Induction method of duckweed spore formation

Abstract

The invention relates to a duckweed spore formation induction method which comprises the following steps: pretreating duckweed plants to obtain duckweed to be cultured; spraying the to-be-cultured duckweed according to a preset period by adopting a gibberellin solution, and stopping spraying when the to-be-cultured duckweed forms spores, so as to obtain the cultured duckweed. The to-be-cultured duckweed is regularly sprayed with gibberellin, so that the duckweed can be effectively induced to form spores, and no adverse effect is caused to duckweed plants. By means of the method, the duckweed resource breed conservation quality can be effectively improved, and remarkable effects are brought to cultivation of excellent duckweed quality and reduction of breed conservation cost.

Description

Technical Field

[0001] The invention relates to the technical field of Azolla culture, in particular to a method for inducing Azolla spore formation. Background Art

[0002] Azolla is a fern-algae symbiotic aquatic plant. The cyanobacteria that live symbiotically in its leaf cavity have a strong ability to fix nitrogen. This enables it to rapidly expand its biomass through asexual reproduction in nutrient-deficient freshwater environments and plays an important role in the nitrogen cycle of freshwater ecosystems. In addition to asexual reproduction, Azolla can also reproduce sexually and form spore fruits under certain conditions.

[0003] At present, due to the fact that seedlings mostly adopt asexual vegetative propagules, and the suitable growth temperature of Azolla is 20-35 degrees Celsius, that is, it is difficult to grow above 40 degrees Celsius or below 5 degrees Celsius, under natural conditions, Azolla is difficult to survive the summer and winter. Therefore, in summer and winter, after the death of Azolla, a large amount of asexual vegetative propagules are required for production and application, and the required vegetative propagules can only be reproduced by artificial measures in summer and winter, resulting in higher seedling propagation costs and long-distance transportation costs in different places. And the use of spore fruit does not require artificial propagation in summer and winter, and the spore fruit is small in size and easy to transport, which can effectively reduce the cost of reproduction and transportation. However, due to the different conditions required for the spore fruit produced by each strain of Azolla, the Azolla strain that can spore in a region is limited.

[0004] Therefore, providing an efficient inducing method for the sporulation of Azolla to promote the sporulation of Azolla is an urgent problem to be solved. Summary of the invention

[0005] In view of this, the present application provides a method for inducing sporulation of Azolla to solve the above problems.

[0006] In a first aspect, the present application provides a method for inducing sporulation of Azolla, comprising the following steps:

[0007] Pre-treating the azolla plants to obtain the azolla to be cultivated;

[0008] The azolla to be cultivated is sprayed with a gibberellin solution according to a preset cycle, and when spores appear in the azolla to be cultivated, the spraying is stopped to obtain the cultivated azolla.

[0009] In some embodiments, the pretreatment of the Azolla plant to obtain the Azolla to be cultivated comprises:

[0010] After washing the duckweed plants in clean water, select duckweed plants that meet the requirements and remove impurities;

[0011] The impurity-removed Algae plants are purified to obtain the Algae to be cultivated.

[0012] In some embodiments, when the impurity-removed Azolla plants are subjected to purification treatment, the impurity-removed Azolla plants are cultured in soil for one week.

[0013] In some embodiments, the gibberellin solution is obtained by diluting a gibberellin mother solution, wherein the preparation process of the gibberellin mother solution comprises:

[0014] Mix gibberellin crystals and anhydrous ethanol in proportion, and stir until they are completely dissolved to obtain an alcohol solution containing gibberellin;

[0015] Deionized water is added to the alcohol solution containing gibberellin, and the mixture is stirred evenly to obtain a gibberellin mother solution; wherein the concentration of the gibberellin mother solution is 500 μg·mL -1 .

[0016] In some embodiments, the treatment concentration of the gibberellin solution is 1-7 μg·mL -1 .

[0017] In some embodiments, the treatment concentration of the gibberellin solution is 7 μg mL -1 .

[0018] In some embodiments, the gibberellin solution is a GA3 solution.

[0019] In some embodiments, the method of spraying the to-be-cultivated duckweed with a gibberellin solution according to a preset cycle, and stopping the spraying when the to-be-cultivated duckweed forms spores, to obtain the cultivated duckweed, comprises:

[0020] The green duckweed to be cultured is placed in a culture pot, the bottom of the pot is covered with sterilized paddy soil, and filled with clean water;

[0021] Under the preset culture conditions, the azolla to be cultured is sprayed with the gibberellin solution, wherein the frequency of spraying is once a week;

[0022] When the Azolla to be cultivated forms spores, spraying is stopped to obtain cultivated Azolla.

[0023] In some embodiments, the culture conditions include:

[0024] The temperature is 20-25℃ and the air humidity is 85-90%.

[0025] In some embodiments, the green duckweed is any one of S. mexicana, S. triangularis and S. microphylla.

[0026] Technical effects of the present invention:

[0027] By regularly spraying the cultivated duckweed with gibberellins, duckweed spores can be effectively induced without causing adverse effects on duckweed plants. In addition, during the cultivation process, duckweed plants are regularly observed for spores, and when spores appear, the spraying of the gibberellins solution is immediately stopped to avoid excessive treatment that has adverse effects on the quality of duckweed spores, thereby increasing the sporulation rate and number of spores of duckweed plants. The overall cultivation method is simple, which can effectively improve the quality of duckweed resources and reduce labor.

[0028] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0030] Figure 1 A flow chart showing a method for inducing sporulation of Azolla sinensis according to an embodiment of the present application is shown. DETAILED DESCRIPTION

[0031] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0032] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0033] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present disclosure.

[0034] The present application embodiment provides a method for inducing sporulation of Azolla. Figure 1 is a flow chart of the steps of the method for inducing sporulation of Azolla according to an embodiment of the present application, such as Figure 1 As shown, the method comprises the following steps:

[0035] S100, pre-treating the duckweed plants to obtain duckweed to be cultivated;

[0036] In this step, it is carried out under natural conditions in a net house greenhouse. Specifically, before formally inducing the sporulation of the green duckweed, it is necessary to first purify the green duckweed, that is, to ensure that there is no contamination from other duckweeds in the subsequent induction process, which affects the induction effect. Among them, the net house greenhouse culture can effectively solve the problem of insects, branches and other litters falling into the culture container caused by the current common outdoor net house culture, and ensure that the growth and purity of the green duckweed are not affected by the outside world.

[0037] S200, spraying the azolla to be cultivated with a gibberellin solution according to a preset cycle, and when spores appear on the azolla to be cultivated, stopping the spraying to obtain the cultivated azolla.

[0038] In this step, sporulation induction is carried out for the pretreated green duckweed to be cultivated. Specifically, it is necessary to use a gibberellin solution to spray the green duckweed to be cultivated within a certain period. It should be noted that gibberellin (GAS) is a class of tetracyclic diterpene carboxylic acid compounds produced by green plants, fungi and bacteria. By spraying the green duckweed to be cultivated with regular gibberellin, the sporulation of the green duckweed can be effectively induced, and the green duckweed plants will not be adversely affected. In addition, during the cultivation process, it is necessary to regularly observe the growth of the green duckweed plants, especially when the sporulation phenomenon occurs, it is necessary to stop the spraying of the gibberellin solution immediately to avoid excessive treatment on the quality of the green duckweed sporulation, thereby improving the sporulation rate and the number of spore fruits of the green duckweed plants.

[0039] In some embodiments, the green duckweed is any one of S. mexicana, S. deltaica and S. microphylla.

[0040] In the present embodiment, the Mexican duckweed has the characteristics of strong cold resistance, good yield, strong nitrogen fixation ability, high sporulation rate, rich nutrition, strong salt tolerance, etc., but poor heat resistance. The small leaf duckweed has the characteristics of strong heat resistance, stable sporulation performance, high sporulation rate, and many female spores, but poor cold resistance. The triangle duckweed has strong nitrogen fixation ability, fast growth rate, high nutritional value, and strong environmental adaptability, and is a good feed raw material for livestock and poultry. Therefore, the present embodiment uses Mexican duckweed, triangle duckweed and small leaf duckweed as the cultivation objects to provide a research method for cultivating duckweed varieties that are resistant to high temperature, resistant to low temperature, fast growth rate and high nutritional value.

[0041] In some of the embodiments, the duckweed plants are pretreated to obtain the duckweed to be cultivated, including: rinsing the duckweed plants in clean water, selecting duckweed plants with required specifications and sizes, and removing impurities; purifying the duckweed plants after impurities removal to obtain the duckweed to be cultivated.

[0042] In this embodiment, first, duckweed of uniform size and specification needs to be selected and placed in a culture basin, and the duckweed and impurities that affect the cultivation of duckweed are removed. Among them, by removing other duckweeds other than duckweed, other algae are prevented from mixing with duckweed to compete for nutrients and space. In the process of inducing sporulation, duckweed needs sufficient nutrients to support its physiological changes, and the presence of duckweed will affect the sporulation effect of duckweed.

[0043] In addition, since duckweed may also carry various microorganisms, such as bacteria and fungi, these biological contaminants need to be removed during the impurity removal process to prevent duckweed diseases. Especially in the subsequent sporulation induction process, the physiological state of duckweed will change, and its own defense mechanism may be affected to a certain extent. By removing the interference of biological contaminants in advance, the normal sporulation of duckweed can be effectively guaranteed. At the same time, since gibberellins are produced by green plants, fungi and bacteria, if impurity removal is not carried out, some microorganisms are likely to decompose gibberellins and change the subsequent induction environment, thereby interfering with the sporulation induction of duckweed.

[0044] It should also be noted that after the impurity removal of the azolla plant, it is necessary to further purify the azolla after the impurity removal, that is, there is a pre-purification process before the formal use of gibberellin spraying. The reason is that the azolla plant may have physical or physiological damage after the impurity removal treatment. Purifying the azolla plant after the impurity removal to ensure that it recovers its normal physiological function is an important prerequisite for effectively inducing spore formation, so that it can better absorb and utilize gibberellin in the future.

[0045] In some embodiments, when the impurity-removed Azolla plants are subjected to purification treatment, the impurity-removed Azolla plants are cultured in soil for one week.

[0046] Specifically, before formally cultivating the green duckweed in this embodiment, it is necessary to use a normal soil culture method to pre-cultivate for one week. During the one week of pre-cultivation, the root system of the green duckweed can gradually adapt to the new soil environment, and during this week, the root cells damaged during the impurity removal can regenerate, providing sufficient material support for the growth and sporulation of the green duckweed during the gibberellin treatment stage. At the same time, after a week of pre-cultivation, the growth progress between the green duckweed individuals will gradually tend to be synchronized, the growth rate of the leaves and the root development program will be relatively close, which is more conducive to the synchronous development of the sporulation process.

[0047] In some embodiments, the gibberellin solution is obtained by diluting the gibberellin mother solution, wherein the preparation process of the gibberellin mother solution includes: mixing gibberellin crystals and anhydrous ethanol in proportion, stirring until completely dissolved, to obtain an alcohol solution containing gibberellin; adding deionized water to the alcohol solution containing gibberellin, stirring evenly to obtain the gibberellin mother solution; wherein the concentration of the gibberellin mother solution is 500 μg·mL -1 .

[0048] In this embodiment, it is necessary to pre-prepare a gibberellin solution for sporulation of green duckweed. The gibberellin solution is obtained by diluting the gibberellin mother liquor according to actual needs. Specifically, the gibberellin crystals are mixed with anhydrous ethanol in proportion, wherein the preferred proportion is to add 1g of gibberellin crystals to every 25ml of anhydrous ethanol, and stir well until completely dissolved, thereby obtaining an alcohol solution containing gibberellin; then, 2L of deionized water is added to the alcohol solution containing gibberellin, and stirred evenly to obtain a concentration of 500μg·mL -1 The gibberellin mother solution can be diluted to the required concentration according to actual requirements during the subsequent cultivation of green duckweed, that is, 1-7μg·mL -1 .

[0049] In some embodiments, the gibberellin solution is a GA3 solution.

[0050] It should be noted that GA3, as an active compound in gibberellins, can accelerate the elongation of duckweed cells, increase the size of duckweed plants and increase the leaf area. A larger leaf area means that duckweed can carry out more photosynthesis, providing energy and material basis for its own growth. In particular, it should be noted that GA3 can stimulate the reproductive growth of duckweed, increase the number of duckweed spores, and thus improve the reproductive efficiency of duckweed.

[0051] In some embodiments, the treatment concentration of the gibberellin solution is 1-7 μg mL -1 .

[0052] In this embodiment, when the gibberellin solution is used to spray the cultured duckweed, the concentration of the gibberellin solution is preferably 1-7 μg·mL -1 At this concentration, the sporulation effect of the cultured duckweed was significantly improved compared to the case without using the gibberellin solution. Specifically, the sporulation rate was 4.67%-15.12%, and the number of sporulations was 3.75-6.32.

[0053] In some of the embodiments, the treatment concentration of the gibberellin solution is 7 μg mL -1 .

[0054] In this embodiment, when the treatment concentration of the gibberellin solution is 7 μg mL -1 When the sporulation effect of Azolla was the best, the sporulation rate was 14.07%-15.12%, and the number of sporulations was 6.23-6.32. There was a significant difference between the Azolla treated without gibberellin.

[0055] In some of the embodiments, the duckweed to be cultivated is sprayed with a gibberellin solution according to a preset cycle, and when spores appear on the duckweed to be cultivated, the spraying is stopped to obtain the cultivated duckweed, comprising: placing the duckweed to be cultivated in a culture pot, and covering the bottom of the pot with sterilized paddy soil and filling it with clean water; spraying the duckweed to be cultivated with a gibberellin solution under preset culture conditions, wherein the spraying frequency is once a week; when spores appear on the duckweed to be cultivated, the spraying is stopped to obtain the cultivated duckweed.

[0056] In the present embodiment, the whole process of cultivating green duckweed is carried out in the greenhouse of the base, and the paddy soil for cultivating green duckweed needs to be sterilized to remove harmful microorganisms therein, so as to provide a clean growth mechanism for cultivating green duckweed. Wherein, the sterilization of paddy soil is realized by high temperature and high pressure, and suitable temperature and pressure can be selected according to actual sterilization requirements, and the present invention will not repeat them here. After the paddy soil sterilized by high temperature and high pressure is placed at the bottom of the culture basin, it is filled with clear water to simulate the growth environment of green duckweed paddy field. Subsequently, the green duckweed to be cultivated is sprayed with the configured gibberellin solution under the preset culture conditions, and its sporulation is observed, and spraying is stopped when sporulation occurs.

[0057] Moreover, when the gibberellin treatment is started, the gibberellin needs to be sprayed according to the preset cycle, and the spraying standard is that the entire azolla can be evenly sprayed with the gibberellin solution, and the gibberellin spraying treatment is carried out once a week on Monday. The growth and physiological activities of azolla have a certain periodicity. The efficiency of once a week treatment can realize the process of azolla from absorption, response to its own physiological state adjustment, which can avoid the stimulation and damage caused by frequent treatment to azolla, and will not make the azolla's response to gibberellin not obvious due to the long interval time.

[0058] In some embodiments, the culture conditions include: a temperature of 20-25° C. and an air humidity of 85-90%.

[0059] In this embodiment, in order to provide the most suitable growth conditions for Azolla, Azolla is preferably cultured at a temperature of 20-25° C. and an air humidity of 85-90%. The growth rate and physiological state of Azolla can be effectively guaranteed in this temperature range and humidity range, especially the humidity environment of 85%-90% is conducive to the formation and maturation of Azolla spores, and the spores are more easily spread and germinated.

[0060] The following will provide detailed descriptions of the sporulation induction of S. mexicana, S. triangularis and S. microphylla by providing examples and comparative examples, respectively.

[0061] It should be noted that the cultivation of each group of examples and comparative examples was carried out under natural conditions in a net house greenhouse, and the amount of duckweed released was the same. After cultivation, the induction effect of each group of duckweed bodies, i.e., the sporulation rate of spore fruit, the number of green duckweed spore fruit and the male-female ratio of spore fruit, as well as the duckweed body size and duckweed body C / N ratio were measured.

[0062] Sporangial fruit conidia rate: Each duckweed body was used as the calculation unit, and the conidia of 100 duckweed bodies were randomly counted in each replicate, and the average of 3 replicates was the final value;

[0063] The number of conidia was calculated based on the number of conidia of each duckweed body. A total of 100 conidia with conidia were counted in each pot, and the average value of 3 repeated pots was taken as the final value.

[0064] Sporangial female-male ratio: calculated based on the number of male and female sporangial fruit of each duckweed body. A total of 100 duckweed bodies with sporangial female-male ratios were counted in each pot, and the average value of 3 repeated pots was taken as the final value.

[0065] Body size of duckweed: The length and width of the duckweed body were measured. The length of the duckweed body was measured based on the longest length of each duckweed body. Thirty duckweed bodies were randomly measured in each repetition, and the average of the three repetitions was the final measurement result. The width of the duckweed body was measured in the same way as the length, and the final average value was taken as the measurement result.

[0066] Mexican Ping:

[0067] Example 1

[0068] S1. Use normal soil culture method to select Mexican duckweed with uniform size and specifications, remove impurities and pre-culture for 1 week;

[0069] S2, using a treatment concentration of 1 μg mL -1 Treat with gibberellin once every Monday and observe the sporulation. Stop spraying when sporulation appears.

[0070] Example 2

[0071] The difference from Example 1 is that the treatment concentration of gibberellin is 3 μg mL -1 .

[0072] Example 3

[0073] The difference from Example 1 is that the treatment concentration of gibberellin is 7 μg mL -1 .

[0074] Comparative Example 1

[0075] Different from Example 1, gibberellin was not used.

[0076] Comparative Example 2

[0077] The difference from Example 1 is that the treatment concentration of gibberellin is 15 μg mL -1 .

[0078] Comparative Example 3

[0079] The difference from Example 1 is that the treatment concentration of gibberellin is 25 μg mL -1 .

[0080] The results are as follows:

[0081] 1. Induction effect:

[0082] (1) Sporulation rate (%)

[0083] As shown in Table 1, Example 3 was treated with gibberellin at a mass concentration of 7 μg mL -1 The sporulation rate was 15.07%. In Example 2, the concentration of gibberellin was 3 μg·mL -1 The sporulation rate was 6.32%. In Example 1, the concentration of gibberellin was 1 μg·mL -1 The sporulation rate was 4.69%;

[0084] Comparative Example 1-3, i.e., the gibberellin treatment concentration is 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rate was 0%.

[0085] Therefore, when the gibberellin treatment concentration is 7 μg·mL -1 The conidia rate was the highest when the concentration was 1 μg·mL -1 The conidia rate and treatment mass concentration were 3 μg·mL -1 The conidia rate was second; while the comparative examples 1-3, i.e., the treatment mass concentration was 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rates of all the strains were 0%, indicating that all strains failed to form sporulation.

[0086] (2) Number of spores

[0087] Mexican water chestnuts treated with 7 μg·mL -1 The number of conidia was 6.32, 3 μg·mL -1 The number of conidia was 4.15, 1 μg·mL -1 The number of conidia was 3.87, 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The number of spores was 0.

[0088] Therefore, the treatment mass concentration in Example 3 is 7 μg·mL -1 The number of spores was the largest, which was significantly different from other embodiments and comparative examples; the treatment mass concentration was 1 μg·mL -1 and 3 μg mL -1 The number of spores is second only to that of

[0089] (3) Male-female ratio

[0090] Mexican duckweed treated with gibberellin at a concentration of 7 μg·mL -1 Sporangia The ratio of male to female was 0.59:1, 3 μg·mL -1 Sporangia The ratio of male to female was 0.52:1, 1 μg·mL -1 Sporangia The ratio of male to female was 0.49∶1, 0 μg·mL -1 , 15μg·mL -1 and 20 μg mL -1 Sporangia The male to female ratio is 0:0.

[0091] Therefore, the treatment mass concentration is 7 μg mL -1 Sporangia The male ratio was the highest, which was significantly different from the control; the treatment concentrations were 1 and 3 μg·mL -1 Sporangia The male ratio was second, and there was no significant difference between the two, but there were extremely significant differences between them and the control ratio.

[0092]

[0093]

[0094] Table 1 The induction effect of gibberellin concentrations on duckweed in Examples 1-3 and Comparative Examples 1-3

[0095] 2. Size of duckweed

[0096] As shown in Table 2, the length and width of the Mexican water chestnut body in Examples 1 to 3 were enhanced, wherein the gibberellin treatment concentration in Example 3 was 7 μg·mL -1The length and width of the duckweed body of Example 1 are the largest, which increases by about 40% compared with the comparative example 1 without adding gibberellin, and the difference with Examples 1 and 2 is also quite significant; the length and width of the duckweed body of Examples 1 and 2 are also relatively increased compared with the comparative example 1, but the difference is small. It can be seen that when Mexican duckweed forms spores, the duckweed body will be relatively larger. Although the length and width of Comparative Examples 2-3 are also relatively increased, the difference with the results of Comparative Example 1 without using gibberellin is small.

[0097]

[0098] Table 2 Schematic diagram of the effect of gibberellin concentration on duckweed size of Examples 1-3 and Comparative Examples 1-3

[0099] 3. C / N ratio

[0100] As shown in Table 3, when the C / N ratio increases to a certain level, the Mexican duckweed can form spores, and the C / N ratio will increase when the Mexican duckweed forms spores. Compared with comparative examples 1-3, the C / N ratios of Examples 1-3 are significantly enhanced. Among them, the gibberellin treatment concentration of Example 3 is 7 μg·mL -1 The C / N ratio is the largest when the gibberellin treatment concentration is 1 and 3 μg·mL, respectively. -1 The C / N ratio of Comparative Examples 1 to 3 is average.

[0101]

[0102] Table 3 Effect of gibberellin concentration on C / N ratio of Examples 1-3 and Comparative Examples 1-3

[0103] Water chestnut:

[0104] Example 4

[0105] S1. Use normal soil culture method to select duckweed with uniform size and specifications, remove impurities and pre-culture for 1 week;

[0106] S2, using a treatment concentration of 1 μg mL -1 Treat with gibberellin once every Monday and observe the sporulation. Stop spraying when sporulation appears.

[0107] Example 5

[0108] The difference from Example 4 is that the treatment concentration of gibberellin is 3 μg·mL -1 .

[0109] Example 6

[0110] The difference from Example 4 is that the treatment concentration of gibberellin is 7 μg mL -1 .

[0111] Comparative Example 4

[0112] Different from Example 4, gibberellin was not used.

[0113] Comparative Example 5

[0114] The difference from Example 4 is that the treatment concentration of gibberellin is 15 μg mL -1 .

[0115] Comparative Example 6

[0116] The difference from Example 4 is that the treatment concentration of gibberellin is 25 μg mL -1 .

[0117] The results are as follows:

[0118] 1. Induction effect:

[0119] (1) Sporulation rate (%)

[0120] As shown in Table 4, Example 6 treated with gibberellin at a mass concentration of 7 μg mL -1 The sporulation rate was 14.97%. In Example 5, the concentration of gibberellin was 3 μg·mL -1 The sporulation rate was 7.31%. In Example 4, the concentration of gibberellin was 1 μg·mL -1 The sporulation rate was 4.67%;

[0121] Comparative Example 4-6, i.e., the gibberellin treatment concentration is 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rate was 0%.

[0122] Therefore, when the gibberellin treatment concentration is 7 μg·mL -1 The conidia rate was the highest when the concentration was 1 μg·mL -1 The conidia rate and treatment mass concentration were 3 μg·mL -1 The conidia rate was second; while the comparative examples 4-6, i.e., the treatment mass concentration was 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rates of all the strains were 0%, indicating that all strains failed to form sporulation.

[0123] (2) Number of spores

[0124] Delta fascicularis treated with 7 μg·mL-1 The number of conidia was 6.27, 3 μg·mL -1 The number of conidia was 4.19, 1 μg·mL -1 The number of conidia was 3.91, 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The number of spores was 0.

[0125] Therefore, the treatment mass concentration in Example 6 is 7 μg·mL -1 The number of spores was the largest, which was significantly different from other embodiments and comparative examples; the treatment mass concentration was 1 μg·mL -1 and 3 μg mL -1 The number of spores is second only to that of

[0126] (3) Male-female ratio

[0127] Delta fascicularis treated with GA 7 μg·mL -1 Sporangia The ratio of male to female was 0.61:1, 3 μg·mL -1 Sporangia The ratio of male to female was 0.56:1, 1 μg·mL -1 Sporangia The ratio of male to female was 0.53:1, 0 μg·mL -1 , 15μg·mL -1 and 20 μg mL -1 Sporangia The male to female ratio is 0:0.

[0128] Therefore, the treatment mass concentration is 7 μg mL -1 Sporangia The male ratio was the highest, which was significantly different from the control; the treatment concentrations were 1 and 3 μg·mL -1 Sporangia The male ratio was second, and there was no significant difference between the two, but there were extremely significant differences between them and the control ratio.

[0129]

[0130] Table 4 The induction effect of gibberellin concentration on duckweed of Example 4-6 and Comparative Example 4-6

[0131] 2. Size of duckweed

[0132] As shown in Table 5, the length and width of the delta-shaped duckweed bodies in Examples 4 to 6 were enhanced, wherein the gibberellin treatment concentration in Example 6 was 7 μg·mL -1The length and width of duckweed body of Example 1 are the largest, which increases by about 40% compared with the comparative example 1 without adding gibberellin, and the difference with Example 4 and Example 5 is also more significant; the length and width of duckweed body of Example 4 and 5 are also relatively increased compared with the comparative example 1, but the difference is small. It can be seen that when duckweed congeals, the duckweed body will be relatively larger. Although the length and width of comparative examples 5-6 are also relatively increased, the difference with the result of comparative example 4 without using gibberellin is small.

[0133]

[0134]

[0135] Table 5 Schematic diagram of the effect of gibberellin concentration on duckweed size of Examples 4-6 and Comparative Examples 4-6

[0136] 3. C / N ratio

[0137] As shown in Table 6, when the C / N ratio increases to a certain level, the duckweed can form spores, and the C / N ratio will increase when the duckweed forms spores. Compared with comparative examples 4-6, the C / N ratios of examples 4-6 are significantly enhanced. Among them, the gibberellin treatment concentration of example 6 is 7 μg·mL -1 The C / N ratio is the largest when the gibberellin treatment concentration is 1 and 3 μg·mL, respectively. -1 The C / N ratio of Comparative Examples 4-6 is average.

[0138]

[0139] Table 6 Effect of gibberellin concentration on C / N ratio of Examples 4-6 and Comparative Examples 4-6

[0140] Xiao Ye Ping:

[0141] Example 7

[0142] S1. Use normal soil culture method to select duckweeds of uniform size and specifications, remove impurities and pre-culture for 1 week;

[0143] S2, using a treatment concentration of 1 μg mL -1 Treat with gibberellin once every Monday and observe the sporulation. Stop spraying when sporulation appears.

[0144] Example 8

[0145] The difference from Example 7 is that the treatment concentration of gibberellin is 3 μg mL -1 .

[0146] Example 9

[0147] The difference from Example 7 is that the treatment concentration of gibberellin is 7 μg mL -1 .

[0148] Comparative Example 7

[0149] Different from Example 7, gibberellin was not used.

[0150] Comparative Example 8

[0151] The difference from Example 7 is that the treatment concentration of gibberellin is 15 μg mL 1 .

[0152] Comparative Example 9

[0153] The difference from Example 7 is that the treatment concentration of gibberellin is 25 μg mL 1 .

[0154] The results are as follows:

[0155] 1. Induction effect:

[0156] (1) Sporulation rate (%)

[0157] As shown in Table 7, Example 9 treated with gibberellin at a mass concentration of 7 μg mL -1 The sporulation rate was 15.12%. In Example 7, the concentration of gibberellin treatment was 3 μg·mL -1 The sporulation rate was 5.29%. In Example 6, the concentration of gibberellin was 1 μg·mL -1 The sporulation rate was 4.83%;

[0158] Comparative Example 7-9, i.e., the gibberellin treatment concentration is 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rate was 0%.

[0159] Therefore, when the gibberellin treatment concentration is 7 μg·mL -1 The conidia rate was the highest when the concentration was 1 μg·mL -1 The conidia rate and treatment mass concentration were 3 μg·mL -1 The conidia rate was second; while the comparative examples 7-9, i.e., the treatment mass concentration was 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The sporulation rates of all the strains were 0%, indicating that all strains failed to form sporulation.

[0160] (2) Number of spores

[0161] In the treatment of gibberellin 7 μg·mL -1 The number of conidia was 6.23, 3 μg·mL -1 The number of conidia was 4.07, 1 μg·mL -1 The number of conidia was 3.75, 0 μg·mL -1 , 15μg·mL -1 and 25 μg mL -1 The number of spores was 0.

[0162] Therefore, the treatment mass concentration in Example 9 is 7 μg·mL -1 The number of spores was the largest, which was significantly different from other embodiments and comparative examples; the treatment mass concentration was 1 μg·mL -1 and 3 μg mL -1 The number of spores is second only to that of

[0163] (3) Male-female ratio

[0164] The concentration of Alpinia scoparia in the treatment of GA was 7 μg·mL -1 The spore-fruit-male ratio was 0.58∶1, 3 μg·mL -1 The spore-fruit-male ratio was 0.49∶1, 1 μg·mL -1 The spore-fruit-male ratio was 0.49:1, 0 μg·mL -1 , 15μg·mL -1 and 20 μg mL -1 The ratio of spore fruits to male fruits is 0:0.

[0165] Therefore, the treatment mass concentration is 7 μg mL -1 The proportion of male spore fruits was the highest, which was significantly different from the control; the treatment mass concentrations were 1 and 3 μg·mL -1 The ratio of spore fruits to males was second, and there was no significant difference between the two, but there were extremely significant differences between them and the control group.

[0166]

[0167] Table 7 Induction effect of gibberellin concentration on duckweed in Example 7-9 and Comparative Example 7-9

[0168] 2. Size of duckweed

[0169] As shown in Table 8, the length and width of the body of the small leaflet in Examples 7 to 9 were enhanced, wherein the gibberellin treatment concentration in Example 9 was 7 μg·mL -1The length and width of the duckweed body of Example 7 are the largest, which increases by about 40% compared with the comparative example 7 without adding gibberellin, and the difference with Examples 7 and 8 is also more significant; the length and width of the duckweed body of Examples 7 and 8 are also relatively increased compared with the comparative example 7, but the difference is small. It can be seen that when duckweed is sporulated, the duckweed body will be relatively larger. Although the length and width of Comparative Examples 8-9 are also relatively increased, the difference is small compared with the results of Comparative Example 7 without using gibberellin.

[0170]

[0171] Table 8 Schematic diagram of the effect of gibberellin concentration on duckweed size of Examples 7-9 and Comparative Examples 7-9

[0172] 3. C / N ratio

[0173] As shown in Table 9, when the C / N ratio increases to a certain level, the duckweed can form spores, and the C / N ratio will increase when the duckweed forms spores. Compared with comparative examples 7-9, the C / N ratios of examples 7-9 are significantly enhanced. Among them, the gibberellin treatment concentration of example 9 is 7 μg·mL -1 The C / N ratio was the largest when the gibberellin concentration was 1 and 3 μg·mL in Example 7 and Example 8, respectively. -1 The C / N ratio of Comparative Examples 7-9 is average.

[0174]

[0175]

[0176] Table 6 Effect of gibberellin concentration on C / N ratio of Examples 7-9 and Comparative Examples 7-9

[0177] In summary, when the gibberellin concentration is 7 μg·mL -1 The culture effect is the best, whether it is the sporulation rate, the number of sporulations, the ratio of males and females, the size of duckweed, and the C / N ratio, all of which are optimal. Moreover, as shown in Tables 1-9, the higher the gibberellin concentration, the better. When the gibberellin concentration increases to a certain extent, it will affect the sporulation of duckweed and inhibit the growth of duckweed. Controlling the gibberellin concentration by the method of the present application can not only effectively improve the seed preservation quality of duckweed resources, bring significant effects for cultivating excellent duckweed quality and reducing seed preservation costs, but also effectively solve the problem of breeding new hybrid varieties and solve the application difficulties in production.

[0178] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but the limitation of the scope of the invention patent cannot be understood as a result. It should be pointed out that, for a person of ordinary skill in the art, several modifications or improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for inducing sporulation of Azolla, characterized in that: The steps include: Pre-treating the azolla plants to obtain the azolla to be cultivated; The azolla to be cultivated is sprayed with a gibberellin solution according to a preset cycle, and when spores appear in the azolla to be cultivated, the spraying is stopped to obtain the cultivated azolla.

2. The method for inducing sporulation of Azolla according to claim 1, characterized in that: The method of pretreating the azolla plant to obtain the azolla to be cultivated comprises: After washing the duckweed plants in clean water, select duckweed plants that meet the requirements and remove impurities; The impurity-removed Algae plants are purified to obtain the Algae to be cultivated.

3. The method for inducing sporulation of Azolla according to claim 2, characterized in that: When the impurity-removed Azolla plants are subjected to purification treatment, the impurity-removed Azolla plants are cultured in soil for one week.

4. The method for inducing sporulation of Azolla according to claim 1, characterized in that: The gibberellin solution is obtained by diluting the gibberellin mother solution, wherein the preparation process of the gibberellin mother solution comprises: Mix gibberellin crystals and anhydrous ethanol in proportion, and stir until they are completely dissolved to obtain an alcohol solution containing gibberellin; Deionized water is added to the alcohol solution containing gibberellin, and the mixture is stirred evenly to obtain a gibberellin mother solution; wherein the concentration of the gibberellin mother solution is 500 μg·mL -1 .

5. The method for inducing sporulation of Azolla according to claim 1, characterized in that: The treatment concentration of the gibberellin solution is 1-7 μg·mL -1 .

6. The method for inducing sporulation of Azolla according to claim 5, characterized in that: The treatment concentration of the gibberellin solution is 7 μg·mL -1 .

7. The method for inducing sporulation of Azolla according to claim 1, characterized in that: The gibberellin solution is a GA3 solution.

8. The method for inducing sporulation of Azolla according to any one of claims 1 to 7, characterized in that: The method comprises spraying the to-be-cultivated duckweed with a gibberellin solution according to a preset cycle, and stopping the spraying when the to-be-cultivated duckweed forms spores, to obtain the cultivated duckweed, comprising: The green duckweed to be cultured is placed in a culture pot, the bottom of the pot is covered with sterilized paddy soil, and filled with clean water; Under the preset culture conditions, the azolla to be cultured is sprayed with the gibberellin solution, wherein the frequency of spraying is once a week; When the Azolla to be cultivated forms spores, spraying is stopped to obtain cultivated Azolla.

9. The method for inducing sporulation of Azolla according to claim 8, characterized in that: The culture conditions include: The temperature is 20-25℃ and the air humidity is 85-90%.

10. The method for inducing sporulation of Azolla according to claim 1, characterized in that: The green duckweed is any one of the duckweed, duckweed and small-leaved duckweed.

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