A method for promoting the increase of the growth points of Leucobryum glaucum leaves, a method for rapid regeneration, and applications thereof

Abstract

The present invention belongs to the technical field of plant regeneration, and specifically relates to a method for promoting the increase of the growth points of Leucobryum glaucum leaves, a method for rapid regeneration, and applications. The present invention provides a method for promoting the increase of the growth points of Leucobryum glaucum leaves and a method for inducing the rapid regeneration of Leucobryum glaucum leaves. The excised leaves of Leucobryum glaucum are treated with brassinolide to promote the generation of new growth points on the excised leaves of Leucobryum glaucum, realizing the rapid growth of Leucobryum glaucum leaves, greatly shortening the cultivation time, and making up for the inherent defect of the slow growth of bryophytes.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant regeneration, and specifically relates to a method for promoting the increase of the growth points of Leucobryum glaucum leaves, a method for rapid regeneration, and applications thereof. Background Art

[0002] Leucobryum glaucum belongs to the family Leucobryaceae and the genus Leucobryum. Its growth state is that the plant body is densely tufted, with a grayish-green color that is greenish-white. The leafy stems of Leucobryum glaucum are erect, up to 8 cm long at most, showing irregular branching growth. The leaves of Leucobryum glaucum are densely arranged, with a sheath-like base, long ovate in shape, gradually becoming lanceolate at the upper part, and often curved to one side; the leaf margins are entire, slightly involute at the upper part. It is dioecious, with a slender seta, reddish-brown; the capsule is conical, arcuately curved. Leucobryum glaucum is mostly found in humid woodlands in mountainous areas south of the Yangtze River Basin in China, and also in southeastern Asia.

[0003] Leucobryum glaucum has characteristics such as strong stress resistance, fast growth rate, and relatively drought tolerance. It is used for landscaping in a low and neat manner, making people feel comfortable and fresh. In recent years, Leucobryum glaucum has been widely used in industries such as home gardening, vertical greening, and landscape greening. However, collecting wild mosses causes great damage to the ecological environment. Since wild mosses grow slowly, once they are removed, it is difficult to recover within 3 to 5 years, and some moss species cannot even recover within 10 years. Therefore, artificial propagation of mosses is extremely important. The slow growth and low yield of Leucobryum glaucum have also been a bottleneck restricting its application. Conducting research on the cultivation and propagation technology of Leucobryum glaucum and improving its yield are the prerequisites for promoting the popularization and application development of Leucobryum glaucum. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for promoting the increase of the growth points of Leucobryum glaucum leaves, a method for rapid regeneration, and applications thereof. By applying the technical solutions provided by the present invention, new growth points can be promoted to be generated on the excised leaves of Leucobryum glaucum, greatly shortening the cultivation time and making up for the inherent defect of slow growth of bryophytes.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] The present invention provides an application of brassinolide in at least one of the following 1) to 3):

[0007] 1) Increasing the number of attachment points of new protonemata on Leucobryum glaucum leaves;

[0008] 2) Increasing the number of gametophytes of Leucobryum glaucum leaves;

[0009] 3) Shortening the cultivation time of Leucobryum glaucum.

[0010] The present invention provides a method for promoting the increase of the growth points of Leucobryum glaucum leaves, including: treating the excised leaves of Leucobryum glaucum with an aqueous solution of brassinolide.

[0011] Preferably, the brassinolide aqueous solution is a solution obtained by diluting brassinolide and sterile water according to a volume ratio of 1:(1900 - 2100).

[0012] Preferably, the Leucobryum glaucum leaves include the leaves intercepted at 4 - 6 mm from the top of the Leucobryum glaucum plant.

[0013] Preferably, before the Leucobryum glaucum leaves are treated with the brassinolide aqueous solution, the Leucobryum glaucum leaves are also washed.

[0014] The present invention provides a method for inducing rapid regeneration of Leucobryum glaucum leaves, comprising the following steps:

[0015] Treating the excised Leucobryum glaucum leaves with the method described in the above technical solution to obtain the treated excised Leucobryum glaucum leaves; culturing the treated excised Leucobryum glaucum leaves to achieve rapid regeneration of the excised Leucobryum glaucum leaves.

[0016] Preferably, during the culturing process, the brassinolide aqueous solution is supplemented in the culture container.

[0017] Preferably, the culture container used in the culturing includes a culture dish; filter paper is placed in the culture dish.

[0018] Preferably, the temperature of the culturing is 22 - 24 °C, and the time of the culturing is 12 - 24 d.

[0019] Preferably, the culturing includes light - dark alternating culturing. In each light - dark alternating culturing cycle, the light culturing time is 12 - 16 h, and the dark culturing time is 8 - 12 h.

[0020] Advantages of the present invention: The present invention provides the application of brassinolide in at least one of the following 1) - 3): 1) increasing the number of attachment points of newly - formed protonemata on Leucobryum glaucum leaves; 2) increasing the number of gametophytes on Leucobryum glaucum leaves; 3) shortening the cultivation time of Leucobryum glaucum. Relying on the totipotency of plant stem cells, the present invention uses brassinolide to promote the development of new attachment points on excised Leucobryum glaucum leaves, generate new growth points, shorten the cultivation time, and improve the cultivation efficiency. Compared with the untreated excised Leucobryum glaucum leaves, the number of newly - formed attachment points on the treated excised leaves increases significantly, with an obvious difference. It can be seen that the technical solution provided by the present invention can promote the rapid growth of excised leaves and make up for the inherent defect of slow growth of bryophytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0022] Figure 1Schematic diagram of the culture method and attachment points of the excised leaves of Leucobryum glaucum in Example 1; where A is the schematic diagram of the culture method of the excised leaves; B is the schematic diagram of the excised leaves after 12 days of treatment, and the arrow indicates the attachment point of the newly formed protonema; C is the schematic diagram of the excised leaves after 24 days of treatment, and the arrow indicates that the newly formed protonema at the attachment point develops into a gametophyte.

[0023] Figure 2 Diagram of the developmental state of the excised leaves of Leucobryum glaucum in Example 1 and Comparative Examples 1-2; where A is the schematic diagram of the plant after 12 days of treatment, and B is the schematic diagram of the plant after 24 days of treatment.

[0024] Figure 3 Data diagram of the growth and development status of the leaves of Leucobryum glaucum in Example 1 and Comparative Examples 1-2, where A is the ratio of new attachment points of the plants after 12 days of treatment of the leaves of Leucobryum glaucum; B is the number of attachment points per millimeter of the excised leaves of Leucobryum glaucum. Detailed implementation mode

[0025] The present invention provides an application of brassinolide in at least one of the following 1)-3):

[0026] 1) Increasing the number of attachment points of the newly formed protonema on the leaves of Leucobryum glaucum;

[0027] 2) Increasing the number of gametophytes on the leaves of Leucobryum glaucum;

[0028] 3) Shortening the cultivation time of Leucobryum glaucum.

[0029] The application of the present invention preferably includes: treating the excised leaves of Leucobryum glaucum with an aqueous solution of brassinolide, and culturing the obtained treated excised leaves of Leucobryum glaucum. The attachment point of the newly formed protonema described in the present invention is the growth point. The brassinolide described in the present invention is preferably an aqueous solution of brassinolide. In the present invention, the excised leaves of Leucobryum glaucum are preferably selected from Leucobryum glaucum plants with good growth status, no impurities, and no pests and diseases, and more preferably the excised leaves are intercepted from the leaf tips of Leucobryum glaucum plants with good growth status, no impurities, and no pests and diseases. The excised leaves described in the present invention preferably include the leaves intercepted at 4-6 mm from the top of the Leucobryum glaucum plant, and more preferably the leaves intercepted at 5 mm from the top of the Leucobryum glaucum leaf. The present invention takes the top leaves to ensure the uniform growth state of the leaves. The tool for intercepting the leaves in the present invention is preferably a sterilized scissors. The substance used for sterilization in the present invention is preferably 75% alcohol.

[0030] In the present invention, before the excised leaves of Leucobryum glaucum are treated with an aqueous solution of brassinolide, it is preferably washed. The washing in the present invention is preferably completed with water or sterile water. The function of the washing described in the present invention is to wash away the soil, residual branches and withered leaves carried by the excised leaves of Leucobryum glaucum.

[0031] The brassinolide aqueous solution described in the present invention is preferably a solution obtained by diluting brassinolide and sterile water according to a volume ratio of 1:(1900-2100), and more preferably a solution obtained by diluting brassinolide and sterile water according to a volume ratio of 1:2000. The brassinolide described in the present invention can promote the development of protonema at the new attachment points of the excised leaves of Leucobryum glaucum, promote the rapid growth of the excised leaves of Leucobryum glaucum, shorten the cultivation time, and improve the cultivation efficiency.

[0032] The present invention provides a method for promoting the increase of the growth points of Leucobryum glaucum leaves, including: treating the excised leaves of Leucobryum glaucum with a brassinolide aqueous solution, and more preferably treating the washed excised leaves of Leucobryum glaucum with a brassinolide aqueous solution.

[0033] The technical solution for selecting the excised leaves of Leucobryum glaucum has been discussed above and will not be elaborated here. The technical solution of the brassinolide aqueous solution has been discussed above and will not be elaborated here. The treatment described in the present invention is preferably to place the excised leaves of Leucobryum glaucum in a culture container containing a brassinolide aqueous solution. In the examples of the present invention, the culture container is a petri dish. After laying filter paper in the petri dish, pour the brassinolide aqueous solution into the filter paper, and then place the excised leaves of Leucobryum glaucum on the filter paper.

[0034] The present invention provides a method for inducing rapid regeneration of Leucobryum glaucum leaves, including the following steps:

[0035] Treating the excised leaves of Leucobryum glaucum with the method described in the above technical solution to obtain the treated excised leaves of Leucobryum glaucum; culturing the treated excised leaves of Leucobryum glaucum to achieve rapid regeneration of the excised leaves of Leucobryum glaucum.

[0036] Using the above technical solution to obtain the treated excised leaves of Leucobryum glaucum, the present invention cultures the treated excised leaves of Leucobryum glaucum to achieve rapid regeneration of the excised leaves of Leucobryum glaucum.

[0037] The present invention has no special limitation on the container used in the cultivation process, and a conventional container can be used. The container used in the cultivation process described in the present invention preferably includes a petri dish. The petri dish described in the present invention preferably has filter paper placed in it before cultivation, and more preferably 2 pieces of filter paper are placed in each petri dish. The present invention has no special limitation on the specifications of the petri dish, and a conventional product can be used. In the examples of the present invention, the petri dish is preferably a petri dish with a diameter of 9 mm.

[0038] The culture dish of the present invention is placed with the processed excised leaves of Leucobryum glaucum and then starts to be cultured. Preferably, during the culture process of the present invention, 4-6 mL of brassinolide aqueous solution is supplemented every 5 days, and more preferably 5 mL of brassinolide aqueous solution is supplemented every 5 days. The purpose of adding and supplementing the brassinolide solution in the present invention is to keep the filter paper moist, provide a suitable humidity environment for the growth and development of the excised leaves of Leucobryum glaucum, and at the same time supplement the nutrient elements required for the growth and development of the excised leaves of Leucobryum glaucum. Preferably, the filter paper in the present invention remains moist without dripping water after being kneaded.

[0039] In the present invention, the temperature of the culture is preferably 22-24 °C, and more preferably 24 °C. The culture time of the present invention is preferably 12-24 d, further preferably 18-24 d, and more preferably 24 d. The culture of the present invention preferably includes alternating light and dark culture. The light culture time in each light and dark alternating culture of the present invention is preferably 12-16 h, and more preferably 16 h. The dark culture time in each light and dark alternating culture of the present invention is preferably 8-12 h, and more preferably 8 h. The culture of the present invention is preferably completed in an incubator. The humidity of the incubator in the present invention is preferably 40-50%, and more preferably 45%. The selection of the culture temperature, light intensity and photoperiod in the present invention is to promote the growth and development of the excised leaves of Leucobryum glaucum.

[0040] The technical solution of the culture in the present invention has been discussed above and will not be repeated here.

[0041] The maintenance and differentiation of plant growth point stem cells are the key to plant development and morphogenesis. The strong totipotency or pluripotency of plant cells also endows plants with strong developmental plasticity and regeneration ability. The present invention provides a method for inducing rapid regeneration of Leucobryum glaucum leaves. Culturing for 24 days can promote the generation of new growth points on the excised leaves, greatly shortening the cultivation time and improving the cultivation efficiency. Compared with the untreated plants, there are obvious differences in the number of newly formed attachment points on the excised leaves after treatment. The present invention discloses a method for accelerating the increase of growth points on Leucobryum glaucum leaves, which is simple in operation, stable and reliable, promotes the rapid growth of excised leaves, and makes up for the inherent defect of slow growth of bryophytes. The present invention utilizes the leaves as explants and the function of brassinolide, and based on the totipotency of plant cells, shortens the cultivation time of Leucobryum glaucum. The technical solution provided by the present invention forms the key technology for large-scale production of Leucobryum glaucum, which is highly operable, has a short cultivation time, is more suitable for accelerating the propagation of Leucobryum glaucum leaves, and provides theoretical support for its large-scale production and application.

[0042] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0043] Example 1, Comparative Example 1, and Comparative Example 2 are Leucobryum glaucum of the same batch with good growth status, no impurities, and no diseases and pests.

[0044] Example 1

[0045] (1) Cut off the 5-mm leaves at the top of the selected 60 Leucobryum glaucum plants and put them into a glass dish filled with clear water. Stir well to wash away impurities.

[0046] (2) Trim two pieces of filter paper to a suitable size and lay them overlapping on the culture dish to increase water retention. Moisten the filter paper with 5 mL of brassinolide solution. The brassinolide solution is obtained by diluting brassinolide and sterile water in a volume ratio of 1:2000.

[0047] (3) Use tweezers to pick out the leaves treated in step (1) and place them flat on the filter paper moistened in step (2).

[0048] (4) Put the culture dish obtained in step (3) into an incubator suitable for the growth of Leucobryum glaucum and culture for 24 days. The culture temperature is 24 °C, the light intensity is 60 - 80 lx, and the photoperiod is 16 h of light and 8 h of darkness. The humidity in the incubator is 45.3%. During the culture process, regularly supplement 5 mL of brassinolide solution every five days to keep the filter paper moist.

[0049] Comparative Example 1

[0050] (1) Cut off the 5-mm leaves at the top of the selected 60 Leucobryum glaucum plants and put them into a glass dish filled with clear water. Stir well to wash away impurities.

[0051] (2) Trim two pieces of filter paper to a suitable size and lay them overlapping on the culture dish to increase water retention. Moisten the filter paper with 5 mL of 6-benzylaminopurine solution. The 6-benzylaminopurine solution is obtained by diluting 6-benzylaminopurine and water in a volume ratio of 1:1000.

[0052] (3) Use tweezers to pick out the leaves treated in step (1) and place them flat on the filter paper moistened in step (2).

[0053] (4) Put the culture dish obtained in step (3) into an incubator suitable for the growth of Leucobryum glaucum and culture for 24 days. The culture temperature is 25 °C, the light intensity is 60 - 80 lx, and the photoperiod is 16 h of light and 8 h of darkness. The humidity in the incubator is 45.3%. During the culture process, regularly supplement 5 mL of 6-benzylaminopurine solution every five days to keep the filter paper moist.

[0054] Comparative Example 2

[0055] (1) Cut off the 5-mm leaves at the top of the selected 60 Leucobryum glaucum plants and put them into a glass dish filled with clear water. Stir well to wash away impurities.

[0056] (2) Trim two pieces of filter paper to a suitable size, overlap and lay them flat on the culture dish to increase water retention, and moisten the filter paper with 5 mL of clear water.

[0057] (3) Use forceps to pick out the leaves processed in step (1) and place them flat on the filter paper moistened in step (2).

[0058] (4) Place the culture dish obtained in step (3) in an incubator suitable for the growth of Leucobryum glaucum and culture for 24 d. The culture temperature is 25 °C, the light intensity is 60 - 80 lx, and the photoperiod is 16 h of light and 8 h of darkness. The humidity in the incubator is 45.3%. During the culture process, replenish 5 mL of clear water regularly every five days to keep the filter paper moist.

[0059] Schematic diagram of the in vitro leaf culture method and attachment point of Example 1. The schematic diagram of the in vitro leaf culture method is shown in Figure Figure 1 . The growth and development of Leucobryum glaucum at different stages in the incubator for Example 1 and Comparative Examples 1 - 2 are as Figure 2 shown. The quantitative comparison of the growth and development status of Leucobryum glaucum in Example 1 and Comparative Examples 1 - 2 can be seen in Figure 3 and Table 1. Figure 3 Processed using the software Graphpad prism 8.0. The data is expressed as Means ± SEM and analyzed using Student's t - test. The significance of differences is indicated by asterisks. * corresponds to p < 0.05, ** corresponds to p < 0.01, *** corresponds to p < 0.001, and **** corresponds to p < 0.0001.

[0060] When calculating the ratio of the number of new attachment points grown by the plants, the number of new attachment points grown by the plants in Example 1 is regarded as 1.

[0061] Number of attachment points per millimeter of in vitro leaf = Number of leaf attachment points / Leaf length (mm)

[0062] The results are shown in Table 1 and Figure 3 .

[0063] Table 1 Data analysis of the growth and development status of Leucobryum glaucum in Example 1 and Comparative Examples 1 - 2

[0064]

[0065] According to Table 1, Figures 1 to 3 it can be seen that the difference in the number of growth points of the new protonema between the brassinolide treatment group in Example 1 and Comparative Examples 1 and 2 is extremely significant. Brassinolide treatment greatly increases the growth rate of in vitro Leucobryum glaucum leaves.

[0066] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for promoting the increase of the growing points of Leucobryum glaucum leaves, characterized in that, Comprising: The excised leaves of Leucobryum glaucum are treated with an aqueous solution of brassinolide; the aqueous solution of brassinolide is a solution obtained by diluting brassinolide and sterile water according to a volume ratio of 1:(1900 - 2100); the leaves of Leucobryum glaucum are the leaves intercepted at 4 - 6 mm from the top of the Leucobryum glaucum plant.

2. The method according to claim 1, characterized in that, Before the leaves of Leucobryum glaucum are treated with the aqueous solution of brassinolide, it further includes washing the leaves of Leucobryum glaucum.

3. The method according to claim 1, characterized in that, The aqueous solution of brassinolide is a solution obtained by diluting brassinolide and sterile water according to a volume ratio of 1:2000; the leaves of Leucobryum glaucum are the leaves intercepted at 5 mm from the top of the Leucobryum glaucum plant.

4. A method for inducing the rapid regeneration of Leucobryum glaucum leaves, characterized in that, Including the following steps: Treating the excised leaves of Leucobryum glaucum with the method according to any one of claims 1 - 3 to obtain the treated excised leaves of Leucobryum glaucum; culturing the treated excised leaves of Leucobryum glaucum to achieve rapid regeneration of the excised leaves of Leucobryum glaucum.

5. The method for rapid regeneration according to claim 4, characterized in that, During the culturing process, an aqueous solution of brassinolide is supplemented in the culture container.

6. The method for rapid regeneration according to claim 4 or 5, characterized in that, The culture container used in the culturing includes a culture dish; filter paper is placed in the culture dish.

7. The method for rapid regeneration according to claim 4, characterized in that, The temperature of the culturing is 22 - 24 °C, and the time of the culturing is 12 - 24 d.

8. The method for rapid regeneration according to claim 4, characterized in that, The culturing includes light - dark alternating culturing, and in each light - dark alternating culturing cycle, the light culturing time is 12 - 16 h, and the dark culturing time is 8 - 12 h.

Images