Cultivation method of tomato seeds

Abstract

The invention relates to the technical field of agriculture, in particular to a tomato seed cultivation method. Comprising the following steps: S1, cleaning and disinfecting male and female parent seeds, sowing the disinfected male and female parent seeds, spraying a detoxification agent, and performing illumination culture; s2, stem tips of top growing points are cut and placed on an induction culture medium to be subjected to induction culture, and tomato seedlings are obtained; s3, performing virus detection on the leaves of the tomato seedlings to obtain virus-free male parent and female parent single plants; s4, carrying out propagation expanding; s5, putting the male and female parent seedlings obtained by propagation on a rooting culture medium for culture, and performing acclimatization and transplanting to obtain virus-free male and female parent seedlings; s6, planting the obtained virus-free male and female parent seedlings, and hybridizing to obtain the tomato seeds. The tomato seeds obtained by the cultivation method disclosed by the invention do not contain viruses, and have very high propagation coefficient, survival rate and germination rate.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technology, and particularly to a method for cultivating tomato seeds. Background Art

[0002] In the field of agricultural science and technology, tomatoes, as a widely cultivated vegetable crop, the quality of their seeds has a decisive impact on agricultural production. The health status of tomato seeds directly affects the growth potential of seedlings, the yield of crops, and the quality of fruits. However, tomato seeds are vulnerable to various viruses under natural conditions, and these viruses can be transmitted through seeds, resulting in virus-infected offspring plants, affecting their growth and development, and even causing serious yield reduction.

[0003] Plant viruses are transmitted to new host plants through main vector organisms, including insects, nematodes, and fungi. The main vector insects for transmitting viruses are those with piercing-sucking mouthparts, and more than 80% of plant viruses are transmitted through vector insects. The types of plant viruses transmitted by nematodes and fungi are relatively few, mainly transmitted by nematodes and fungi in the soil, such as viruses of the genus Nepovirus, viruses of the genus Furovirus, and viruses of the genus Potexvirus transmitted by Olpidium brassicae.

[0004] Seeds and seedlings (including asexual propagation materials such as seed potatoes and seed bulbs) carrying viruses are the main reasons for the occurrence of plant virus diseases in the seedling stage and the early stage, and achieve rapid and long-distance transmission through seeds and seedlings

[10] . The diseased plants caused by seeds and seedlings carrying viruses become the primary infection sources, and are transmitted multiple times in the field through vector insects or agricultural operations, resulting in the outbreak and epidemic of virus diseases in the middle and late stages, causing serious yield and quality losses.

[0005] Seed transmission is an important primary infection source for many viruses with a seed transmission mode, and these viruses are then transmitted through vector insects. In this way, plant viruses can cause outbreaks and epidemics even when the seed transmission rate is very low. Although the role of seed-transmitted viruses in plant virus epidemiology has been widely recognized, little is known about which host and virus characteristics interact to determine the efficiency of seed transmission.

[0006] To solve this problem, this patent provides a brand-new method for cultivating tomato seeds, aiming to cultivate virus-free tomato seeds with a high reproduction rate through a series of scientific steps. This method includes cleaning and disinfecting the parental seeds, sowing them in nutrient pots and spraying a detoxifying agent, light cultivation, and subsequent steps such as shoot tip cutting, induction culture, virus detection, propagation, strong seedling cultivation, and acclimatization transplantation. Through these steps, the viruses inside and outside the seeds can be effectively removed, so as to obtain virus-free parental individual plants, and then virus-free tomato seeds can be harvested through hybridization. Summary of the Invention

[0007] In view of the deficiencies existing in the above-mentioned prior art, the present invention provides a cultivation method for tomato seeds.

[0008] At present, tomato seeds can only remove the viruses on the seed surface through soaking, but cannot remove the viruses carried inside the seeds. There is still a possibility of virus outbreak after the seedlings are planted. This technology can remove the viruses inside and outside the seeds, ensure that the seeds are completely virus-free, and completely eliminate the problem of virus-carrying seeds.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is:

[0010] A cultivation method for tomato seeds, comprising the following steps:

[0011] S1. Clean and disinfect the parental seeds to obtain disinfected parental seeds; sow the disinfected parental seeds in nutrient bowls respectively, spray a detoxifying agent, and cultivate under light;

[0012] S2. Disinfect the top growing point, cut the shoot tip and place it on an induction medium for induction for two months to obtain tomato seedlings;

[0013] S3. Take the leaves of the above tomato seedlings for virus detection. If there are still viruses, repeat the steps of spraying the detoxifying agent and light cultivation in step S1 and step S2 until virus-free parental single plants are obtained;

[0014] S4. Put the buds of the obtained virus-free parental single plants into a propagation medium for propagation;

[0015] S5. Put the propagated parental seedlings on a rooting medium for strong seedling cultivation for one month, place them in a hardening greenhouse for hardening transplantation for one week to obtain virus-free parental seedlings;

[0016] S6. Plant the obtained virus-free parental seedlings in an isolation net room, carry out hybridization, and harvest the seeds, which are the tomato seeds.

[0017] Preferably, the disinfection method in S1 is: soak the parental seeds in clear water for 0.5 - 2h respectively, then soak them in an 8 - 12% aqueous solution of trisodium phosphate for 20 - 50min, wash them with sterile water, and dry them; the spraying method is: spray a detoxifying agent with a concentration of 0.2 - 1.2g / L at the roots, leaves, and top positions of tomato seedlings, and the spraying amount is 5 - 12mL / plant; the detoxifying agent is any one of tannic acid aqueous solution, salicylic acid aqueous solution, and citric acid aqueous solution; preferably, the detoxifying agent is tannic acid aqueous solution.

[0018] Tannic acid is a natural polyphenolic compound commonly found in the plant kingdom. It has certain antibacterial and antioxidant functions and has the advantages of being renewable, environmentally friendly and safe. The main effects of spraying tannic acid during the cultivation of tomato seeds include improving the disease resistance of seeds, promoting seed germination and enhancing the antioxidant capacity of seeds. Specifically: (1) Improving the disease resistance of seeds: Tannic acid has antibacterial and antiviral effects, can inhibit the growth of pathogenic bacteria, and at the same time can also inhibit the activity of key enzymes of viruses, reducing the disease threat suffered by seeds during germination. These characteristics enable tannic acid to effectively reduce the invasion of pathogens during the seed cultivation and germination processes, and improve the virus detoxification efficiency of seeds; (2) Promoting seed germination: Tannic acid can improve the physiological state of seeds, promote the germination speed and uniformity of seeds. By spraying tannic acid, the germination rate and emergence rate of seeds can be significantly increased, thus ensuring that crops can obtain better growth conditions in the initial growth stage. In addition, tannic acid can also enhance the nutrient absorption of seeds, further promoting the germination and growth of seeds; (3) Enhancing the antioxidant capacity of seeds: Tannic acid has antioxidant effects, can scavenge free radicals, and reduce the damage of oxidative stress to cells. This antioxidant capacity helps to protect seed cells from oxidative damage, improve the overall health status and growth potential of seeds, and can help seeds resist oxidative stress, thereby reducing the adverse effects of environmental stress on seed germination and growth, protecting seeds from damage, extending the storage period of seeds, and ensuring their normal germination and growth after sowing is of great significance.

[0019] Preferably, the light intensity in S1 is 8000 - 12000 Lux, the daily light exposure is 14 - 18 h, the dark period is 6 - 10 h, the temperature of the light incubator is 35 - 40 °C, and the humidity is 70 - 80%.

[0020] Preferably, the formula of the induction medium in S2 is MS + 0.3 - 0.8 mg / L 6-benzylaminopurine + 20 - 40 g / L sucrose + 6 - 8 g / L agar + 5 - 10 g / L antiviral nutrient solution.

[0021] In order to remove the viruses carried inside the seeds, there is still a possibility of virus outbreak after the seedlings are planted. Adding an antiviral nutrient solution during the induction culture process can remove the viruses inside and outside the seeds, and at the same time inhibit the amplification of viruses in the plant body, improve the shoot tip virus detoxification efficiency, ensure that the seeds are completely virus-free, and completely eliminate the problem of virus-carrying seeds. The present invention has developed a nutrient solution that can enhance the antiviral ability and at the same time promote the growth and development of tomatoes.

[0022] Preferably, the viruses in S3 include brown rugose fruit virus, mosaic virus, TY virus, and Verticillium wilt virus.

[0023] Preferably, the formula of the multiplication medium in S4 is MS + 0.005 - 0.02 mg / L thidiazuron + 0.05 - 0.2 mg / L indolebutyric acid + 20 - 40 g / L sucrose + 6 - 8 g / L agar.

[0024] Preferably, the formula of the rooting medium in S5 is 1 / 2MS + 0.05 - 0.2 mg / L naphthaleneacetic acid + 20 - 40 g / L sucrose + 6 - 8 g / L agar.

[0025] Preferably, the composition of the MS medium is as follows: potassium nitrate 1.6 - 1.9 g / L, ammonium nitrate 1.5 - 1.8 g / L, potassium dihydrogen phosphate 0.1 - 0.2 g / L, magnesium sulfate 0.2 - 0.6 g / L, calcium chloride 0.3 - 0.7 g / L, potassium iodide 0.8 - 1.2 g / L, manganese sulfate 0.01 - 0.1 g / L, sodium molybdate 0.1 - 0.5 mg / L, copper sulfate 0.01 - 0.06 mg / L, cobalt chloride 0.01 - 0.07 mg / L, disodium ethylenediaminetetraacetate 0.02 - 0.06 g / L, inositol 0.05 - 0.3 g / L, glycine 1 - 3 mg / L, thiamine hydrochloride 0.1 - 0.4 mg / L, pyridoxine hydrochloride 0.3 - 1 mg / L, and the pH is 5.8.

[0026] Preferably, the antiviral nutrient solution is composed of the following raw materials in parts by weight: 1 - 4 parts by weight of zeatin, 2 - 5 parts by weight of betaine, 1 - 3 parts by weight of wolfberry polysaccharide, 1 - 3 parts by weight of pullulan, 2 - 5 parts by weight of fennel extract, 0.5 - 2 parts by weight of ammonium humate, and 30 - 80 parts by weight of water.

[0027] Polysaccharide antiviral agents have the characteristics of safety, greenness, high efficiency, etc., and are known as "plant vaccines". The carbohydrate structure of wolfberry polysaccharide and its medicinal value of immune regulation make it have the potential to be used as an immune inducer to prevent and control plant virus diseases.

[0028] The Lycium barbarum polysaccharide, pullulan polysaccharide substances and fennel extract contained in the antiviral nutrient solution prepared by the present invention have immunomodulatory effects, can enhance the resistance of tomato seedlings to viruses. These components, as "plant vaccines", help induce the plant to produce defense responses, reduce virus infection, thereby reducing the spread of viruses during the tissue culture process and improving the detoxification efficiency; the zeatin and betaine nutrients in the antiviral nutrient solution provide essential nutrients for the seedlings, promote their healthy growth and development. These substances help the seedlings grow better under sterile conditions, improving their growth rate and quality; the nutrients and plant extracts in the nutrient solution act together to help improve the overall health of the seedlings, thereby improving the growth quality and health status of the seedlings. Healthy seedlings are more likely to produce more buds and roots during the propagation stage, thus increasing the propagation coefficient; in addition, virus-free parent single plants can reduce the spread of viruses and other pathogens during hybridization, thereby reducing the occurrence of hybrid plants and improving the purity of seeds and the consistency of field performance.

[0029] Preferably, the preparation method of the fennel extract is as follows: Wash and dry fennel, crush it, and pass through a 40-60 mesh sieve to obtain fennel powder; Mix the fennel powder with a 70-90 wt% ethanol aqueous solution at a mass ratio of 1:25-35, extract at 65-80 °C for 2-4 h, filter, and retain filtrate 1; Add water 20-30 times the mass of the fennel powder to the filter residue, extract at 50-70 °C, ultrasonic frequency 30-60 kHz, and ultrasonic power 100-300 W for 1-3 h, filter, and retain filtrate 2. Combine the two filtrates, concentrate under reduced pressure to 8-12% of the volume, dry, bake until the moisture content is below 4%, crush, and pass through a 30-50 mesh sieve to obtain the fennel extract.

[0030] Preferably, the conditions for acclimatization of seedlings in S5 are: light intensity 8000-12000 Lux, daily light 14-18 h, darkness 6-10 h, environmental relative humidity 75-80%, and temperature 23-28 °C.

[0031] A tomato seed is prepared by using the cultivation method of the tomato seed described above.

[0032] The beneficial effects of the present invention:

[0033] The present invention provides a method for cultivating tomato seeds, which includes strictly cleaning and disinfecting the parent seeds, spraying and detoxifying with a specific detoxifying agent such as a tannic acid aqueous solution, and cultivating in an induction medium with an antiviral nutrient solution added, thereby finally obtaining virus-free tomato seeds. The tomato seeds cultivated by the above method are virus-free, the paternal material and the maternal material are obtained by tissue culture and propagation, have the same genes, high seedling consistency, and no foreign plants, and have a high reproduction coefficient, germination rate, and survival rate, providing an efficient and reliable tomato seed cultivation solution for agricultural production. DETAILED DESCRIPTION

[0034] The above content of the present invention is further described in detail below in conjunction with specific implementation modes, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0035] Introduction of some raw materials in this application:

[0036] Tomato A is a virus-resistant tomato variety, Matsuda Ruby, purchased from Suqian Lvpeng Seed Technology Co., Ltd.

[0037] Tomato B is a virus-resistant tomato variety, Chunxi, purchased from Shouguang Huaze Seed Co., Ltd.

[0038] Example 1

[0039] A method for cultivating tomato seeds comprises the following steps:

[0040] S1. Take tomato A as the female parent and tomato B as the male parent, sow the seeds of the parents in nutrient pots respectively, and when the tomato seedlings grow to 20 cm, spray the roots, leaves and tops of the tomato seedlings with a concentration of 1.0 g / L tannic acid aqueous solution, with a spraying amount of 8 mL / plant, and culture them in a culture box under white light for 2 weeks; the light intensity is 10000 Lux, the light is 16 hours per day, the dark is 8 hours, the temperature of the light culture box is 38°C, and the humidity is 75%;

[0041] S2 After the seedlings grow to a height of 40 cm, cut off 5 cm of the top growth point, disinfect with 5% sodium hypochlorite on a clean bench for 15 minutes, wash with sterile water, cut off 0.5 mm of the stem tip under a dissecting microscope, put it on an induction medium for induction for two months, and obtain tomato seedlings; the induction medium formula is MS + 0.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6.5 g / L agar;

[0042] In S3, when the height of the above tomato seedlings reaches 7 cm, take the leaves for virus detection until no brown wrinkled fruit virus, mosaic virus, TY virus, and Verticillium wilt virus are detected in the leaves. If there are still viruses, repeat the spraying of the detoxifying agent, light cultivation in step S1, and step S2 until virus-free male and female parent single plants are obtained;

[0043] In S4, after cutting the buds of the obtained virus-free male and female parent single plants under a laminar flow hood, place them in a multiplication medium for multiplication. The formula of the multiplication medium is MS + 0.01 mg / L thidiazuron + 0.1 mg / L indolebutyric acid + 30 g / L sucrose + 6.5 g / L agar;

[0044] In S5, place the multiplied male and female parent seedlings on a rooting medium for strong seedling cultivation. The formula of the rooting medium is 1 / 2MS + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 6.5 g / L agar. The cultivation time is one month, the seedling height is 10 cm, and the root length is 5 cm. Place them in a hardening-off greenhouse for one week of hardening-off and transplanting to obtain virus-free male and female parent seedlings; The conditions for hardening-off: the light intensity is 10000 Lux, the daily light is 16 h, the darkness is 8 h, the environmental relative humidity is 80%, and the temperature is 25 °C;

[0045] In S6, plant the obtained virus-free male and female parent seedlings in an isolation net room for hybridization. The isolation net room is a 60-mesh insect-proof net, and harvest the seeds, which are the tomato seeds.

[0046] The composition of the MS medium is as follows: potassium nitrate 1.8 g / L, ammonium nitrate 1.6 g / L, potassium dihydrogen phosphate 0.16 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.45 g / L, potassium iodide 1.0 g / L, manganese sulfate 0.02 g / L, sodium molybdate 0.2 mg / L, copper sulfate 0.03 mg / L, cobalt chloride 0.025 mg / L, disodium ethylene diamine tetraacetate 0.04 g / L, inositol 0.1 g / L, glycine 2 mg / L, thiamine hydrochloride 0.2 mg / L, pyridoxine hydrochloride 0.6 mg / L, and the pH is 5.8.

[0047] Example 2

[0048] A cultivation method for tomato seeds, comprising the following steps:

[0049] S1. Using tomato A as the female parent and tomato B as the male parent, soak the seeds of both parents in clean water for 1 h, then soak them in a 10% aqueous solution of trisodium phosphate for 30 min, wash them with sterile water, and air-dry them to obtain disinfected parent seeds. Sow the disinfected parent seeds in nutrient pots respectively. When the tomato seedlings grow to 20 cm, spray an aqueous solution of tannic acid with a concentration of 1.0 g / L at the roots, leaves, and top positions of the tomato seedlings. The spraying amount is 8 mL / plant, and then place them in an incubator with white light illumination for 2 weeks. The light intensity is 10000 Lux, with 16 h of light and 8 h of darkness per day. The temperature of the light incubator is 38 °C, and the humidity is 75%.

[0050] S2. After the seedling height reaches 40 cm, cut off the top growing point of 5 cm. Disinfect it with 5% sodium hypochlorite for 15 min in a laminar flow hood, wash it with sterile water, and cut a 0.5-mm shoot tip under a dissecting microscope. Place it on an induction medium for induction for two months to obtain tomato seedlings. The formula of the induction medium is MS + 0.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6.5 g / L agar.

[0051] S3. When the height of the above tomato seedlings reaches 7 cm, take the leaves for virus detection until no brown wrinkled fruit virus, mosaic virus, TY virus, or Verticillium wilt virus is detected in the leaves. If there are still viruses, repeat the steps of spraying the detoxifying agent and light cultivation in step S1 and step S2 until virus-free parent single plants are obtained.

[0052] S4. After cutting the buds of the obtained virus-free parent single plants in a laminar flow hood, place them on a multiplication medium for multiplication. The formula of the multiplication medium is MS + 0.01 mg / L thidiazuron + 0.1 mg / L indolebutyric acid + 30 g / L sucrose + 6.5 g / L agar.

[0053] S5. Place the multiplied parent seedlings on a rooting medium for strong seedling cultivation. The formula of the rooting medium is 1 / 2MS + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 6.5 g / L agar. The cultivation time is one month, with a seedling height of 10 cm and a root length of 5 cm. Place them in a greenhouse for acclimatization and transplanting for one week to obtain virus-free parent seedlings. The conditions for acclimatization: The light intensity is 10000 Lux, with 16 h of light and 8 h of darkness per day, the relative environmental humidity is 80%, and the temperature is 25 °C.

[0054] S6. Plant the obtained virus-free parent seedlings in an isolation net room for hybridization. The isolation net room is a 60-mesh insect-proof net. Harvest the seeds, which are the tomato seeds.

[0055] The composition of the MS medium is as follows: potassium nitrate 1.8 g / L, ammonium nitrate 1.6 g / L, potassium dihydrogen phosphate 0.16 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.45 g / L, potassium iodide 1.0 g / L, manganese sulfate 0.02 g / L, sodium molybdate 0.2 mg / L, copper sulfate 0.03 mg / L, cobalt chloride 0.025 mg / L, disodium ethylene diamine tetraacetate 0.04 g / L, inositol 0.1 g / L, glycine 2 mg / L, thiamine hydrochloride 0.2 mg / L, pyridoxine hydrochloride 0.6 mg / L, and the pH is 5.8.

[0056] Example 3

[0057] A cultivation method for tomato seeds comprises the following steps:

[0058] S1. Using tomato A as the female parent and tomato B as the male parent, soaking the seeds of the parent plants in clear water for 1 h respectively, then soaking them in a 10% aqueous solution of trisodium phosphate for 30 min, washing with sterile water, and air-drying to obtain disinfected parent plant seeds; sowing the disinfected parent plant seeds in nutrient pots respectively. When the tomato seedlings grow to 20 cm, spraying an aqueous solution of tannic acid with a concentration of 1.0 g / L at the root, leaf, and top positions of the tomato seedlings, with a spraying amount of 8 mL / plant, and placing them in an incubator with white light illumination for 2 weeks; the illumination intensity is 10000 Lux, with 16 h of illumination and 8 h of darkness per day, and the temperature of the illumination incubator is 38 °C and the humidity is 75%;

[0059] S2. After the seedlings grow to a height of 40 cm, cutting off the top growing point of 5 cm, disinfecting it with 5% sodium hypochlorite for 15 min in a clean bench, washing with sterile water, cutting a 0.5-mm shoot tip under a dissecting microscope, and placing it on an induction medium for induction for two months to obtain tomato seedlings; the formula of the induction medium is MS + 0.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6.5 g / L agar + 8 g / L antiviral nutrient solution;

[0060] S3. When the height of the above-mentioned tomato seedlings reaches 7 cm, taking leaves for virus detection until no brown wrinkled fruit virus, mosaic virus, TY virus, or verticillium wilt virus is detected in the leaves. If there are still viruses, repeat the steps of spraying the detoxifying agent and light cultivation in step S1 and step S2 until virus-free male and female parent single plants are obtained;

[0061] S4. After cutting off the buds of the obtained virus-free male and female parent single plants in a clean bench, placing them on a propagation medium for propagation. The formula of the propagation medium is MS + 0.01 mg / L thidiazuron + 0.1 mg / L indolebutyric acid + 30 g / L sucrose + 6.5 g / L agar;

[0062] S5 Place the multiplied parent seedlings on the rooting medium for strong seedling cultivation. The formula of the rooting medium is 1 / 2MS + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 6.5 g / L agar. The cultivation time is one month. The seedling height is 10 cm and the root length is 5 cm. Place them in the acclimatization greenhouse for one week of acclimatization and transplanting to obtain virus-free parent seedlings. The conditions for acclimatization are as follows: the light intensity is 10000 Lux, the daily light exposure is 16 h, the darkness is 8 h, the environmental relative humidity is 80%, and the temperature is 25°C.

[0063] S6 Plant the obtained virus-free parent seedlings in the isolation net room for hybridization. The isolation net room is a 60-mesh insect-proof net. Harvest the seeds, which are the tomato seeds.

[0064] The composition of the MS medium is as follows: potassium nitrate 1.8 g / L, ammonium nitrate 1.6 g / L, potassium dihydrogen phosphate 0.16 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.45 g / L, potassium iodide 1.0 g / L, manganese sulfate 0.02 g / L, sodium molybdate 0.2 mg / L, copper sulfate 0.03 mg / L, cobalt chloride 0.025 mg / L, disodium ethylene diamine tetraacetate 0.04 g / L, inositol 0.1 g / L, glycine 2 mg / L, thiamine hydrochloride 0.2 mg / L, pyridoxine hydrochloride 0.6 mg / L, and the pH is 5.8.

[0065] The antiviral nutrient solution is composed of the following raw materials in parts by weight: 2 parts by weight of zeatin, 3 parts by weight of betaine, 2 parts by weight of wolfberry polysaccharide, 2 parts by weight of pullulan polysaccharide, 1 part by weight of ammonium humate, and 50 parts by weight of water mixed together.

[0066] Example 4

[0067] A cultivation method for tomato seeds, comprising the following steps:

[0068] S1. Using tomato A as the female parent and tomato B as the male parent, soak the parent seeds in clear water for 1 h respectively, then soak them in a 10% aqueous solution of trisodium phosphate for 30 min, wash them with sterile water, and dry them to obtain disinfected parent seeds. Sow the disinfected parent seeds in nutrient pots respectively. When the tomato seedlings grow to 20 cm, spray a detoxifying agent with a concentration of 1.0 g / L at the roots, leaves, and top positions of the tomato seedlings. The spraying amount is 8 mL / plant, and place them in an incubator with white light illumination for 2 weeks. The light intensity is 10000 Lux, the daily light exposure is 16 h, the darkness is 8 h, the temperature of the light incubator is 38°C, and the humidity is 75%.

[0069] After the seedling grows to a height of 40 cm, cut off the top growing point of 5 cm. Disinfect it with 5% sodium hypochlorite for 15 minutes in a laminar flow hood, then wash it with sterile water. Under a dissecting microscope, cut a 0.5-mm shoot tip and place it on an induction medium for two months to obtain tomato seedlings. The formula of the induction medium is MS + 0.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6.5 g / L agar + 8 g / L antiviral nutrient solution.

[0070] When the height of the above-mentioned tomato seedlings reaches 7 cm, take the leaves for virus detection until no brown wrinkled fruit virus, mosaic virus, TY virus, and Verticillium wilt virus are detected in the leaves. If there are still viruses, repeat the steps of spraying the detoxifying agent and light culture in step S1 and step S2 until virus-free male and female parent plants are obtained.

[0071] After cutting off the buds of the obtained virus-free male and female parent plants in a laminar flow hood, place them on a multiplication medium for multiplication. The formula of the multiplication medium is MS + 0.01 mg / L thidiazuron + 0.1 mg / L indolebutyric acid + 30 g / L sucrose + 6.5 g / L agar.

[0072] Place the multiplied male and female parent seedlings on a rooting medium for strong seedling cultivation. The formula of the rooting medium is 1 / 2 MS + 0.1 mg / L naphthaleneacetic acid + 30 g / L sucrose + 6.5 g / L agar. The cultivation time is one month, the seedling height is 10 cm, and the root length is 5 cm. Place them in a hardening-off greenhouse for one week of hardening-off and transplanting to obtain virus-free male and female parent seedlings. The conditions for hardening-off: the light intensity is 10000 Lux, the daily light is 16 h, the darkness is 8 h, the environmental relative humidity is 80%, and the temperature is 25 °C.

[0073] Plant the obtained virus-free male and female parent seedlings in an isolation net room for hybridization. The isolation net room is a 60-mesh insect-proof net. Harvest the seeds, which are the tomato seeds.

[0074] The composition of the MS medium is as follows: potassium nitrate 1.8 g / L, ammonium nitrate 1.6 g / L, potassium dihydrogen phosphate 0.16 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.45 g / L, potassium iodide 1.0 g / L, manganese sulfate 0.02 g / L, sodium molybdate 0.2 mg / L, copper sulfate 0.03 mg / L, cobalt chloride 0.025 mg / L, disodium ethylenediaminetetraacetate 0.04 g / L, inositol 0.1 g / L, glycine 2 mg / L, thiamine hydrochloride 0.2 mg / L, pyridoxine hydrochloride 0.6 mg / L, and the pH is 5.8.

[0075] The detoxifying agent is any one of tannic acid aqueous solution, salicylic acid aqueous solution, and citric acid aqueous solution.

[0076] The antiviral nutrient solution is composed of the following raw materials in parts by weight: 2 parts by weight of zeatin, 3 parts by weight of betaine, 2 parts by weight of wolfberry polysaccharide, 2 parts by weight of pullulan polysaccharide, 3 parts by weight of fennel extract, 1 part by weight of ammonium humate, and 50 parts by weight of water are mixed.

[0077] The preparation method of the fennel extract is as follows: Wash the fennel, dry it, crush it, and pass through a 50-mesh sieve to obtain fennel powder; Mix the fennel powder and 80wt% ethanol aqueous solution in a mass ratio of 1:30, extract at 70°C for 2.5 h, filter, and retain filtrate 1; Add water 25 times the mass of the fennel powder to the filter residue, extract at 60°C, ultrasonic frequency 40 kHz, and ultrasonic power 200 W for 1.5 h, filter, and retain filtrate 2. Combine the two filtrates, concentrate under reduced pressure to 10% of the volume, dry, bake until the water content is below 4%, crush, and pass through a 40-mesh sieve to obtain the fennel extract.

[0078] Comparative Example 1

[0079] It is basically the same as Example 1, and the main difference is in S1: Using A tomato as the female parent and B tomato as the male parent, sow the seeds of the male and female parents in nutrient pots respectively. When the tomato seedlings grow to 20 cm, spray a detoxifying agent with a concentration of 1.0 g / L at the root, leaf, and top positions of the tomato seedlings, with a spraying amount of 8 mL / plant, and place them in an incubator with white light illumination for 2 weeks; The light intensity is 10,000 Lux, with 16 h of light and 8 h of darkness per day, the temperature of the light incubator is 38°C, and the humidity is 75%; The detoxifying agent is a citric acid aqueous solution.

[0080] Comparative Example 2

[0081] It is basically the same as Example 1, and the main difference is in S1: Using A tomato as the female parent and B tomato as the male parent, sow the seeds of the male and female parents in nutrient pots respectively. When the tomato seedlings grow to 20 cm, spray a detoxifying agent with a concentration of 1.0 g / L at the root, leaf, and top positions of the tomato seedlings, with a spraying amount of 8 mL / plant, and place them in an incubator with white light illumination for 2 weeks; The light intensity is 10,000 Lux, with 16 h of light and 8 h of darkness per day, the temperature of the light incubator is 38°C, and the humidity is 75%; The detoxifying agent is a salicylic acid aqueous solution.

[0082] Test Example 1

[0083] The virus detection method is as follows: Use RT-PCR for virus detection. The RNA of tomatoes is extracted using a kit, and the reverse transcription method is carried out according to the kit instructions. According to the sequences reported in Genebank, forward and reverse primers are designed through Primer 5 software, and PCR is carried out for amplification. The control group is cultivated by the conventional method, without spraying tannic acid aqueous solution and without adding antiviral nutrient agent during the cultivation process. The results are shown in Table 1.

[0084] Table 1 Virus Detection Results

[0085] Detoxification efficiency, % Example 1 85％ Example 2 87％ Example 3 91％ Example 4 95％ Comparative Example 1 65％ Comparative Example 2 63％ Control Group 35％

[0086] As can be seen from Table 1, when the seeds cultivated by the present method are sown, the resulting seedling plants are detected to be free of brown wrinkled fruit virus, mosaic virus, TY virus, and Verticillium wilt virus. Therefore, tomato seeds obtained by sowing through the cultivation method of the present invention can yield virus-free tomato seeds.

[0087] Test Example 2

[0088] Reproductive performance test: Select tomato parental seeds purchased in the same batch, randomly divide them into 7 groups, with 100 seeds in each group, and plant them according to the methods in Examples 1-4 and Comparative Examples 1-2 respectively. The control group was not sprayed with tannic acid aqueous solution and no antiviral nutrient was added during the cultivation process to obtain experimental samples. The germination rate and reproductive coefficient were statistically analyzed. The reproductive coefficient = total number of formed buds / number of transferred buds. The results are shown in Table 2.

[0089] Table 2 Reproduction Results

[0090] Germination rate, % Survival rate, % Propagation coefficient Example 1 88％ 92％ 5.4 Example 2 92％ 95％ 5.8 Example 3 95％ 97％ 6.7 Example 4 98％ 100％ 7.3 Comparative Example 1 71％ 81％ 4.2 Comparative Example 2 64％ 72％ 3.7 Control Group 55％ 60％ 2.7

[0091] From the above results, it can be seen that the tomato seeds cultivated by the present invention hardly contain viruses, and at the same time have a very high reproductive coefficient, with very high survival rate and germination rate. Comparing Examples 1-4, it can be seen that adding antiviral nutrients during the cultivation process can not only reduce the virus rate inside the tomatoes, but also increase the reproductive coefficient and survival rate of the tomatoes. The reason is that the Lycium barbarum polysaccharide, pullulan polysaccharide substances, and fennel extract contained in the antiviral nutrient solution prepared by the present invention have an immunomodulatory effect, which can enhance the resistance of tomato seedlings to viruses. These components, as "plant vaccines", help induce the plant to produce a defense response, reduce virus infection, thereby reducing the spread of viruses during tissue culture and improving the detoxification efficiency; the zeatin and betaine nutrients in the antiviral nutrient solution provide essential nutrients for the seedlings, promoting their healthy growth and development. These substances help the seedlings grow better under sterile conditions, improving their growth rate and quality; the nutrients and plant extracts in the nutrient solution act together to help improve the overall health of the seedlings, thereby improving the growth quality and health status of the seedlings. Healthy seedlings are more likely to produce more buds and roots during the propagation stage, thus increasing the reproductive coefficient; in addition, virus-free parental single plants can reduce the spread of viruses and other pathogens during hybridization, thereby reducing the occurrence of heterozygous plants and improving the purity of the seeds and the consistency of field performance.

[0092] Further comparison between Example 1 and Comparative Examples 1-2 shows that using tannic acid as a detoxifying agent has better effects than salicylic acid and citric acid. The reason is that tannic acid is a natural polyphenolic compound with antibacterial and antiviral effects. It can inhibit the growth of pathogenic bacteria and reduce the disease threat to seeds during germination. This property enables tannic acid to effectively reduce pathogen infestation and improve the detoxification efficiency during seed cultivation. Tannic acid can improve the physiological state of seeds and promote the germination speed and uniformity of seeds. By spraying tannic acid, the germination rate and emergence rate of seeds can be significantly increased, thus ensuring better growth conditions for crops in the initial growth stage. Tannic acid helps reduce the possibility of virus outbreaks after planting by enhancing the disease resistance of seeds.

Claims

1. A method for cultivating tomato seeds, characterized in that: The steps include: S1. Clean and disinfect the parent seeds to obtain sterilized parent seeds; sow the sterilized parent seeds in nutrient pots, spray with detoxifying agent, and culture under light; S2 cut the top growth point and disinfect it, cut the stem tip and put it on the induction medium for induction for two months to obtain tomato seedlings; S3 taking leaves of the above-mentioned tomato seedlings for virus testing until virus-free male and female plants are obtained; S4 placing the obtained buds of the virus-free parental plant into a propagation medium for propagation; S5: placing the parent seedlings obtained by propagation on a rooting medium for seedling cultivation, placing them in a seedling hardening greenhouse for seedling hardening and transplanting, and obtaining virus-free parent seedlings; S6: Planting the virus-free parent seedlings obtained above, performing hybridization, and harvesting seeds, which are the tomato seeds.

2. The method for cultivating tomato seeds according to claim 1, characterized in that: The detoxifying agent in S1 is any one of tannic acid aqueous solution, salicylic acid aqueous solution and citric acid aqueous solution.

3. The method for cultivating tomato seeds according to claim 1, characterized in that: The light intensity in S1 is 8000-12000 Lux, with 14-18 hours of light and 6-10 hours of darkness per day. The temperature of the light incubator is 35-40° C. and the humidity is 70-80%.

4. The method for cultivating tomato seeds according to claim 1, characterized in that: The induction medium formula in the S2 is MS+0.3-0.8 mg / L 6-benzylaminopurine+20-40 g / L sucrose+6-8 g / L agar+5-10 g / L antiviral nutrient solution.

5. The method for cultivating tomato seeds according to claim 1, characterized in that: The expansion medium formula in the S4 is MS+0.005-0.02 mg / L thidiazuron+0.05-0.2 mg / L indolebutyric acid+20-40 g / L sucrose+6-8 g / L agar.

6. The method for cultivating tomato seeds according to claim 1, characterized in that: The rooting medium formula in S5 1 / 2MS+0.05-0.2mg / L naphthaleneacetic acid+20-40g / L sucrose+6-8g / L agar.

7. The method for cultivating tomato seeds according to any one of claims 4 to 6, characterized in that: The composition of the MS culture medium is as follows: 1.6-1.9 g / L potassium nitrate, 1.5-1.8 g / L ammonium nitrate, 0.1-0.2 g / L potassium dihydrogen phosphate, 0.2-0.6 g / L magnesium sulfate, 0.3-0.7 g / L calcium chloride, 0.8-1.2 g / L potassium iodide, 0.01-0.1 g / L manganese sulfate, 0.1-0.5 mg / L sodium molybdate, 0.01-0.06 mg / L copper sulfate, 0.01-0.07 mg / L cobalt chloride, 0.02-0.06 g / L disodium ethylenediaminetetraacetic acid, 0.05-0.3 g / L inositol, 1-3 mg / L glycine, 0.1-0.4 mg / L thiamine hydrochloride, 0.3-1 mg / L pyridoxine hydrochloride, and the pH is 5.

8.

8. The method for cultivating tomato seeds according to claim 4, characterized in that: The antiviral nutrient solution is composed of the following raw materials in parts by weight: 1-4 parts by weight of zeatin, 2-5 parts by weight of betaine, 1-3 parts by weight of wolfberry polysaccharide, 1-3 parts by weight of pullulan, 2-5 parts by weight of fennel extract, 0.5-2 parts by weight of ammonium humate, and 30-80 parts by weight of water.

9. The method for cultivating tomato seeds according to claim 1, characterized in that: The conditions for hardening the seedlings in S5 are: light intensity 8000-12000 Lux, daily light 14-18 hours, dark 6-10 hours, environmental relative humidity 75-80%, and temperature 23-28°C.

10. A tomato seed, characterized in that: The tomato seeds are prepared by the tomato seed cultivation method according to any one of claims 1 to 9.