erythromyces serrulate that were cleaned in step 3, have good growth and regular morphology, and soak them in the nutrient solution prepared in step 4 for 2.5 hours (activation process of Erythromyces serrulate), then clean the nutrient solution on the surface of Erythromyces serrulate with distilled water, and sow them neatly on the above substrate block (two rows of cuttings on the side) for cultivation (the photoperiod in the incubator is 16 hours / 8 hours, and the light intensity is 150 μmol / m 2 / s, day / night temperature of 25°C / 15°C, relative humidity of 60%, and observation and recording of changes in the overall morphology of the plants. Three replicates were set up. During the entire incubation process, the substrate blocks did not come into contact with the nutrient solution used to soak the Erythromyces dentata.

[0080] The results showed that the soaking-washing-cultivation method in Comparative Example 2 was inferior to the cultivation method in Example 2 in which the substrate block was immersed in the nutrient solution and continuously supplied with nutrients, both in terms of the time for the emergence of new individuals of Erythromyces serrulate and the number of new individuals increased.

[0081] Comparative Example 3

[0082] The only difference from Example 1 is that the solution prepared in step 4 is Beneche, Part, BG 11 , Hogland, IAA (0.05 mg / L, 0.01 mg / L), 6-BA (0.05 mg / L, 0.01 mg / L), sucrose aqueous solution (mass concentration 2%, 4%, 10%), glucose aqueous solution (mass concentration 2%, 10%), fructose aqueous solution (mass concentration 2%, 4%, 10%);

[0083] Among them, Beneche's preparation method: NH4NO3 200mg / L, KH2PO4 100mg / L, MgSO4·7H2O 100mg / L, CaCl2 100mg / L, FeCl3·6H2O trace, and make up to 1L with sterile water.

[0084] Preparation method of Part: NH4NO3 2500mg / L, KH2PO4 830mg / L, MgSO4·7H2O 740mg / L, and make up to 1L with sterile water.

[0085] BG 11 Preparation method: KH2PO4 40 mg / L, NaNO3 1500 mg / L, Na2CO3 20 mg / L, Na2EDTA 1 mg / L, CaCl2 36 mg / L, MgSO4·7H2O 70 mg / L, citric acid 6 mg / L, ferric citrate ammonium 6 mg / L, and make up to 1 L with sterile water.

[0086] Hogland's preparation method: KH2PO4 180mg / L, Ca(NO3)2·4H2O 110mg / L, MgSO4·7H2O 140mg / L, KNO3 11mg / L, H3BO3 2.86mg / L, Na2C 10 H 14 O8N2·2H2O 2.68 mg / L, ZnSO4·7H2O 2.3 mg / L, CuSO4·5H2O 0.07 mg / L, MnSO4·H2O 1.54 mg / L, FeSO4·7H2O 1.98 mg / L, and make up to 1 L with sterile water.

[0087] The results showed that the effect of activating Erythromyces dentata with the above solution was far inferior to that of activating Erythromyces dentata with a 4% glucose solution; the subsequent emergence of new individuals of Erythromyces dentata took a longer time and the number of new individuals was significantly smaller.

[0088] Comparative Example 4

[0089] Step 1 is the same as step 1 in Example 1.

[0090] Step 2 is the same as step 2 in Example 1.

[0091] Step 3 is the same as step 3 in Example 1.

[0092] Step 4: The activation solution is a 4% glucose solution. The specific preparation method of the 4% glucose solution is the same as that in Example 1.

[0093] Step 5: Place the substrate block in a tissue culture bottle filled with 60 mL of 4% glucose solution; select the single plant of Erythromyces serrulate that was cleaned in step 3, has good growth and regular morphology, and soak it in the activation solution (4% glucose solution) prepared in step 4 for 2.5 hours (activation process of Erythromyces serrulate), and then sow the single plant of Erythromyces serrulate neatly on the above substrate block (two rows of cuttings on the side) for cultivation (the photoperiod in the incubator is 16 hours / 8 hours, and the light intensity is 150 μmol / m 2 / s, day / night temperature of 25°C / 15°C, and relative humidity of 60%. That is, compared with Example 1, this comparative example omitted the step of washing after soaking in the activation solution, and the single Erythromyces dentata plantlets soaked in the activation solution were directly sown on the substrate block.

[0094] Results: Contamination with Erythromyces costatus began to appear on the 7th day of culture.

[0095] In summary, the Erythromyces serrulate plants obtained using the method of Example 1 of the present invention not only achieved a 100% survival rate, but also had the most significant greenness and weight gain. In addition, new individuals appeared earlier, and the number of new individuals was significantly greater than that of Example 2, as shown in Table 1. The method of Example 1 of the present invention has a very good effect on promoting the growth and development of the drought-tolerant moss Erythromyces serrulate.

[0096] Table 1

[0097]

[0098]

[0099] The method of artificially spraying glucose can promote the growth of plants and make them strong, but it is difficult to avoid the mildew and contamination of materials on the other hand. Moreover, the addition of sugar sources has always been considered unsuitable for the artificial cultivation of Erythromyces serrulate. The present invention soaks Erythromyces serrulate in a 4% glucose solution for 2-3 hours for a short period of activation, then cleans the activated Erythromyces serrulate, and then places it into a matrix block filled with water. During the entire treatment process, the matrix block is guaranteed not to contact the 4% glucose solution. Ultimately, it has a good promoting effect on the growth and development of the drought-resistant moss Erythromyces serrulate, and a large number of gametophytes can be obtained in a short period of time to meet the needs of later experiments.

[0100] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for rapidly obtaining a large number of gametophytes of the drought-tolerant moss Erythromyces dentata, characterized in that: The following steps are involved: Soak Erythromyces dentata in the activation solution for 2-3 hours, then wash it and sow it on the substrate block for cultivation; The activation solution is a glucose solution; The mass concentration of glucose in the glucose solution is 4%, and the solvent is water.

2. The method according to claim 1, characterized in that The cleaning specifically includes: repeatedly rinsing with distilled water until no activation solution remains on the surface of the Erythromyces costatus.

3. The method according to claim 1, characterized in that The matrix blocks are composed of coconut husk, peat and lignin as main raw materials.

4. The method according to claim 1, wherein The culture was performed as follows: the photoperiod was 16 hours / 8 hours, and the light intensity was 150 μmol / m 2 / s, day / night temperature is 25℃ / 15℃, and indoor relative humidity is 60%.

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