Method for culturing gametophyte of Amblystegium griffithii

Abstract

The invention provides a method for culturing gametophyte of Amblystegium giganteum, and belongs to the technical field of bioengineering. The method for culturing the gametophyte of the Amblystegium giganteum comprises the following steps that stem segments of the Amblystegium giganteum are laid on a culture medium to be cultured, the length of the stem segments is 1.8-2.2 cm, and the culture medium is sphagnum moss. According to the method, regeneration of the gametophyte of the hypnum macrophyllum can be promoted, a large-scale production system of the hypnum macrophyllum can be established, and the method is simple and efficient and provides theoretical support for a cultivation and propagation system of the hypnum macrophyllum.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for culturing macrophylla gametophytes. Background Art

[0002] There are many species of bryophytes, which are the second largest plant group. They are a type of plant with high research value, ornamental value and economic value. Many bryophytes have been found to have medicinal value and are used clinically. Some mosses have become raw materials for over-the-counter drugs. Rhodobryumgiganteum belongs to the genus Rhodobryumgiganteum of the family Mossaceae, also known as heart-turning grass. It has a wide distribution range, and most of them grow in the humus surface layer or thin soil on the rock surface with high humidity under the forest. Rhodobryumgiganteum is large in size, 2-5cm high, sparsely clustered, with creeping underground stems, upright above-ground stems, and leaves clustered in rosettes at the top of the stems. Rhodobryumgiganteum not only has a high appreciation value in moss landscape applications, but is also an important medicinal bryophyte, which has a good effect on the treatment of cardiovascular diseases. The preparations of Rhodobryumgiganteum used clinically have certain effects on the treatment of angina pectoris, lowering blood pressure, increasing coronary flow, and relieving chest tightness.

[0003] As the application value of warm-land moss is gradually explored, the demand for landscape and medicinal purposes is in short supply. However, the warm-land moss on the market mainly comes from farmers' planting and wild collection. Long-term wild collection behavior not only destroys the ecology, but also cannot guarantee the quality of wild collection. Therefore, the development of cultivation and reproduction technology for warm-land moss is conducive to the establishment of a large-scale production system for warm-land moss. At present, there is no mature cultivation and reproduction technology for warm-land moss, and there is a lack of relevant research. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for culturing macrophylla gametophytes, which can significantly increase the number of newly generated gametophytes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for culturing gametophytes of Macrophylla sphaerocephala comprises the following steps: spreading stem segments of Macrophylla sphaerocephala flat on a culture substrate for culturing, wherein the stem segments are 1.8 to 2.2 cm in length and the culture substrate is peat moss.

[0007] Preferably, the macrophylla is warm-region macrophylla.

[0008] Preferably, the culture temperature is 16-20°C or 24-26°C.

[0009] Preferably, the light cycle of the culture is 12 h light + 12 h dark, and the intensity of the light is 4500-6500 Lux or 600 Lux.

[0010] Preferably, the peat moss is obtained by mixing dry peat moss with water, and the dry peat moss and water are mixed at a mass volume ratio of 1 g:15-25 mL.

[0011] Preferably, the dry peat moss is sterilized before use, and the sterilization conditions are: temperature of 121° C., pressure of 0.1 MPa, and sterilization time of 20 min.

[0012] Preferably, during the culture process, water is supplemented every 5 days, and the volume of the supplemented water is 15 mL per 500 g of culture medium.

[0013] Another object of the present invention is to provide application of the above method in increasing the number of newly formed gametophytes of Macrophylla.

[0014] Another object of the present invention is to provide application of the above method in increasing the length of newly born gametophytes of Macrophylla.

[0015] Another object of the present invention is to provide application of the above method in maintaining excellent traits of Macrophylla.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The invention provides a method for culturing the gametophyte of Macrophylla, which uses peat moss as a culture matrix to promote the regeneration of the gametophyte of Macrophylla, and is beneficial to establishing a large-scale production system of warm-region Macrophylla. The method provided by the invention is simple and efficient, and provides theoretical support for the cultivation and propagation system of Macrophylla. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The dry sphagnum moss used in Example 1 and the peat substrate used in Comparative Example 1, the scale bar is 1 cm;

[0019] Figure 2 This is a schematic diagram of the culture site of the warm-land macrophylla, where A is a complete plant of the warm-land macrophylla, and B is a cut stem segment. The scale bar is 1 cm.

[0020] Figure 3 The newly formed gametophytes of warm-land macrophylla under different culture medium conditions, the scale bar is 1 cm;

[0021] Figure 4 The changes in the length and number of newly born gametophytes of warm-land macrophylla under different culture medium conditions, where A is the change in gametophyte length, B is the change in gametophyte number, and asterisks indicate significant differences, where * corresponds to p<0.05, ** corresponds to p<0.01, *** corresponds to p<0.001, **** corresponds to p<0.0001, and ns corresponds to no difference;

[0022] Figure 5 The effect of culture temperature on the number and length of regenerated gametophytes of warm-land macrophylla, where the left figure shows the change in the number of gametophytes, and the right figure shows the change in the length of gametophytes. Asterisks indicate significant differences, where * corresponds to p<0.05, ** corresponds to p<0.01, *** corresponds to p<0.001, **** corresponds to p<0.0001, and ns corresponds to no difference;

[0023] Figure 6 Figure 2 shows the effect of light intensity on the number and length of regenerated gametophytes of M. cyrtonema. The left figure shows the change in the number of gametophytes, and the right figure shows the change in the length of gametophytes. Asterisks indicate significant differences, where * corresponds to p<0.05, ** corresponds to p<0.01, *** corresponds to p<0.001, **** corresponds to p<0.0001, and ns corresponds to no difference. DETAILED DESCRIPTION

[0024] The present invention provides a method for culturing gametophytes of a macrophylla, comprising the following steps: laying a stem segment of a macrophylla flat on a culture substrate for culturing, wherein the stem segment has a length of 1.8 to 2.2 cm and the culture substrate is peat moss. The macrophylla in the present invention is preferably a warm-land macrophylla.

[0025] In the present invention, the peat moss is obtained by mixing dry peat moss with water, and the dry peat moss and water are mixed at a mass volume ratio of 1g:15-25mL, and the preferred volume ratio is 1g:18-22mL. In the present invention, a peat moss matrix in a completely water-absorbed state can be obtained by mixing dry peat moss with water. As an implementable method, the peat moss matrix in a completely water-absorbed state refers to a peat moss matrix that is completely soaked and absorbed with water after the peat moss is mixed with water, and water does not drip out.

[0026] The dry peat moss in the present invention is sterilized before use, and the sterilization conditions are: temperature of 121°C, pressure of 0.1MPa, and sterilization time of 20min. The dry peat moss in the present invention is dried after sterilization and then mixed with water to prepare the peat moss culture matrix. As an practicable method, the dry peat moss in the present invention is sterilized and dried at 65°C for 8-12h.

[0027] The present invention uses dry peat moss as a culture matrix, which is relatively loose and has strong air permeability and water retention capacity. When it is in a completely water-absorbing state, the humidity is high, so that the stem segments of the large-leaf moss are in a stuffy state, and the stem segments of the large-leaf moss can quickly obtain more gametophytes.

[0028] In the present invention, the culture temperature is 16-20°C or 24-26°C. The culture temperature provided by the present invention is helpful to increase the length of the newly formed gametes.

[0029] In the present invention, the light cycle of the culture is 12h light + 12h dark, the intensity of the light is 4500-6500Lux, preferably 5000-6000Lux, and the intensity of the light in the present invention can also be 600Lux. The light intensity has a significant effect on the growth length of the newly formed gametes, and the light intensity provided by the present invention can significantly increase the growth rate of the newly formed gametes.

[0030] In the present invention, water is added every 5 days during the culture process, and 15 mL of water is added for every 500 g of culture medium. The present invention can meet the water required for normal growth of the plant by regularly adding water during the culture process.

[0031] The invention also provides application of the method in increasing the number of newly-born gametophytes of macrophylla.

[0032] The invention also provides application of the method in increasing the length of newly-born gametophytes of Moss macrophylla.

[0033] The present invention also provides application of the method in maintaining excellent traits of macrophylla.

[0034] The dry peat moss in the specific embodiment of the present invention is caterpillar sphagnum moss imported from Chile (purchased from Tai'an Hetai Agricultural Technology Co., Ltd.), and the peat soil is Danish Pinner peat soil.

[0035] In the specific embodiment of the present invention, the experimental data were plotted and processed using Graphpadprism 8.0, and the data were expressed as Mean ± SEM for the data analyzed by Student's t-test, and the difference was indicated by an asterisk, where * corresponds to p < 0.05, ** corresponds to p < 0.01, *** corresponds to p < 0.001, **** corresponds to p < 0.0001, and ns corresponds to no difference.

[0036] 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.

[0037] Example 1

[0038] A method for culturing macrophylla gametophytes, comprising the following steps:

[0039] In this embodiment, the dry peat moss is caterpillar sphagnum moss;

[0040] The dry peat moss (Sphagnumpalustre) was sterilized at 121°C and 0.1 MPa pressure for 20 min. After the sterilization, it was dried at 65°C for 10 h to obtain a dry peat moss substrate.

[0041] Dry peat moss substrate (such as Figure 1Mix the mixture with water in a mass volume ratio of 1g:20mL and place it 4-5cm thick to obtain the culture matrix. Smooth the surface for later use. Select the plants of warm-land macrophylla with good morphology and robustness, and take the 2cm stem segment in the middle of the stem (as shown in the figure on the right). Figure 2 As shown), the stem segments were spread flat on the surface of the above-mentioned culture substrate for culture, and the culture conditions were: temperature 16°C, photoperiod 12h light + 12h dark, and light intensity 5000Lux; during the culture process, water was supplemented every 5 days, and the supplemented water was 15mL for 500g of culture substrate.

[0042] Example 2

[0043] A method for culturing macrophylla gametophytes, the difference between this embodiment and embodiment 1 is that the culturing temperature is 20°C.

[0044] Example 3

[0045] A method for culturing macrophylla gametophytes, the difference between this embodiment and embodiment 1 is that the culturing temperature is 25°C.

[0046] Example 4

[0047] A method for culturing macrophylla gametophytes, the difference between this embodiment and embodiment 1 is that the light intensity is 600 Lux.

[0048] Example 5

[0049] A method for culturing macrophylla gametophytes, the difference between this embodiment and embodiment 1 is that the light intensity is 6000 Lux.

[0050] Comparative Example 1

[0051] A method for culturing macrophylla gametophytes, the difference between this comparative example and Example 1 is that the culture medium is peat soil (such as Figure 1 It is obtained by mixing the precipitate (as shown in the middle left figure) with water in a mass volume ratio of 1g:20mL.

[0052] Comparative Example 2

[0053] A method for culturing macrophylla gametophytes. The difference between this comparative example and Example 1 is that the culturing temperature is 22°C.

[0054] Test Example 1

[0055] Effect of culture medium on newly formed gametophytes of Moss macrophylla.

[0056] The methods of Example 1 (recorded as dry peat moss) and Comparative Example 1 (recorded as peat soil) were adopted to culture the gametophytes of Macrophylla: different culture substrates were placed in culture boxes of 16×14×7.5 cm, 4 stem segments were placed in each box, and each method was repeated for 5 boxes.

[0057] 1. After 14 days of culture, 10 plants were randomly sampled from each treatment, and the length and number of newly formed gametes were recorded. The results are shown in Tables 1 and Figure 3 , Figure 4 shown.

[0058] Table 1 Gametophyte regeneration data of stem segments of Macrophylla spp. (14 days)

[0059]

[0060] In Example 1, the culture matrix was prepared using dry sphagnum moss, and the average length of the newly regenerated gametophytes of the warm-land macrophylla was 0.88 cm, and the average number of newly regenerated gametophytes was 1.3. The average length of the newly regenerated gametophytes in Comparative Example 1 was 0.858 cm, and the average number of newly regenerated gametes was 1. The length of the newly regenerated gametophytes in Example 1 was slightly higher than that in Comparative Example 1, and the method provided in Example 1 can obviously induce multiple newly regenerated gametophytes.

[0061] 2. After 21 days and 28 days of culture, 10 plants were randomly sampled from each treatment, and the length and number of newly formed gametes were recorded. The results are shown in Tables 2, 3, Figure 3 , Figure 4 shown.

[0062] Table 2 Gametophyte regeneration data of stem segments of Macrophylla spp. (21 days)

[0063]

[0064]

[0065] Table 3 Gametophyte regeneration data of stem segments of Macrophylla spp. (28 days)

[0066]

[0067] After the plants grew for 28 days, the average length of the newly born gametophytes in Example 1 was 1.982 cm, and the average number of newly born gametophytes was 1.6. The average length of the newly born gametophytes in Comparative Example 1 was 1.951 cm, and the average number of newly born gametophytes was 1. There was no significant change in the length of the newly born gametophytes in the two cultivation methods of Example 1 and Comparative Example 1. The treatment method of Example 1 can induce multiple newly born gametophytes, which is significantly different from Comparative Example 1. The method provided by the present invention can significantly increase the number of newly born gametophytes in the stem segments of warm-land giant moss.

[0068] Test Example 2

[0069] Effect of culture temperature on newly formed gametophytes of Moss macrophylla.

[0070] The methods of Example 1, Example 2, Example 3 and Comparative Example 2 were used to culture the gametophytes of Macrophylla: different culture substrates were placed in 16×14×7.5 cm culture boxes, 4 stem segments were placed in each box, and each method was repeated for 5 boxes. After 14 days, 21 days, and 28 days of culture, 5 plants were randomly sampled from each treatment, and the length and number of newly formed gametophytes in each treatment were recorded. The results are shown in FIG. Figure 5 shown.

[0071] according to Figure 5 It can be seen that temperature has no significant effect on the number of regenerated gametophytes in the stem segments of warm-land macrophylla, but significantly affects the length of the newly formed gametophytes. After 28 days of culture, the average length of the gametophytes in the method provided in Example 1 reached 2.12 cm, the average length of the gametophytes in Example 2 reached 1.86 cm, and the average length of the gametophytes in Example 3 reached 1.88 cm. The average length of the gametophytes cultured in Comparative Example 2 was 1.34 cm. The changes in the growth length of the gametophytes are shown in Table 4, and the changes in the number of gametophyte regeneration are shown in Table 5.

[0072] Table 4 Changes in gametophyte growth length at different temperatures (cm)

[0073]

[0074] Table 5 Changes in the number of gametophytes at different temperatures (units)

[0075]

[0076] Test Example 3

[0077] Effect of light intensity on newly formed gametophytes of Moss macrophylla.

[0078] The method of Example 1 and Example 4 was used to culture the gametophytes of Macrophylla: different culture substrates were placed in 16×14×7.5 cm culture boxes, 4 stem segments were placed in each box, and each method was repeated 5 boxes. After 14 days, 21 days, and 28 days of culture, 8 plants were randomly sampled from each treatment, and the length and number of newly formed gametophytes in each treatment were recorded. The results are shown in Table 1. Figure 6 , as shown in Table 6 and Table 7.

[0079] according to Figure 6 As shown in Table 6 and Table 7, the light intensity (5000Lux and 600Lux) provided by the present invention can quickly obtain regenerated gametes, and the number of regenerated gametes under different light intensities is not much different. When the light intensity is 600Lux, the regenerated gametes obtained in the same culture time are longer. The present invention can significantly improve the growth rate of gametes.

[0080] Table 6 Changes in gametophyte growth length under different light intensities (cm)

[0081]

[0082]

[0083] Table 7 Changes in the number of gametophytes grown under different light intensities (individuals)

[0084]

[0085] 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 method for culturing gametophytes of Macrophylla, characterized in that: The method comprises the following steps: laying the stem segments of the macrophylla on a culture substrate for culture, wherein the length of the stem segments is 1.8 to 2.2 cm, and the culture substrate is peat moss.

2. The method according to claim 1, characterized in that: The macrophylla described is a warm-region macrophylla.

3. The method according to claim 1, characterized in that The culture temperature is 16-20°C or 24-26°C.

4. The method according to claim 1, characterized in that The light cycle of the culture is 12 hours of light + 12 hours of darkness, and the intensity of the light is 4500-6500 Lux or 600 Lux.

5. The method according to claim 1, characterized in that: The peat moss is obtained by mixing dry peat moss with water, and the dry peat moss and water are mixed at a mass volume ratio of 1 g:15-25 mL.

6. The method according to claim 5, characterized in that The dry peat moss is sterilized before use, and the sterilization conditions are: temperature of 121° C., pressure of 0.1 MPa, and sterilization time of 20 min.

7. The method according to claim 1, characterized in that During the culture process, water was supplemented every 5 days, and the volume of the supplemented water was 15 mL per 500 g of culture medium.

8. Use of the method according to any one of claims 1 to 7 in increasing the number of newly formed gametophytes of Macrophylla.

9. Use of the method according to any one of claims 1 to 7 in increasing the length of newly born gametophytes of Macrophylla.

10. Use of the method according to any one of claims 1 to 7 in maintaining excellent traits of Macrophylla.

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