Preparation method of detoxified sweet potato seedling

Abstract

The application provides a preparation method of detoxified sweet potato seedlings, comprising the following cultivation steps: washing sweet potatoes, placing the sweet potatoes in a sterilization liquid to accelerate germination, disinfecting the newly born stem tips, placing the stem tips in a culture medium I to culture, and obtaining sweet potato stem tips; when the sweet potato stem tips grow 4-5 leaves, the stem tip meristem is inoculated into a culture medium II, and the culture is carried out to 8-9 stem segments, and sweet potato seedlings are obtained; the sweet potato seedlings are transplanted to a seedling raising tray containing a nutrient medium, and the seedlings are cultured in an artificial climate chamber for 30-35 days, and then the seedlings are transplanted into soil for breeding. The preparation method of the detoxified sweet potato seedlings significantly improves the growth condition of the sweet potato seedlings, increases the plant height, the number of root systems and the length of root systems of the sweet potato seedlings, and significantly improves the net photosynthetic rate (Pn) and the stomatal conductance (Gs) of the detoxified sweet potato seedlings, which is beneficial to the healthy growth of the sweet potato in the seedling stage.

Description

Technical Field

[0001] This invention relates to the field of planting, and specifically to a method for preparing virus-free sweet potato seedlings. Background Technology

[0002] Sweet potato, a plant belonging to the genus Ipomoea in the legume family, is also called yam. The description of sweet potato in the *Compendium of Materia Medica*, compiled by Li Shizhen, a Ming Dynasty physician, quotes the original text from Yang Fu's *Record of Strange Things* from the Eastern Han Dynasty: "Sweet potatoes come from Jiaoguang (Guangdong and Guangxi) in the south. People plant them in February and harvest them in October. Their roots resemble taro, and some are very large. The larger ones are like goose eggs, and the smaller ones are like chicken or duck eggs. Peel off the purple skin, and the flesh is pure white like fat. Southerners use it as a substitute for rice; the fruit, whether steamed or roasted, is fragrant and delicious. Initially very sweet, it becomes slightly less sweet after a while, especially when exposed to wind." Meanwhile, the *Compendium of Materia Medica* also quotes the original text from *Southern Plants and Trees* by Ji Han of the Jin Dynasty: "Sweet potato, a type of yam or taro. Its roots and leaves are also like taro. The root is as large as a fist, and when steamed, it tastes like yam, but is not very cold in nature. Those in Zhuyai who do not farm only grow this, steaming, slicing, drying, and harvesting it to supplement their food, calling it 'sweet potato grain.' People in the coastal areas are long-lived, also because they do not eat grains but sweet potatoes." This text describes sweet potato as a grain crop, an important economic crop, and highly popular with consumers. Currently, the cultivation of sweet potato seedlings faces the following problems: Sweet potatoes are asexually propagated crops, mainly through stems and tubers. During cultivation, the stems and leaves are easily infected by aphids, planthoppers, and other virus-carrying pests. Once infected, the virus enters the tubers through the nutrient transport system. Through years of continuous planting, the amount and types of viruses carried by the tubers gradually increase, seriously affecting the yield and quality of sweet potatoes. More than 30 types of sweet potato viruses have been reported worldwide, with sweet potato geminitrovirus and sweet potato chlorosis virus having a significant impact on sweet potato production. Symptoms of above-ground viral diseases in sweet potatoes mainly include leaf spots, mosaic patterns, leaf curling, leaf wrinkling, and leaf yellowing, with leaf spots being the most prevalent, such as purple feathery spots, purple spots, and yellow spots. Symptoms on tubers are mainly blackish-brown or yellowish-brown cracks. Given that no highly resistant sweet potato varieties have been developed domestically to date, and there are no highly effective pesticides for controlling sweet potato viral diseases, promoting the application of virus-free sweet potato seedlings is currently an effective method for controlling sweet potato viral diseases and improving sweet potato yield and quality. Traditional virus-free sweet potato seedling production is divided into three to four stages: the first year involves planting seed potatoes in a laboratory greenhouse; the second year involves field planting and harvesting high-quality seed potatoes; and the third to fourth years involve cultivating seedlings from these high-quality seed potatoes, which become commercial seedlings for sweet potato production. This method is time-consuming and highly susceptible to viral infection during the seedling stage, leading to decreased sweet potato quality, reduced yield, and seed degeneration. Summary of the Invention

[0003] Based on the existing technology, the technical problem to be solved by the present invention is to provide a method for preparing virus-free sweet potato seedlings, so as to solve the above-mentioned problem.

[0004] The technical solution of the present invention:

[0005] A method for preparing virus-free sweet potato seedlings includes the following steps:

[0006] S1: Wash the sweet potatoes, place them in a sterilization solution to promote sprouting, disinfect the new shoot tips, and culture them in culture medium I for 5-10 days to obtain sweet potato shoot tips;

[0007] S2: When the sweet potato stem tip grows 5-7 leaves, the stem tip meristem is inoculated into culture medium II and cultured until 8-9 stem segments are obtained to obtain sweet potato seedlings;

[0008] S3: Transplant sweet potato seedlings into seedling trays containing nutrient substrate, cultivate them in an artificial climate chamber for 30-35 days, and then transplant the seedlings into sterilized soil for propagation.

[0009] Furthermore, in S1, the total mass percentage of methyl thiophanate and λ-cyhalothrin in the sterilization solution is 0.02%-0.04%, and the sterilization solution is composed of methyl thiophanate and λ-cyhalothrin in a mass ratio of 1:0.1-0.2.

[0010] Further, in S1, the sprouting process involves placing sweet potatoes in a high-temperature sterilized mixed substrate for sprouting. The mixed substrate comprises coconut coir, perlite, Terminalia chebula powder, and acarbose in a mass ratio of 1-1.2:1-1.5:0.4-0.5:0.1-0.3. The sprouting temperature is 30-36℃, and the temperature is maintained for 14-16 hours. Then, the temperature is lowered to 25-27℃, and the temperature is maintained for 6-10 days.

[0011] Furthermore, the particle size of the Terminalia chebula powder is 30-40 micrometers.

[0012] Further, S1, the stem tip disinfection involves rinsing the stem tip with water 2-4 times, soaking it in 75% alcohol for 10-12 seconds, sterilizing it by soaking it in 1-1.5% sodium hypochlorite solution for 10-15 minutes, and rinsing it with water 3-5 times.

[0013] Further, S1, the culture medium I comprises the following raw materials in parts by weight: NH4H2PO3 5-7 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 3-4 g / L, Ca(NO3)2·4H2O 2-3 g / L, FeSO4·7H2O 2-2.5 g / L, IAA 0.2-0.4 g / L, cysteine ​​0.1-0.3 g / L, brassinolide 1-1.5 g / L and 6-furfurylaminopurine 0.1-0.2 g / L.

[0014] Furthermore, S1, the culture conditions of the culture medium are as follows: mix the culture medium raw materials, dilute with distilled water 8-10 times, add 3-4 g / L of agar, adjust the pH value to 5.6-6.0, autoclave at 100℃-135℃ for 40-50 min, and solidify at room temperature.

[0015] Furthermore, S2, the culture medium II comprises the following raw materials in parts by weight: KNO3 7-9 g / L, KH2PO4 2-4 g / L, NAA 0.5-0.6 g / L, 6-BA 0.4-0.5 g / L, IAA 0.2-0.3 g / L and GA3 0.1-0.3 g / L.

[0016] Furthermore, the artificial climate chamber is used for cultivation at a temperature of 26-30℃, a light intensity of 1000-1200 lux, and a light cycle of 8-8.5 h / d.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The method for preparing virus-free sweet potato seedlings in this invention significantly improves the growth of sweet potato seedlings, increasing plant height, root number, and root length. Photosynthetic characteristic experiments show that the net photosynthetic rate (Pn) and stomatal conductance (Gs) of the virus-free sweet potato seedlings prepared by this method are significantly improved. This improvement in these two indicators indicates an increased ability of the sweet potato seedlings to synthesize organic matter using light energy and a higher net photosynthetic rate. It also indicates that the plant absorbs more carbon dioxide and produces more carbohydrates during photosynthesis, thereby improving photosynthetic efficiency and seedling growth rate. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] The raw material used in this embodiment, methyl thiophanate, has the molecular formula C0.05. 12 H 14 N4O4S2, with a relative molecular weight of 342.39 and CAS number 23564-05-8, was purchased from Shanghai Ruichu Biotechnology Co., Ltd.; λ-cyhalothrin: molecular formula C 23 H 19 C lF3NO3, with a relative molecular mass of 449.85 and a purity of 95%, was purchased from Mengcheng Technology (Shanghai) Co., Ltd.; IAA: full name indoleacetic acid, chemical formula C 10 H9NO2, with a relative molecular weight of 175.184, was purchased from Hubei Bolan Chemical Co., Ltd.; NAA (1-Naphthaleneacetic acid): full name 1-naphthaleneacetic acid, molecular formula C 12 H 10 O2, with a relative molecular weight of 175.184, was purchased from Hubei Bolan Chemical Co., Ltd.; 6-BA: full name 6-benzylaminopurine, molecular formula C 12 H 11 N5, with a relative molecular weight of 225.26, was purchased from Shanghai Guchen Biotechnology Co., Ltd.; GA3 (Gibberellin A3): full name gibberellin, chemical formula is C 19 H 22 O6, with a relative molecular weight of 346.37, was purchased from Jiangxi Xinruifeng Biochemical Co., Ltd.

[0022] Example 1

[0023] The preparation of a virus-free sweet potato seedling includes the following steps:

[0024] Step (1): Wash the sweet potatoes with clean water and place them in a sterilization solution to germinate. After sterilization and germination in the sterilization solution, disinfect the new stem tips. First, rinse the stem tips with water 2-4 times. Second, soak them in 75% alcohol for 10 seconds, then sterilize them by soaking in 1% sodium hypochlorite solution for 10 minutes, and rinse them with water 3 times. After sterilization, place them in a high-temperature sterilized mixed substrate to germinate. The mixed substrate includes coconut coir, perlite, Terminalia chebula powder, and acarbose in a mass ratio of 1:1:0.4:0.1. The Terminalia chebula powder has a particle size of 30 micrometers. The germination temperature is 30℃, and the holding time is 14 hours. Then, the temperature is lowered to 25℃ and the holding time is 6 days. Finally, the sweet potatoes are placed in culture medium I for cultivation.

[0025] The disinfectant solution is composed of methyl thiophanate and λ-cyhalothrin in a mass ratio of 1:0.1, and the total mass percentage of methyl thiophanate and λ-cyhalothrin in the disinfectant solution is 0.02%.

[0026] Culture medium I comprises the following raw materials in parts by weight: NH4H2PO3 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 3 g / L, Ca(NO3)2·4H2O 2 g / L, FeSO4·7H2O 2 g / L, IAA 0.2 g / L, cysteine ​​0.1 g / L, brassinolide 1 g / L and 6-furfurylaminopurine 0.1 g / L.

[0027] Culture medium conditions: Mix the culture medium raw materials, dilute with distilled water 8 times, add 3g / L agar, adjust the pH to 5.6, autoclave at 100℃ for 40min, and solidify at room temperature.

[0028] Step (2): Five leaves grow from the sweet potato stem tip. The stem tip meristem is inoculated into culture medium II and cultured until eight stem segments are formed to obtain sweet potato seedlings. Culture medium II includes the following raw materials by weight: KNO3 7 g / L, KH2PO4 2 g / L, NAA 0.5 g / L, 6-BA 0.4 g / L, IAA 0.2 g / L and GA3 0.1 g / L.

[0029] Step (3): Transplant the sweet potato seedlings into a seedling tray containing nutrient substrate and cultivate them in an artificial climate chamber for 30 days. The temperature of the artificial climate chamber is 26℃, the light intensity is 1000 lux, and the light cycle is 8h / d. Then transplant the seedlings into sterilized soil for propagation.

[0030] Example 2

[0031] The preparation of a virus-free sweet potato seedling includes the following steps:

[0032] Step (1): Wash the sweet potatoes with clean water and place them in a sterilization solution to germinate. After sterilization and germination in the sterilization solution, disinfect the new stem tips. First, rinse the stem tips with water 4 times. Second, soak them in 75% alcohol for 12 seconds. Then, sterilize them by soaking them in 1.5% sodium hypochlorite solution for 15 minutes. Rinse them with water 5 times. After sterilization, place them in a high-temperature sterilized mixed substrate to germinate. The mixed substrate includes coconut coir, perlite, Terminalia chebula powder, and acarbose in a mass ratio of 1.2:1.5:0.5:0.3. The Terminalia chebula powder has a particle size of 40 micrometers. The germination temperature is 36℃ and the holding time is 16 hours. Then, the temperature is lowered to 27℃ and the holding time is 10 days. Finally, the sweet potatoes are placed in culture medium I for cultivation.

[0033] The disinfectant solution is composed of methyl thiophanate and lambda-cyhalothrin in a mass ratio of 1:0.2, and the total mass percentage of methyl thiophanate and lambda-cyhalothrin in the disinfectant solution is 0.04%.

[0034] Culture medium I comprises the following raw materials in parts by weight: 7 g / L NH4H2PO3, 2 g / L KH2PO4, 4 g / L MgSO4·7H2O, 3 g / L Ca(NO3)2·4H2O, 2.5 g / L FeSO4·7H2O, 0.4 g / L IAA, 0.3 g / L cysteine, 1.5 g / L brassinolide, and 0.2 g / L 6-furfurylaminopurine.

[0035] Culture medium conditions: Mix the culture medium raw materials, dilute with distilled water 10 times, add 4 g / L agar, adjust the pH to 6.0, autoclave at 135℃ for 50 min, and solidify at room temperature.

[0036] Step (2): Seven leaves grow from the sweet potato stem tip. The stem tip meristem is inoculated into culture medium II and cultured until nine stem segments are formed to obtain sweet potato seedlings. Culture medium II includes the following raw materials by weight: KNO3 9 g / L, KH2PO4 4 g / L, NAA 0.6 g / L, 6-BA 0.5 g / L, IAA 0.3 g / L and GA3 0.3 g / L.

[0037] Step (3): Transplant the sweet potato seedlings into a seedling tray containing nutrient substrate and cultivate them in an artificial climate chamber for 35 days. The temperature of the artificial climate chamber is 30℃, the light intensity is 1200 lux, and the light cycle is 8.5h / d. Then transplant the seedlings into sterilized soil for propagation.

[0038] Example 3

[0039] The preparation of a virus-free sweet potato seedling includes the following steps:

[0040] Step (1): Wash the sweet potatoes with clean water and place them in a sterilization solution to germinate. After sterilization and germination in the sterilization solution, disinfect the new stem tips. First, rinse the stem tips with water three times. Second, soak them in 75% alcohol for 11 seconds, then sterilize them by soaking in 1.2% sodium hypochlorite solution for 12 minutes, and rinse them with water four times. After sterilization, place them in a high-temperature sterilized mixed substrate to germinate. The mixed substrate includes coconut coir, perlite, Terminalia chebula powder, and acarbose in a mass ratio of 1.1:1.3:0.4:0.2. The Terminalia chebula powder has a particle size of 35 micrometers. The germination temperature is 34℃, and the holding time is 15 hours. Then, the temperature is lowered to 26℃ and the holding time is 8 days. Finally, the sweet potatoes are placed in culture medium I for cultivation.

[0041] The disinfectant solution is composed of methyl thiophanate and lambda-cyhalothrin in a mass ratio of 1:0.1, and the total mass percentage of methyl thiophanate and lambda-cyhalothrin in the disinfectant solution is 0.03%.

[0042] Culture medium I comprises the following raw materials in parts by weight: NH4H2PO3 6 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 3.5 g / L, Ca(NO3)2·4H2O 2.5 g / L, FeSO4·7H2O 2.2 g / L, IAA 0.3 g / L, cysteine ​​0.2 g / L, brassinolide 1.2 g / L, and 6-furfurylaminopurine 0.2 g / L.

[0043] Culture medium conditions: Mix the culture medium raw materials, dilute with distilled water 9 times, add 3.5 g / L agar, adjust the pH to 5.8, autoclave at 120℃ for 45 min, and solidify at room temperature.

[0044] Step (2): When the sweet potato stem tip grows 6 leaves, the stem tip meristem is inoculated into culture medium II and cultured until 8 stem segments are obtained to obtain sweet potato seedlings; wherein, culture medium II includes the following raw materials by weight: KNO3 is 8 g / L, KH2PO4 is 3 g / L, NAA is 0.5 g / L, 6-BA is 0.4 g / L, IAA is 0.2 g / L and GA3 is 0.2 g / L.

[0045] Step (3): Transplant the sweet potato seedlings onto a seedling tray containing nutrient substrate and cultivate them in an artificial climate chamber for 33 days. The temperature of the artificial climate chamber is 28℃, the light intensity is 1100 lux, and the light cycle is 8h / d. Then transplant the seedlings into sterilized soil for propagation.

[0046] Comparative Example 1

[0047] The difference between Comparative Example 1 and Example 3 is that the germination step in the sterilization solution is removed in step 1, while the other methods and steps are the same as in Example 3.

[0048] Comparative Example 2

[0049] The difference between Comparative Example 2 and Example 3 is that the ingredients of the mixed matrix in step 1 are excluding Terminalia chebula powder and acarbose, while the other methods and steps are the same as in Example 3.

[0050] Comparative Example 3

[0051] The difference between Comparative Example 3 and Example 3 is that the composition of culture medium I was modified to be the same as that of culture medium II, and the sterilization solution was modified to be a 75% alcohol solution by mass. Other methods and steps were the same as in Example 3.

[0052] test

[0053] Experiment 1: Rooting culture experiment. Healthy sweet potatoes were selected, and large and robust root tubers were chosen. The sweet potato variety was a commercially available variety (Yanshu 25). Rooting culture was carried out using the methods of Examples 1-3 and Comparative Examples 1-3. Five equilibrium experiments were conducted for each culture. After 21 days of culture, the average rooting parameters were statistically analyzed at a uniform time (see Table 1). The number of roots was measured using the ruler method.

[0054] Table 1

[0055]

[0056]

[0057] Note: a and b indicate significant differences with P < 0.05.

[0058] Experiment 2: Photosynthetic characteristics experiment. Leaves at the 3rd-4th phylum were selected as the test subjects. A Yanxin-1102 portable photosynthesis system was used to measure photosynthetic parameters, including net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (C), during the period of 10:30-11:30 on a sunny day. i An open gas path was adopted, and the photosynthetically active radiation was set at 1200 μmol / L. -2 ·s -1 Five equilibrium experiments were conducted for each culture. After 20-21 days of culture, the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (C) of the leaves were statistically analyzed at a uniform time. i ), see Table 2 for specific data.

[0059] Table 2

[0060]

[0061] Note: a and b indicate significant differences with P < 0.05.

[0062] Rooting culture experiments showed that the virus-free sweet potato seedlings cultured in Examples 1-3 had better plant height, root number, root length, and overall growth than those in Comparative Examples 1-3. Removing the germination step in the sterilization solution significantly reduced plant height, root number, root length, and overall growth. Furthermore, altering the composition of the mixed substrate and the sterilization solution significantly affected the growth of the sweet potato seedlings.

[0063] The photosynthetic characteristic experiment showed that the net photosynthetic rate (Pn) and stomatal conductance (Gs) of the virus-free sweet potato seedlings cultivated in Examples 1-3 were significantly improved. Net photosynthetic rate (Pn) directly reflects the ability of sweet potato seedlings to synthesize organic matter using light energy. The higher the net photosynthetic rate, the more carbon dioxide the plant absorbs during photosynthesis, and the more carbohydrates it produces, thus increasing biomass. This indicates that the virus-free sweet potato seedlings cultivated in Examples 1-3 significantly increased their ability to synthesize organic matter while also increasing biomass. The significant increase in stomatal conductance (Gs) indicates that the sweet potato seedlings enhanced their CO2 absorption and water use efficiency, thereby improving photosynthetic efficiency and seedling growth rate. In Comparative Examples 1-3, omitting the germination step in the sterilization solution, changing the composition of the mixed substrate, and altering the composition of the sterilization solution all significantly reduced the net photosynthetic rate (Pn) and stomatal conductance (Gs) of the sweet potato seedlings, affecting their photosynthetic efficiency. Therefore, the methods for preparing virus-free sweet potato seedlings in Examples 1-3 are beneficial to the growth of seedlings, and can effectively improve the photosynthetic efficiency of seedlings, increase their plant height and root number.

[0064] The above description is merely an example of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing virus-free sweet potato seedlings, characterized in that, The cultivation steps include the following: S1: Wash the sweet potatoes, place them in a sterilization solution to promote sprouting, disinfect the new shoot tips, and culture them in culture medium I for 5-10 days to obtain sweet potato shoot tips; The disinfectant solution is composed of methyl thiophanate and lambda-cyhalothrin in a mass ratio of 1:0.1-0.2, and the total mass percentage of methyl thiophanate and lambda-cyhalothrin in the disinfectant solution is 0.02%-0.04%. The sprouting process involves placing sweet potatoes in a high-temperature sterilized mixed substrate for sprouting. The mixed substrate includes coconut coir, perlite, Terminalia chebula powder with a particle size of 30-40 micrometers, and acarbose in a mass ratio of 1-1.2:1-1.5:0.4-0.5:0.1-0.

3. The germination temperature is 30-36℃, and the holding time is 14-16 hours. Then, the temperature is lowered to 25-27℃ and the holding time is 6-10 days. The culture medium I comprises the following raw materials in parts by weight: NH4H2PO3 5-7 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 3-4 g / L, Ca(NO3)2·4H2O 2-3 g / L, FeSO4·7H2O 2-2.5 g / L, IAA 0.2-0.4 g / L, cysteine ​​0.1-0.3 g / L, brassinosteroids 1-1.5 g / L, and 6-furfurylaminopurine 0.1-0.2 g / L; S2: When the sweet potato stem tip grows 4-5 leaves, the stem tip meristem is inoculated into medium II and cultured until 8-9 stem segments are formed to obtain sweet potato seedlings; The culture medium II comprises the following raw materials in parts by weight: KNO3 7-9 g / L, KH2PO4 2-4 g / L, NAA 0.5-0.6 g / L, 6-BA 0.4-0.5 g / L, IAA 0.2-0.3 g / L and GA3 0.1-0.3 g / L; S3: Transplant sweet potato seedlings into seedling trays containing nutrient substrate, cultivate them in an artificial climate chamber for 30-35 days, and then transplant the seedlings into soil for propagation.

2. The method for preparing virus-free sweet potato seedlings according to claim 1, characterized in that, Step S1, the disinfection of the new shoot tip, involves rinsing the shoot tip with water 2-4 times, then immersing it in a 1-1.5% sodium hypochlorite solution for 10-15 minutes for sterilization, and rinsing it with water 3-5 times after sterilization.

3. The method for preparing virus-free sweet potato seedlings according to claim 1, characterized in that, Step S3: The temperature of the artificial climate chamber is 26-30℃, the light intensity is 1000-1200 lux, and the light cycle is 8-8.5h / d.

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