A method for producing and protecting seeds of Artemisia annua

Abstract

The present invention provides a method for seed production and conservation of Artemisia annua. By controlling fruit processing, seed preparation, sugar induction and light induction of germination conditions, and seedling hardening and shading control, the key conservation technology of Artemisia annua is effectively realized. During the entire cultivation process, no drug damage, poisoning or other chemical effects are caused to the cultivation medium and seedlings. The method plays a positive role in the utilization process of Artemisia annua as an important medicinal plant, has good development, utilization and promotion and application prospects in the utilization of similar plants, and is the first of its kind in China.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for producing and protecting seeds of Artemisia annua. Background Art

[0002] Triosteum himalayanum is a perennial herbaceous plant of the genus Triosteum in the family Caprifoliaceae. It is coarsely hairy and has a thick, conical rhizome. Its stem is erect and solitary, and its leaves are opposite and sessile, fused at the base. The stem runs through the center, which is why it gets its name. Triosteum himalayanum has a terminal spike-like inflorescence with numerous flowers but low fruit set. Mature fruits contain three seeds. It is primarily found in the Qinling Mountains and southwestern mountainous regions of my country at altitudes of 3,000-4,000 meters. It typically grows on moist slopes, beneath hard-leaved broadleaf forests and cool-temperate coniferous forests, or in mountain shrubs and grasses. The entire herb is used as a medicinal herb. It has a bitter and cold nature and is used for diuresis, detumescence, and menstrual regulation. It also has a certain therapeutic effect on women's irregular menstruation and pain caused by exertion, and has good development potential and value.

[0003] Preliminary investigations revealed that while C. australis has a wide distribution, it is scattered and patchy, with no dominant populations in the wild. Wild resources are small, and changes in forest stands and human disturbances can easily lead to poor plant growth or death. Currently, there are few reports on the cultivation of C. australis in China. This is partly due to a lack of knowledge about the plant itself, and partly because C. australis is found at high altitudes, requiring demanding seed preparation and germination conditions. Consequently, there has long been no technical support for its introduction and cultivation.

[0004] Therefore, it is necessary to provide a method for seed production and conservation of the sedge, and through the study of artificial propagation methods of this resource, it is urgent to carry out conservation and restoration under mature technology. It has good development, utilization and promotion prospects for similar plants. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present invention provides a method for seed production and conservation of Artemisia annua.

[0006] The present application provides a method for seed production and conservation of Artemisia annua, comprising the following steps:

[0007] S1: collecting the mature fruits of Ribes australis and drying them until the fruit weight loss reaches 25%, and then taking them out for seed production;

[0008] S2: After rubbing the seeds of the Rhizoma Cibotii until they are separated from the pericarp and fall off, rinse the seeds with sterile water until the purity reaches over 80%. Immediately place the rinsed seeds in a sterile Petri dish lined with filter paper at an ambient temperature of 18°C. Add a sucrose solution with a molar concentration of 5‰ until the filter paper is completely soaked. Induce germination under a light condition of 800 lux for 24 hours.

[0009] S3: After the radicle of the germinated seedlings has elongated, rinse the seedlings three times with sterile water and transfer them to the surface of a sterilized substrate. Under the conditions of 2000 lux for 24 hours, the air temperature is maintained at 18°C ​​and the air humidity is maintained at 60%, and the seedling morphology is induced until true leaves grow;

[0010] S4: Transplant the true leaf seedlings to an outdoor environment with a shade degree of 50%-75%, maintain the air temperature at 12-28℃ and the air humidity at 50%-75% until the seedlings mature.

[0011] Furthermore, the drying in S1 is gradient drying; the humidity of the gradient drying is: 40%, 30%, and 20% in sequence; and the time interval of the gradient drying is 48 hours.

[0012] Furthermore, the drying in S1 is shade drying, and the ripe fruits are kept in an environment away from direct sunlight until the skin shrinks.

[0013] Furthermore, the components of the sterilized matrix in S3 include: leaf mold with a 4-mesh sieve; the moisture content of the sterilized matrix is ​​60%-70%.

[0014] Furthermore, the kneading in S2 is a one-step kneading.

[0015] Furthermore, the method further comprises:

[0016] S5: Spray methyl thiophanate solution every 20 days after planting to protect the seedlings; the methyl thiophanate solution is a 1000-fold dilution of 50% wettable powder.

[0017] Beneficial technical effects of the present invention:

[0018] 1. The method provided by the present invention is mainly aimed at the fruit processing and conservation technology of the clematis serrata. Through the gradient dehydration of the fruit, seed production, seedling establishment and determination of seedling hardening conditions, the conservation and growth of the clematis serrata are effectively guaranteed.

[0019] 2. The method provided by the present invention restricts the water restriction conditions for seed production of C. australis, provides a method for inducing germination with a low-concentration sucrose solution, limits the conditions for light-induced seedling morphogenesis, and simultaneously restricts the environmental conditions for seedling hardening. This method can improve the germination rate and seedling survival rate of C. australis seeds, providing an important guarantee for the research and development of this plant resource.

[0020] 3. The method provided by the present invention is relatively simple to operate, and does not cause phytotoxicity, poisoning or other chemical effects on the cultivation medium and seedlings. It plays a positive role in the utilization of important medicinal plants and has good prospects for development, utilization and promotion of applications. DETAILED DESCRIPTION

[0021] The following describes alternative embodiments of the present invention in more detail. Although alternative embodiments of the present invention are described, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Instead, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0022] For the sake of clarity, the invention is described in detail below through the following Example 1. Unless otherwise specified, the experimental materials and reagents used in the following Example 1 were purchased from the market.

[0023] Example 1

[0024] A method for seed production and conservation of Artemisia annua, comprising the following steps:

[0025] (1) In 2021, the red and mature fruits of Rhizoma Cibotii were collected around Yulong Snow Mountain in Lijiang City, Yunnan Province. The collected fruits were gradient dried in a constant humidity drying cabinet with controlled humidity of 40%, 30%, and 20% at intervals of 48 hours. After the fruit weight loss reached 25%, they were taken out for seed production.

[0026] (2) After rubbing until the seeds of the Rhizoma Coptidis are separated from the pericarp and fall off, rinse with sterile water three times. The rinsed seeds are immediately placed in groups of 50 seeds each in a sterile culture dish covered with filter paper at an ambient temperature of 18°C. Sucrose solution with a molar concentration of 5‰ is added until the filter paper is completely soaked. Germination is induced under relatively dark full light conditions (800 lux, 24 h).

[0027] (3) After the radicle of the germinated seedlings has elongated, rinse the seedlings with sterile water three times until the seed length reaches more than 80%, and transfer them to the surface of a sterilized substrate containing leaf mold passed through a 4-mesh sieve. Maintain normal full light conditions (2000 lux, 24 h), keep the substrate volume moisture content at 60%-70%, the air temperature at 18°C, and the air humidity at 60%, and induce the seedling morphology until true leaves grow.

[0028] (4) Transplant the true leaf seedlings to an outdoor environment with a shade degree of 50%, maintain the air temperature at 12-28°C and the air humidity at 50%-75%, spray methyl thiophanate solution (1000 times solution of 50% wettable powder) every 20 days, and protect until the seedlings are established.

[0029] Example 2

[0030] According to the method for seed production and conservation of C. australis provided in Example 1, in step (1), after collecting C. australis fruits, they are placed at room temperature to dry and avoid direct sunlight until the peel shrinks and the fruit mass loss reaches 25%, and then steps (2) to (4) of Example 1 are implemented.

[0031] Comparative Example 1

[0032] According to the method for seed production and conservation of Artemisia annua provided in Example 1, after implementing step (1) of Example 1, in step (2), rubbing the seeds until the seed purity reaches more than 80%, rinsing with sterile water, and then implementing the subsequent operations of step (2) of Example 1 and steps (3) and (4).

[0033] Comparative Example 2

[0034] According to the method for producing and preserving Artemisia scoparia seeds provided in Example 1, after implementing step (1) of Example 1, in step (2), the concentrations of the sucrose solution in the control group were 5%, 1%, and 1‰, and another control group was set up with sterile water instead of the 5‰ sucrose solution, and the seed germination was observed. Thereafter, the subsequent operations of step (2) of Example 1 and steps (3) and (4) were implemented.

[0035] Comparative Example 3

[0036] According to the method for seed production and conservation of C. australis provided in Example 1, after implementing step (1) of Example 1, a dark environment (0 lux, 24 hours) and a normal light environment (1500 lux, 2000 lux, 2500 lux, 24 hours) were selected to observe the effect of light intensity on C. australis seed germination. Thereafter, steps (3) and (4) of Example 1 were implemented.

[0037] Comparative Example 4

[0038] According to the method for seed production and conservation of Artemisia annua provided in Example 1, steps (1) to (3) of Example 1 were implemented, and true leaf seedlings were transplanted to environments with shading degrees of 0%, 25%, and 75%, respectively, for seedling hardening. The growth status of the seedlings was observed after 360 days.

[0039] Table 1 Effects of different fruit treatments on the germination rate of Rhizoma Cibotii seeds

[0040] Case Fruit processing methods Germination rate (%) Example 1 Gradient drying <![CDATA[73.33±5.03 a ]]> Example 2 Directly dry in the shade <![CDATA[54.67±3.06 b ]]>

[0041] Table 1 shows that the germination rate of fruits directly dried in the shade decreased after seed production and subsequent germination. This suggests that, if conditions permit, gradient drying can be beneficial for seed germination. If conditions are truly impractical, direct drying at room temperature is also acceptable, but the germination rate will be significantly reduced.

[0042] Table 2 Effects of different seed production methods on the germination rate of Rhizoma Cibotii seeds

[0043] Case Seed treatment method Germination rate (%) Example 1 One-step rubbing followed by rinsing <![CDATA[73.33±5.03 a ]]> Comparative Example 1 Rub repeatedly <![CDATA[31.33±4.16 b ]]>

[0044] As shown in Table 2, repeated kneading significantly reduced seed germination rates. Therefore, during the seed production process for C. australis, attention should be paid to the integrity of the seed coat. While kneading once followed by multiple rinsing is acceptable, repeated kneading damages the seed coat, preventing germination. This is because the seed coat of C. australis is covered with a thin waxy layer, which can be removed by rinsing after a single kneading step. Repeated kneading, however, can damage the seed coat and, consequently, the embryo structure, preventing germination.

[0045] Table 3 Effects of different concentrations of sucrose solution on the germination rate of Codonopsis pilosula seeds

[0046]

[0047] Table 3 shows that the addition of a sucrose solution at a molar concentration of 5‰ during the germination of C. australis seeds can stimulate seed germination. Sugar, as an intracellular signaling substance, has a certain stimulating effect on seed germination. However, excessive concentrations can alter cell membrane permeability and inhibit seed germination. A 5‰ sucrose solution can stimulate C. australis seed germination and is therefore suitable for widespread use in practical applications.

[0048] Table 4 Effects of different light induction intensities on the germination rate of Rhizoma Coptidis seeds

[0049]

[0050] As can be seen from Table 4, during the germination process of C. australis seeds, the germination rate is regulated by light signals. Both dark and strong light environments will significantly reduce the germination rate of seeds, which is not conducive to seed germination. Under weak light conditions (800 lux), the germination rate is the highest.

[0051] Table 5 Effects of different shading intensities on the seedling rate of Ribes australis

[0052]

[0053] It can be seen from Table 5 that although the changes in light intensity caused by shading have little effect on the seedling rate, they have a certain effect on the morphological development of C. australis seedlings. Moderate shading can promote the jointing of C. australis plants and produce more pairs of leaves, thereby improving the efficiency of photosynthesis.

[0054] In summary, the method for seed production and conservation of the Artemisia annua provided by the present invention is relatively simple to operate. By controlling fruit processing, seed preparation, germination conditions of sugar induction and light induction, and seedling hardening and shading control, the key technology for conservation of the Artemisia annua is effectively realized. During the entire cultivation process, no drug damage, poisoning or other chemical effects are caused to the cultivation medium and seedlings. The method plays a positive role in the utilization of the Artemisia annua as an important medicinal plant, has good development, utilization and promotion and application prospects in the utilization of similar plants, and is the first of its kind in China.

[0055] The embodiments of the present invention have been described above. The above description is illustrative and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for seed production and conservation of Artemisia annua, characterized in that: The method comprises the following steps: S1: collecting the mature fruits of Ribes australis and drying them until the fruit weight loss reaches 25%, and then taking them out for seed production; The drying is gradient drying; the humidity of the gradient drying is: 40%, 30%, 20%; the time interval of the gradient drying is 48 hours; S2: After kneading the seeds of the Rhizoma Cibotii fruit until they are separated from the pericarp and fall off, the seeds are rinsed with sterile water until the purity reaches over 80%. The rinsed seeds are immediately placed in a sterile Petri dish lined with filter paper at an ambient temperature of 18°C. A sucrose solution with a molar concentration of 5‰ is added until the filter paper is completely soaked. Germination is induced under a light condition of 800 lux for 24 hours. S3: After the radicle of the germinated seedlings has elongated, rinse the seedlings three times with sterile water and transfer them to the surface of a sterilized substrate. Under the conditions of 2000 lux for 24 hours, the air temperature is maintained at 18°C ​​and the air humidity is maintained at 60%, and the seedling morphology is induced until true leaves grow; S4: Transplant the true leaf seedlings to an outdoor environment with a shade degree of 50%-75%, maintain the air temperature at 12-28℃ and the air humidity at 50%-75% until the seedlings mature.

2. The method according to claim 1, characterized in that The drying in S1 is shade drying, and the ripe fruits are dried in an environment away from direct sunlight until the skin shrinks.

3. The method according to claim 1, characterized in that The components of the sterilized matrix in S3 include: leaf mold with a 4-mesh sieve; the moisture content of the sterilized matrix is ​​60%-70%.

4. The method according to claim 1, wherein The method further comprises: S5: Spray methyl thiophanate solution every 20 days after planting to protect the seedlings; the methyl thiophanate solution is a 1000-fold dilution of 50% wettable powder.

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