Method for annual propagation of virus-free sweet potato seedlings by water culture

Abstract

The application provides a method for annual propagation of virus-free sweet potato seedlings by water culture, which comprises the following steps: washing the tissue culture seedlings detected free of virus and then soaking the seedlings in a treatment solution; transplanting the pretreated tissue culture seedlings to a seedling plate in a water tank, pouring the first nutrient solution into the water tank to promote the seedlings to rapidly root, and maintaining a certain temperature, humidity and light in the water culture room; replacing the nutrient solution in the water tank with the second nutrient solution after a certain period of growth, and regularly replacing the second nutrient solution; after the tissue culture seedlings grow into plants with a certain stem node, cutting the stem node segments, soaking the stem node segments in the treatment solution, and then repeating step (2) to obtain cutting seedlings, and spraying the plants with the growth-promoting solution to promote the growth of branches; repeating step (3) to culture the plants with the cut stem segments and the cutting seedlings, and regularly spraying the plants with the foliar nutrient solution and the insecticide. The propagation method is used for the propagation of virus-free seedlings by water culture, and the propagation of the seedlings is simple, efficient and easy to manage, and the annual and efficient propagation of the virus-free sweet potato seedlings can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of crop cultivation, and relates to sweet potato seedling raising technology, in particular to a method for annual propagation of virus-free sweet potato seedlings by water culture. BACKGROUND

[0002] Sweet potato (Ipomoea batatas L.) is a plant of the Convolvulaceae family and the genus Ipomoea, and is originated from tropical South America. The tuberous roots of sweet potato can be eaten fresh, processed into vermicelli, or used as feed and industrial raw material to produce ethanol and modified starch. The industrial chain of sweet potato is long and has great development potential. Sweet potato is propagated by cutting and grafting the seedlings grown from the tuberous roots, which belongs to asexual propagation. After multiple generations of planting, virus will accumulate, resulting in yield reduction or even no yield. Studies have shown that the growth potential of virus-free seedlings is significantly enhanced, and the number of branches and stem diameter are significantly better than those of non-virus-free plants. The yield-increasing effect is significant. Therefore, breeding and popularizing virus-free seedlings is the key to high yield of sweet potato. Virus-free test tube seedlings (tissue culture seedlings) can be obtained by culturing the stem tips of about 0.2-0.3 mm of sweet potato on tissue culture medium.

[0003] The propagation of virus-free sweet potato seedlings mainly includes three stages. The first stage is to propagate virus-free tissue culture seedlings by tissue culture. The second stage is to transplant the tissue culture seedlings to the substrate for acclimation in the room. The third stage is to transplant the acclimated seedlings to the greenhouse or net shed for grafting and propagation, and to harvest the tubers as seed potatoes for the next year's seedling raising. The propagation of virus-free sweet potato tissue culture seedlings mainly involves cutting the stem segments with axillary buds on a clean bench, and transferring them to a culture medium containing MS and growth regulators for propagation. This method has the following problems: first, the traditional propagation of virus-free seedlings can only be carried out in a clean bench in the room, which requires high technical requirements for the operators, otherwise batch contamination may occur; second, the culture medium is generally MS, which requires 14 kinds of analytical pure chemical reagents, white sugar and carrageenan (or agar), and the preparation process is complicated. In addition, high-temperature and high-pressure sterilization is required, which is costly. Third, under tissue culture conditions, sweet potato seedlings grow slowly due to the limited space in the bottle. One seedling can only propagate 3-4 seedlings in about 30 days, and the seedlings are weak. In order to ensure the survival rate, the seedlings cannot be directly transplanted to the net shed or field, but need to be transplanted to the substrate in the room for acclimation first, and then transplanted to the outdoor.

[0004] Currently, some researchers have proposed pre-culturing (acclimatizing) test-tube seedlings in a substrate for later use, followed by grafting and propagation in greenhouses and polytunnels. In greenhouses, seedlings are transplanted into 50-cell trays using vermiculite as a substrate, with nutrient solution added to the bottom of the trays for cultivation. In polytunnels, seedlings are directly planted in raised beds. However, this method is limited by external weather conditions and cannot achieve year-round propagation. Furthermore, the risk of aphid infestation and viral infection increases when seedlings grow outdoors. Additionally, some researchers have proposed directly transplanting sweet potato tissue culture seedlings to a netted greenhouse nursery to improve transplanting efficiency, eliminating the indoor acclimatization process. However, the survival rate depends on weather conditions, requiring an atmospheric temperature of 19–24℃ and a soil temperature of 17–23℃. Pre-transplanting watering is necessary to achieve a soil moisture content of 13–14%. If the temperature is too high, different densities of holes need to be punched in the film at different times to regulate the temperature and humidity under the greenhouse film. However, the large fluctuations in external temperature and humidity make this practically difficult to implement.

[0005] Therefore, traditional methods of propagating virus-free sweet potato seedlings have problems such as high cost, long time consumption, and low efficiency. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide a method for year-round hydroponic propagation of virus-free sweet potato seedlings. This propagation method uses hydroponics to propagate virus-free seedlings, which can reduce the amount of tissue culture seedlings used in the first stage and eliminate the second stage of acclimatization process, making seedling propagation simple, efficient and easy to manage, and can achieve year-round efficient propagation of virus-free sweet potato seedlings.

[0007] To achieve the above objectives, the present invention provides a method for year-round hydroponic propagation of virus-free sweet potato seedlings, comprising:

[0008] (1) Pretreatment of tissue culture seedlings

[0009] Remove the bottom roots of the tissue culture seedlings that have been tested and found to be virus-free, wash the plants, and then soak the cut surfaces in the treatment solution.

[0010] (2) Transplanting of tissue culture seedlings

[0011] Inject the first nutrient solution into the water tank, with the liquid level of the first nutrient solution 1-2 cm above the seedling board above the water tank. Transplant the pretreated tissue culture seedlings onto the seedling board, ensuring that the cut end of the tissue culture seedling is submerged in the first nutrient solution. Maintain a certain temperature, humidity, and light exposure above the seedling board in the room. After a certain period of growth, replace the nutrient solution in the water tank with the second nutrient solution and replace the second nutrient solution regularly.

[0012] (3) Splicing and propagation

[0013] After the tissue culture seedlings have grown into plants with certain stem nodes, they are cut into segments with 2 to 4 stem nodes until the plant has 2 to 4 stem nodes remaining. The leaves of the cut segments are removed, and the remaining segments are sprayed with growth promoting solution. The stem segments are soaked in the treatment solution and then the step (2) is repeated for culture treatment to obtain the cut seedlings.

[0014] (4) Growth Management

[0015] Repeat step (3) with the remaining plants and the cut seedlings after the stem nodes have been pruned, and regularly spray the leaves with nutrient solution and insecticide.

[0016] In the technical solution adopted in this invention, after cleaning the tissue culture seedlings that have been tested and found to be virus-free, soaking them in a treatment solution can promote root growth, improve disease resistance, and ensure survival rate. The inventors utilize the characteristic that sweet potato plants can grow indefinitely under suitable temperature, light, and nutrient conditions. They provide light sources, maintain suitable temperature and humidity for sweet potato growth, provide nutrients through hydroponics to facilitate absorption by the sweet potato roots, and simultaneously regulate plant development to promote branching growth and axillary bud seedling formation. They then graft and transplant stem segments with axillary buds to rapidly propagate virus-free seedlings. This propagation method, through repeated cultivation in steps (2) and (3), can not only propagate virus-free seedlings year-round, but is also simple to operate, low in cost, and highly efficient. Compared to the traditional tissue culture propagation method, for virus-free seedlings propagated through this technology, grafting plants with 2-3 segments can directly transplant them into a net greenhouse without the need for indoor substrate acclimatization, further saving time and improving propagation efficiency.

[0017] As a technical solution of the present invention, the pretreatment of the tissue culture seedlings includes using RT-PCR or qRT-PCR to detect the virus removal status of the tissue culture seedlings, then cutting off the roots, removing the yellowed old leaves at the bottom, pruning the tissue culture seedlings into segments, rinsing with running water, and soaking the bottom cuts in the treatment solution for no more than 48 hours.

[0018] As a technical solution of the present invention, the treatment solution comprises 80-120 mg / L of vitamin C, 100-150 mg / L of disodium copper ethylenediaminetetraacetate, 400-600 mg / L of 50 wt.% chlorobromoisocyanuric acid, and 4-6 mg / L of aminoethyl ester. The first nutrient solution comprises a modified 1 / 20 MS mineral nutrient solution ratio, 2-4 mg / L of abscisic acid, 4-6 mg / L of aminoethyl ester, and 1-20 mg / L of 50 wt.% chlorobromoisocyanuric acid. The second nutrient solution comprises a modified 1 / 4 MS mineral nutrient solution ratio, 4-6 mg / L of aminoethyl ester, and 1-20 mg / L of 50% chlorobromoisocyanuric acid. The growth promoter... The influent includes 10-35 mg / L of aminoethyl ester, 10-15 mg / L of 6-benzyladenine, 36-72 μg / L of brassinolide, 20-30 mg / L of gibberellin, and 10-20 mg / L of sodium nitrophenolate. The foliar nutrient solution includes 1.00-2.50 g / L of NH4NO3, 1.0-3.0 g / L of KNO3, 0.1-1.0 g / L of CaCl2, 0.1-1.0 g / L of MgSO4, 0.2-1.0 g / L of 50 wt.% chlorobromoisocyanuric acid, and 2-3 mL / L of 0.5 wt.% chitosan. The insecticide includes 1-10 wt.% cypermethrin. As a technical solution of the present invention, the pH of the first nutrient solution is adjusted to 5.8-6.2 using hydrochloric acid, and the first nutrient solution is pumped into the water tank.

[0019] As a technical solution of the present invention, after planting the tissue culture seedlings on the seedling plate, the seedling plate is covered with a transparent plastic cover and cultured for 5 to 7 days. After 20 days of culture, the nutrient solution in the water tank is replaced with the second nutrient solution. The second nutrient solution is replaced every 14 to 21 days.

[0020] As a technical solution of the present invention, the water tank is maintained at a temperature of 20-25°C, a humidity of 60-80%, a light intensity of 4000-6000 lex, and a light cycle of 10 hours of light followed by 14 hours of darkness.

[0021] As a technical solution of the present invention, the foliar nutrient solution is sprayed once every 7 to 14 days, and the insecticide is fumigated once every 30 days.

[0022] As a technical solution of the present invention, the seedling board is a perforated black polypropylene board with a thickness of 0.5-1.0 cm, a hole diameter of 1 cm, a hole spacing of 4-6 cm, and a cross is made at the tape position of the hole after sealing the hole with black tape.

[0023] As a technical solution of the present invention, the water tank is made of white polypropylene boards spliced ​​and glued together. The interior of the water tank is divided into two compartments by using white polypropylene boards as partitions, and liquid inlets and liquid outlets are respectively provided in the different compartments.

[0024] As a technical solution of the present invention, multiple water tanks are arranged on a frame, and the frame is made of multiple metal pipes arranged horizontally and vertically and fixed to form a multi-layer structure, with 2 to 4 LED tubes in each layer. Attached Figure Description

[0025] Figure 1 The frame used in the method for year-round hydroponic propagation of virus-free sweet potato seedlings according to the present invention.

[0026] Figure 2 This is a schematic diagram of part of the frame used in the method for year-round hydroponic propagation of virus-free sweet potato seedlings according to the present invention.

[0027] Figure 3 The water tank used in the method for year-round hydroponic propagation of virus-free sweet potato seedlings according to the present invention.

[0028] Figure 4 This is a seedling board used in the method for year-round hydroponic propagation of virus-free sweet potato seedlings according to the present invention.

[0029] Figure 5 This is the growth of potatoes after soaking in DA-6 in Example 2.

[0030] Figure 6 This is the second example of potato growth after soaking in DA-6 in Example 2.

[0031] Figure 7 This shows the growth of sweet potatoes after ABA soaking in Example 2.

[0032] Figure 8 This shows the growth of the plant in Example 3.

[0033] Figure 9 This is the condition of the transplanted potato 15 test tube seedlings after 6 days of hydroponic growth in Example 4.

[0034] Figure 10 This is the condition of the cut seedlings in Example 4 after 10 days of cultivation in the first nutrient solution.

[0035] Figure 11 This is the result of culturing the cut seedlings of potato 15 for 30 days in Example 4.

[0036] Component conformity specifications

[0037] 100 - Shelf; 10 - Water tank; 101 - Horizontal pipe; 103 - Vertical pipe; 11 - Polypropylene board; 13 - Liquid outlet; 15 - Liquid inlet; 30 - Seedling board; 31 - Hole Detailed Implementation

[0038] This invention provides a method for year-round hydroponic propagation of virus-free sweet potato seedlings, which is applicable to year-round propagation of virus-free sweet potato seedlings, including pretreatment of tissue culture seedlings, transplanting of tissue culture seedlings, grafting propagation, and growth management.

[0039] The pretreatment of tissue culture seedlings includes removing the bottom roots of virus-free seedlings, washing the plants, and then soaking the cut surfaces in the treatment solution. Virus removal in tissue culture seedlings can be detected using RT-PCR or qRT-PCR, primarily detecting sweet potato leaf curl virus (SPLCV), sweet potato chlorosis dwarf virus (SPCSV), and sweet potato feather mottle virus (SPFMV). The tissue culture seedlings used in the implementation examples include Mianshu 15, Mianshu 16, Mianshu 25, Shangshu 19, and Putuo 32. After removing the roots and yellowed old leaves from the bottom of the virus-free seedlings, the seedlings are trimmed into segments of approximately 10cm. After rinsing with running water, the bottom cut surfaces are soaked in the treatment solution for no more than 48 hours; prolonged soaking can cause phytotoxicity.

[0040] The treatment solution includes 80–120 mg / L of vitamin C, 100–150 mg / L of disodium ethylenediaminetetraacetate (EDTA), 400–600 mg / L of 50 wt.% chlorobromoisocyanuric acid, and 4–6 mg / L of aminoethyl ester. Further, the treatment solution includes 100 mg / L of vitamin C, 125 mg / L of disodium ethylenediaminetetraacetate (EDTA), 500 mg / L of 50 wt.% chlorobromoisocyanuric acid, and 4–6 mg / L of aminoethyl ester. Soaking tissue culture seedlings in this treatment solution after pruning can sterilize, promote root growth, and prevent browning. This treatment solution has bactericidal, root-promoting, and browning-preventing effects, and is easy to store; it can still be used after 60 days at room temperature without mold or moss growth.

[0041] Transplanting tissue culture seedlings involves injecting the first nutrient solution into a water tank, with the liquid level of the first nutrient solution 1-2 cm above the planting board. The pretreated tissue culture seedlings are then transplanted onto the planting board, ensuring that the cut end of the seedling is submerged in the first nutrient solution. A certain temperature, humidity, and light exposure above the planting board are maintained in the room. After a certain period of growth, the nutrient solution in the water tank is replaced with the second nutrient solution, which is then replaced regularly.

[0042] The first nutrient solution uses hydrochloric acid to adjust the pH to 5.8–6.2. It is pumped into the water tank, ensuring the solution level is at least 1 cm below the seedling tray. The water tank is maintained at a temperature of 20–25°C and humidity of 60–80%. The light intensity at a depth of approximately 10–15 cm above the seedling tray is maintained at 4000–6000 Lex, with a photoperiod of 10 hours of light followed by 14 hours of darkness. If the water temperature from outdoors is low in winter, the first nutrient solution can be heated to approximately 18°C ​​before being added to the water tank, followed by transplanting and cultivation of seedlings.

[0043] The first nutrient solution comprises a modified 1 / 20 MS mineral nutrient solution ratio, 2–4 mg / L abscisic acid, 4–6 mg / L aminoethylhexanoate, and 1–20 mg / L 50 wt.% chlorobromoisocyanuric acid. More specifically, the first nutrient solution comprises a 1 / 20 MS mineral nutrient solution ratio, 2–4 mg / L abscisic acid, 4–6 mg / L aminoethylhexanoate, and 10 mg / L 50 wt.% chlorobromoisocyanuric acid. Aminoethylhexanoate (chemical name: 2-N,N-ethylaminoethylhexanoate, (DA-6)) is a cytokinin-type plant growth regulator that effectively regulates plant growth by increasing the activity of plant peroxidase and nitrate reductase, promoting chlorophyll content, and increasing photosynthetic efficiency, thereby promoting root development and improving yield and quality. Abscisic acid (also known as S-inducer, ABA) is a plant hormone that inhibits growth. It can induce leaf abscission, cause buds to enter a dormant state, promote the formation of tubers and roots in potatoes and sweet potatoes, and inhibit cell elongation. Furthermore, it transmits signals in plant responses to abiotic stress, enhancing drought and salt tolerance, and strengthening cold and frost resistance. In other words, amino acid esters and abscisic acid have different mechanisms of action; the former promotes growth, while the latter inhibits it. The inventors of this invention cleverly utilize amino acid esters and abscisic acid for hydroponic seedling propagation, promoting rapid rooting of stem segments, maintaining a high survival rate, and accelerating growth.

[0044] The preparation method for the first nutrient solution is as follows. Taking a specific formula as an example, prepare a 25L solution multiplied 20 times in mother liquor tank 1: add 47.50g of nitrogen and potassium water-soluble fertilizer, 31.25mL of urea ammonium nitrate solution, 4.25g of potassium dihydrogen phosphate, 9.25g of magnesium sulfate heptahydrate, 10g of calcium nitrate, and 0.4g of DA-6 in sequence. Prepare a 25L solution multiplied 20 times in mother liquor tank 2, weighing out 3.125g and dissolving it. Prepare a 25L solution multiplied 20 times in mother liquor tank 3: dissolve 5.00g of 50w.% chlorobromoisocyanuric acid directly, and weigh out 1.00g of ABA, dissolve it with a small amount of alkali, and then pour it into the mother liquor tank. After diluting according to the ratio, transfer it to the nutrient solution tank, add hydrochloric acid to adjust the pH to 5.8-6.2, start the water pump and UV sterilization lamp, and then transfer it to the water tank for cultivating sweet potato seedlings, ready for use.

[0045] After transplanting tissue culture seedlings or stem segment seedlings to the planting board, cover the board with a transparent plastic lid to maintain humidity. After 5-7 days, once rooting begins, uncover the lid and continue cultivation. After 20 days, replace the nutrient solution in the water tank with a second nutrient solution, which should be changed every 14-21 days. The second nutrient solution consists of a 1 / 4 MS mineral nutrient solution ratio, 4-6 mg / L of aminoethyl ester, and 1-20 mg / L of 50% chlorobromoisocyanuric acid. Alternatively, the second nutrient solution may consist of a 1 / 4 MS mineral nutrient solution ratio, 4-6 mg / L of aminoethyl ester, and 10 mg / L of 50% chlorobromoisocyanuric acid. The second nutrient solution primarily provides the nutrients required for growth.

[0046] The preparation method of the second nutrient solution is the same as that of the first nutrient solution. The components and dosages are shown in Table 1.

[0047] The first and second nutrient solutions were modified from the conventional MS mineral nutrient solution formulation, as shown in Table 1. Agricultural potassium nitrate, urea ammonium nitrate, potassium dihydrogen phosphate, calcium nitrate, and magnesium sulfate heptahydrate were used to ensure that the molar ratios of macroelements such as nitrogen, phosphorus, potassium, calcium, and magnesium were consistent with those of conventional MS. EDTA chelated micronutrient water-soluble fertilizer was added at 0.125 mL / L. This modification not only does not affect the growth of sweet potato seedlings, but also simplifies the preparation process and reduces the cost to only 40-45% of that of conventional MS.

[0048] Table 1. MS mineral nutrient solution ratio for the first and second nutrient solutions.

[0049]

[0050] The method for year-round hydroponic propagation of virus-free sweet potato seedlings of the present invention uses multiple water tanks 10, which are arranged on a frame 100. The frame is constructed of multiple metal pipes arranged horizontally and vertically and fixed to form a multi-layer structure (e.g., ...). Figure 1(As shown). The frame can be made of aluminum alloy or stainless steel tubing, with a thickness sufficient to support the weight of the water tank, nutrient solution, and tissue culture seedlings. The tubing size can be 4cm x 4cm. Considering the number of layers, the horizontal tubing 101 is cut to 120cm in length and connected to the vertical tubing 103 with screws after drilling holes inside, forming one compartment on the long side of the frame. The number of compartments (frame length) is determined by the area of ​​the hydroponic room. Alternatively, the horizontal tubing 101 can be cut to 70-80cm in length and connected to the vertical tubing 103 with screws after drilling holes inside, forming the wide side of the frame. A horizontal bar is installed every 4cm to form a plane supporting the weight of the nutrient solution tank. The frame is designed with four layers, each spaced 50-55cm apart, with the bottom layer approximately 70cm from the ground. The horizontal bars support the weight of the water tank, nutrient solution, planting board, and seedlings. After placing one end against the wall, the other end of the shelf should be 1.5–2.0m away from the wall. Each shelf should have a net height of 45–50cm, with the bottom shelf 70cm high. The shelves should be spaced 0.8–1.0m apart to facilitate movement and operation. The shelves can be made of aluminum alloy or stainless steel tubing, with a thickness sufficient to support the weight of the water tank, nutrient solution, and seedlings. Each shelf should have 2–4 LED lights. Figure 2 As shown, the bottom of each shelf is a water tank, and the top is equipped with light tubes. Full-spectrum LED light tubes are used as the light source. The light tubes are evenly distributed on the top of each shelf. The light intensity at a position d (10-15cm) above the seedling board can reach 4000-6000 lex. The setting is 10h light / 14h darkness.

[0051] like Figure 3As shown, the water trough 10 used for transplanting tissue culture seedlings can be made of white polypropylene boards spliced ​​and glued together. The interior of the water trough 10 is divided into two compartments by white polypropylene boards 11 as partitions, with inlets 15 and outlets 13 in each compartment. Specifically, the water trough is made of white polypropylene boards 10 glued together, and the thickness of the white polypropylene boards is 2cm. The boards are cut to a width of 11cm. The length of the boards is based on the total length of the horizontal sides of the frame to form the long side of the water trough, and the width of the boards is based on the width of the frame to form the wide side of the water trough. The boards need to be about 2-4cm shorter than the long and wide sides of the frame to facilitate placement on the horizontal support of the frame. The length and width of the boards are determined according to the dimensions after the water trough is glued together. A polypropylene board 11 is glued to the bottom of the water tank 10 in the middle to form a partition. The inlet 15 and outlet 13 (approximately 2.5cm in diameter) of the water tank 10 are located on opposite sides of the partition, with the inlet 15 on the wider side wall and the outlet 13 at the bottom, allowing for more even water intake and output. A 2.5cm diameter PVC pipe is connected to the inlet 15. A valve is installed at the inlet 15 of each water tank, and a 2.5cm diameter PVC pipe is connected to the outlet 13. A rubber piston is used to plug the outlet 13 to store the nutrient solution. A fine wire mesh is used to prevent root debris from clogging the pipes during drainage, with one end inserted into the outlet 13 and the other end clipped to the water tank's filter screen. A 2cm thick polypropylene strip is glued to the wall of the water tank, 2cm from the top, to hold the seedling tray.

[0052] The planting board 30 is made of perforated black polypropylene board. The thickness of the planting board 30 is 0.5-1.0cm, the diameter of the holes is 1cm, and the spacing between the holes is 4-6cm. After sealing the holes with black tape, a cross is made at the tape position of the hole 31 to help stabilize the newly planted sweet potato seedlings and allow them to expand automatically as the stem thickness increases.

[0053] The propagation process involves cutting the tissue culture seedlings into segments with 2 to 4 segments after they have grown into plants with certain stem nodes. This process continues until the plant has 2 to 4 stem nodes remaining. The leaves of the cut segments are removed, and the remaining plants are sprayed with a growth-promoting solution. The cut ends of the stem segments are then soaked in a treatment solution, and step (2) is repeated for further cultivation to obtain the cut seedlings.

[0054] The growth-promoting solution includes 10–35 mg / L of aminoethyl ester, 10–15 mg / L of 6-benzyladenine, 36–72 μg / L of brassinolide, 20–30 mg / L of gibberellin, and 10–20 mg / L of sodium nitrophenolate. Further, the growth-promoting solution includes 20 mg / L of aminoethyl ester, 10–15 mg / L of 6-benzyladenine, 36–72 μg / L of brassinolide, 20–30 mg / L of gibberellin, and 10–20 mg / L of sodium nitrophenolate. This growth-promoting solution is beneficial for promoting the growth of axillary buds in the aboveground parts and forming new branches. Furthermore, when the tissue culture seedlings have grown to 5-10 stem nodes (with axillary buds), cut off 1-3 stem segments with 2-3 stem nodes below the top. The mother plant retains 2-4 stem nodes and removes the leaves so that new plants can grow from the axillary buds of the stem nodes for the next cutting propagation. After grafting, spray the mother plant with a growth promoter and transplant for propagation.

[0055] Growth management includes repeating step (3) with the remaining plants and cut seedlings after the stem nodes have been pruned, and regularly spraying foliar nutrient solution and pesticides.

[0056] The foliar nutrient solution includes 1.00–2.50 g / L of NH4NO3, 1.0–3.0 g / L of KNO3, 0.1–1.0 g / L of CaCl2, 0.1–1.0 g / L of MgSO4, 0.2–1.0 g / L of 50 wt.% chlorobromoisocyanuric acid, and 2–3 mL / L of 0.5 wt.% chitosan. Further, the foliar nutrient solution includes 1.65 g / L of NH4NO3, 1.9 g / L of KNO3, 0.332 g / L of CaCl2, 0.37 g / L of MgSO4, 0.2–1.0 g / L of 50 wt.% chlorobromoisocyanuric acid, and 2–3 mL / L of 0.5 wt.% chitosan. This foliar nutrient solution effectively provides the nutrients needed for the growth of the aboveground stems and leaves of virus-free seedlings, while also improving virus resistance. Insecticides include cypermethrin at a concentration of 1–10 wt.%. More specifically, insecticides include cypermethrin at a concentration of 5 wt.% to control aphids. Of course, other aphid-controlling agents, such as imidacloprid, can also be used, with dosage according to the instructions. Foliar nutrient solution is sprayed every 7–14 days, and insecticide fumigation is performed once every 30 days. After transplanting cut seedlings, a noticeable root system will appear approximately 3–7 days later. After 20 days, the second nutrient solution is replaced, and thereafter replaced every 14–21 days. Foliar nutrient solution is sprayed every 7–14 days. After each thinning, a growth-promoting solution is sprayed once, and fumigation is performed once every 30 days to prevent aphids from entering the hydroponic room and spreading viruses.

[0057] To better illustrate the purpose, technical solution, and beneficial effects of this invention, the invention will be further described below with reference to specific embodiments. It should be noted that the methods described below are further explanations of this invention and should not be construed as limiting it.

[0058] Example 1 - Pretreatment of Tissue Culture Seedlings

[0059] Tissue culture seedlings of varieties Mianshu 15, Mianshu 16, Mianshu 25, Shangshu 19, and Putuo 32, which were free of sweet potato leaf curl virus (SPLCV), sweet potato chlorosis dwarf virus (SPCSV), and sweet potato feather mottle virus (SPFMV) as detected by RT-PCR, were selected. Roots were removed, and yellowed old leaves at the base were removed. The seedlings were then trimmed into sections approximately 10 cm in length, rinsed under running water, and soaked in a pretreatment solution at the bottom cuts overnight, but not exceeding 48 hours. The treatment solution consisted of 100 mg / L vitamin C, 125 mg / L copper disodium EDTA, 500 mg / L 50 wt.% chlorobromoisocyanuric acid, and 4 mg / L aminoethyl ester.

[0060] Example 2 - Preparation of the first and second nutrient solutions

[0061] (1) Using segments of potato 15 and potato 16 with one axillary bud as materials, they were first soaked in the pretreatment solution for 12 hours. 2 mg / L, 4 mg / L, 6 mg / L, and 8 mg / L LDA-6 were added to a modified 1 / 20 MS mineral nutrient solution, respectively, with 0.2 mg / L of the traditional rooting agent naphthaleneacetic acid (NAA) as the control (CK). The results after 6 days of hydroponics are as follows... Figure 5 As shown, the top row represents sweet potato 15 and the bottom row represents sweet potato 16. It can be seen that compared to the control (CK), DA-6 significantly promotes rooting of sweet potato stem segments, outperforming CK in the 2–8 mg / L range, but showing the best effect in the 4–6 mg / L range. Treatment of sweet potato segments with the conventional rooting agent NAA began rooting after 3 days, the earliest rooting, but root elongation was slow, and roots were short and weak. A comprehensive analysis of root quantity, root length, and root diameter, consistent with morphological appearance, showed that the 4–6 mg / L DA-6 treatment produced the best rooting quality. Further... Figure 6 As shown (the size of the spheres is proportional to time, representing 9d, 12d, and 15d respectively), comparing the changes in various indicators from 5 to 15 days, it is evident that DA-6 has the characteristic of first promoting root elongation and then promoting an increase in root number. After 15 days of hydroponics, compared with the control, the number of roots in each treatment increased by 51.94–76.45%, root length increased by 40.95–85.46%, root diameter increased by 17.78–38.73%, and root weight was 1.8–2.6 times that of the control.

[0062] (2) Using stem segments of sweet potato 32 with 2-3 nodes (without leaves) as material, the segments were first soaked in pretreatment solutions for 12 hours. CK1 consisted of modified 1 / 20 MS mineral nutrient solution; CK2 consisted of modified 1 / 20 MS mineral nutrient solution + 0.2 mg / L NAA; and ABA treatment consisted of modified 1 / 20 MS mineral nutrient solution + 1-4 mg / L ABA (including 1, 2, 3, and 4 mg / L). 100 stem segments were bundled together and soaked in each treatment solution, with the nodes submerged 1-2 cm. Vigorous segments with the apex were uniformly soaked in CK1, while middle and lower segments were randomly bundled and soaked in CK2 and ABA treatment solutions. Results showed that low concentrations of ABA had a strong ability to promote rooting of sweet potato stem segments, with 2 mg / L ABA showing the best performance. Further... Figure 7 As shown, after 3 days of treatment with 2 mg / LABA, the potato 32 plant grew 2-3 cm of very healthy white roots. The segmental rooting was even better than the apical rooting of CK1. At the same time, the ABA treatment was also better than the NAA treatment of CK2. The difference was even more obvious after 5 days.

[0063] Based on the above research, the inventors of this invention developed a first nutrient solution comprising a modified 1 / 20 MS base nutrient solution, and ABA, DA-6, and chlorobromoisocyanuric acid to promote root growth and prevent disease. The first nutrient solution is a 1 / 20 MS mineral nutrient solution ratio, 2 mg / L abscisic acid, 5 mg / L aminoethyl ester, and 10 mg / L 50 wt.% chlorobromoisocyanuric acid. The second nutrient solution is a 1 / 4 MS mineral nutrient solution ratio, 5 mg / L aminoethyl ester, and 10 mg / L 50% chlorobromoisocyanuric acid.

[0064] Example 3 - Preparation of growth promotion solution

[0065] First, prepare stock solutions of 6-benzyladenine (6-BA), brassinolide (BR), and gibberellin (GA3). For 100 mL of 10 mg / mL 6-BA: Weigh 1 g, dissolve it in a few drops of 10% NaOH, and then add purified water to 100 mL. For 2 mL of 12.5 mg / mL BR: Add 2 mL of anhydrous ethanol to a bottle containing 25 mg of BR.

[0066] 100mL GA3 (20mg / mL): Weigh 2g, dissolve in a few drops of 10% NaOH, and add purified water to 100mL. To prepare 10L of growth promoter solution, add 0.2g DA-6, 10mL 6-BA stock solution, 30μL BR stock solution, 10mL GA3 stock solution, 2g 50% chlorobromoisocyanuric acid, and 0.2g sodium nitrophenolate. Pinch the top of one side of the same hydroponic plant and spray with the growth promoter solution; do not pinch the top of the other side. Figure 8The results showed that the left-side branches of the plant, even without topping, maintained strong apical dominance, inhibiting the development of axillary buds into plants. Topping the right-side branches (circled in red) weakened the inhibitory effect of the apical buds on axillary buds, resulting in the emergence of five branches after spraying with a growth-promoting solution, thus improving propagation efficiency. Therefore, a growth-promoting solution containing 20 mg / L aminoethyl ester, 10–15 mg / L 6-benzyladenine, 36–72 μg / L brassinolide, 20–30 mg / L gibberellin, and 10–20 mg / L sodium nitrophenolate can be selected.

[0067] Example 4 - Year-round hydroponic propagation of virus-free sweet potato seedlings

[0068] (1) The first nutrient solution from Example 2 was poured into the water tank, and the pH was adjusted to 6.0 using hydrochloric acid. The level of the first nutrient solution was about 1 cm below the seedling plate. The tissue culture seedlings pretreated in Example 1 were transplanted onto the seedling plate in the water tank. The plate was covered with a transparent plastic cover for 5 days of moist cultivation, and then the cover was removed. The indoor temperature was maintained at 25°C, the humidity at 70%, and the light intensity at a plane 15 cm above the plate was 5000 lux, with a photoperiod of 10 hours of light and 14 hours of darkness. The survival rate of the five varieties of test-tube seedlings (Mian Shu 15, Mian Shu 16, Mian Shu 25, Shang Shu 19, and Putu Shu 32) after transplanting into hydroponics was all above 90%. Furthermore, as Figure 9 As shown, after 6 days of hydroponic growth, the transplanted potato 15 test tube seedlings showed good growth in both the above-ground parts (left image) and the root system (right image).

[0069] (2) When the plants from step (1) have grown to 5-10 stem nodes (with axillary buds), cut off the top and 1-3 stem segments with 2-3 stem nodes below it. Retain 2-4 stem nodes from the mother plant and remove the leaves so that new plants can grow from the stem nodes for the next cutting propagation. After grafting, spray the mother plant with the growth-promoting solution of Example 3. Pour the first nutrient solution into the new water tank, with the liquid level of the first nutrient solution about 1 cm away from the seedling board. Remove all the leaves from the cut top and stem segments, soak them in the pretreatment solution for 12 hours, and then transplant them to the seedling board for cultivation. 600 stem segments (including the top) were cut from each of the 5 varieties. 100 plants were planted in the CK (conventional 1 / 20MS mineral nutrient solution + 0.2 mg / L naphthaleneacetic acid) and the first nutrient solution, with 3 replicates. The time required for 80% rooting and the seedling rate after 14 days of cultivation were recorded. The results are shown in Table 2. The results in Table 2 show that the first nutrient solution not only promoted rooting but also increased the seedling survival rate of stem segments, which was 7.45%–14.11% higher than the control (CK). There were significant differences in seedling survival rates among varieties; for example, the seedling survival rates of Mianshu 15, Mianshu 16, and Putuo 32 reached over 90% in the first nutrient solution. Figure 10 As shown, the roots of the cut potato stem segments 15 grew healthily and vigorously after being cultured in the first nutrient solution for 10 days.

[0070] Table 2 Comparison of root-promoting effects of cut seedlings

[0071]

[0072] (3) Whether it is the mother seedling or the cut seedling in step (2), after transplanting and hydroponics, replace the first nutrient solution with the second nutrient solution about 20 days later. Replace the second nutrient solution every 20 days thereafter, and spray a foliar nutrient solution every 10 days (the foliar nutrient solution consists of 1.65 g / L NH4NO3, 1.9 g / L KNO3, 0.332 g / L CaCl2, 0.37 g / L MgSO4, and 0.2–1.0 g / L 50 wt.% chlorobromoisocyanuric acid). After each cutting, spray the growth-promoting solution from Example 3 twice, with a 10-day interval between sprays. Simultaneously, fumigate with 5 wt.% cypermethrin insecticide every 30 days. This method can yield sweet potato seedlings with good plant growth. Figure 11 As shown, the seedlings of Mianshu 15 grew well after 30 days of cultivation.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, it is not limited to those listed in the embodiments. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for annual propagation of virus-free seedling of sweet potato by water culture, characterized by, The method comprises the following steps: (1) Pre-treatment of the tissue culture seedlings The tissue culture seedlings without virus are removed from the bottom roots, washed, and then the incisions are soaked with a treatment solution, which comprises 80-120 mg / L of vitamin C, 100-150 mg / L of copper ethylenediaminetetraacetate disodium salt, 400-600 mg / L of 50 wt.% chlorobromoisocyanuric acid, and 4-6 mg / L of amaranth; (2) Transplanting of the tissue culture seedlings A first nutrient solution is injected into a water tank, the liquid surface of the first nutrient solution is 1-2 cm away from the seedling plate above the water tank, the pre-treated tissue culture seedlings are transplanted onto the seedling plate, and the incisions of the tissue culture seedlings are ensured to be immersed in the first nutrient solution. The indoor temperature, humidity, and light above the seedling plate are maintained, the nutrient solution in the water tank is replaced with a second nutrient solution after a certain period of growth, and the second nutrient solution is replaced regularly. The first nutrient solution comprises a modified 1 / 20 MS mineral nutrient solution, 2-4 mg / L of abscisic acid, 4-6 mg / L of amaranth, and 1-20 mg / L of 50 wt.% chlorobromoisocyanuric acid. The second nutrient solution comprises a modified 1 / 4 MS mineral nutrient solution, 4-6 mg / L of amaranth, and 1-20 mg / L of 50 wt.% chlorobromoisocyanuric acid. The pH value of the first nutrient solution is adjusted to 5.8-6.2 with hydrochloric acid, and the first nutrient solution is input into the water tank by a water pump; (3) Cutting and propagation After the tissue culture seedlings grow into plants with a certain number of stems, the plants are cut into segments with 2-4 stems, until there are 2-4 stems left on the plants. The cut segments are removed from the leaves, the remaining plants are sprayed with a growth-promoting solution, the stem segments are soaked with a treatment solution, and then step (2) is repeated for culture and treatment, to obtain cut seedlings. The growth-promoting solution comprises 10-35 mg / L of amaranth, 10-15 mg / L of 6-benzyladenine, 36-72 µg / L of brassinolide, 20-30 mg / L of gibberellin, and 10-20 mg / L of sodium nitrophenol; (4) Growth management The remaining plants with cut stems and the cut seedlings are cultured by repeating step (3), and are regularly sprayed with a foliar nutrient solution and an insecticide. The foliar nutrient solution comprises 1.00-2.50 g / L of NH4NO3, 1.0-3.0 g / L of KNO3, 0.1-1.0 g / L of CaCl2, 0.1-1.0 g / L of MgSO4, 0.2-1.0 g / L of 50 wt.% chlorobromoisocyanuric acid, and 2-3 mL / L of 0.5 wt.% chitosan. The insecticide comprises cypermethrin with a concentration of 1-10 wt.%.

2. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, The pre-treatment of the tissue culture seedlings comprises detecting the virus removal of the tissue culture seedlings by RT-PCR or qRT-PCR, removing the roots, removing the yellow and old leaves at the bottom, trimming the tissue culture seedlings into segments, and then washing with water and soaking the incisions at the bottom with the treatment solution for no more than 48 h.

3. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, After the tissue culture seedlings are planted on the planting board, the planting board is covered with a transparent plastic cover, and after 5-7 days of culture, the cover is removed and the culture is continued, and after 20 days of culture, the nutrient solution in the water tank is replaced with the second nutrient solution, and the second nutrient solution is replaced every 14-21 days.

4. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, The temperature in the water tank is maintained at 20-25°C, the humidity is 60-80%, the light intensity is 4000-6000 lux, and the light cycle is 10 hours of light and 14 hours of darkness.

5. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, The leaf nutrient solution is sprayed every 7-14 days, and the insecticide is fumigated every 30 days.

6. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, The planting board is a black polypropylene board with holes, the thickness of the planting board is 0.5-1.0 cm, the diameter of the holes is 1 cm, the spacing between the holes is 4-6 cm, and the holes are sealed with black tape and a cross is drawn on the tape at the hole position.

7. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, The water tank is made of white polypropylene boards spliced and adhered together, the inside of the water tank is divided into two compartments by white polypropylene boards as partitions, and liquid inlets and outlets are provided in different compartments.

8. The method for annual propagation of virus-free sweet potato seedling in water culture according to claim 1, characterized in that, Multiple water tanks are arranged on a shelf, the shelf is formed by horizontally and vertically arranging and fixing multiple metal pipes to form a multi-layer structure, and 2-4 LED light tubes are provided on each layer.

Images