Efficient acclimatization and transplantation method for sweet potato virus-free test-tube plantlets

Abstract

The invention discloses an efficient acclimatization and transplantation method for sweet potato virus-free test-tube plantlets, which comprises the following steps: removing fibrous roots from sweet potato virus-free test-tube plantlets which are subcultured for 20-30 days through virus detection, treating the sweet potato virus-free test-tube plantlets for 24 hours through an acclimatization nutrient solution, directly planting the sweet potato virus-free test-tube plantlets into a transplantation matrix, keeping the temperature of a culture area at 28 + / -2 DEG C, keeping the relative humidity at 75-80%, and culturing for 3 days under weak light, and supplementing carbon dioxide to the culture area until the concentration is 1000-1200ppm, culturing for 20-30 days under normal illumination, transferring the sweet potato virus-free test-tube plantlet with the survival zone transplanting matrix into a hole tray, filling a seedling culture matrix to ensure that each hole of the matrix is full, watering, carrying out shading treatment for 3-5 days, and transferring to a normal illumination condition for culturing, so as to obtain the sweet potato virus-free test-tube plantlet. According to the method, the seedling age of virus-free seedling transplanting is reduced to 20-30 days, the cost is saved, the transplanting survival rate is remarkably increased, the key technical obstacle from test-tube plantlets to propagation seedlings is broken through, and the sweet potato virus-free test-tube plantlets can be quickly applied to production.

Description

Technical Field

[0001] The invention belongs to the field of sweet potato virus-free test tube seedling hardening and transplanting, and particularly relates to a method for efficiently hardening and transplanting sweet potato virus-free test tube seedlings. Background Art

[0002] As an asexually propagated crop, sweet potato often degenerates due to viral infection during long-term cultivation, severely impacting yield and quality. In particular, sweet potato virus disease (SPVD), caused by the symbiotic infection of sweet potato feathery mottle virus and sweet potato chlorotic stunt virus, has a significant impact on sweet potato yield, resulting in yield losses exceeding 90% or even total crop failure. Shoot tip virus removal is currently the only effective treatment for sweet potato virus diseases, and producing high-quality, virus-free tissue culture seedlings is crucial for the healthy and sustainable development of the sweet potato industry. Our team previously conducted specialized research on the cultivation of weak regeneration genotypes and the removal of SPVD viruses, each of which has been granted national invention patents and has become a key technology for the cultivation of virus-free sweet potato seedlings. However, as growers have become increasingly receptive to virus-free seedlings, it has also been discovered that many lack the technology to transplant virus-free in vitro seedlings. This makes it difficult for them to directly import high-generation virus-free seedlings from seedling cultivation units for propagation, forcing them to purchase production seedlings from seedling companies. The problem is that existing seedling companies are small in scale and insufficient in number, and the production of virus-free and expanded seedlings is small, which is difficult to meet market demand, resulting in large-scale growers being unable to purchase virus-free seedlings for sweet potato production in a timely manner.

[0003] Efficient seedling hardening and transplanting technology is a key step in the production and utilization of virus-free seedlings. Existing technologies have low transplant survival rates, primarily due to insufficient standardization and poor repeatability, which prevent them from guaranteeing high survival rates. The initial cost of producing virus-free sweet potato test-tube seedlings is very high, and failure to achieve a high survival rate results in significant losses and significantly increases subsequent costs. Simplifying and standardizing the transplanting technology for virus-free sweet potato test-tube seedlings and improving the survival rate are urgent technical challenges that need to be addressed for the large-scale industrial application of virus-free sweet potato seedlings. Summary of the Invention

[0004] In order to establish a stable and reliable seedling hardening and transplanting technology, promote the large-scale application of sweet potato virus-free seedlings, and make up for the shortcomings of existing technologies, the present invention provides a method for efficient seedling hardening and transplanting of sweet potato virus-free test tube seedlings, which reduces the seedling age of virus-free seedlings for transplanting to 20-30 days, saves costs, and significantly improves the transplanting survival rate, thus breaking through the key technical barriers from test tube seedlings to propagation seedlings, and promoting the industrial application of sweet potato virus-free test tube seedlings.

[0005] The invention is achieved through the following technical scheme: a method for efficiently hardening and transplanting virus-free sweet potato test tube seedlings, comprising the following steps: removing fibrous roots of virus-free sweet potato test tube seedlings that have been subcultured for 20-30 days after virus detection, treating the seedlings with a hardening nutrient solution for 24 hours, and directly planting the seedlings in a transplanting medium; maintaining a temperature of 28±2°C and a relative humidity of 75-80% in a culture area; cultivating the seedlings under weak light for 3 days, then supplementing the culture area with a carbon dioxide concentration of 1000-1200 ppm, and cultivating the seedlings under normal light for 20-30 days; transferring the surviving virus-free sweet potato test tube seedlings with the transplanting medium into a plug tray, filling the plug tray with a seedling culture medium to ensure that each hole is full of the medium, watering the plug tray, shading the plug tray for 3-5 days, and then transferring the plug tray to a normal light condition for cultivation.

[0006] As a preferred embodiment of the method for efficiently hardening and transplanting the virus-free sweet potato test tube seedlings of the present invention, the method for preparing the hardening nutrient solution is as follows: Weigh 500 mg of calcium nitrate, 250 mg of potassium nitrate, 250 mg of magnesium sulfate, and 40 mg of ammonium nitrate, dissolve them in ultrapure water and make up the volume to 500 ml to prepare solution 1; weigh 11 mg of ferrous sulfate heptahydrate and 15 mg of disodium ethylenediaminetetraacetic acid, dissolve them in pure water and mix well to make up the volume to 400 ml to prepare solution 2; weigh 1 mg of naphthaleneacetic acid, dissolve it in anhydrous ethanol, and make up the volume to 100 mg with ultrapure water to prepare solution 3; mix solution 1, solution 2, and solution 3 in a volume ratio of 5:4:1, and mix well to obtain the seedling hardening nutrient solution.

[0007] As a preferred embodiment of the method for efficiently hardening and transplanting the virus-free sweet potato test tube seedlings of the present invention, the method for preparing the transplanting matrix is ​​as follows: Select vermiculite with a particle size of 2-3 mm, add 5 g of Bacillus subtilis, 3 g of Trichoderma harzianum and 200 ml of tap water per liter of vermiculite, stir evenly and put into a sealed barrel, and ferment under normal sunlight conditions for 3 days; then add 100 ml of nutrient solution per liter of vermiculite, stir and mix to obtain the transplanting medium.

[0008] The nutrient solution was prepared as follows: 1200 mg of potassium nitrate, 800 mg of ammonium nitrate, 180 mg of magnesium sulfate, and 100 mg of potassium dihydrogen phosphate were weighed, dissolved in ultrapure water, and the volume was adjusted to 1000 ml to prepare the nutrient solution.

[0009] As a preferred embodiment of the method for efficient hardening and transplanting of virus-free sweet potato test tube seedlings described in the present invention: the weak light culture refers to a 30 W full-spectrum LED lamp installed on the culture rack, and during the culture stage, the light intensity is 1000-1200 lx.

[0010] As a preferred embodiment of the method for efficient hardening and transplanting of virus-free sweet potato test tube seedlings described in the present invention: the normal light culture refers to a 30 W full-spectrum LED lamp installed on the culture rack, and during the culture stage, the light intensity is 3300-3500 lx.

[0011] As a preferred embodiment of the method for efficiently hardening and transplanting the virus-free sweet potato test tube seedlings of the present invention, the preparation method of the seedling culture medium is as follows: Vermiculite, coconut coir, and garden soil were mixed in a volume ratio of 1:3:1. 50 g of Bacillus subtilis and 50 g of Trichoderma harzianum were added to every 100 L of the mixture. 20 L of water was added and stirred. The mixture was covered with plastic film and placed in a greenhouse under sunlight at room temperature for 5 days to prepare the seedling medium.

[0012] The invention has the beneficial effects of reducing the transplanting age of virus-free sweet potato test tube seedlings to 20-30 days, saving costs, significantly improving the transplanting survival rate, breaking through the key technical barriers from test tube seedlings to propagated seedlings, facilitating the rapid application of virus-free test tube seedlings in production, and meeting the needs of seed potato seedling breeding enterprises to provide high-generation virus-free seedlings, as well as meeting the needs of large-scale growers to construct seedling nurseries. Compared with existing methods, the invention has good repeatability, high technical maturity, and a clear and simple operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram of the growth of virus-free sweet potato test tube seedlings in transplanting medium; Figure 2 This is a diagram of the growth of virus-free sweet potato test tube seedlings in the seedling medium. DETAILED DESCRIPTION

[0014] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0015] Example 1: Efficient hardening and transplanting of virus-free sweet potato test tube seedlings

[0016] The virus-free sweet potato test tube seedlings that have been subcultured for 20-30 days after virus detection were removed from the fibrous roots and treated with seedling nutrient solution for 24 hours. They were then directly planted in the transplanting medium. The temperature in the culture area was maintained at 28±2℃ and the relative humidity was maintained at 75-80%. They were cultured under weak light for 3 days, and then the carbon dioxide concentration in the culture area was supplemented to 1000-1200 ppm. They were cultured under normal light for 20-30 days (see Figure 1 ), transfer the virus-free sweet potato seedlings with transplanting matrix into the hole tray, fill the seedling matrix to ensure that each hole is full, water, shade for 3-5 days, and then transfer to normal light conditions for cultivation (see Figure 2 ).

[0017] The preparation method of the seedling hardening nutrient solution is as follows: weighing 500 mg of calcium nitrate, 250 mg of potassium nitrate, 250 mg of magnesium sulfate, and 40 mg of ammonium nitrate, dissolving them in ultrapure water to a volume of 500 ml, to prepare solution 1; weighing 11 mg of ferrous sulfate heptahydrate and 15 mg of disodium ethylenediaminetetraacetic acid, dissolving them in pure water, mixing them evenly, and adjusting the volume to 400 ml, to prepare solution 2; weighing 1 mg of naphthaleneacetic acid, dissolving it in anhydrous ethanol, and adjusting the volume to 100 mg with ultrapure water, to prepare solution 3; and mixing solution 1, solution 2, and solution 3 in a volume ratio of 5:4:1, and mixing them evenly to obtain the seedling hardening nutrient solution.

[0018] The preparation method of the transplanting matrix is ​​as follows: vermiculite with a particle size of 2-3 mm is selected, 5 g of Bacillus subtilis and 3 g of Trichoderma harzianum are added per liter of vermiculite, and 200 ml of tap water are added, the mixture is stirred evenly, and the mixture is placed in a sealed barrel, and fermented under normal sunlight conditions for 3 days; then, 100 ml of nutrient solution is added per liter of vermiculite, and the mixture is stirred evenly to obtain the transplanting matrix.

[0019] The nutrient solution was prepared as follows: 1200 mg potassium nitrate, 800 mg ammonium nitrate, 180 mg magnesium sulfate, and 100 mg potassium dihydrogen phosphate were weighed and dissolved in ultrapure water to a volume of 1000 ml.

[0020] The low-light culture is as follows: refers to a 30 W full-spectrum LED lamp installed on the culture rack. During this culture stage, only one light needs to be turned on, and the light intensity is 1000-1200 lx.

[0021] Normal lighting refers to the 30 W full-spectrum LED light installed on the culture rack. During this culture stage, all three lights are turned on, and the light intensity is 3300-3500 lx.

[0022] The seedling medium preparation method is as follows: vermiculite, coconut coir, and garden soil are mixed in a volume ratio of 1:3:1, 50 g of Bacillus subtilis and 50 g of Trichoderma harzianum are added to every 100 L of the mixture, 20 L of water is added, stirred and mixed, covered with plastic film, and left in a greenhouse under sunlight at room temperature for 5 days to obtain the seedling medium.

[0023] Verification experiment 1: Effects of hardening nutrient solution on the growth and survival rate of virus-free sweet potato test tube seedlings

[0024] Weigh 500 mg of calcium nitrate, 250 mg of potassium nitrate, 250 mg of magnesium sulfate, and 40 mg of ammonium nitrate, dissolve them in ultrapure water to a volume of 500 ml to obtain solution 1; weigh 11 mg of ferrous sulfate heptahydrate and 15 mg of disodium ethylenediaminetetraacetic acid, dissolve them in pure water, mix well, and adjust the volume to 400 ml to obtain solution 2; weigh 1 mg of naphthaleneacetic acid, dissolve it in anhydrous ethanol, and adjust the volume to 100 mg with ultrapure water to obtain solution 3; mix solution 1, solution 2, and solution 3 in a volume ratio of 5:4:1 and mix well to obtain a seedling hardening nutrient solution. After removing the roots of virus-free sweet potato test tube seedlings from subcultures for 20-30 days, they were incubated in a hardening nutrient solution and an aqueous solution for 24 hours, then transplanted into a transplanting medium. The incubation area was maintained at a temperature of 28 ± 2°C and a relative humidity of 75-80% under low light conditions for 3 days. Afterwards, the incubation area was supplemented with a carbon dioxide concentration of 1000-1200 ppm and incubated under normal light conditions. Root formation and survival rates were assessed after 10 days (see Table 1). The control group (water) used the conventional hydroponic method, replacing the hardening nutrient solution with water. All other conditions remained unchanged. The variety used in this experiment was Pushu 32, a popular fresh food variety on the market.

[0025] Table 1 shows the growth and survival rate of virus-free sweet potato seedlings treated with nutrient solution for 24 h.

[0026] .

[0027] The results showed that adding nutrients, iron salts, and a root-inducing plant growth regulator to the hardened nutrient solution helped accelerate root development and enhance plant growth in virus-free sweet potato seedlings. After 10 days of cultivation, the plants grew robustly, with new leaves and a well-developed root system. In contrast, the hydroponic control (water) treatment resulted in relatively weak plants with smaller leaves, slower overall growth, and less developed root systems. The survival rate was 15% lower than that of the hardened nutrient solution treatment.

[0028] Verification experiment 2: Effects of transplanting medium on root rot and survival rate of virus-free sweet potato test tube seedlings

[0029] Vermiculite with a particle size of 2-3 mm was used. 5 g of Bacillus subtilis and 3 g of Trichoderma harzianum were added to 200 ml of tap water per liter of vermiculite. The mixture was stirred thoroughly and placed in a sealed barrel for fermentation under normal sunlight for 3 days. Then, 100 ml of a nutrient solution (1200 mg of potassium nitrate, 800 mg of ammonium nitrate, 180 mg of magnesium sulfate, and 100 mg of potassium dihydrogen phosphate, dissolved in ultrapure water to a volume of 1000 ml) was added per liter of vermiculite and stirred thoroughly to prepare the transplanting medium. Plants treated with the nutrient solution were then transplanted into the transplanting medium. The culture area was maintained at a temperature of 28 ± 2°C and a relative humidity of 75-80% under low light conditions for 3 days. Subsequently, the culture area was supplemented with a carbon dioxide concentration of 1000-1200 ppm and incubated under normal light conditions. After 20 days, root rot, survival rate, and plant growth were assessed (see Table 2 below). The transplanting medium for control group 1 was vermiculite, for control group 2 it was garden soil, and for control group 3 it was coconut coir. Other conditions remained unchanged. The variety used in the experiment was Pushu 32, a popular fresh food variety on the market.

[0030] Table 2 shows the root development and survival rate of virus-free sweet potato seedlings on different transplanting substrates

[0031] .

[0032] The results showed that the transplanting substrates in the control group 1-3, without any treatment, were not suitable for transplanting virus-free sweet potato test-tube seedlings. The bacterial strains surviving in the transplanting substrates may have weak resistance, which had a severe impact on young and tender virus-free sweet potato test-tube seedlings, resulting in poor root development and root rot, with a survival rate of no more than 55%. However, the substrate treated with biological bacteria was suitable for transplanting virus-free sweet potato test-tube seedlings. In particular, the addition of nutrient solution provided sufficient nutrition for the test-tube seedlings that survived the transplant and took root. Not only did the root system develop well, but the aboveground part grew strong, the leaves were larger, and many new leaves grew. This significantly improved the transplanting survival rate, reaching 98%, which is conducive to the transplanting and reproduction of virus-free sweet potato test-tube seedlings.

[0033] Verification experiment 3: Effects of seedling medium on the survival rate and growth of virus-free sweet potato test tube seedlings

[0034] Vermiculite, coconut coir, and garden soil were mixed in a volume ratio of 1:3:1. 50 g of Bacillus subtilis and 50 g of Trichoderma harzianum were added to every 100 L of the mixture. 20 L of water was added and stirred thoroughly. Cover with plastic film and leave in a greenhouse under sunlight at room temperature for 5 days to prepare the seedling medium. Surviving virus-free test tube sweet potato seedlings were transferred to plug trays with the medium, filled with the medium until each well was completely filled, watered, and shaded for 3-5 days before culturing under normal light conditions. Survival rates and growth were assessed after 20 days (see Table 3 below). The control groups consisted of untreated vermiculite (Control 1), coconut coir (Control 2), and garden soil (Control 3). The fresh food variety Pushu 32 was used in the experiment.

[0035] Table 3 shows the growth and survival of the virus-free sweet potato test tube seedlings 10 days after transplantation

[0036] .

[0037] The results showed that the mixed substrate treated with the biological strains was highly suitable for the growth of virus-free sweet potato seedlings in vitro. It not only promoted root development but also plant growth, providing sufficient nutrients for plant growth. However, the untreated control group showed a slightly lower survival rate due to nutrient deficiencies and the influence of bacteria. Some surviving plants died after transplantation, and showed slow and weak growth. Therefore, the combined and treated substrate was suitable for the propagation of virus-free sweet potato seedlings in vitro, achieving a 100% survival rate without damaging the root system.

[0038] The above description is only an explanation of the preferred embodiment of the present invention. Any modifications or changes made to the related invention under the same invention should be included in the scope of protection intended by the present invention.

Claims

1. A method for efficiently hardening and transplanting virus-free sweet potato test tube seedlings, characterized in that: The virus-free sweet potato test tube seedlings that have been subcultured for 20-30 days after virus detection have their fibrous roots removed and treated with seedling hardening nutrient solution for 24 hours. They are then directly planted in transplanting substrates. The temperature in the culture area is maintained at 28±2°C, and the relative humidity is maintained at 75-80%. They are cultured under weak light for 3 days, and then the carbon dioxide concentration in the culture area is supplemented to 1000-1200 ppm. After culturing under normal light for 20-30 days, the surviving virus-free sweet potato test tube seedlings with transplanting substrates are transferred to hole trays, filled with seedling substrate to ensure that each hole is full of substrate, watered, and shaded for 3-5 days. Then, they are transferred to normal light conditions for culture.

2. a kind of sweet potato virus-free test tube seedling efficient hardening and transplanting method according to claim 1, it is characterized in that: The preparation method of the seedling hardening nutrient solution is as follows: Weigh 500 mg of calcium nitrate, 250 mg of potassium nitrate, 250 mg of magnesium sulfate, and 40 mg of ammonium nitrate, dissolve them in ultrapure water and make up the volume to 500 ml to prepare solution 1; weigh 11 mg of ferrous sulfate heptahydrate and 15 mg of disodium ethylenediaminetetraacetic acid, dissolve them in pure water and mix well to make up the volume to 400 ml to prepare solution 2; weigh 1 mg of naphthaleneacetic acid, dissolve it in anhydrous ethanol, and make up the volume to 100 mg with ultrapure water to prepare solution 3; mix solution 1, solution 2, and solution 3 in a volume ratio of 5:4:1, and mix well to obtain the seedling hardening nutrient solution.

3. a kind of sweet potato virus-free test tube seedling efficient hardening and transplanting method according to claim 1, it is characterized in that: The preparation method of the transplanting matrix is ​​as follows: Select vermiculite with a particle size of 2-3 mm, add 5 g of Bacillus subtilis and 3 g of Trichoderma harzianum per liter of vermiculite, and 200 ml of tap water, stir evenly, and put it into a sealed bucket. Ferment under normal sunlight conditions for 3 days; then add 100 ml of nutrient solution per liter of vermiculite and stir to mix well to prepare the transplanting medium; The nutrient solution was prepared as follows: 1200 mg of potassium nitrate, 800 mg of ammonium nitrate, 180 mg of magnesium sulfate, and 100 mg of potassium dihydrogen phosphate were weighed, dissolved in ultrapure water, and the volume was adjusted to 1000 ml to prepare the nutrient solution.

4. a kind of sweet potato virus-free test tube seedling efficient hardening and transplanting method according to claim 1, it is characterized in that: The weak light culture refers to a 30 W full-spectrum LED lamp installed on the culture rack. During the culture stage, the light intensity is 1000-1200 lx.

5. a kind of sweet potato virus-free test tube seedling efficient hardening and transplanting method according to claim 1, it is characterized in that: The normal light culture refers to a 30 W full-spectrum LED lamp installed on the culture rack, and during the culture stage, the light intensity is 3300-3500 lx.

6. A method for efficiently hardening and transplanting sweet potato virus-free test tube seedlings according to claim 1, characterized in that: The preparation method of the seedling-raising matrix is ​​as follows: Vermiculite, coconut coir, and garden soil were mixed in a volume ratio of 1:3:

1. 50 g of Bacillus subtilis and 50 g of Trichoderma harzianum were added to every 100 L of the mixture. 20 L of water was added and stirred. The mixture was covered with plastic film and placed in a greenhouse under sunlight at room temperature for 5 days to prepare the seedling medium.

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