A kind of stevia tissue culture method

Abstract

The present application relates to the technical field of stevia cultivation, and specifically discloses a stevia tissue culture method. The method comprises the following steps: I. obtaining stevia adventitious buds; II. polyploid induction; III. rooting culture; IV. hardening and transplanting to obtain stevia plants; the method for polyploid induction comprises the following steps: soaking the stevia adventitious buds in a cyanoacrylate solution containing an oxidant, washing with a reducing agent solution, and washing with water; the cyanoacrylate content in the cyanoacrylate solution is 10-15 mg / L. The method of the present application has the advantages of high induction rate of stevia adventitious buds and low mortality.

Description

Technical Field

[0001] The present application relates to the technical field of stevia cultivation, and more specifically, to a stevia tissue culture method. Background Art

[0002] Stevia plays a very important role in the fields of food and pharmaceuticals, mainly because it is rich in active ingredients, such as glycosides and flavonoids. The sweetness of steviol glycosides is about 200-300 times that of sucrose, but the calories are only 1 / 300 of sucrose.

[0003] At present, tissue culture of stevia includes the step of polyploidy mutation breeding. The content of steviol glycosides, plant stress resistance and growth of stevia obtained by mutation breeding are significantly improved. The methods of artificially inducing polyploidy include physical mutagenesis, chemical mutagenesis and biological mutagenesis. Among them, chemical mutagenesis is widely used because of its advantages of stable mutagenesis direction and high efficiency.

[0004] The commonly used chemical mutagen is colchicine, which mainly achieves polyploid mutation breeding by destroying cell mitosis. However, when colchicine is used as a mutagen, there are the disadvantages of high plant mortality and expensive reagents; for example, the induction rate of stevia adventitious buds soaked in a 150mg / L colchicine solution for 7-21 days is 13.3-20.8%, but the mortality rate of adventitious buds is as high as 39.6-77.8%. There are also related studies that use dinitroaniline herbicides (such as trifluralin and amosulfuron) as mutagens to carry out polyploid mutation breeding of stevia, which can reduce the mortality of stevia plants after mutation breeding and ensure a certain induction rate; for example, the induction rate of stevia adventitious buds soaked in a 50mg / L trifluralin solution for 12-48h is 15.7-28.1%, and the mortality rate is reduced to 27.8-56.2%.

[0005] However, despite this, the mortality rate of adventitious buds after polyploid mutation breeding is still high, so it is necessary to provide a method for stevia mutation breeding to further reduce the mortality rate of adventitious buds during polyploid mutation breeding. Summary of the invention

[0006] In order to further reduce the mortality rate of adventitious buds during polyploid mutation breeding, the present application provides a stevia tissue culture method.

[0007] The present application provides a stevia tissue culture method using the following technical solution:

[0008] A stevia tissue culture method comprises the following steps:

[0009] 1. Obtaining stevia adventitious buds;

[0010] II. Inducing polyploidy of the stevia adventitious buds to obtain surviving stevia polyploid adventitious buds;

[0011] III. performing rooting culture on the surviving polyploid adventitious buds of Stevia rebaudiana to obtain Stevia rebaudiana tissue culture seedlings;

[0012] IV, hardening and transplanting the stevia tissue culture seedlings to obtain stevia plants;

[0013] The method for inducing polyploidy in step II comprises the following steps: soaking in an oxidant solution, soaking the stevia adventitious buds in a cyanoacrylate solution, washing with a reducing agent solution, and washing with water; the cyanoacrylate content in the cyanoacrylate solution is 10-15 mg / L.

[0014] In the related art, a large dose of treatment agent is generally added to treat adventitious buds to ensure a high induction rate, but this method also brings a high mortality rate of adventitious buds. The present application adopts the above technical scheme, mainly using cyanoacrylate as a mutagen to achieve polyploid induction. In this scheme, stevia adventitious buds are treated with a lower dose of cyanoacrylate to achieve the purpose of reducing their mortality rate. However, when only a low dose of cyanoacrylate is used to treat stevia adventitious buds, there is often a problem of low induction rate. Therefore, a low dose of cyanoacrylate is used in combination with an oxidant to treat stevia adventitious buds, so that the mortality rate of stevia adventitious buds is low and the induction rate is improved. The oxidant may act on the cells to a certain extent, so that the cell permeability is enhanced, and the activity of mitosis-related proteins and enzymes is further reduced to improve the effect of polyploid induction and the induction rate; but at the same time, compared with the cell damage of cyanoacrylate, the cell damage of the oxidant is relatively low, thereby reducing the mortality rate of stevia adventitious buds. After the stevia adventitious buds are treated by this method, they are further washed with a reducing agent to prevent the residual oxidant and cyanoacrylate from continuously damaging the cells. This method is a relatively mild chemical method for inducing polyploidy, which can take into account both the polyploidy induction rate and the adventitious bud survival rate.

[0015] Optionally, the cyanoacrylate is selected from any one or more of methyl α-cyanoacrylate and ethyl α-cyanoacrylate.

[0016] Optionally, the content of the oxidant in the oxidant solution is 0.8-1.2 wt %.

[0017] By adopting the above technical solution, polyploid induction can be achieved with the cooperation of an appropriate amount of oxidant. Too high an oxidant content will also cause greater cell damage, significantly increasing the mortality rate of stevia adventitious buds; while too little oxidant will directly affect the induction rate.

[0018] Optionally, in step II, the stevia adventitious buds are soaked in the cyanoacrylate solution for 24-48 hours.

[0019] Optionally, the oxidant is selected from any one or more of hydrogen peroxide and ozone.

[0020] By adopting the above technical solution, when the above oxidants are used in appropriate amounts to treat stevia adventitious buds, the damage to the cells is relatively small and too many other impurities, especially metal impurities, are not introduced.

[0021] Optionally, the oxidant is hydrogen peroxide.

[0022] By adopting the above technical solution, the experiment found that when hydrogen peroxide was used as the oxidant, the polyploid induction rate and survival rate of Stevia adventitious buds were better.

[0023] Optionally, the reducing agent content in the reducing agent solution is 1.5-3wt%.

[0024] Optionally, the reducing agent is selected from any one or more of astaxanthin, tea polyphenols, and tannin.

[0025] Optionally, the method for obtaining stevia adventitious buds comprises the following steps: obtaining stem segments with axillary buds and / or terminal buds from stevia plants, cutting them into pieces of 0.5-1.5 cm in size after disinfection and sterilization, absorbing moisture and placing them on an adventitious bud culture medium to culture until the height of the tissue culture seedlings is 3-4 cm; removing the leaves of the tissue culture seedlings and cutting the remaining parts into stem segments of 0.8-1.2 cm, placing them on an adventitious bud culture medium for culture, and selecting stevia adventitious buds with a length of 0.3-0.8 cm for polyploid induction.

[0026] Optionally, the components of the adventitious bud culture medium include: MS culture medium, 0.3-0.8 mg / L 6-BA and 0.15-0.75 mg / L NAA.

[0027] Optionally, the rooting culture method in step III includes the following steps: placing the surviving polyploid adventitious buds of Stevia rebaudiana on a rooting medium and culturing until the root length of the Stevia rebaudiana tissue culture seedlings is 5-7 cm.

[0028] Optionally, the culture conditions for rooting culture include: temperature of 24-26°C, humidity of 65-75%, light intensity of 1500-2000 μmol / (m 2 ·s), and the photoperiod is 10-14h / d.

[0029] Optionally, the rooting medium components include: MS medium, 0.2-0.5 mg / L IBA and 0.1-0.5 mg / L NAA.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. The present application uses a low concentration of mutagen combined with an oxidant to treat stevia adventitious buds to achieve the purpose of reducing the mortality rate of adventitious buds and ensuring the induction rate of adventitious buds; in this scheme, cyanoacrylate is specially selected as a polyploid mutagen. Compared with other mutagens, after cooperating with oxidants, it has excellent effects of increasing the induction rate and reducing the mortality rate.

[0032] 2. The cyanoacrylate content in the cyanoacrylate solution of the present application is 10-15 mg / L, and the oxidant content in the oxidant solution is 0.8-1.2 wt %, so as to achieve the purpose of better reducing the mortality rate of adventitious buds and improving the induction rate of adventitious buds.

[0033] 3. The method of the present application is to first treat the adventitious buds with an oxidant, then treat the adventitious buds with a mutagen, and finally neutralize the oxidant with a reducing agent to eliminate the continuous cell damage caused by the oxidant. This treatment sequence is necessary, and no step can be omitted, otherwise it will lead to poor tissue culture results. DETAILED DESCRIPTION

[0034] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0035] IBA refers to indolebutyric acid; NAA refers to naphthylacetic acid; 6-BA refers to 6-benzylaminopurine, a cytokinin.

[0036] Example

[0037] Example 1

[0038] A stevia tissue culture method, comprising the following specific steps:

[0039] I. Obtaining Stevia adventitious buds, the specific steps are:

[0040] Take the young stem segments with axillary buds and terminal buds from the stevia plant with scissors, remove the leaves on both sides of the stem segments, and use the remaining parts as explants for tissue culture, and then sterilize the explants. The steps of the sterilization treatment are: wash the stem segments with axillary buds and terminal buds with leaves removed with mild running water, soak them in 5wt% washing powder water for 15 minutes, wash them with running water, sterilize them with 70Vol.% ethanol solution for 10 seconds, wash them with sterile water for 3 times, soak them in 0.2wt% sodium hypochlorite solution for 8 minutes, and wash them with sterile water for 5 times, that is, the sterilization is completed.

[0041] After disinfection and sterilization, the explants were cut into pieces of about 1 cm in size, dried and placed on an adventitious bud culture medium until the tissue culture seedlings were 3-4 cm; after removing the leaves, the remaining parts were cut into 0.8 cm stem segments, placed on an adventitious bud culture medium and cultured for 14 days, and stevia adventitious buds of about 0.5 cm in length were selected for polyploid induction. The adventitious bud culture medium components are: MS medium, 0.3 mg / L 6-BA and 0.15 mg / L NAA.

[0042] II. Inducing polyploidy of stevia adventitious buds and cultivating them to obtain surviving stevia polyploid adventitious buds. The specific steps are: preparing a 0.8 wt% hydrogen peroxide solution with water as solvent, preparing a 10 mg / L α-cyanoacrylate ethyl solution with acetone as solvent, and preparing a 1.5 wt% tea polyphenol solution with water as solvent.

[0043] The stevia adventitious buds with a length of 0.3-0.8 cm obtained in step I were soaked in a 0.8 wt % hydrogen peroxide solution for 30 min, then soaked in a 10 mg / L α-cyanoacrylate ethyl solution for 48 h, and finally soaked in a 1.5 wt % tea polyphenol solution for 30 min and then washed with water 5 times to remove the residual solution, and the water was dried with filter paper.

[0044] The Stevia multi-ligand adventitious buds obtained after polyploid induction were placed in an adventitious bud culture medium for 4 weeks, and the mortality rate of the adventitious buds was calculated. The adventitious bud culture medium components include: MS medium, 0.3 mg / L 6-BA and 0.15 mg / L NAA.

[0045] III. Perform rooting culture on the surviving polyploid stevia buds to obtain stevia tissue culture seedlings. The specific steps are as follows: the surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 1-2 cm, the chromosome ploidy of the mutant plants is identified, and the polyploid induction rate is calculated. The surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 5-7 cm, and then the subsequent seedling hardening and transplanting are carried out. The conditions for rooting culture are: temperature 25±1°C, humidity 65%, light intensity 1500μmol / (m 2 ·s) under light intensity of 14 h / d. The rooting medium composition was: MS medium, 0.2 mg / L IBA and 0.1 mg / L NAA.

[0046] IV. Hardening and transplanting the stevia tissue culture seedlings to obtain stevia plants. The specific steps are: placing the stevia tissue culture seedlings in a culture room under a natural scattered light environment. The culture bottle containing the stevia tissue culture seedlings is irradiated under natural scattered light for 3 days before the bottle mouth is opened, and then the bottle mouth of the culture bottle is opened and covered with a transparent sealed bag to continue hardening for 2 days. The environmental conditions of the whole process are: humidity 70-80%, temperature 25±1°C, and hardening is completed.

[0047] Use tweezers to take out the stevia tissue culture seedlings that have been hardened and transplant them into black sterile planting bags with a substrate volume ratio of humus soil: vermiculite = 1:1. Transplant one tissue culture seedling into each planting bag, cover the tissue culture seedlings with a transparent sealing bag, and place them in a constant temperature and light incubator for 10 days. The culture conditions are: humidity 70-80%, temperature 25±1°C. Then remove the transparent sealing bag, continue to culture for 7 days, then transplant them into planting pots, and place them in a greenhouse for 2 weeks.

[0048] Example 2

[0049] A stevia tissue culture method, comprising the following specific steps:

[0050] I. Obtaining Stevia adventitious buds, the specific steps are:

[0051] Use scissors to take the tender stem segments with axillary buds and terminal buds from the stevia plant, remove the leaves on both sides of the stem segments, and use the remaining parts as explants for tissue culture, which are then sterilized. The steps of sterilization are the same as those in Example 1.

[0052] After disinfection and sterilization, the explants were cut into pieces of about 1 cm in size, dried and placed on an adventitious bud culture medium until the tissue culture seedlings were 3-4 cm; after removing the leaves, the remaining parts were cut into stem segments of about 1 cm in length, placed on an adventitious bud culture medium and cultured for 14 days, and stevia adventitious buds of about 0.5 cm in length were selected for polyploid induction. The adventitious bud culture medium components are: MS culture medium, 0.5 mg / L 6-BA and 0.5 mg / L NAA.

[0053] II. Inducing polyploidy of stevia adventitious buds and cultivating them to obtain surviving stevia polyploid adventitious buds. The specific steps are: preparing a 1wt% hydrogen peroxide solution with water as solvent, preparing a 12mg / L α-methyl cyanoacrylate (CAS No.: 137-05-3, C5H5NO2) solution with acetone as solvent, and preparing a 2.5wt% astaxanthin solution with water as solvent.

[0054] The Stevia adventitious buds with a length of about 0.5 cm obtained in step I were soaked in a 1 wt% hydrogen peroxide solution for 25 min, then soaked in a 12 mg / L α-methyl cyanoacrylate solution for 36 h, and finally soaked in a 2.5 wt% astaxanthin solution for 25 min and washed with water 5 times to remove the residual solution, and the water was dried with filter paper.

[0055] The Stevia multi-ligand adventitious buds obtained after polyploid induction were placed in an adventitious bud culture medium for 4 weeks, and the mortality rate of the adventitious buds was calculated. The components of the adventitious bud culture medium were the same as above.

[0056] III. Rooting culture is performed on the surviving polyploid stevia buds to obtain stevia tissue culture seedlings. The specific steps are as follows: the surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 1-2 cm, the chromosome ploidy of the mutant plants is identified, and the polyploid induction rate is calculated. The surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 5-7 cm, and then the subsequent seedling hardening and transplanting are performed. The conditions for rooting culture are: temperature 25±1°C, humidity 70%, light intensity 1800μmol / (m 2 ·s) under light intensity of 12h / d. The rooting medium composition was: MS medium, 0.3mg / L IBA and 0.3mg / L NAA.

[0057] IV. Hardening and transplanting the stevia tissue culture seedlings to obtain stevia plants. The specific steps are the same as those in Example 1.

[0058] Example 3

[0059] A stevia tissue culture method, comprising the following specific steps:

[0060] I. Obtaining Stevia adventitious buds, the specific steps are:

[0061] Use scissors to take the tender stem segments with axillary buds and terminal buds from the stevia plant, remove the leaves on both sides of the stem segments, and use the remaining parts as explants for tissue culture, which are then sterilized. The steps of sterilization are the same as those in Example 1.

[0062] After disinfection and sterilization, the explants were cut into pieces of about 1 cm in size, dried and placed on an adventitious bud culture medium until the tissue culture seedlings were 3-4 cm; after removing the leaves, the remaining parts were cut into stem segments of about 1 cm in length, placed on an adventitious bud culture medium and cultured for 14 days, and stevia adventitious buds of about 0.5 cm in length were selected for polyploid induction. The adventitious bud culture medium components are: MS culture medium, 0.8 mg / L 6-BA and 0.75 mg / L NAA.

[0063] II. Inducing polyploidy of stevia adventitious buds and cultivating them to obtain surviving stevia polyploid adventitious buds. The specific steps are: preparing a 1.2 wt% hydrogen peroxide solution with water as solvent, preparing a 15 mg / L α-cyanoacrylate solution with acetone as solvent, and preparing a 3 wt% tannin solution with water as solvent.

[0064] The Stevia adventitious buds with a length of about 0.5 cm obtained in step I were soaked in a 1.2 wt% hydrogen peroxide solution for 20 min, then soaked in a 15 mg / L α-cyanoacrylate solution for 24 h, and finally soaked in a 3 wt% tannin solution for 20 min and then washed with water 5 times to remove the residual solution, and the water was dried with filter paper.

[0065] The Stevia multi-ligand adventitious buds obtained after polyploid induction were placed in an adventitious bud culture medium for 4 weeks, and the mortality rate of the adventitious buds was calculated. The components of the adventitious bud culture medium were the same as above.

[0066] III. Perform rooting culture on the surviving polyploid stevia buds to obtain stevia tissue culture seedlings. The specific steps are as follows: the surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 1-2 cm, the chromosome ploidy of the mutant plants is identified, and the polyploid induction rate is calculated. The surviving polyploid stevia buds are placed on a rooting medium and cultured until the roots of the stevia tissue culture seedlings are 5-7 cm, and then the subsequent seedling hardening and transplanting are carried out. The conditions for rooting culture are: temperature 25±1°C, humidity 75%, light intensity 2000μmol / (m 2 ·s) for 10 h / d. The rooting medium consisted of MS medium, 0.5 mg / L IBA and 0.5 mg / L NAA.

[0067] IV. Hardening and transplanting the stevia tissue culture seedlings to obtain stevia plants. The specific steps are the same as those in Example 1.

[0068] Embodiment 4-5

[0069] The difference between Example 4-5 and Example 2 is that the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution is different, specifically: the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution of Example 4 is 10 mg / L, and the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution of Example 5 is 15 mg / L.

[0070] Embodiment 6-9

[0071] The difference between Examples 6-9 and Example 2 is that the content of the oxidant in the oxidant solution is different, specifically: the oxidant content in the oxidant solution of Example 6 is 0.5wt%, the oxidant content in the oxidant solution of Example 7 is 0.8wt%, the oxidant content in the oxidant solution of Example 8 is 1.2wt%, and the oxidant content in the oxidant solution of Example 9 is 1.5wt%.

[0072] Comparative Example

[0073] Comparative Example 1

[0074] A stevia tissue culture method, which is different from Example 2 in that the method of polyploid induction in step II is different; step II specifically comprises:

[0075] The polyploidy induction and cultivation of the stevia adventitious buds are carried out to obtain surviving stevia polyploid adventitious buds. The specific steps are: 12 mg / L α-methyl cyanoacrylate solution is prepared with acetone as a solvent. The stevia adventitious buds with a length of about 0.5 cm obtained in step I are soaked in the 12 mg / L α-methyl cyanoacrylate solution for 36 hours, washed with water 5 times to remove the residual solution, and dried with filter paper.

[0076] The Stevia multi-ligand adventitious buds obtained after polyploid induction were placed in an adventitious bud culture medium and cultured for 4 weeks, and the adventitious bud mortality rate was calculated. The components of the adventitious bud culture medium were the same as those in Example 2.

[0077] Comparative Example 2

[0078] A stevia tissue culture method, which is different from Example 2 in that the method of polyploid induction in step II is different; step II specifically comprises:

[0079] The polyploidy of stevia adventitious buds is induced and cultivated to obtain surviving polyploid stevia adventitious buds. The specific steps are: preparing a 1wt% hydrogen peroxide solution with water as a solvent and preparing a 12mg / L α-methyl cyanoacrylate solution with acetone as a solvent. The stevia adventitious buds with a length of about 0.5cm obtained in step I are soaked in a 1wt% hydrogen peroxide solution for 25min, then soaked in a 12mg / L α-methyl cyanoacrylate solution for 36h, then washed with water 5 times to remove the residual solution, and the water is dried with filter paper.

[0080] The Stevia multi-ligand adventitious buds obtained after polyploid induction were placed in an adventitious bud culture medium and cultured for 4 weeks, and the adventitious bud mortality rate was calculated. The components of the adventitious bud culture medium were the same as those in Example 2.

[0081] Comparative Examples 3-4

[0082] A stevia tissue culture method, which is different from Example 2 in that the method of polyploid induction in step II is different; step II specifically comprises:

[0083] The difference between Comparative Examples 3-4 and Example 2 is that the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution is different, specifically: the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution of Comparative Example 3 is 7 mg / L, and the content of methyl α-cyanoacrylate in the methyl α-cyanoacrylate solution of Comparative Example 4 is 20 mg / L.

[0084] Comparative Example 5

[0085] A stevia tissue culture method, which differs from Example 2 in that, when polyploid induction is performed in step II, α-methyl cyanoacrylate is replaced with an equal weight of colchicine; step II specifically comprises:

[0086] Stevia rebaudiana adventitious buds are induced to polyploidy and cultivated to obtain surviving stevia rebaudiana polyploidy adventitious buds. The specific steps are: prepare a 1wt% hydrogen peroxide solution with water as solvent, prepare a 12mg / L colchicine solution with ethanol as solvent, and prepare a 2.5wt% astaxanthin solution with water as solvent. The stevia rebaudiana adventitious buds with a length of about 0.5cm obtained in step I are soaked in a 1wt% hydrogen peroxide solution for 25min, then soaked in a 12mg / L colchicine solution for 36h, and finally soaked in a 2.5wt% astaxanthin solution for 25min and then washed 5 times with water to remove the residual solution, and the water is blotted with filter paper. The stevia rebaudiana multi-ligand adventitious buds obtained after polyploidy induction are placed in an adventitious bud culture medium and cultured for 4 weeks, and the adventitious bud mortality rate is calculated. The adventitious bud culture medium components are the same as in Example 2.

[0087] Comparative Example 6

[0088] A stevia tissue culture method, which differs from Example 2 in that, when polyploid induction is performed in step II, α-methyl cyanoacrylate is replaced with an equal weight of trifluralin; step II specifically comprises:

[0089] Stevia adventitious buds are induced to polyploidy and cultivated to obtain surviving stevia polyploid adventitious buds. The specific steps are: prepare a 1wt% hydrogen peroxide solution with water as solvent, prepare a 12mg / L trifluralin solution with acetone as solvent, and prepare a 2.5wt% astaxanthin solution with water as solvent. The stevia adventitious buds with a length of about 0.5cm obtained in step I are soaked in a 1wt% hydrogen peroxide solution for 25min, then soaked in a 12mg / L trifluralin solution for 36h, and finally soaked in a 2.5wt% astaxanthin solution for 25min and then washed 5 times with water to remove the residual solution, and the water is dried with filter paper. The stevia multi-ligand adventitious buds obtained after polyploidy induction are placed in an adventitious bud culture medium and cultured for 4 weeks, and the adventitious bud mortality rate is calculated. The adventitious bud culture medium components are the same as in Example 2.

[0090] Comparative Example 7

[0091] A stevia tissue culture method, which differs from Example 2 in that, when polyploid induction is performed in step II, methyl α-cyanoacrylate is replaced with oryzalin of equal weight; step II specifically comprises:

[0092] Stevia adventitious buds are induced to polyploidy and cultivated to obtain surviving stevia polyploid adventitious buds. The specific steps are: prepare a 1wt% hydrogen peroxide solution with water as solvent, prepare a 12mg / L oryzalin solution with acetone as solvent, and prepare a 2.5wt% astaxanthin solution with water as solvent. The stevia adventitious buds with a length of about 0.5cm obtained in step I are soaked in a 1wt% hydrogen peroxide solution for 25min, then soaked in a 12mg / L oryzalin solution for 36h, and finally soaked in a 2.5wt% astaxanthin solution for 25min and then washed 5 times with water to remove the residual solution, and the water is dried with filter paper. The stevia multi-ligand adventitious buds obtained after polyploidy induction are placed in an adventitious bud culture medium and cultured for 4 weeks, and the adventitious bud mortality rate is calculated. The adventitious bud culture medium components are the same as in Example 2.

[0093] 1. Polyploidy induction rate and mortality rate

[0094] 60 explants were selected from each group and stevia tissue culture was carried out using different implementation schemes. The specific results of polyploid induction rate and mortality rate are shown in Table 1.

[0095] Among them, induction rate % = number of mutant plants / number of treated adventitious buds × 100%; mortality rate % = number of dead plants / number of treated adventitious buds × 100%.

[0096] Table 1 Polyploid induction rate and mortality rate in different implementation schemes

[0097]

[0098]

[0099] As can be seen from the data results in Table 1, the present application provides a stevia tissue culture method with a high polyploid mutation rate and a low mortality rate, which achieves this purpose by cooperating with an oxidant mutagen. Among them, Example 2 and Comparative Examples 1-2 prove that during the induction process, the cells need to be treated with an oxidant solution first to change the cell permeability to a certain extent, so that the mutagen can efficiently enter the cell later to exert a mutagenic effect; and the oxidant further interacts with the mutagen after entering the cell, and may affect the function of related enzymes and proteins in the cell, so as to cooperate with the mutagen and improve the induction rate; at the same time, a low dose of the mutagen can further reduce its cytotoxicity and reduce the mortality rate of adventitious buds after induction.

[0100] The data results of Example 2, Examples 4-5 and Comparative Examples 3-4 show that: in this scheme, the concentration of methyl α-cyanoacrylate in the cyanoacrylate solution has a relatively large effect on the mutagenesis result, and the content of methyl α-cyanoacrylate in the cyanoacrylate solution is recommended to be in the range of 10-15 mg / L; if the concentration of methyl α-cyanoacrylate is too low, it will directly affect the mutagenesis rate, and if the concentration of methyl α-cyanoacrylate is too high, the mortality rate of adventitious buds will be high.

[0101] In this method, the amount of oxidant used also has a certain influence on the results. The data of Examples 2 and 6-9 reflect that the concentration of the oxidant in the oxidant solution is recommended to be in the range of 0.8-1.2wt%: if the concentration of the oxidant is too high, it will cause cell damage itself, resulting in a higher mortality rate of adventitious buds; if the concentration of the oxidant is too low, the effect on cell permeability is weak, and the resistance of the mutagen to enter the cell becomes greater, thereby affecting the induction rate. The advantage of using an oxidant as a factor affecting cell permeability is that it can be further removed more easily through the action of a reducing agent, thereby avoiding continued cell damage.

[0102] In addition, this scheme further compares the effects of colchicine, oryzalin and fluralin through the settings of comparative examples 5-7, and the results show that colchicine, oryzalin and fluralin have poor effects compared with methyl α-cyanoacrylate. It may be that in the process of cooperation between mutagens and oxidants, the relevant regulation process has great differences, making it difficult for several mutagens in the comparative examples to achieve good cooperation with oxidants, thereby significantly affecting the mutagenesis results and the effect of reducing mortality.

[0103] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for stevia tissue culture, characterized in that: The following steps are involved:

1. Obtaining stevia adventitious buds; II. Inducing polyploidy of the stevia adventitious buds to obtain surviving stevia polyploid adventitious buds; III. performing rooting culture on the surviving polyploid adventitious buds of Stevia rebaudiana to obtain Stevia rebaudiana tissue culture seedlings; IV, hardening and transplanting the stevia tissue culture seedlings to obtain stevia plants; The method of polyploid induction in step II comprises the following steps: soaking the stevia adventitious buds in an oxidant solution, soaking the stevia adventitious buds in a cyanoacrylate solution for 24-48 hours, washing with a reducing agent solution, and washing with water; The cyanoacrylate content in the cyanoacrylate solution is 10-15 mg / L; the oxidant content in the oxidant solution is 0.8-1.2 wt %; the cyanoacrylate is selected from any one or more of α-methyl cyanoacrylate and α-ethyl cyanoacrylate.

2. The method for stevia tissue culture according to claim 1, characterized in that: The oxidant is selected from any one or more of hydrogen peroxide and ozone.

3. The method for stevia tissue culture according to claim 1, characterized in that: The oxidant is hydrogen peroxide.

4. The method for stevia tissue culture according to claim 1, characterized in that: The reducing agent content in the reducing agent solution is 1.5-3 wt %.

5. The method for stevia tissue culture according to claim 4, characterized in that: The reducing agent is selected from any one or more of astaxanthin, tea polyphenols and tannin.

6. The method for stevia tissue culture according to claim 1, characterized in that: The method for obtaining stevia adventitious buds comprises the following steps: obtaining stem segments with axillary buds and / or terminal buds from stevia plants, cutting them into 0.5-1.5 cm in size after disinfection and sterilization, absorbing moisture and placing them on an adventitious bud culture medium to culture until the height of the tissue culture seedlings is 3-4 cm; the remaining parts after removing the leaves of the tissue culture seedlings are cut into 0.8-1.2 cm stem segments, placing them on an adventitious bud culture medium for culture, and selecting stevia adventitious buds with a length of 0.3-0.8 cm for polyploid induction.

7. The method for stevia tissue culture according to claim 1, characterized in that: The rooting culture method in step III comprises the following steps: placing the surviving polyploid adventitious buds of Stevia rebaudiana on a rooting medium and culturing until the root length of the Stevia rebaudiana tissue culture seedlings is 5-7 cm.

8. The method for stevia tissue culture according to claim 7, characterized in that: The culture conditions for rooting culture include: temperature of 24-26°C, humidity of 65-75%, light intensity of 1500-2000 μmol / (m 2 ·s), and the photoperiod is 10-14h / d.

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