Angelica sinensis plant tissue regeneration culture method

Abstract

The invention discloses a plant tissue regeneration culture method of angelica sinensis. The method comprises the following steps: obtaining disinfected angelica sinensis seeds, carrying out germination culture on the disinfected angelica sinensis seeds on an induction culture medium to obtain tissue culture seedlings, obtaining hypocotyl of the tissue culture seedlings as explants, then carrying out callus induction culture on the explants on a callus culture medium to obtain calluses, and carrying out tissue culture on the calluses. Performing adventitious bud induction culture on the callus on a subculture medium to obtain adventitious buds, and performing rooting induction culture on the adventitious buds on a rooting culture medium to obtain a complete plant; the angelica sinensis seedlings cultivated by the method are high in survival rate and are not limited by seasons and regions, namely, a large batch of angelica sinensis seedlings can be cultivated, and the method is easy to operate, low in production cost and free of environmental pollution.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a plant tissue regeneration and cultivation method for angelica sinensis. Background Art

[0002] Angelica sinensis (Oliv.) Diels is a perennial herbaceous plant of the Apiaceae family, mainly produced in southeastern Gansu, with Minxian producing the most and producing the best quality, followed by Yunnan, Sichuan, Shaanxi and other provinces. Its dried root is a commonly used Chinese herbal medicine, which has the effects of nourishing blood, regulating menstruation and relieving pain, moistening the intestines, and improving immunity. Angelica sinensis can be used not only for treating diseases, but also for health care and beauty. It is also one of the main medicinal plants for China's export and foreign exchange earnings, and its demand is increasing year by year.

[0003] At present, angelica mainly adopts traditional propagation methods for breeding or seedling raising, but the traditional propagation method has a low propagation coefficient and the seedlings grow slowly, especially in the early growth stage. The seedlings are more sensitive to environmental conditions and are easily attacked by pests and diseases as well as the influence of adverse climatic conditions, resulting in a high seedling loss rate in the seedling raising process, which cannot meet the needs of market development and is difficult to achieve large-scale production. Summary of the invention

[0004] The object of the present invention is to provide a plant tissue regeneration culture method for Angelica sinensis, which is not restricted by seasons and regions, has a high survival rate of the obtained seedlings, and can produce Angelica sinensis seedlings throughout the year only with corresponding laboratory facilities and technical conditions, thus meeting market demand and realizing large-scale production.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The present invention discloses a plant tissue regeneration and culture method for Angelica sinensis, the method comprising:

[0007] S1: obtaining sterilized Angelica sinensis seeds, germinating and culturing the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, and obtaining hypocotyls of the tissue culture seedlings as explants;

[0008] S2: performing callus induction culture on the explant on a callus culture medium to obtain callus tissue;

[0009] S3: performing adventitious bud induction culture on the callus on a subculture medium to obtain adventitious buds;

[0010] S4: performing root induction culture on the adventitious buds on a rooting medium to obtain a complete plant.

[0011] In a possible implementation manner, before obtaining the sterilized Angelica sinensis seeds, the process further includes obtaining Angelica sinensis seeds and subjecting the Angelica sinensis seeds to a germination treatment.

[0012] In a possible implementation manner, the germination treatment includes: soaking the angelica seeds at a preset temperature for a preset time, wherein the preset temperature is 25 to 35° C., and the preset time is 12 to 18 hours.

[0013] In a possible implementation manner, the induction medium in step S1 includes MS medium, agar and sucrose.

[0014] In a possible implementation manner, the callus culture medium in step S2 includes a first basal culture medium and a first plant growth regulator, and the first plant growth regulator is 6-benzylaminopurine and / or naphthylacetic acid.

[0015] In a possible implementation manner, the callus culture medium satisfies at least one of the following conditions:

[0016] The first basal culture medium includes MS culture medium;

[0017] In the callus culture medium, the first plant growth regulator is 6-benzylaminopurine and naphthylacetic acid;

[0018] In the callus culture medium, the concentration of 6-benzylaminopurine is 3-4 mg / L;

[0019] In the callus culture medium, the concentration of naphthylacetic acid is 0.4-0.6 mg / L.

[0020] In a possible implementation, the subculture culture medium in step S3 includes a second basal culture medium and a second plant growth regulator, and the second plant growth regulator includes at least one of 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid.

[0021] In a possible embodiment, the subculture medium satisfies at least one of the following characteristics:

[0022] The second basic culture medium includes 2 / 3MS culture medium, wherein the 2 / 3MS culture medium refers to a culture medium obtained by reducing the macroelements in the MS culture medium to 2 / 3 of the original ones;

[0023] The second plant growth regulator is 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid;

[0024] In the subculture medium, the concentration of 6-benzylaminopurine is 0.4-0.6 mg / L;

[0025] In the subculture medium, the concentration of naphthylacetic acid is 0.05-0.15 mg / L;

[0026] In the subculture culture medium, the concentration of indolebutyric acid is 0.1-0.3 mg / L.

[0027] In a possible implementation manner, the rooting medium in step S4 includes a third basal medium and a third plant growth regulator, and the third plant growth regulator includes at least one of 6-benzylaminopurine, indolebutyric acid and activated carbon.

[0028] In a possible implementation manner, the rooting medium satisfies at least one of the following characteristics:

[0029] The third basic culture medium includes 1 / 2MS culture medium, wherein the 1 / 2MS culture medium refers to a culture medium obtained by reducing the macroelements in the MS culture medium by half;

[0030] In the rooting medium, the third plant growth regulator is 6-benzylaminopurine, indolebutyric acid and activated carbon;

[0031] In the rooting medium, the concentration of 6-benzylaminopurine is 0.3-0.5 mg / L;

[0032] In the rooting medium, the concentration of indolebutyric acid is 0.1-0.2 mg / L;

[0033] In the rooting medium, the concentration of the activated carbon is 0.4-0.6 g / L.

[0034] In a possible implementation manner, during the cultivation process of steps S1-S4, light cultivation is required, and the light cultivation satisfies at least one of the following characteristics:

[0035] The illumination duration during the illumination culture process is 12 to 16 hours;

[0036] The light intensity during the light culture process is 3000-5000 lx;

[0037] The temperature during the light culture process is 20-26°C.

[0038] In a possible implementation manner, during the culturing process of steps S1-S2, dark culturing is required first and then light culturing, and the method includes at least one of the following features:

[0039] During the cultivation process of steps S1-S2, the dark cultivation time is 5 to 7 days;

[0040] During the cultivation process of steps S1-S2, the temperature used for dark cultivation is 20-26°C;

[0041] The pH value of the culture medium in steps S1-S4 is maintained at 5-6.

[0042] In a possible implementation manner, the obtaining of the sterilized Angelica sinensis seeds comprises: obtaining Angelica sinensis seeds and sterilizing the Angelica sinensis seeds to obtain sterilized Angelica sinensis seeds, wherein during the sterilization of the Angelica sinensis seeds, the Angelica sinensis seeds need to be soaked in a sterilization solution for a preset time, and the method satisfies at least one of the following characteristics:

[0043] The preset soaking time of the angelica seeds is 8 to 16 minutes;

[0044] The concentration of the sterilization solution is 2-4%;

[0045] The sterilization solution is a sodium hypochlorite solution.

[0046] Based on the above technical scheme, the present application has the following beneficial effects: the technical scheme of the present invention provides a plant tissue regeneration culture method of Angelica sinensis, the method comprising: obtaining disinfected Angelica sinensis seeds, germinating and culturing the disinfected Angelica sinensis seeds on an induction culture medium to obtain tissue culture seedlings, obtaining the hypocotyls of the tissue culture seedlings as explants, and then performing callus induction culture on the explants on a callus culture medium to obtain callus tissue, and then performing adventitious bud induction culture on the callus tissue on a subculture culture medium to obtain adventitious buds, and finally performing rooting induction culture on the adventitious buds on a rooting culture medium to obtain complete plants; the Angelica sinensis seedlings cultivated by the method have a high survival rate and are not restricted by seasons and regions, that is, a large number of Angelica sinensis seedlings can be cultivated, and the method is easy to operate, has a low production cost, does not pollute the environment, and a stable and efficient tissue regeneration culture system is established by the method, which can realize large-scale production, and also provides a flexible technical platform for variety improvement and germplasm innovation of Angelica sinensis, and can easily introduce advanced technical means such as modern genetic engineering to cultivate new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the embodiments, wherein the same components are represented by the same reference numerals. Obviously, the drawings described below are only some embodiments of the present invention, and it is possible for ordinary technicians in this field to obtain other drawings based on these drawings without creative work.

[0048] Figure 1 The present invention provides a schematic flow chart of a method for plant tissue regeneration and cultivation of Angelica sinensis.

[0049] Figure 2This is the growth process of the Angelica sinensis seeds of the 14th experimental group in Example 1 of the present invention after being inoculated into the induction medium, wherein, sequence number 1 is the Angelica sinensis inoculation period, sequence number 2 is the Angelica sinensis first true leaf growth period, and sequence number 3 is the Angelica sinensis growth period.

[0050] Figure 3 This is the callus formation process of the first experimental group in Example 2 of the present invention, wherein Figure 3 A is the callus inoculation period, Figure 3 B is the stage of callus expansion and differentiation into seedlings.

[0051] Figure 4 This is the differentiation and proliferation process of the adventitious buds of the first experimental group in Example 3 of the present invention, wherein sequence number 1 is the Angelica inoculation period, sequence number 2 is the Angelica proliferation period, and sequence number 3 is the Angelica growth period.

[0052] Figure 5 This is the process of rooting and transplanting Angelica sinensis tissue culture seedlings into the substrate in the second experimental group in Example 4 of the present invention, wherein Figure 5 A is the complete plant, Figure 5 B is the plant transplanted into the substrate.

[0053] Figure 6 This is a plant growth diagram of the plants of the second experimental group in Example 4 of the present invention after being transplanted into the substrate and grown for 3 months. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and therefore cannot be understood as limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0055] It should be noted that the terms "first", "second", etc. in the specification, claims and drawings of the present invention are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention can be implemented in an order other than the following diagrams or the following descriptions. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0056] This embodiment provides a plant tissue regeneration culture method of Angelica sinensis. Figure 1 As shown, the method includes:

[0057] S1: obtaining sterilized Angelica sinensis seeds, germinating and culturing the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, and obtaining hypocotyls of the tissue culture seedlings as explants;

[0058] In some embodiments, obtaining the sterilized Angelica sinensis seeds includes: obtaining Angelica sinensis seeds and sterilizing the Angelica sinensis seeds to obtain sterilized Angelica sinensis seeds. By soaking the seeds in a sterilizing solution for sterilization, bacteria and fungi on the surface of the seeds can be killed and inhibited, thereby preventing the cultivated seedlings from developing diseases and thus reducing the survival rate of the seedlings.

[0059] Specifically, the disinfection treatment includes: soaking the angelica seeds in a sterilization solution for a preset time to obtain disinfected angelica seeds.

[0060] Specifically, after soaking the angelica seeds in the sterilization solution for a preset time, sterile water is used to wash the angelica seeds to obtain sterilized angelica seeds. Furthermore, the washing times are 2-3 times.

[0061] In some embodiments, before disinfecting the angelica seeds, ethanol is used to pre-disinfect the angelica seeds to obtain pre-disinfected angelica seeds. During the pre-disinfection process of the angelica seeds, the angelica seeds need to be soaked in ethanol for a preset time, the concentration of ethanol is 75%, and the preset time is 30 seconds.

[0062] Specifically, during the pre-disinfection process of the angelica seeds, after the angelica seeds are soaked in ethanol for a preset time, sterile water is used to wash the angelica seeds to obtain pre-disinfected angelica seeds. Furthermore, the washing times are 2-3 times.

[0063] In some embodiments, the preset time for soaking the angelica seeds is 8 to 16 minutes. The preset time can be any point value within the above range, which is not enumerated here. If the soaking time is too long, the contamination rate of the angelica seeds will be reduced, but the germination rate of the seeds will be lower, while if the soaking time is too short, it will not have a sterilization effect. Therefore, the above time is limited so that the seeds can be completely sterilized and have a higher germination rate. Preferably, the preset time for soaking the angelica seeds is 16 minutes.

[0064] In some embodiments, the concentration of the sterilizing solution is 2-4%. The concentration of the sterilizing solution can be any point value within the above range, which is not enumerated here. By limiting the concentration of the sterilizing solution, the problem of low seed germination rate caused by too high concentration of the sterilizing solution is avoided, and the problem of high seed contamination rate caused by too low concentration of the sterilizing solution is also avoided. Preferably, the concentration of the sterilizing solution is 4%.

[0065] In some embodiments, the sterilization solution is a sodium hypochlorite solution. The present invention uses a sodium hypochlorite solution as a sterilization solution, which is environmentally friendly and non-toxic to the human body. Compared with using mercuric chloride for disinfection, the toxicity rate to seeds is lower.

[0066] In some embodiments, before obtaining the sterilized angelica seeds, the step further includes obtaining the angelica seeds and accelerating the germination of the angelica seeds. Accelerating the germination of the angelica seeds can improve the vitality of the seeds and promote their germination.

[0067] Specifically, the germination treatment includes: soaking the angelica seeds at a preset temperature for a preset time, the preset temperature is 25 to 35°C, and the preset time is 12 to 18 hours. The preset temperature and the preset time can be any point value within the above range, which are not enumerated here. Preferably, the preset temperature is 30°C and the preset time is 16 hours. If the preset temperature is too high or too low, it will affect the respiration of the seeds and the conversion of nutrients, thereby affecting the germination rate. The appropriate preset time can help the seeds fully absorb water, break the dormancy state, and further improve the germination rate of the seeds. Therefore, the appropriate preset time and preset temperature range can improve the germination rate of the seeds.

[0068] In some embodiments, the induction medium in step S1 includes MS medium, agar and sucrose. The induction medium of the present application is mainly MS medium, and tissue culture seedlings can be induced without adding any plant growth regulators, and the germination rate is as high as 98%, which significantly reduces the cost.

[0069] Specifically, in the induction medium, the concentration of agar is 5 to 7 g / L. Preferably, the concentration of agar is 6 g / L.

[0070] Specifically, in the induction medium, the concentration of sucrose is 25-35 g / L. Preferably, the concentration of sucrose is 30 g / L.

[0071] S2: Callus induction culture of the explant on the callus medium to obtain callus tissue. The main purpose of inoculating the explant on the callus medium is to form callus tissue, so that the cells in the hypocotyl can divide and differentiate, and then produce more buds, that is, one hypocotyl can form multiple plants, and the yield of the obtained seedlings is higher.

[0072] In some embodiments, the callus culture medium includes a first basal culture medium and a first plant growth regulator, and the first plant growth regulator is 6-benzylaminopurine and / or naphthylacetic acid. 6-benzylaminopurine can promote cell division and induce the occurrence of callus, and naphthylacetic acid can also promote cell division and growth, thereby promoting the formation and growth of callus. The first plant growth regulator is 6-benzylaminopurine and / or naphthylacetic acid, which can induce the formation, growth and proliferation of callus, thereby producing more buds, and the yield of the obtained seedlings is higher.

[0073] In some embodiments, the first basal culture medium comprises MS medium.

[0074] Specifically, the first basic culture medium also includes agar at a concentration of 6 g / L and sucrose at a concentration of 30 g / L.

[0075] In some embodiments, in the callus culture medium, the first plant growth regulator is 6-benzylaminopurine and naphthylacetic acid. Under the synergistic effect of 6-benzylaminopurine and naphthylacetic acid, the formation and growth of callus tissue can be further stimulated, avoiding the occurrence of callus tissue vitrification or browning phenomenon that may be caused by the lack of one of the hormones.

[0076] In some embodiments, in the callus culture medium, the concentration of 6-benzylaminopurine is 3 to 4 mg / L. The concentration of 6-benzylaminopurine can be any point value within the above range, which is not enumerated here. By limiting the concentration of 6-benzylaminopurine, it is avoided that the concentration of 6-benzylaminopurine is too high, resulting in too many cell divisions and difficulty in growth, and the formed callus tissue may show browning or vitrification. At the same time, it is avoided that the concentration of 6-benzylaminopurine is too low, resulting in a decrease in induction rate. Preferably, the concentration of 6-benzylaminopurine in the callus culture medium is 3 mg / L.

[0077] In some embodiments, in the callus culture medium, the concentration of naphthaleneacetic acid is 0.4-0.6 mg / L. The concentration of naphthaleneacetic acid can be any point value within the above range, which is not enumerated here. By limiting the concentration range of naphthaleneacetic acid, the problem of reduced induction rate caused by too low concentration is avoided, and the problem of easy browning of callus tissue caused by too high concentration is avoided. Preferably, the concentration of naphthaleneacetic acid is 0.5 mg / L.

[0078] S3: The callus tissue is cultured on a subculture medium for adventitious bud induction to obtain adventitious buds.

[0079] In some embodiments, the subculture medium includes a second basal medium and a second plant growth regulator, and the second plant growth regulator includes at least one of 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid. Adventitious bud induction culture using the above-mentioned second plant growth regulator can promote the transformation of callus tissue into buds, improve differentiation efficiency, and also increase the proliferation multiple, avoiding the occurrence of vitrification and browning.

[0080] In some embodiments, the second basal culture medium includes 2 / 3MS culture medium, which refers to a culture medium obtained by reducing the macroelements in the MS culture medium to 2 / 3 of the original. In the adventitious bud differentiation stage, excessive macroelements in the MS culture medium will cause the leaves of the grown seedlings to turn yellow, and will also inhibit the differentiation of callus tissue into buds and reduce the germination rate; while too few macroelements in the MS culture medium will cause the leaves of the grown seedlings to be small, the growth rate to be slow, and may also be accompanied by the occurrence of vitrification and browning.

[0081] Specifically, the second basic culture medium also includes agar and sucrose, wherein the concentration of agar is 6 g / L, and the concentration of sucrose is 30 g / L.

[0082] In some embodiments, the second plant growth regulator is 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid. Under the synergistic effect of 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid, cell division and growth are further promoted, proliferation multiples are increased, and differentiation of callus into buds is promoted, avoiding the occurrence of vitrification and browning.

[0083] In some embodiments, in the subculture medium, the concentration of 6-benzylaminopurine is 0.4-0.6 mg / L. The concentration of 6-benzylaminopurine can be any point value within the above range, which is not enumerated here. Preferably, the concentration of 6-benzylaminopurine is 0.5 mg / L.

[0084] In some embodiments, in the subculture medium, the concentration of naphthaleneacetic acid is 0.05-0.15 mg / L. The concentration of naphthaleneacetic acid can be any point value within the above range, which is not enumerated here. Preferably, the concentration of naphthaleneacetic acid is 0.1 mg / L.

[0085] In some embodiments, in the subculture medium, the concentration of indolebutyric acid is 0.1 to 0.3 mg / L. The concentration of indolebutyric acid can be any point value within the above range, which is not enumerated here. Preferably, the concentration of indolebutyric acid is 0.2 mg / L. By limiting the concentration of the above-mentioned plant growth regulator types, it is avoided that the concentration of 6-benzylaminopurine is too high and inhibits the differentiation of buds. At the same time, the concentration of naphthaleneacetic acid is limited to be lower than the concentration of 6-benzylaminopurine. At this time, it is more conducive to the differentiation of buds, and the growth of the grown seedlings is better, while avoiding the problem that too high indolebutyric acid may reduce the germination rate.

[0086] S4: The adventitious buds are induced to root on a rooting medium to obtain complete plants.

[0087] In some embodiments, the rooting medium includes a third basic medium and a third plant growth regulator, and the third plant growth regulator includes at least one of 6-benzylaminopurine, indolebutyric acid and activated carbon. Indolebutyric acid can promote the division and elongation of plant cells, which is helpful for the root growth of adventitious buds. At the same time, during the rooting culture, the addition of activated carbon can adjust the hormone content in the culture medium to keep it at an appropriate level, thereby further promoting the rooting of adventitious buds, and the addition of 6-benzylaminopurine further stimulates the growth and division of plant root cells, thereby increasing the number and growth rate of the root system, so that the rooting rate is increased to 89%.

[0088] In some embodiments, the third basal medium includes 1 / 2MS medium, which refers to a medium obtained by reducing the macroelements in the MS medium by half. The MS medium with the macroelements reduced by half is used as the basal medium to avoid excessive salt concentration in the medium inhibiting the growth and development of the root system.

[0089] In some embodiments, in the rooting medium, the third plant growth regulator is 6-benzylaminopurine, indolebutyric acid and activated carbon. Under the synergistic effect of 6-benzylaminopurine, indolebutyric acid and activated carbon, the root growth of adventitious buds is facilitated, and the rooting rate of the plant is significantly improved.

[0090] In some embodiments, in the rooting medium, the concentration of 6-benzylaminopurine is 0.3-0.5 mg / L. The concentration of 6-benzylaminopurine can be any point value within the above range, which is not enumerated here. Preferably, in the rooting medium, the concentration of 6-benzylaminopurine is 0.3 mg / L.

[0091] In some embodiments, in the rooting medium, the concentration of indolebutyric acid is 0.1-0.2 mg / L. The concentration of indolebutyric acid can be any point value within the above range, which is not enumerated here. Preferably, in the rooting medium, the concentration of indolebutyric acid is 0.2 mg / L.

[0092] In some embodiments, in the rooting medium, the concentration of activated carbon is 0.4 to 0.6 g / L. The concentration of activated carbon can be any point value within the above range, which is not enumerated here. Preferably, in the rooting medium, the concentration of activated carbon is 0.5 g / L. By limiting the concentrations of the above-mentioned 6-benzylaminopurine, indolebutyric acid and activated carbon, cell division, enlargement and elongation can be promoted, which helps the division and differentiation of root cells, thereby promoting rooting, and indolebutyric acid can increase the speed and quantity of rooting to a certain extent.

[0093] In some embodiments, during the culturing process of steps S1 to S4, light culturing is required.

[0094] In some embodiments, the illumination duration during the illumination culture process is 12 to 16 hours. The illumination duration can be any point value within the above range, which is not enumerated here. Preferably, the illumination duration during the illumination culture process is 13.5 to 14.5 hours.

[0095] In some embodiments, the light intensity during the light culture process is 3000-5000 lx. The light intensity can be any point value within the above range, which is not enumerated here. The use of appropriate light duration and intensity promotes the growth and differentiation of plants, improves the reproduction rate and quality of plants, and avoids excessive light intensity or excessive light duration, which may cause photoinhibition and photooxidation in plant tissues, thereby affecting the growth and development of plants.

[0096] In some embodiments, the temperature during the light culture process is 20-26°C. The temperature can be any point value within the above range, which is not enumerated here. Preferably, the temperature during the light culture process is 22-23°C. The use of a suitable temperature can provide a suitable growth environment so that cell division and differentiation can proceed normally.

[0097] In some embodiments, during the cultivation process of steps S1-S2, dark cultivation is required first and then light cultivation. During the cultivation process, dark cultivation is performed first and then light cultivation is performed to avoid direct light exposure of the callus tissue, which is prone to browning, and can also promote the growth of the callus tissue. The method of dark cultivation first and then light cultivation is helpful to improve the germination rate of seeds.

[0098] Specifically, the dark culture time is 5 to 7 days. Preferably, the dark culture time is 6 days. The number of days of dark culture is limited to avoid too long dark culture days which may lead to slow callus growth and reduce germination rate.

[0099] In some embodiments, the temperature used for dark culture is 20-26° C. The temperature can be any point value within the above range, which is not enumerated here. The temperature used for dark culture is 22-23° C. The use of a suitable temperature can provide a suitable growth environment so that cell division and differentiation can proceed normally.

[0100] In some embodiments, the pH value of the culture medium in steps S1-S4 is maintained at 5 to 6. A suitable pH value can inhibit the growth of harmful microorganisms and prevent the browning of plant tissues during growth. Preferably, the pH value of the culture medium in steps S1-S4 is maintained at 5.8.

[0101] The following describes a specific embodiment of the present application in combination with the above scheme.

[0102] Example 1: Seed induction culture.

[0103] (1) Seed germination and disinfection: 1800 high-quality and full Angelica sinensis seeds were selected as samples, and the Angelica sinensis seeds were placed in a constant temperature water bath set at a certain preset temperature and soaked for a preset time (germination time) to obtain germinated Angelica sinensis seeds; the germinated Angelica sinensis seeds were placed in an ultra-clean workbench, soaked in 75% ethanol for 30 seconds, and rinsed with sterile water 2-3 times to obtain pre-sterilized Angelica sinensis seeds; the pre-sterilized Angelica sinensis seeds were soaked in a sodium hypochlorite solution for a preset time, and rinsed with sterile water 2-3 times to obtain 1800 disinfected Angelica sinensis seeds.

[0104] (2) Induction culture: 1800 sterilized Angelica sinensis seeds were divided into 18 experimental groups and cultured in 18 50 ml culture bottles for induction culture. Each culture bottle contained 100 sterilized Angelica sinensis seeds and an induction medium. The components of the induction medium were MS medium, 30 g / L sucrose and 6 g / L agar. The pH of the medium was 5.8. Each culture bottle containing Angelica sinensis seeds was first cultured in the dark for 7 days and then transferred to light culture. After 28 days of light culture, the growth of the tissue culture seedlings of the experimental group in each culture bottle was observed, and the germination rate and contamination rate in each culture bottle were calculated to obtain the corresponding experimental results. The conditions for light culture were 14±0.5h / d of light per day, 3000-5000lx of light intensity, and the temperatures for dark culture and light culture were both 22-23°C. The composition of the induction medium for each group was the same, but the germination time, sodium hypochlorite concentration, and immersion time in the sodium hypochlorite solution (disinfection time) were different for each experimental group, see Table 1 for details.

[0105] The calculation method of germination rate: germinated angelica seeds / total amount of inoculated angelica seeds;

[0106] When the inoculated angelica seeds are infected by fungi or bacteria, they are judged to be contaminated. The contamination rate is calculated as: contaminated angelica seeds / total number of inoculated angelica seeds.

[0107] (3) Experimental results: As shown in Table 1, as the concentration of sodium hypochlorite solution decreases, the contamination rate of Angelica sinensis seeds gradually increases. However, a long disinfection time will result in a low germination rate. Therefore, in order to achieve a disinfection effect while ensuring a certain germination rate, a 4% concentration of sodium hypochlorite and a soaking time of 16 min were selected. The germination time will affect the germination rate of the seeds. 16 h was selected as the optimal germination time. In addition, Figure 2 As shown, Figure 2This is the induction process of the tissue culture seedlings of the 14th experimental group, where serial number 1 is the inoculation period of Angelica sinensis, serial number 2 is the period when Angelica sinensis grows its first true leaf, and serial number 3 is the growth period of Angelica sinensis. It can be seen that the seeds after disinfection began to germinate and grow, and the seedlings were strong, the leaves were green, and the roots were white, straight and smooth.

[0108] Table 1: Effects of different germination times, sodium hypochlorite concentrations, and immersion time in sodium hypochlorite solution on seed germination rate.

[0109]

[0110]

[0111] Example 2: Selection of callus culture medium

[0112] (1) Experimental method: The tissue culture seedlings obtained under the culture conditions in the 14th experimental group were used as samples, and the hypocotyls of the samples were taken as explants. Three experimental groups were set up, with 120 hypocotyls in the first experimental group, 92 hypocotyls in the second experimental group, and 105 hypocotyls in the third experimental group. The hypocotyls in the three experimental groups were inoculated into callus culture medium, respectively, and cultured in the dark for 7 days, and then transferred to light culture. After 28 days of culture, the induction rate of each experimental group was calculated to obtain the corresponding experimental results. The conditions of light culture were 14±0.5h / d, light intensity was 3000-5000lx, the temperature of dark culture and light culture was 22-23°C, the components of callus culture medium of each group were MS medium + 30g / L sucrose + 6g / L agar, 6-benzylpurine and 0.5mg / L naphthaleneacetic acid, but the concentration of 6-benzylpurine in the callus culture medium of each experimental group was different, the concentration of 6-benzylpurine in the first experimental group was 3mg / L, the concentration of 6-benzylpurine in the second experimental group was 5mg / L, and the concentration of 6-benzylpurine in the third experimental group was 2mg / L, and the experimental results were shown in Table 2. The pH value in the callus culture medium was 5.8.

[0113] Induction rate (%) = number of hypocotyls producing callus / total number of inoculated hypocotyls × 100%

[0114] (2) Experimental results: As shown in Table 2, the callus culture medium of each experimental group was able to induce callus tissue. When the concentration of 6-benzylpurine was 2 mg / L, 3 mg / L to 5 mg / L, the callus induction rates of Angelica sinensis were 37.14%, 69.16% and 45.65%, respectively. It can be seen that when the concentration of 6-benzylpurine was high or 6-BA was low, it was not conducive to the formation of callus tissue. Therefore, the callus culture effect of the callus culture medium obtained when the concentration of 6-benzylpurine was 3 mg / L was better. Figure 3 The formation process of callus tissue in the first experimental group. Figure 3 It can be seen that with the extension of culture time, the callus tissue grows and expands rapidly, among which, Figure 3 A is the callus inoculation period, Figure 3 B is the stage of callus expansion and differentiation into seedlings.

[0115] Table 2: Effects of different first plant growth regulator concentration combinations on callus induction

[0116]

[0117] Example 3: Selection of subculture medium

[0118] (1) Experimental method: The callus tissue obtained under the culture conditions in Example 2 was used as a sample and divided into three experimental groups. The number of callus tissues in each experimental group was 60. The experiment was repeated 3 times. The samples were inoculated into a subculture medium with a pH of 5.8. After 28 days of light culture under the conditions of a temperature of 22-23°C, a light duration of 14±0.5h / d and a light intensity of 3000-5000lx, the growth potential of the adventitious buds in each experimental group was observed, and the proliferation multiples were calculated to obtain the experimental results. The components of the subculture medium of the three experimental groups all included 30g / L sucrose + 6g / L agar + 0.5mg / L 6-benzylpurine + 0.1mg / L naphthaleneacetic acid + 0.2mg / L indolebutyric acid. The difference was that the first experimental group also included 2 / 3MS culture medium, the second experimental group also included MS culture medium, and the third experimental group also included 1 / 2MS culture medium. The calculation formula for the proliferation multiples: Proliferation multiples = number of adventitious buds / total amount of callus tissue

[0119] (2) Experimental results: As shown in Table 3, the higher or lower the concentration of macroelements in the subculture medium, the lower or slower the germination rate of adventitious buds, the lower the proliferation multiples, and the vitrification or browning phenomenon will occur. Therefore, it is best to select 2 / 3MS medium as the basic medium of the subculture medium. Figure 4 As shown, the leaves of the adventitious buds obtained in the first experimental group were dark green and grew well, wherein serial number 1 was the angelica inoculation period, serial number 2 was the angelica proliferation period, and serial number 3 was the angelica growth period.

[0120] Table 3 Effects of different subculture media on subculture proliferation of Angelica sinensis adventitious buds

[0121]

[0122]

[0123] Note: The results of ANOVA in the table are represented by letters, and different capital letters indicate extremely significant differences (P<0.01).

[0124] Example 4: Selection of rooting medium

[0125] (1) Experimental method: The adventitious buds obtained under the culture conditions in Example 3 were used as samples and divided into three experimental groups. The number of samples in each experimental group was 100. The samples were inoculated into a rooting medium with a pH of 5.8 and cultured under the conditions of a temperature of 22-23°C, a light duration of 14±0.5 h / d, and a light intensity of 3000-5000 lx. After 28 days, the growth of the plants was observed and the rooting rate was calculated. The components of the rooting medium of the first experimental group were 1 / 2MS medium + 30g / L sucrose + 6g / L agar + 0.5mg / L6-benzylpurine + 0.1mg / L indolebutyric acid + 0.5g / L activated carbon, the components of the rooting medium of the second experimental group were 1 / 2MS medium + 30g / L sucrose + 6g / L agar + 0.3mg / L6-benzylpurine + 0.2mg / L indolebutyric acid + 0.5g / L activated carbon, and the components of the rooting medium of the third experimental group were MS medium + 30g / L sucrose + 6g / L agar + 0.5mg / L6-benzylpurine + 0.1mg / L indolebutyric acid + 0.5g / L activated carbon.

[0126] The formula for calculating the rooting rate is: number of rooted adventitious buds / total number of adventitious buds × 100%

[0127] (2) Experimental results: From the results in Table 4, it can be seen that the rooting rate using 1 / 2MS medium is significantly higher than that using conventional MS medium. Taking 1 / 2MS medium as the control, the rooting rate of the second experimental group is higher than that of the first and third experimental groups, and reaches a significant level. Figure 5 The plants cultivated by the second experimental group, such as Figure 5 As shown in A, the seedlings after rooting culture are growing well, with strong and vigorous root systems. Figure 5 B is the plant after transplantation. The transplanting process is as follows: the plants obtained from the second experimental group were taken out after hardening for 3-5 days, and the culture medium remaining on the roots was washed with water. They were then soaked in a systemic fungicide aqueous solution for 1-2 minutes before transplanting. The composition of the transplanting medium was peat soil: coconut bran: perlite = 5:3:1 (volume ratio). Figure 5 B is the growth of the plant after it was transplanted into the substrate and grown for 45 days. Figure 6 This is the growth of the plants after they were transplanted into the substrate and grown for 3 months. It can be seen that the growth of the transplanted plants obtained by the cultivation method of the present application is good.

[0128] Table 4: Effects of different rooting media on rooting of adventitious buds

[0129]

[0130] It can be seen from the above embodiments that the present invention has the following beneficial effects:

[0131] The invention sets the germination time in the seed germination and disinfection process to 16 hours, limits the concentration of the sodium hypochlorite solution to 4%, and the soaking time in the sodium hypochlorite solution to 16 minutes, thereby significantly reducing the contamination rate, so that the contamination rate is maintained between 2% and 4%, and the induction medium is mainly based on MS, and sterile tissue culture seedlings can be induced without adding any plant growth regulator, and the germination rate of the seeds is increased to 98%, and then the concentration of 6-benzylpurine in the callus medium is adjusted, so that the callus medium can form good callus tissue on the hypocotyl, and the induction rate of the callus tissue is increased to 69.16. %, on this basis, further regulating the components of the subculture medium, so that the germination rate of the adventitious buds is high, and the proliferation multiple is high, the proliferation coefficient is as high as 5 or more, and then regulating the concentrations of 6-benzylpurine and indolebutyric acid in the rooting medium, significantly improving the rooting rate of the adventitious buds, the rooting rate is as high as 89%, the obtained plants grow well and have vigorous and strong root systems; the angelica seedlings cultivated by the above method have a high survival rate after transplanting, and are not restricted by seasons and regions, that is, a large number of angelica seedlings can be cultivated, which effectively solves the problem of large-scale production of angelica seedlings, and the method is easy to operate, has a low production cost, and does not pollute the environment.

[0132] What is described above are only some embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art should understand that the present invention may be subject to various changes and improvements, and any modifications, equivalent substitutions and improvements made in accordance with the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for plant tissue regeneration and culture of Angelica sinensis, characterized in that: The method comprises: S1: obtaining sterilized Angelica sinensis seeds, germinating and culturing the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, and obtaining hypocotyls of the tissue culture seedlings as explants; S2: performing callus induction culture on the explant on a callus culture medium to obtain callus tissue; S3: performing adventitious bud induction culture on the callus on a subculture medium to obtain adventitious buds; S4: performing root induction culture on the adventitious buds on a rooting medium to obtain a complete plant.

2. The method according to claim 1, characterized in that Before obtaining the sterilized angelica seeds, the method also includes obtaining angelica seeds and performing a germination treatment on the angelica seeds.

3. The method according to claim 2, characterized in that The germination treatment comprises: soaking the angelica seeds at a preset temperature for a preset time, wherein the preset temperature is 25 to 35° C. and the preset time is 12 to 18 hours.

4. The method according to claim 1, characterized in that: The induction medium in step S1 includes MS medium, agar and sucrose.

5. The method according to claim 1, characterized in that The callus culture medium in step S2 includes a first basal culture medium and a first plant growth regulator, wherein the first plant growth regulator is 6-benzylaminopurine and / or naphthylacetic acid.

6. The method according to claim 5, characterized in that The callus culture medium at least satisfies at least one of the following conditions: The first basal culture medium includes MS culture medium; In the callus culture medium, the first plant growth regulator is 6-benzylaminopurine and naphthylacetic acid; In the callus culture medium, the concentration of 6-benzylaminopurine is 3-4 mg / L; In the callus culture medium, the concentration of naphthylacetic acid is 0.4-0.6 mg / L.

7. The method according to claim 1, characterized in that The subculture culture medium in step S3 includes a second basal culture medium and a second plant growth regulator, wherein the second plant growth regulator includes at least one of 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid.

8. The method according to claim 1, characterized in that The subculture medium satisfies at least one of the following characteristics: The second basic culture medium includes 2 / 3MS culture medium, wherein the 2 / 3MS culture medium refers to a culture medium obtained by reducing the macroelements in the MS culture medium to 2 / 3 of the original ones; The second plant growth regulator is 6-benzylaminopurine, naphthylacetic acid and indolebutyric acid; In the subculture medium, the concentration of 6-benzylaminopurine is 0.4-0.6 mg / L; In the subculture medium, the concentration of naphthylacetic acid is 0.05-0.15 mg / L; In the subculture culture medium, the concentration of indolebutyric acid is 0.1-0.3 mg / L.

9. The method according to any one of claims 1 to 8, characterized in that: The rooting medium in step S4 includes a third basal medium and a third plant growth regulator, and the third plant growth regulator includes at least one of 6-benzylaminopurine, indolebutyric acid and activated carbon.

10. The method according to claim 9, characterized in that The rooting medium meets at least one of the following characteristics: The third basic culture medium includes 1 / 2MS culture medium, wherein the 1 / 2MS culture medium refers to a culture medium obtained by reducing the macroelements in the MS culture medium by half; In the rooting medium, the third plant growth regulator is 6-benzylaminopurine, indolebutyric acid and activated carbon; In the rooting medium, the concentration of 6-benzylaminopurine is 0.3-0.5 mg / L; In the rooting medium, the concentration of indolebutyric acid is 0.1-0.2 mg / L; In the rooting medium, the concentration of the activated carbon is 0.4-0.6 g / L.

11. The method according to any one of claims 1 to 8, characterized in that: During the cultivation process of steps S1-S4, light cultivation is required, and the light cultivation meets at least one of the following characteristics: The illumination duration during the illumination culture process is 12 to 16 hours; The light intensity during the light culture process is 3000-5000 lx; The temperature during the light culture process is 20-26°C.

12. The method according to any one of claims 1 to 8, characterized in that: In the culturing process of steps S1-S2, dark culturing is required first and then light culturing. The method includes at least one of the following characteristics: During the cultivation process of steps S1-S2, the dark cultivation time is 5 to 7 days; During the cultivation process of steps S1-S2, the temperature used for dark cultivation is 20-26°C; The pH value of the culture medium in steps S1-S4 is maintained at 5-6.

13. The method according to claim 12, characterized in that The method of obtaining the sterilized angelica seeds comprises: obtaining angelica seeds and sterilizing the angelica seeds to obtain sterilized angelica seeds. During the sterilization process of the angelica seeds, the angelica seeds need to be soaked in a sterilization solution for a preset time. The method meets at least one of the following characteristics: The preset soaking time of the angelica seeds is 8 to 16 minutes; The concentration of the sterilization solution is 2-4%; The sterilization solution is a sodium hypochlorite solution.

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