A method for preparing virus-free seedling of sweet potato by axillary bud differentiation

Abstract

The present application belongs to the field of agricultural technology, and particularly relates to a method for efficiently preparing virus-free sweet potato seedlings by axillary bud differentiation. The present application uses the representative variety "Xiguahong" of sweet potato as the material, selects the healthy stem segments of "Xiguahong" cultivated in the open air, removes the leaves, obtains the aseptic stem segments after disinfection. The stem segments are inoculated on the axillary bud differentiation formula for 20 days, and at least 2 or more axillary buds can be obtained from each stem segment. The bud tips of the aseptic seedlings of "Xiguahong" are peeled off and inoculated on the stem tip meristem formula to obtain the regenerated seedlings. Virus detection is performed on the regenerated sweet potato, and the virus-free sweet potato seedlings can be obtained. The virus-free seedlings are inoculated on the clump plant formula for growth culture, and a large number of virus-free sweet potato seedlings can be obtained. The axillary buds induced by the method have a shape more consistent with the plant bud meristem model, which is convenient for the peeling of the bud tip meristem. The number of regenerated virus-free sweet potato seedlings obtained under the same cost is significantly increased, the efficiency of obtaining virus-free seedlings is improved, and the production cost of virus-free sweet potato seedlings is reduced.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a method for preparing virus-free sweet potato seedlings using plant tissue culture technology. Background Technology

[0002] sweet potato( Dioscorea esculenta (Lour.) Burkill is a perennial twining vine belonging to the Dioscoreaceae family. Native to South America, it has been cultivated in my country for over 400 years. Currently, the annual planting area of ​​sweet potatoes in my country is approximately 4.6 million hectares. 2 China's sweet potato cultivation area accounts for more than half of the global sweet potato area. The total fresh sweet potato production is approximately 100 million tons, accounting for over 80% of the world's total fresh sweet potato production. In daily production and cultivation, sweet potatoes are propagated asexually, primarily through stems and tubers. The succulent nature of sweet potato stems and leaves makes them susceptible to bites from aphids, planthoppers, and other virus-carrying pests. Once infected by viruses during feeding, these pests first replicate extensively within the plant, eventually infecting the entire plant through vascular bundles and plasmodesmata. Continuous planting of virus-infected sweet potatoes for many years leads to a gradual increase in the amount and types of viruses carried in the tubers, ultimately resulting in varietal degeneration, severely impacting sweet potato yield and quality, and causing significant losses for farmers. According to incomplete statistics, the annual losses in my country due to planting virus-infected sweet potatoes reach as high as 4 billion yuan.

[0003] As of 2020, more than 30 types of sweet potato viruses have been confirmed, with about 20 of them found in China. The main viruses affecting sweet potato production are sweet potato geminitrovirus, potato virus Y, and sweet potato chlorosis dwarf virus. Viruses cause the following effects on the above-ground parts of the sweet potato: abnormal leaf color (purple feathery spots, purple spots, yellow spots, mosaic, yellowing, etc.), leaf deformities (leaf curling, wrinkling, etc.), and abnormal plant development (dwarfing, wrinkling, etc.). The effects of viruses on the underground parts of the sweet potato are often manifested as reduced tuber yield and the appearance of dark brown or yellowish-brown cracks inside or on the surface of the tubers. Currently, neither virus-resistant sweet potato varieties have been developed domestically nor internationally, and effective measures for controlling sweet potato viral diseases are also lacking. Therefore, promoting virus-free sweet potato seedlings in production practice is currently the most effective method to solve various sweet potato viral diseases, increase sweet potato yield, and improve sweet potato quality.

[0004] Currently, the main method used domestically and internationally is to obtain virus-free seedlings through explant shoot tip detoxification, and to obtain large quantities of virus-free seedlings through rapid propagation. The current standard DB65T4164-2018 has the following disadvantages: (1) Direct disinfection of explants leads to a high contamination rate in shoot tip development into virus-free seedlings; (2) Only the growing point of a sweet potato plant can be used for shoot tip peeling, and a large number of sweet potato growing points are required when obtaining a large number of virus-free seedlings; (3) Because the leaf bud differentiation of the growing point is obvious and the leaves are large, it is easy to make operational errors during the peeling process, resulting in secondary infection of the bud meristem with the virus; (4) Sweet potato virus-free seedlings are propagated using growing points and stem segments as propagation materials (amplification coefficient ≥1:0.05), and the amplification coefficient is low. When a small number of virus-free seedlings are obtained, it takes a longer time to expand the virus-free seedling population; (5) Explant disinfection requires the use of toxic substances such as 0.1% mercuric chloride. Summary of the Invention

[0005] To overcome the shortcomings of existing methods, this invention provides a method for efficiently obtaining virus-free seedlings by utilizing the differentiation of sweet potato axillary buds. This method can significantly improve the amplification coefficient of virus-free sweet potato seedlings and obtain a large number of virus-free sweet potato seedlings in a short period of time.

[0006] Specifically, the present invention provides a method for obtaining virus-free seedlings by utilizing sweet potato axillary bud differentiation, comprising the following steps: (1) Stem segment preparation: Select sweet potato stem tips grown in the open field, remove all leaves except the growing point, and keep the stem segments for later use; (2) Stem segment disinfection: The stem segments obtained in step (1) are first soaked in 75% alcohol for 5-6 minutes, and then washed with sterile water; then, they are soaked in 4% NaClO disinfectant for 5-7 minutes, washed with sterile water, and then dried with sterile paper. The stem segments are kept for later use. (3) Axillary bud differentiation culture: After disinfection, the stem segments were inoculated onto the axillary bud differentiation culture medium. First, induction culture was carried out to induce the stem segments to grow and form more stem nodes and axillary buds. Then, under dark conditions, the sweet potato leaves were induced to fall off naturally, leaving the stems and axillary buds. (4) Bud tip meristem stripping: After taking out the sweet potato stem segment containing axillary buds, place it under a microscope, select the axillary buds and strip the stem tip; (5) Inducing shoot tip to obtain regenerated seedlings: The stripped shoot tips are inoculated into shoot tip meristem culture medium and induced to grow into sweet potato seedlings; Preferably, the sweet potato of the present invention belongs to the genus Dioscorea in the family Convolvulaceae; preferably, the sweet potato variety is "Watermelon Red".

[0007] Preferably, the axillary bud differentiation culture medium is formulated as follows: pH 6.0, MS medium supplemented with NAA 0.05~0.1mg / L, TDZ 0.01~0.1mg / L, chlorpyrifos 0.01~0.03mg / L, vitamin C 50mg / L, and cephalosporin 250mg / L.

[0008] Preferably, the bud tip meristem culture medium is formulated with pH 6.0, MS medium supplemented with NAA 0.1~0.5 mg / L and 6-BA 0.5~1.0 mg / L.

[0009] In one embodiment of the present invention, the above method further includes: (6) Conduct virus testing, that is, test all obtained sweet potato virus-free seedlings for viruses; (7) To induce single sweet potato seedlings to grow into clump plants, the obtained sweet potato seedlings are inoculated into a bud induction medium and induced to grow axillary buds of single sweet potato seedlings to form clump plants.

[0010] Preferably, the bud induction medium is formulated as follows: pH 6.0, MS medium with NAA 0.01~0.1 mg / L + TDZ 0.01~0.05 mg / L.

[0011] In one embodiment of the present invention, the requirements for stem segment preparation in step (1) are as follows: the stem segment must include a growth point and multiple stem nodes. Retaining the growth point ensures that the explant can grow normally after disinfection, and stem nodes are more conducive to the differentiation and induction of axillary bud growth points, thereby increasing the amplification coefficient of the original material.

[0012] In one embodiment of the present invention, the stem segment disinfection requirements in step (2) are as follows: Before disinfection, the stem segments need to be cleaned to remove impurities from the surface of the explant. After cleaning, soak in sterile water containing 200 mg / L cephalosporin for 4-6 hours. Then, soak the stem segments in 75% alcohol for 5-7 minutes, rinse with sterile water, and then soak in 4% NaClO disinfectant solution for 5-7 minutes. Finally, rinse with sterile water and blot dry with sterile paper. The stem segments are reserved for later use.

[0013] In one embodiment of the present invention, axillary bud differentiation culture is performed in step (3). During the axillary bud differentiation culture process, it is first necessary to maintain a light intensity of 16000 lux, a photoperiod of 16 hours, and a temperature of 22 degrees Celsius. oCultured in C environment for about 15 days to induce axillary bud differentiation. The longer the culture time in this environment, the more axillary buds differentiate, but the smaller the axillary bud growth point, making it difficult to separate the shoot tip later. Once axillary buds appear at the stem nodes, they should be promptly transferred to a dark environment for about 10 days of culture. Prolonged exposure to darkness, under the induction of TDZ (Dietary Therapy-Induced Variation), causes the leaves to gradually turn yellow. Due to the lack of light induction, photomorphogenesis of the axillary buds at the stem nodes is inhibited. At this time, the leaves formed from the differentiation of bud primordia remain in the leaf primordia state.

[0014] In one embodiment of the present invention, the bud tip meristem is peeled off in step (4). Because a prolonged dark environment inhibits photomorphogenesis at the axillary bud growth point, the bud is in a state of low differentiation. During peeling, excessive cutting is unnecessary; the growth point can be directly selected under a stereomicroscope and peeled off. This process avoids secondary viral infections caused by excessive cutting.

[0015] In one embodiment of the present invention, step (5) of inducing shoot tip regeneration involves inoculating the stripped shoot tips onto a growth medium, the medium formulation of which is based on standard DB65T4164-2018. The growth medium is grown under a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o Cultivate in C environment for 20 days to induce shoot tip growth and form sweet potato seedlings.

[0016] In one embodiment of the present invention, step (7) induces single sweet potato seedlings to form clump-forming plants, that is, the obtained sweet potato seedlings are inoculated into a shoot induction medium, and the culture environment has a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o C. Inducing the growth of axillary buds from individual sweet potato seedlings to form clustered plants. This process can rapidly propagate virus-free sweet potato seedlings in a short time and maximize the propagation coefficient of virus-free seedlings.

[0017] In one embodiment of the present invention, step (6) involves virus detection, that is, testing all the sweet potato virus-free seedlings obtained in step (5) for viruses. For details of the detection method, please refer to standard NY / T 402-2016.

[0018] Preferably, the axillary bud differentiation culture medium in step (3) consists of MS medium supplemented with NAA 0.05~0.2mg / L, TDZ 0.01~0.1mg / L, chlorpyrifos 0.01~0.03mg / L, vitamin C 50mg / L, and cephalosporin 250mg / L.

[0019] Preferably, the growth medium for shoot tip meristem in step (5) consists of MS medium supplemented with 0.5~1.0 mg / L 6-BA and 0.1~0.5 mg / L NAA.

[0020] Preferably, step (7) involves adding NAA 0.01~0.1 mg / L and TDZ 0.01~0.05 mg / L to MS medium for bud induction.

[0021] This invention involves first culturing the axillary buds of sterilized sweet potato growing points under light conditions to induce the formation of numerous axillary buds in the leaf axils of stem segments. Then, the plants are cultured in complete darkness, causing all leaves to fall off, leaving only the axillary bud growing points and the main stem portion. This simplifies the stem tip stripping process and effectively reduces secondary viral infection of the bud meristem. After seedling formation, the bud meristem induces the growth of individual plants into clump-forming plants, increasing the amplification coefficient from 1:0.05 to 1:1.37. Beneficial effects

[0022] Compared with the prior art, the present invention has the following significant advantages: (1) The amplification coefficient is ≥1:200, which can achieve the amplification of ≥200 virus-free seedlings in a short time; (2) The contamination rate is low. The explants are disinfected first to obtain sterile seedlings, and then the sterile seedlings are peeled off, which greatly reduces the contamination rate. (3) The viral infection rate is low because the axillary bud growth point is at a low degree of differentiation, so there is no need to cut and peel it, thus avoiding secondary viral infection caused by the operation process.

[0023] (4) The cost is low, and the time and reagent costs are lower than those of the existing schemes, but the number of virus-free sweet potatoes obtained is much greater than that of the existing schemes.

[0024] (5) Because it does not involve highly toxic substances such as 0.1% mercuric chloride, the operation is safer. Attached Figure Description

[0025] Figure 1 Traditional methods for obtaining virus-free sweet potato seedlings. Group A: Field selection of sweet potato stem tips; Group B: Separation of sweet potato bud meristem; Group C: Development of bud meristem into seedlings; Group D: Virus-free sweet potato seedlings. Requirements for field selection of sweet potato stem tips in Group A: a) Select vigorous, disease-free plants; b) Select the top stem tip of the vine; c) The selected growth point must meet certain criteria; d) Retain the sweet potato stem tip growth point and remove excess leaves. Separation of bud meristem in Group B: a) Cutting the growth point; b) Separating the leaves and leaf primordia at the growth point; c) Cutting the bud meristem. Group C: Cultivating the apical meristem of the sweet potato explant, which develops into sweet potato seedlings after 30 days; Group D: Virus-free sweet potato seedlings. Traditional methods can only expand to a 1:1 ratio of explant to virus-free seedling to form single virus-free seedlings.

[0026] Figure 2Virus detection results of sweet potato virus-free seedlings obtained by traditional methods. From top to bottom: Sweet potato feather mottle virus (SPFMV), sweet potato cork virus (SPICV), sweet potato chlorotic dwarf virus (SPCSV), sweet potato yellow dwarf virus (SPYDV), and sweet potato latent virus (SPLV). The bands at approximately 1000bp in the gel image show the virus-carrying status of the virus-free seedlings obtained by traditional methods.

[0027] Figure 3 Contamination rate of sweet potato shoot tip explant culture. The results showed that disinfection with 75% alcohol for 5-7 min and 4% NaClO for 5-7 min could significantly reduce the contamination rate of shoot tip explants.

[0028] Figure 4 Sweet potato shoot tip regeneration rate. The results showed that disinfection with 75% alcohol for 5-6 minutes and disinfection with 4% NaClO for 5-6 minutes could significantly improve the shoot tip regeneration rate.

[0029] Figure 5 Virus detoxification rate of sweet potatoes. The results showed that the virus detoxification rates of sweet potatoes in Case 1, 2 and 3 were significantly higher than those in Case 1, with Case 1 having the highest virus detoxification rate.

[0030] Figure 6 A method for efficiently obtaining virus-free sweet potato seedlings through axillary bud differentiation. The method involves: A) selecting sweet potato stem tips in the field; B) sweet potato axillary bud differentiation culture; C) peeling the axillary bud meristem; D) the development of the bud meristem into seedlings; and E) the induced clustered sweet potato plants. A. Requirements for selecting sweet potato stem tips in the field: a) Select vigorous, disease-free plants; b) Select the top stem tip of the vine; c) The selected growth point must meet certain requirements; d) Retain the sweet potato stem tip growth point and remove excess leaves. B. Sweet potato axillary bud differentiation culture: After 10-15 days of culture under adapted light conditions, numerous axillary buds sprout from the sweet potato stem nodes. After the axillary buds develop, promptly transfer them to a dark environment. Around 20-25 days later, all leaves fall off, leaving only the axillary bud basal growth point. C. Axillary bud meristem tip stripping; a. Magnified view of sweet potato axillary buds; each stem node of the sweet potato contains at least one axillary bud; b. Direct selection of axillary buds; c. Direct stripping of the axillary bud apical meristem; d. Magnified view of stripped axillary bud apical meristem; e. Removal and inoculation of the axillary bud meristem (arrow). D. Cultivation of the axillary bud apical meristem; sweet potato seedlings develop after 30 days. E. Inoculation of single sweet potato seedlings onto a clump-forming plant induction medium; after 45 days, clump-forming virus-free sweet potato plants with ≥3 plants are formed. A method for efficiently obtaining virus-free seedlings through sweet potato axillary bud differentiation: Explants are expanded to form clump-forming virus-free sweet potato seedlings.

[0031] Figure 7Comparing the virus test results of the virus-free seedlings obtained in Case 1: from top to bottom, they are sweet potato feather mottle virus (SPFMV), sweet potato cork virus (SPICV), sweet potato chlorosis dwarf virus (SPCSV), sweet potato yellow dwarf virus (SPYDV), and sweet potato latent virus (SPLV). The test results show that some virus-free seedlings still contain the virus and have not been completely devirulentized. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] MS medium was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: M519-100L; 75% alcohol was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: E99945; 4% NaClO was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: S953735; NAA was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: N8010; TDZ was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: T8050; 6-BA was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: A8170; The chlorpyrifos was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: IF0890; Vitamin C (Vc) was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: A8101; The cephalosporin was purchased from Nanjing Zhengyang Biotechnology Co., Ltd., product number: IC5100; Calculation formula: Shoot tip contamination rate = Number of contaminated shoot tips / Total number of inoculated shoot tips (300) × 100%; Shoot tip regeneration rate = Number of shoot tip explants in seedlings / Total number of inoculated shoot tips (300) × 100%; Virus-free seedling amplification coefficient = Number of virus-free seedlings: Total number of virus-free sweet potato seedlings in the first subgeneration; Sweet potato virus elimination rate = negative plants / total number of virus-free seedlings to be tested; Example 1

[0034] A method for obtaining virus-free seedlings using sweet potato axillary bud differentiation includes the following steps: (1) Stem segment preparation: Select the stem tips of "Watermelon Red" sweet potato grown in the open field, remove all leaves except the growing point, and keep the stem segments for later use; (2) Stem segment disinfection: The "watermelon red" stem segments obtained in step (1) are first soaked in 75% alcohol for 5 minutes, and then washed with sterile water. Then they are soaked in 4% NaClO disinfectant for 5 minutes, washed with sterile water, and then dried with sterile paper. The stem segments are kept for later use. (3) Axillary bud differentiation culture: After disinfection, the stem segments were inoculated into axillary bud differentiation medium (pH 6.0, MS + NAA 0.05 mg / L + TDZ 0.01 mg / L + chlorpyrifos 0.01 mg / L + Vc 50 mg / L + cephalosporin 250 mg / L). In the early stage, the light intensity was 16000 lux, the photoperiod was 16 h, and the temperature was 22°C. o Cultivate in a C environment for about 15 days to induce stem segment growth and the formation of more stem nodes and axillary buds. Later, at a temperature of 22°C... o Cultivate in the dark environment of C for about 10 days to induce the sweet potato leaves to fall off naturally, leaving the stems and axillary buds; (4) Bud tip meristem stripping: After taking out the stem segments of "Watermelon Red" sweet potato containing axillary buds, place them under a stereomicroscope and select axillary buds and strip the stem tips in the field of view with a magnification of 40x. (5) Induction of shoot tip regeneration: The stripped shoot tips were inoculated into growth medium (pH 6.0, MS + NAA 0.1 mg / L + 6-BA 0.5 mg / L) and grown under light intensity of 16000 lux, photoperiod of 16 h, and temperature of 22°C. o Cultivate in C environment for 20 days to induce shoot tip growth and form sweet potato seedlings; (6) Perform virus testing, that is, test all the virus-free sweet potato seedlings obtained in step (5) for viruses; (7) To induce single sweet potato seedlings into clump-forming plants, the sweet potato seedlings obtained in step (6) were inoculated into a shoot induction medium (pH 6.0, MS + NAA 0.01 mg / L + TDZ 0.01 mg / L). The induction was carried out under a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o Cultivating sweet potato seedlings in a C environment for about 15 days induces the growth of axillary buds in individual seedlings, forming clustered plants. Example 2

[0035] A method for efficiently obtaining virus-free seedlings using sweet potato axillary bud differentiation includes the following steps: (1) Stem segment preparation: Select the stem tips of "Watermelon Red" sweet potato grown in the open field, remove all leaves except the growing point, and keep the stem segments for later use; (2) Stem segment disinfection: The "watermelon red" stem segments obtained in step (1) are first soaked in 75% alcohol for 6 minutes, and then washed with sterile water. Then they are soaked in 4% NaClO disinfectant for 6 minutes, washed with sterile water, and then dried with sterile paper. The stem segments are kept for later use. (3) Axillary bud differentiation culture: After disinfection, the stem segments were inoculated into axillary bud differentiation medium (pH 6.0, MS + NAA 0.1 mg / L + TDZ 0.05 mg / L + chlorpyrifos 0.02 mg / L + Vc 50 mg / L + cephalosporin 250 mg / L). In the early stage, the light intensity was 16000 lux, the photoperiod was 16 h, and the temperature was 22°C. o Cultivate in a C environment for about 15 days to induce stem segment growth and the formation of more stem nodes and axillary buds. Later, at a temperature of 22°C... o Cultivate in the dark environment of C for about 10 days to induce the sweet potato leaves to fall off naturally, leaving the stems and axillary buds; (4) Bud tip meristem stripping: After taking out the stem segments of "Watermelon Red" sweet potato containing axillary buds, place them under a stereomicroscope and select axillary buds and strip the stem tips in the field of view with a magnification of 40x. (5) Inducing shoot tip regeneration: The stripped shoot tips were inoculated into growth medium (pH 6.0, MS + NAA). 0.3 mg / L + 6-BA 0.75 mg / L), under light intensity of 16000 lux, photoperiod of 16 h, and temperature of 22°C. o Cultivate in C environment for 20 days to induce shoot tip growth and form sweet potato seedlings; (6) Conduct virus testing, that is, test all the virus-free sweet potato seedlings obtained in step (5) for viruses; (7) To induce single sweet potato seedlings into clump-forming plants, the sweet potato seedlings obtained in step (6) were inoculated into a shoot induction medium (pH 6.0, MS + NAA 0.05 mg / L + TDZ 0.03 mg / L). The inoculation was carried out under a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o Cultivating sweet potato seedlings in a C environment for about 15 days induces the growth of axillary buds in individual seedlings, forming clustered plants.

[0036] Implementation Case 3

[0037] A method for efficiently obtaining virus-free seedlings using sweet potato axillary bud differentiation includes the following steps: (1) Stem segment preparation: Select the stem tips of "Watermelon Red" sweet potato grown in the open field, remove all leaves except the growing point, and keep the stem segments for later use; (2) Stem segment disinfection: The "watermelon red" stem segments obtained in step (1) are first soaked in 75% alcohol for 7 minutes, and then washed with sterile water. Then they are soaked in 4% NaClO disinfectant for 7 minutes, washed with sterile water, and then dried with sterile paper. The stem segments are kept for later use. (3) Axillary bud differentiation culture: After disinfection, the stem segments were inoculated into axillary bud differentiation medium (pH 6.0, MS + NAA 0.1 mg / L + TDZ 0.1 mg / L + chlorpyrifos 0.03 mg / L + Vc 50 mg / L + cephalosporin 250 mg / L). In the early stage, the light intensity was 16000 lux, the photoperiod was 16 h, and the temperature was 22°C. o Cultivate in a C environment for about 15 days to induce stem segment growth and the formation of more stem nodes and axillary buds. Later, at a temperature of 22°C... o Cultivate in the dark environment of C for about 10 days to induce the sweet potato leaves to fall off naturally, leaving the stems and axillary buds; (4) Bud tip meristem stripping: After taking out the stem segments of "Watermelon Red" sweet potato containing axillary buds, place them under a stereomicroscope and select axillary buds and strip the stem tips in the field of view with a magnification of 40x. (5) Inducing shoot tip regeneration: The stripped shoot tips were inoculated into growth medium (pH 6.0, MS + NAA). 0.5 mg / L + 6-BA 1.0 mg / L), under light intensity of 16000 lux, photoperiod of 16 h, and temperature of 22°C. o Cultivate in C environment for 20 days to induce shoot tip growth and form sweet potato seedlings; (6) Conduct virus testing, that is, test all the virus-free sweet potato seedlings obtained in step (5) for viruses; (7) To induce single sweet potato seedlings into clump-forming plants, the sweet potato seedlings obtained in step (6) were inoculated into a shoot induction medium (pH 6.0, MS + NAA 0.1 mg / L + TDZ 0.05 mg / L). The inoculation was carried out under a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o Cultivating sweet potato seedlings in a C environment for about 15 days induces the growth of axillary buds in individual seedlings, forming clustered plants.

[0038] Comparative Example 1

[0039] The control group used sweet potato virus-free in vitro seedling culture and rapid propagation technology (DB65T4164-2018), including the following steps: (1) Variety selection: Select the sweet potato variety "Watermelon Red" which is currently widely planted in the market.

[0040] (2) Selection of parts: Select tender stem tips with strong regeneration ability.

[0041] (3) Pretreatment of explants: Select tender stem tips of about 3cm from the field vines, number them, and tie them separately. Indoors, remove the leaves from the stem tips taken from the field, then rinse with tap water (with detergent) for 2-3 minutes (to lightly remove dirt attached to the surface of the material), then add 1-2 mL of Tween (the ideal surfactant with strong cleaning ability; it can also be added to the sterilization solution during surface sterilization to improve the sterilization effect) and wash for 2-3 minutes. Finally, rinse with running tap water for 1-2 minutes. Drain and set aside for use.

[0042] (4) Sterilization of explants: Place the pretreated sweet potato stem tips into a 100mL Erlenmeyer flask (or a 100mL beaker), soak in 70% ethanol for 30s, rinse with sterile water, then soak in 0.1% mercuric chloride solution for 8 minutes, shaking the flask several times to ensure a good sterilization effect. Then, pour out the mercuric chloride solution (store it in a brown bottle), rinse three times with sterile water, shaking constantly to remove residual mercuric chloride and reduce toxicity to meristems. After cleaning, place the stem tips in a petri dish to remove excess water.

[0043] (5) Extraction and inoculation of shoot tip meristem: Place the sterilized shoot tip under a 40x double-sided dissecting microscope. Hold the shoot tip material with forceps in your left hand and use a scalpel in your right hand to peel off the leaf primordia embedded in the shoot tip meristem. For sweet potato virus-free culture, it is advisable to extract the material with 1-2 leaf primordia attached, about 0.4 mm in size. When cutting, try not to cut other tissues connected to the shoot tip meristem. Then, inoculate the peeled shoot tip meristem onto the induction medium. At a temperature of 28°C... o At C, with a relative humidity of 50%, a photoperiod of 16 h, and a light intensity of 3000 lux, plants were cultured in an induction medium (pH 5.8, MS + 6-BA 2 mg / L + TDZ 1 mg / L) for 20 days until the shoot tip differentiation plants regenerated. Each regenerated plant was then used as a separate line for propagation.

[0044] (6) Virus-free seedling testing: When more than 10 virus-free seedlings are produced by rapid propagation of the cut segments, 5 seedlings from each line are tested for the virus. Virus-positive test-tube seedlings are eliminated, and the best line that meets the characteristics of the variety and is high-yielding is selected, thereby screening out high-grade virus-free test-tube seedlings.

[0045] (7) Second rapid propagation: Under aseptic conditions, the advanced virus-free test-tube seedlings were cut into stem segments with 1 to 2 leaf nodes and inoculated onto the rapid propagation medium. The propagation was carried out at a temperature of 28°C. oAt room temperature (C), relative humidity 50%, photoperiod 16 h, light intensity 3000 lux, and after 30 days of subculture on medium (pH 5.8, MS + NAA 0.1 mg / L + 6-BA 1.0 mg / L) to allow shoot tip differentiation and plant regeneration, each regenerated plant was used as a separate line for propagation. Seedlings with 5–7 leaves were then ready for the next round of subculture.

[0046] Comparative Case 1 was used as a control group for implementation Cases 1, 2, and 3 for comparative study.

[0047] Shoot tips of explants are prone to contamination during culture due to incomplete sterilization. A comparison was made between Case 1 (shoot tip contamination rate 42.3%), Implementation Case 1 (shoot tip contamination rate 12%), Implementation Case 2 (shoot tip contamination rate 4%), and Implementation Case 3 (shoot tip contamination rate 2.3%). Compared to the existing standard implementation case 1, Implementation Cases 1, 2, and 3 significantly reduced the contamination rate, avoiding losses caused by contamination. Figure 3 ).

[0048] Changes in disinfection methods have varying degrees of impact on the activity of shoot apical meristems, ultimately affecting their regeneration rate. See also... Figure 4 The study compared Case 1 (shoot tip meristem regeneration rate 79.6%), Implementation Case 1 (shoot tip meristem regeneration rate 96.3%), Implementation Case 2 (shoot tip meristem regeneration rate 89.3%), and Implementation Case 3 (shoot tip meristem regeneration rate 80.7%). Compared to the existing implementation standard, Implementation Case 1, Implementation Cases 1 and 2 significantly reduced the contamination rate while significantly increasing the shoot tip meristem regeneration rate. (See Table 1) Axillary bud induction in explants increased the number of shoot tip meristem copies in the experimental cases, increasing the sweet potato virus-free seedling amplification coefficient from 1:0.05 to ≥1:1.37. This improved the efficiency of obtaining virus-free sweet potato seedlings by at least 27 times (Table 1).

[0049] ; Table 1: Effects of different methods on the acquisition of virus-free sweet potato seedlings See Figure 5 The study compared Case 1 (detoxification rate 11.36%), Implementation Case 1 (detoxification rate 26.35%), Implementation Case 2 (detoxification rate 27.32%), and Implementation Case 3 (detoxification rate 20.11%). Compared to the existing standard implementation case 1, Implementation Cases 1 and 2 significantly reduced the contamination rate while significantly increasing the regeneration rate of shoot tip meristem (see [reference]). Figure 5 (Table 1).

[0050] Compared to Comparative Case 1, Cases 1, 2, and 3 all added two key steps: axillary bud differentiation culture and induction of basal plants. Figure 6(Medium B). Axillary bud differentiation culture not only induced the germination of axillary buds at stem nodes, increasing the amount of shoot meristem available for obtaining virus-free seedlings, but also eliminated the need for leaf removal from the axillary bud growth points, avoiding secondary viral infection during operation and significantly improving the virus-free rate. Simultaneously, advanced sterilization culture of explants before shoot tip meristem removal avoided contamination caused by incomplete sterilization, significantly reducing the contamination rate of shoot tip culture. Clump-forming plant culture enabled the differentiation of single-plant to multi-plant sweet potato virus-free seedlings, significantly improving the efficiency of virus-free sweet potato seedlings.

[0051] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing virus-free sweet potato seedlings through axillary bud differentiation, characterized in that, The method includes the following steps: (1) Stem segment preparation: Select sweet potato stem tips grown in the open field, remove all leaves except the growing point, and keep the stem segments for later use; the stem segments must include the growing point and multiple stem nodes; (2) Stem segment disinfection: The stem segments obtained in step (1) are first soaked in 75% alcohol for 5-6 minutes, and then washed with sterile water; then, they are soaked in 4% NaClO disinfectant for 5-7 minutes, washed with sterile water, and then dried with sterile paper. The stem segments are kept for later use. (3) Axillary bud differentiation culture: After disinfection, the stem segments were inoculated onto the axillary bud differentiation medium and cultured under light intensity of 16000 lux, photoperiod of 16 h, and temperature of 22°C. o Cultured in C environment for 15 days to induce axillary bud differentiation, induce stem segment growth to form more stem nodes and axillary buds, and then cultured in the dark for about 10 days to induce sweet potato leaves to fall off naturally, leaving stems and axillary buds; (4) Bud tip meristem stripping: After taking out the sweet potato stem segment containing axillary buds, place it under a microscope, select the axillary buds and strip the stem tip; (5) Inducing shoot tip to obtain regenerated seedlings: The stripped shoot tips are inoculated into shoot tip meristem culture medium and induced to grow into sweet potato seedlings; (6) Virus testing: Test the obtained virus-free sweet potato seedlings for viruses; (7) Inducing single sweet potato seedlings into clump plants: The obtained sweet potato seedlings were inoculated into a bud induction medium and induced to grow axillary buds of single sweet potato seedlings to form clump plants; The axillary bud differentiation medium is formulated as follows: pH 6.0, MS medium, NAA 0.05~0.1 mg / L, TDZ 0.01~0.1 mg / L, chlorpyrifos 0.01~0.03 mg / L, vitamin C 50 mg / L, and cephalosporin 250 mg / L. The formula for the shoot tip meristem culture medium consists of pH 6.0, MS medium, and NAA 0.1~0.5 mg / L and 6-BA 0.5~1.0 mg / L; The bud induction medium is formulated with pH 6.0, MS medium, and NAA 0.01~0.1 mg / L and TDZ 0.01~0.05 mg / L.

2. The method according to claim 1, characterized in that, The sweet potato variety mentioned is "Watermelon Red".

3. The method according to claim 1, characterized in that, Step (5) involves inducing shoot tip regeneration by inoculating the detached shoot tips onto a growth medium. The medium formulation is based on standard DB65T4164-2018, with a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o Sweet potato seedlings were induced to grow from shoot tips by culturing them in a C environment for 20 days.

4. The method according to any one of claims 1-3, characterized in that, Step (7) induces single sweet potato seedlings to become clump-forming plants: the obtained sweet potato seedlings are inoculated into a bud induction medium, and the culture environment is characterized by a light intensity of 16000 lux, a photoperiod of 16 h, and a temperature of 22°C. o C induces the growth of axillary buds in individual sweet potato seedlings, forming clustered plants.

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