Nutrient solution for sugar-free tissue culture of sweet potato virus-free seedlings

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a nutrient solution for sugar-free tissue culture of sweet potato virus-free seedlings. The sugar-free tissue culture method comprises the following steps: stripping 2-3 leaf primordia on the tip of a sweet potato stem tip, performing stem tip rapid propagation culture, screening out detoxified sweet potato seedlings, transplanting the detoxified sweet potato seedlings into a vermiculite substrate, and adding a nutrient solution for culture; in the culture process, carbon dioxide gas is introduced. The nutrient solution is prepared from the following raw materials in parts by mass: 1.6 to 3.2 parts of calcium nitrate, 0.1 to 0.5 part of monopotassium phosphate and 1000 parts of water. The invention provides a nutrient solution formula of a sugar-free group cultivation technology, the cost is reduced, the pollution rate is reduced, sweet potato seedlings are cultivated to be stronger, and common planters can cultivate the sweet potato seedlings without specific technicians.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a nutrient solution for sugar-free tissue culture sweet potato virus-free seedlings. Background Technology

[0002] Sweet potatoes are susceptible to viral infections (such as SPFMV and SPLV), leading to varietal degeneration. Traditional sweet potato tissue culture is currently the most mature technology for cultivating sweet potato seedlings. This method utilizes plant tissue culture for rapid propagation and production of virus-free seedlings. The procedure involves first obtaining 2-3 leaf primordia from the tip of a sweet potato seedling, then cultivating them to obtain virus-free seedlings. These seedlings are then expanded and propagated using traditional tissue culture techniques to obtain a large quantity of high-quality virus-free seedlings for use as virus-free seed stock. This technology is conducted in a laboratory and is not limited by seasonal factors. The seedlings are transplanted into specially designed tissue culture bottles and grown in a nutrient solution containing sugar. The basal medium is primarily MS (Murashige & Skoog), supplemented with sucrose (30 g / L) and agar (6-8 g / L), along with growth hormones such as NAA. However, this traditional sweet potato tissue culture method has the following problems:

[0003] Poor quality and high contamination rate of sweet potato seedlings: Although the presence of sugar provides the necessary carbon for seedling growth, sugar also easily breeds microorganisms and bacteria. Most viruses originate from sugar-containing nutrient solutions, resulting in poor-quality seedlings contaminated with bacteria and viruses, leading to a yield reduction of nearly 60%, severely impacting production. Furthermore, the size of traditional tissue culture bottles significantly hinders the upward growth of sweet potato seedlings, restricting their development.

[0004] High cost: Traditional sweet potato tissue culture seedling technology uses expensive tissue culture bottles, and the cost of sugar-containing nutrient solutions is also high. In addition, the transplanting of sweet potato seedlings requires high temperature and high pressure sterilization and sterile ultra-clean benches, which leads to high electricity costs, equipment wear and tear, and high time costs. Furthermore, it requires professional technicians to complete the operation, and the cost of professional technicians is also high.

[0005] Complex operation: Traditional tissue culture technology can only be carried out in a sterile environment. Because the operation requires no virus contamination, it requires highly skilled workers to handle, making it difficult to promote.

[0006] Given the high cost, technical difficulty, and high contamination rate of traditional tissue culture techniques due to the presence of sugar, it is necessary to propose a sugar-free tissue culture method for virus-free sweet potato seedlings. Summary of the Invention

[0007] Based on the above, the present invention provides a nutrient solution for sugar-free tissue culture sweet potato virus-free seedlings.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a sugar-free nutrient solution for cultivating virus-free sweet potato seedlings. By mass, the raw materials consist of 1.6-3.2 parts calcium nitrate, 0.1-0.5 parts potassium dihydrogen phosphate, and 1000 parts water.

[0010] In a preferred embodiment of the present invention, the raw materials consist of 1.6 to 3.2 parts calcium nitrate, 0.1 to 0.5 parts potassium dihydrogen phosphate, and 1000 parts water by mass.

[0011] In some embodiments of the present invention, the sugar-free nutrient solution is obtained by mixing the raw materials according to the mass proportions.

[0012] This invention also provides a sugar-free tissue culture method for virus-free sweet potato seedlings. Two to three leaf primordia are peeled off from the tip of the sweet potato stem and then propagated through rapid stem tip culture. The virus-free sweet potato seedlings are selected and transplanted onto a vermiculite substrate, and the above-mentioned nutrient solution is added for culture. During the culture process, carbon dioxide gas is introduced.

[0013] This invention uses vermiculite as a substrate, replacing agar and MS medium in traditional tissue culture, which can reduce costs by 70%.

[0014] In a preferred embodiment of the present invention, the mass ratio of nutrient solution to vermiculite matrix is ​​1:2.

[0015] In a preferred embodiment of the present invention, the culture time is 28 days.

[0016] In a preferred embodiment of the present invention, the concentration of carbon dioxide gas in the culture environment is 1400 ppm.

[0017] In the actual cultivation of virus-free sweet potato seedlings, CO2 gas released from a CO2 cylinder can be used to replace the sugar in the nutrient solution as the carbon source for the growth of sweet potato seedlings.

[0018] In a preferred embodiment of the present invention, the ambient temperature is 23-27°C and the ambient humidity is 60% during the cultivation process.

[0019] In a preferred embodiment of the present invention, during the cultivation process, the light intensity is 2000 lux and the light duration is 24 hours.

[0020] In the actual cultivation of virus-free sweet potato seedlings, an automated control system can be used to achieve real-time synchronization and cloud-based regulation of CO2, temperature, and humidity data.

[0021] The present invention discloses the following technical effects:

[0022] This invention provides a sugar-free nutrient solution formula for tissue culture, reducing costs and contamination rates, resulting in stronger sweet potato seedlings that can be cultivated by ordinary growers without the need for specialized technicians. Compared to traditional tissue culture methods, this invention significantly shortens the cultivation cycle, substantially increases the yield of virus-free sweet potato seedlings, reduces the annual yield loss due to virus contamination, and lessens the burden on growers, thereby increasing growers' income, lowering sweet potato costs, and potentially reducing market prices, thus contributing to rural revitalization. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Photograph of traditional sweet potato virus-free seedling tissue culture (containing sugar agar medium and nutrient solution) in Comparative Example 1.

[0025] Figure 2 This is a photograph of the sweet potato virus-free seedlings obtained after 4 weeks of culture using the traditional sweet potato virus-free seedling tissue culture method in Comparative Example 1.

[0026] Figure 3 This is a photograph of the sugar-free tissue culture sweet potato virus-free seedlings in Example 1 of the present invention.

[0027] Figure 4 This is a photograph of miniature potatoes that grew from the sugar-free tissue culture sweet potato virus-free seedlings at 4 weeks in Example 1 of the present invention. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] This invention uses a sugar-free nutrient solution to promote the growth of sweet potato seedlings. Sugar-free sweet potato tissue culture uses a nutrient solution that contains no sugar, releasing it via a CO2 gas cylinder to replace sugar (the carbon element required for plant growth). The specific components of the nutrient solution are potassium dihydrogen phosphate and calcium nitrate. This invention uses different ratios of potassium dihydrogen phosphate and calcium nitrate in a comparative experiment using orthogonal distributions. The ratios are: calcium nitrate (0-3.2g), potassium dihydrogen phosphate (0-0.5g), and 1kg of water. Each ratio is replicated three times. After one month, various indicators of the virus-free sweet potato seedlings (plant height, root system, leaves, dry weight, fresh weight, etc.) are observed to obtain the optimal nutrient solution ratio, thereby improving the growth efficiency of sweet potato seedlings and solving current technical challenges (high cost, high technical difficulty, and high contamination rate). The orthogonal experimental nutrient solution ratios are shown in Table 1 below.

[0034] Table 1

[0035]

[0036]

[0037] This invention improves the nutrient solution by removing the sugar from the nutrient solution used in traditional sweet potato seedling cultivation techniques. Since sweet potato seedlings require CO2 gas to promote growth, this invention modifies the cultivation equipment, resulting in lower equipment costs and sufficient ventilation. Furthermore, the absence of sugar, which can cause virus production in sweet potato seedlings, eliminates the need for sterile environments during transplantation; it can be done in a greenhouse without gloves, making the process simple and quick. In addition, this invention's new sugar-free tissue culture technique has shown that some sweet potato seedlings have developed root systems into miniature sweet potatoes, which is impossible in traditional tissue culture. These miniature sweet potatoes, or microtubules, can be directly used as seed stock for seedling cultivation, providing significant assistance and technical insights for the research of microtubule seed stock.

[0038] The method of the present invention has the following advantages:

[0039] Cost reduction: By changing the culture bottle, eliminating sugar as a nutrient solution, replacing agar with vermiculite as the seedling substrate, eliminating aseptic operation equipment, and promoting tissue culture operations to ordinary growers, the final cost of one sweet potato seedling grown using the sugar-free tissue culture technology of this invention is half that of sweet potato seedlings grown using traditional tissue culture technology. The cost of the sugar-free culture medium of this invention is 0.05 yuan / seedling (0.10 yuan / seedling for traditional tissue culture).

[0040] Reduced contamination rate: Compared to the 60% contamination rate under traditional seedling technology, the sweet potato virus-free seedlings cultivated by the sugar-free tissue culture technology of this invention have a 0% survival rate, significantly increasing yield. Furthermore, the average height and weight of each sweet potato seedling are higher than those of traditional tissue culture seedlings.

[0041] Reduced technical difficulty: Traditional tissue culture technology requires operation in a sterile environment and highly skilled personnel. The sugar-free tissue culture technology of this invention can be carried out under normal greenhouse conditions, and ordinary growers can also get started directly, saving time and increasing social value.

[0042] Miniature sweet potatoes: Miniature sweet potatoes, when used as seed stock, are almost indistinguishable from traditional, larger sweet potatoes in terms of seed quantity and quality. Due to their smaller size and weight, the transportation cost of the seed stock can be effectively reduced. In the novel sugar-free tissue culture technology provided by this invention, it was observed that the roots of some virus-free sweet potato seedlings had already grown into small sweet potatoes, which is impossible to achieve in traditional tissue culture. These small sweet potatoes, i.e., miniature sweet potatoes, can be directly used as seed stock to cultivate seedlings, which greatly assists in the research of miniature sweet potato seed stock and provides technical insights.

[0043] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0044] The vermiculite matrix used in this embodiment of the invention is composed of vermiculite with a particle size of 1 mm.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] After removing 2-3 leaf primordia from the tip of the sweet potato stem, the leaf primordia were cultured through stem tip rapid propagation and then subjected to virus testing. Virus-free sweet potato seedlings (2-3 leaves with a radius of about 3 cm) and plants with a length of about 2 nodes were selected and transplanted onto vermiculite substrate. Nutrient solution was added and cultured in a sealed environment for 4 weeks. The nutrient solution consisted of calcium nitrate, potassium dihydrogen phosphate, and water, with the concentrations of each component being 0.32% calcium nitrate and 0.05% potassium dihydrogen phosphate. The mass ratio of nutrient solution to vermiculite substrate was 1:2. During the cultivation process, carbon dioxide gas was released through a carbon dioxide gas cylinder to ensure that the concentration of carbon dioxide gas in the cultivation environment was 1400 ppm. At the same time, the ambient temperature was maintained at 25℃, the ambient humidity at 60%, the light intensity at 2000 lux, and the light duration at 24h. Watering was carried out regularly during the cultivation period (watering once every three days for the first two weeks and once a week for the last two weeks, with 2-3L of water each time). The experiment was observed, and experimental data (plant height, number of leaves, etc.) were recorded weekly.

[0048] Comparative Example 1

[0049] The only difference from Example 1 is that the culture medium (vermiculite matrix + nutrient solution) is replaced with the traditional basal culture medium (MS medium + 30 g / L sucrose and 7 g / L agar), and the operation is carried out under sterile conditions, using culture bottles for seedling cultivation in a closed environment.

[0050] Comparative Example 2

[0051] The only difference from Example 1 is that the addition of potassium dihydrogen phosphate to the nutrient solution is omitted; all other steps and parameters are the same as in Example 1.

[0052] Comparative Example 3

[0053] The only difference from Example 1 is that the addition of calcium nitrate to the nutrient solution is omitted; all other steps and parameters are the same as in Example 1.

[0054] Table 2 shows the indicators of virus-free sweet potato seedlings obtained after 4 weeks of sugar-free tissue culture in Examples 1 and 1-3:

[0055] Table 2

[0056] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Have small sweet potatoes grown? yes no yes yes Percentage of sweet potatoes that grew into small ones (%) 60 0 10 10 Transplanting acclimatization period 10 30 10 10 Survival rate (%) 98 95 98 98 Plant height 19 9 14 17 Number of leaves 6.5 4 5.7 6.1 leaf area 466 269 358 447 Fresh weight of stems and leaves 18 5 11 16 Dry weight of stems and leaves 6 2 4 5

[0057] The percentage of sweet potatoes that grow into small sweet potatoes (%) = number of virus-free sweet potato seedlings that grow into small sweet potatoes / total number of virus-free sweet potato seedlings * 100.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A sugar-free nutrient solution for cultivating virus-free sweet potato seedlings, characterized in that, The raw materials consist of 1.6 to 3.2 parts calcium nitrate, 0.1 to 0.5 parts potassium dihydrogen phosphate, and 1000 parts water by mass.

2. The sugar-free nutrient solution for cultivating virus-free sweet potato seedlings according to claim 1, characterized in that, The raw materials consist of 1.6 to 3.2 parts calcium nitrate, 0.1 to 0.5 parts potassium dihydrogen phosphate, and 1000 parts water by mass.

3. A sugar-free tissue culture method for virus-free sweet potato seedlings, characterized in that, Includes the following steps: After removing 2-3 leaf primordia from the tip of the sweet potato stem, the seedlings are selected through rapid stem tip propagation and transplanted onto a vermiculite substrate. The nutrient solution described in claim 1 or 2 is added for cultivation. During the cultivation process, carbon dioxide gas is introduced.

4. The method for sugar-free tissue culture of virus-free sweet potato seedlings according to claim 3, characterized in that, The mass ratio of nutrient solution to vermiculite matrix is ​​1:

2.

5. The method for sugar-free tissue culture of virus-free sweet potato seedlings according to claim 3, characterized in that, The culture period is 28 days.

6. The sugar-free tissue culture method for virus-free sweet potato seedlings according to claim 3, characterized in that, The concentration of carbon dioxide gas in the culture environment was 1400 ppm.

7. The method for sugar-free tissue culture of virus-free sweet potato seedlings according to claim 3, characterized in that, During cultivation, the ambient temperature is 23–27℃ and the ambient humidity is 60%.

8. The method for sugar-free tissue culture of virus-free sweet potato seedlings according to claim 3, characterized in that, During cultivation, the light intensity was 2000 lux and the light duration was 24 hours.

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