Method for promoting increase of epiphytic points of gametophytes of pogonatum pogonatum, method for regenerating gametophytes and application of gametophytes

Abstract

The invention belongs to the technical field of plant regeneration, and particularly relates to a method for promoting increase of epiphytic points of gametophytes of pogonatum pogonatum, a method for regenerating the gametophytes and application of the gametophytes. The invention provides application of 6-benayl aminopurine in promoting increase of the number of neonatal insertion points of gametophytes of pogonatum aloides and / or shortening cultivation time. According to the method, a 6-benzylaminopurine solution is used for treating a pogonatum pogonatum gametophyte stem segment. Maintenance and differentiation of plant epiphytic point stem cells are the key of plant development and morphogenesis, the 6-benayl aminopurine solution can promote the development of pogonatum aloides gametophyte stem cells to form new epiphytic points, the regeneration of pogonatum aloides gametophytes is induced, and the culture time of pogonatum aloides broken stem propagation is shortened. According to the method, a broken stem propagation technology is combined with hormone treatment, so that the cultivation time is greatly shortened, and the cultivation efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant regeneration, and in particular relates to a method for promoting the increase of white hair moss gametophyte attachment points, a method for gametophyte regeneration and an application thereof. Background Art

[0002] There are many species of mosses in the world, about 22,400 species. Most of the mosses are tiny, simple in structure and varied in form. By combining the growth characteristics of mosses with the art of gardening and horticulture, we can create a new type of moss landscape with natural beauty. In addition, mosses have strong adaptability, are basically free of pests and diseases, are evergreen, grow neatly and do not need to be pruned. The landscape after landscaping can be maintained for a long time, greatly saving manpower and material resources, and has a good development prospect.

[0003] Leucobryum glaucum belongs to the Leucobryaceae family and the genus Leucobryum. The plant grows in dense mats and is green with a whitish gray-green color. The leaf stems of Leucobryum glaucum are upright and can be up to 8 cm long, showing an irregular branching growth state. The leaves of Leucobryum glaucum are densely grown, with a sheath-like base, long ovate, and gradually lanceolate at the top, often deflected to one side; the leaf margins are entire, and the top is slightly involute. Leucobryum glaucum is dioecious, with a slender, reddish-brown spore stalk; the spore capsule is conical and arched. Leucobryum is common in moist woodlands in the mountainous areas south of the Yangtze River Basin in China, and also in southeastern Asia. Leucobryum has the characteristics of strong stress resistance, fast growth rate, and relative drought tolerance. It is used for landscaping, low and neat, giving people a comfortable and fresh feeling. In recent years, Leucobryum has been widely used in industries such as home gardening, three-dimensional greening, and garden landscape greening, but collecting wild mosses has caused great damage to the ecological environment. Artificially cultivated white-haired moss has slow growth and low yield, which is the bottleneck restricting the application of moss plants. Therefore, conducting research on the cultivation and reproduction technology of white-haired moss and increasing the yield of white-haired moss are the prerequisites for promoting the popularization and application of white-haired moss.

[0004] According to the differences in propagation materials and methods, the propagation methods of mosses are generally divided into three types in research and production: division propagation, stem cutting propagation and tissue culture. Among them, stem cutting propagation has a larger propagation coefficient than division propagation, and can extend the utilization period of moss blocks. Most mosses can obtain new plants by stem cutting propagation. However, the cultivation time of stem cutting propagation of white-haired moss is long. Summary of the invention

[0005] The purpose of the present invention is to provide a method for promoting the increase of white-haired moss gametophyte attachment points and a method and application of inducing white-haired moss gametophyte regeneration, thereby inducing white-haired moss gametophyte regeneration and shortening the cultivation time of white-haired moss stem fragmentation reproduction.

[0006] The invention provides the use of 6-benzylaminopurine in promoting the increase of the number of new attachment points of white hair moss gametophytes and / or shortening the cultivation time.

[0007] The invention provides a method for promoting the increase of white-hair moss gametophyte attachment points, comprising: treating the white-hair moss gametophyte stem segments with the 6-benzylaminopurine solution described in the above technical scheme.

[0008] Preferably, the molar concentration of 6-benzylaminopurine in the 6-benzylaminopurine solution is 48-52 nM.

[0009] Preferably, the method for obtaining the stem segment includes cutting off the top 5 mm of the gametophyte of the white hair moss and the yellowed and aged tissue at the bottom to obtain the stem segment.

[0010] Preferably, the length of the stem segment is 0.8 to 1.2 cm.

[0011] Preferably, before treating the stem segment with the 6-benzylaminopurine solution, the method further comprises washing the stem segment.

[0012] The present invention provides a method for inducing the regeneration of white-haired moss gametophytes, comprising the following steps:

[0013] After the gametophyte of white-haired moss is treated by the method described in the above technical solution, the gametophyte of white-haired moss is cultured and induced to regenerate.

[0014] Preferably, the culture temperature is 22-24° C., and the culture time is 12-24 days.

[0015] Preferably, the culture includes light and dark culture; the light culture time in each light and dark culture cycle is 12 to 16 hours, and the dark culture time is 8 to 12 hours; the light intensity of the culture is 60 to 80 lx.

[0016] Preferably, during the culture process, the 6-benzylaminopurine solution is supplemented in the culture container every 5 days.

[0017] Beneficial effects of the present invention: The present invention provides an application of 6-benzylaminopurine in promoting the increase in the number of new attachment points of white-haired moss gametophytes and / or shortening the cultivation time. The maintenance and differentiation of plant attachment point stem cells are the key to plant development and morphological construction. The 6-benzylaminopurine solution of the present invention can promote the development of white-haired moss gametophyte stem cells to form new attachment points, induce white-haired moss gametophyte regeneration, and shorten the cultivation time of white-haired moss stem cutting reproduction. The present invention greatly shortens the cultivation time and improves the cultivation efficiency by combining stem cutting reproduction technology with hormone treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1Schematic diagram of the plant and the attachment point after the stem cutting treatment in Example 1; wherein A is a schematic diagram of the cultivation method after the stem cutting; B is a schematic diagram of the gametophyte after the stem cutting; C is a schematic diagram of the stem segment of the attachment point of the new gametophyte, and the arrow points to the attachment point of the new gametophyte;

[0020] Figure 2 The gametophyte development states at different stages of Example 1 and Comparative Examples 1-2 are shown in Figure 1; A is a schematic diagram of the plant after 12 days of treatment; B is a schematic diagram of the plant after 24 days of treatment;

[0021] Figure 3 It is a data chart of the growth and development status of Example 1 and Comparative Examples 1-2, wherein A is the ratio of new growth points grown by the plants after 12 days of treatment; B is the number of growth points on the 24th day of treatment; and C is the plant area on the 24th day of treatment. DETAILED DESCRIPTION

[0022] The present invention provides an application of 6-benzylaminopurine in promoting the increase in the number of new attachment points of white-hair moss gametophytes and / or shortening the cultivation time. The application of the present invention preferably comprises: treating white-hair moss gametophyte stem segments with a solution of 6-benzylaminopurine to obtain treated stem segments; and culturing the treated stem segments to induce white-hair moss gametophyte regeneration.

[0023] Cytokinins play an important role in the growth and development of plants, such as cell division, leaf senescence, apical dominance, nutrient signal transmission, and regulation of biological and abiotic stresses. Rational use of cytokinins can effectively improve the progress of plant development. The maintenance and differentiation of plant stem cells at the attachment point is the key to plant development and morphological construction. The strong omnipotence or pluripotency of plant cells also gives plants stronger developmental plasticity and regeneration ability, produces branches and root systems, and can grow multiple attachment points at the same time; it also enables plants to produce new stem cell centers and attachment points during the recovery process after damage; it can also enable plant somatic cells to have extremely strong regeneration ability in vivo and in vitro to cope with environmental changes. The technical solution provided by the present invention combines stem cutting propagation technology with cytokinin treatment, and uses cytokinin 6-benzylaminopurine solution to promote the development of white hair moss gametophyte stem cells to form new attachment points, thereby improving gametophyte regeneration.

[0024] The present invention has no special limitation on the source of the 6-benzylaminopurine, and conventional products can be used.

[0025] The invention provides a method for promoting the increase of white-hair moss gametophyte attachment points, comprising: treating the white-hair moss gametophyte stem segments with the 6-benzylaminopurine solution described in the above technical scheme.

[0026] In the present invention, the method for obtaining the stem segment preferably includes cutting off the top 5 mm of the white-haired moss gametophyte and the yellowed and aged tissue at the bottom to obtain the stem segment. The present invention preferably cuts the stem segment from the white-haired moss gametophyte in good growth state. In the present invention, the top of the white-haired moss gametophyte is cut off to suppress apical dominance and promote the increase of new attachment points; the yellowed tissue at the bottom is removed because the yellowed tissue at the bottom has aged and died and will not regenerate. The present invention does not specifically limit the tools for obtaining the stem segments, and conventional tools can be used. The tools are preferably disinfected before use. The tools used in the present invention are preferably scissors, and the scissors are preferably disinfected with 75% alcohol before use. The length of the stem segment of the present invention is preferably 0.8 to 1.2 cm, and more preferably 1 cm. The present invention selects gametophyte stem segments as explants because of their simple operation.

[0027] After obtaining the stem segment, the present invention preferably uses 6-benzylaminopurine solution to treat the stem segment, and also includes cleaning the stem segment. The present invention does not specifically limit the cleaning method, as long as it is ensured to be clean. The cleaning of the present invention is preferably completed using distilled water (dH2O). The purpose of the cleaning of the present invention is to remove the carried soil, broken branches and stems. In an embodiment of the present invention, it is preferred that distilled water is placed in a culture dish and then the stem segment is placed in it for thorough stirring to wash away impurities. The diameter of the culture dish is preferably 9 cm.

[0028] In the present invention, the molar concentration of the 6-benzylaminopurine solution is preferably 48 to 52 nM, more preferably 50 nM. The unit represented by nM in the present invention is nmol / L. The 6-benzylaminopurine solution in the present invention can promote the development of white hair moss gametophyte stem cells to form new attachment points at a specific concentration, improve gametophyte regeneration, and shorten the cultivation time of white hair moss stem fragment reproduction.

[0029] The present invention provides a method for inducing the regeneration of white-haired moss gametophytes, comprising the following steps:

[0030] After the gametophyte of the white-haired moss is treated by the method for increasing the gametophyte attachment points described in the above technical solution, the gametophyte of the white-haired moss is cultured to induce the regeneration of the gametophyte.

[0031] In the present invention, the container for the culture application preferably includes a culture dish. Before the culture is carried out, the present invention preferably places filter paper in the culture dish, and the number of filter papers placed is preferably 2. After placing the filter paper, the present invention preferably pours 5 mL of 6-benzylaminopurine solution with a molar concentration of 50 nM on the filter paper in the culture dish, and then places the treated stem segment for culture. The culture temperature of the present invention is preferably 22 to 24°C, more preferably 24°C; the culture time is preferably 12 to 24 days, more preferably 16 to 24 days, and further preferably 20 to 24 days, more preferably 24 days.

[0032] The culture described in the present invention preferably includes light and dark culture; the time of light culture in each light and dark culture cycle of the present invention is preferably 12 to 16 hours, more preferably 14 to 16 hours, and more preferably 16 hours; the time of dark culture is preferably 8 to 12 hours, more preferably 8 to 10 hours, and more preferably 8 hours. Each light and dark culture cycle described in the present invention is 24 hours. The light intensity of the culture described in the present invention is preferably 60 to 80 lx, more preferably 65 to 75 lx, and more preferably 70 lx. The settings of the culture temperature, time, and light intensity of the present invention create suitable conditions for the growth of white hair moss gametophytes.

[0033] In the culture process of the present invention, preferably every 5 days, 6-benzylaminopurine solution is supplemented in the culture dish to keep the filter paper moistening. The concentration of 6-benzylaminopurine solution of the present invention is preferably 50nM. In an embodiment of the present invention, the diameter of the culture dish is 9cm, and preferably every 5 days, 5mL of 6-benzylaminopurine solution is supplemented in the culture process to keep the filter paper moistening. The effect of supplementing 6-benzylaminopurine solution is to keep the filter paper moistening, provide suitable humidity for the growth of white hair moss gametophyte, and promote the growth of white hair moss gametophyte cells simultaneously.

[0034] The invention discloses a method for accelerating the increase of attachment points of ornamental moss white-haired moss gametophytes and expanding reproduction. The method for accelerating the increase of attachment points of white-haired moss gametophytes provided by the invention can promote the gametophytes to produce new attachment points after 24 days of cultivation in the laboratory, has strong operability, greatly shortens the cultivation time, and improves the cultivation efficiency. The technical scheme provided by the invention is more suitable for accelerating the expansion and propagation of white-haired moss gametophytes. Compared with untreated plants, the number of new attachment points of white-haired moss gametophytes after treatment is significantly different. The scheme is simple to operate, stable and reliable, and can promote the rapid growth of gametophytes. The invention explores the conditions for promoting the rapid growth of gametophytes through the research on the rapid propagation and cultivation technology of white-haired moss, and makes up for the inherent defect of slow growth of moss plants. The technical scheme provided by the invention forms the key technology for large-scale production of white-haired moss, and provides theoretical support for its large-scale production and application.

[0035] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] 240 white-hair moss gametophytes with good growth status, no impurities, and no diseases and insect pests were selected as treatment materials and divided into four groups on average, of which 60 strains were used as experimental materials for Example 1, 60 strains were used as experimental materials for Comparative Example 1, 60 strains were used as experimental materials for Comparative Example 2, and 60 strains were used as experimental materials for Comparative Example 3.

[0037] Example 1

[0038] (1) Each selected white-haired moss gametophyte plant was about 3 cm in height. The selected 60 white-haired moss gametophytes were used as treatment materials for stem cutting treatment, specifically: removing the top 5 mm tissue and the bottom yellow tissue of the white-haired moss gametophyte plant, cutting the middle 1 cm plant, and placing the cut plant in a glass dish filled with distilled water and stirring it thoroughly to wash away impurities.

[0039] (2) Trim two pieces of filter paper to a suitable size and overlap them on a culture dish to increase water retention. Wet the filter paper with 5 mL of 50 nM 6-benzylaminopurine solution. The diameter of the culture dish is 9 cm.

[0040] (3) Use tweezers to pick up the plant treated in step (1) and place it flat on the filter paper moistened in step (2).

[0041] (4) The culture dish obtained in step (3) was placed in an incubator suitable for the growth of white hair moss and cultured for 24 days. The humidity of the incubator was 45.3%. The culture temperature was 24°C, the light intensity was 60-80 lx, and the photoperiod was 16 hours of light and 8 hours of darkness. In order to keep the filter paper moist, 5 mL of 6-benzylaminopurine solution was added every 5 days during the culture process.

[0042] Comparative Example 1

[0043] The same as Example 1, the only difference is that the 6-benzylaminopurine solution is replaced by a calcium prohexadione solution. The calcium prohexadione solution is a solution obtained by diluting calcium prohexadione with distilled water at a volume ratio of 1:1500.

[0044] Comparative Example 2

[0045] Same as Example 1, the only difference is that clean water is used instead of 6-benzylaminopurine solution.

[0046] Comparative Example 3

[0047] The same as Example 1, the only difference is that the 6-benzylaminopurine solution is replaced by the brassinolide solution. The brassinolide solution is obtained by diluting brassinolide with water at a volume ratio of 1:2000.

[0048] The schematic diagram of the plant and the attachment point after the gametophyte stem cutting treatment of Example 1 is shown in Figure 1 The growth and development of gametocytes in Example 1 and Comparative Examples 1-2 at different stages of cultivation in the incubator are shown in Figure 2 The ratio of new growth points of plants grown in the incubator after 12 days of cultivation in Example 1 and Comparative Examples 1-2, the number of growth points on the 24th day of cultivation, and the area of ​​plants on the 24th day of cultivation were shown in Table 1. Figure 3and Table 1. The ratio of plants growing new attachment points was calculated according to the formula: The formula is: the ratio of plants growing new attachment points = the number of plants growing new attachment points / the total number of plants in the treatment, and the plant area was measured using the measurement software Graphpad.

[0049] Figure 3 The data were processed using Graphpad Prism 8.0 software, and the data were expressed as Mean ± SEM. The data were analyzed using Student's t-test, and significant differences were indicated by asterisks, ** corresponds to p < 0.01, and **** corresponds to p < 0.0001.

[0050] Table 1 Ratio of new growth points incubated at different times in the incubator of Example 1 and Comparative Examples 1-2

[0051]

[0052] According to Table 1 and Figure 3 It can be seen that the 6-benzylaminopurine treatment group of Example 1 has extremely significant differences in the number of new gametophyte attachment points and plant area with Comparative Examples 1 and 2. The 50 nM 6-benzylaminopurine treatment greatly increases the growth rate of white hair moss after stem cutting.

[0053] The number of white hair moss gametophyte attachment points of Comparative Example 2 and Comparative Example 3 is shown in Table 1. According to Table 1, there is no difference in the number of white hair moss gametophyte attachment points between Comparative Example 2 and Comparative Example 3, which are basically the same, and brassinolide cannot increase the number of white hair moss gametophyte attachment points.

[0054] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of 6-benzylaminopurine in promoting the increase of the number of new attachment points of white hair moss gametophytes and / or shortening the cultivation time.

2. A method for promoting the increase of gametophyte attachment points of white hair moss, characterized in that: include: The gametophyte stem segments of the white-hair moss are treated with the 6-benzylaminopurine solution according to claim 1.

3. The method according to claim 2, characterized in that The molar concentration of 6-benzylaminopurine in the 6-benzylaminopurine solution is 48-52 nM.

4. The method according to claim 2, characterized in that: The method for obtaining the stem segment comprises cutting off the top 5 mm of the gametophyte of the white hair moss and the yellowed and aged tissue at the bottom to obtain the stem segment.

5. The method according to claim 2 or 4, characterized in that: The length of the stem segment is 0.8 to 1.2 cm.

6. The method according to claim 2, characterized in that Before the stem segment is treated with the 6-benzylaminopurine solution, the process also includes washing the stem segment.

7. A method for inducing gametophyte regeneration of white hair moss, characterized in that: The steps include: The gametophyte of white-haired moss is treated by the method described in any one of claims 2 to 6, and then cultured to induce the regeneration of the gametophyte of white-haired moss.

8. The method according to claim 7, characterized in that The culture temperature is 22-24° C., and the culture time is 12-24 days.

9. The method according to claim 7, characterized in that: The culture includes light and dark culture; the light culture time in each light and dark culture cycle is 12 to 16 hours, and the dark culture time is 8 to 12 hours; the light intensity of the culture is 60 to 80 lx.

10. The method according to claim 7, characterized in that During the culture process, the culture container was supplemented with 6-benzylaminopurine solution every 5 days.

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