Method for removing tomato brown fruit crinkling virus in tomato seeds

Abstract

The invention relates to the technical field of seed treatment, in particular to a method for removing tomato brown wrinkled fruit viruses in tomato seeds, which comprises the following steps: S1, heat-moisture treatment: soaking the tomato seeds with viruses in boiling water with the volume 5 times that of the tomato seeds, quickly stirring and cooling to 40-45 DEG C, and draining an aqueous solution; s2, chemical agent treatment and seed filtration: soaking the tomato seeds after the water solution is drained in a 10% Na3PO4 solution of which the volume is 5 times that of the tomato seeds for 3 hours, filtering, and taking the tomato seeds; s3, rinsing: washing the tomato seeds with sterile water for 3-5 times, and draining the sterile water; s4, drying: putting the tomato seeds obtained in the step S3 on a net rack in time, putting the net rack in a drying oven, and treating at 40 DEG C for 72 hours to prevent the seeds from sprouting and mildewing. According to the method, the germination rate and germination potential of the tomato seeds are improved, ToBRFV in the seeds is removed more thoroughly, and the detoxification rate can reach 100%.

Description

Technical Field

[0001] The invention relates to the technical field of seed treatment, in particular to a method for removing tomato brown wrinkled fruit virus from tomato seeds. Background Art

[0002] Tomato is a Solanaceae crop that is widely grown around the world. Its fruit is rich in nutrients such as vitamin C and lycopene, and is deeply loved by people.

[0003] Tomato brown wrinkle fruit virus (TBCV) is a new virus in the genus Tobacco mosaic virus (TMV). It infects over 40 plant species, with tomatoes and peppers being its natural hosts. It is particularly detrimental to tomatoes, affecting fruit coloration and severely reducing yield and quality. To prevent and control the spread of the virus, current tomato seed detoxification technologies include dry heat treatment and chemical treatments such as hydrochloric acid, trisodium phosphate, and sodium hypochlorite. However, none of these methods can completely eliminate the virus from seeds. Even if they do, they significantly affect the germination rate and vigor of tomato seeds, rendering them unsuitable for seed use. This poses a significant risk to the spread of the virus and the normal use of seeds. Therefore, precisely controlling the technical parameters for inactivating the virus to ensure both complete and long-lasting seed detoxification and high seed viability to mitigate risks and ensure production safety remains a pressing challenge. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and propose a method for removing Tomato Brown Rough Fruit Virus from tomato seeds, which can not only completely remove ToBRFV from tomato seeds but also does not reduce the germination rate and vitality of tomato seeds.

[0005] In order to achieve the above object, the technical solutions specifically adopted by the present invention are as follows: A method for removing tomato brown fruit virus from tomato seeds comprises the following steps: S1. Moist heat treatment: Soak the infected tomato seeds in 5 times the volume of boiling water, stir rapidly (>200 rpm), cool to 40-45°C, and filter the aqueous solution. S2. Chemical treatment and seed filtration: After draining the aqueous solution, soak the tomato seeds in 5 times the volume of 10% Na3PO4 solution for 3 h, filter, and take the tomato seeds; S3. Rinse: Rinse the tomato seeds 3-5 times with sterile water and drain the sterile water; S4, drying: promptly place the tomato seeds obtained in step S3 on a wire rack and place in an oven at 40° C. for 72 hours to prevent the seeds from germinating and becoming moldy.

[0006] The present invention not only improves the germination rate and germination potential of tomato seeds, but also removes ToBRFV in the seeds more thoroughly, with the detoxification rate reaching 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings: Figure 1 These are the ToBRFV detection results in tomato seeds using different detoxification treatment methods in the examples of the present invention. DETAILED DESCRIPTION

[0008] The present invention is described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0009] Example 1: In this example, 11 tomato seed detoxification methods were designed, with undetoxified tomato seeds used as controls. Approximately 100 full, virus-infected tomato seeds were used for each treatment. After seed detoxification, ToBRFV was detected in the tomato seeds using TaqMan real-time fluorescence quantitative PCR, and the efficacy of each detoxification method was compared.

[0010] The detoxification method is designed as follows: Treatment 1: 2% HCl treatment for 1 h: Soak the infected tomato seeds in 2% HCl for 60 min, then filter the solution and wash the seeds with sterile water 3 to 5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0011] Treatment 2: 10% Na3PO4 treatment for 3 h: Soak the infected tomato seeds in 10% Na3PO4 for 3 h, then filter the solution and wash the seeds with sterile water 3 to 5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0012] Treatment 3: 2% HCl treatment for 0.5 h: Soak the infected tomato seeds in 2% HCl for 30 min, then filter the solution and wash the seeds with sterile water 3 to 5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0013] Treatment 4: Moist heat treatment: Soak the infected tomato seeds in 5 times the volume of boiling water, stir quickly and cool to 40-45℃, then filter the water, place the seeds on a wire rack and place them in an oven at 40℃ for 72 hours to prevent the seeds from mildew and germination.

[0014] Treatment 5: Moist heat treatment + 2% HCl treatment for 0.5 h: Soak the infected tomato seeds in 5 times the volume of boiling water, and quickly stir and cool to 40-45°C. Then soak the seeds in 2% HCl solution for 30 min. After that, filter the solution and wash the seeds with sterile water for 3-5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0015] Treatment 6: Moist heat treatment + 10% Na3PO4 treatment for 3 h: Soak the infected tomato seeds in 5 times the volume of boiling water, and quickly stir and cool to 40-45°C. Then soak the seeds in 10% Na3PO4 solution for 3 h. After that, filter the solution and wash the seeds with sterile water for 3-5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0016] Treatment 7: Moist heat treatment + 2% HCl treatment for 1 h: Soak the infected tomato seeds in 5 times the volume of boiling water, and quickly stir and cool to 40-45°C. Then soak the seeds in 2% HCl solution for 60 min. After that, filter the solution and wash the seeds with sterile water for 3-5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0017] Treatment 8: 2% HCl treatment for 0.5 h + 10% Na3PO4 treatment for 3 h: Soak the infected tomato seeds in 2% HCl solution for 30 min, then wash them with sterile water for 3 to 5 times, soak them in 10% Na3PO4 solution for 3 h, filter the solution, wash the seeds with sterile water for 3 to 5 times, filter the aqueous solution, and place the seeds on a wire rack in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0018] Treatment 9: 2% HCl treatment for 1 h + 10% Na3PO4 treatment for 3 h: Soak the infected tomato seeds in 2% HCl solution for 60 min, then wash them with sterile water for 3 to 5 times, soak them in 10% Na3PO4 solution for 3 h, filter the solution, wash the seeds with sterile water for 3 to 5 times, filter the aqueous solution, and place the seeds on a wire rack in an oven at 40°C for 72 h to prevent the seeds from mildew and germination.

[0019] Treatment 10: Moist heat treatment + 2% HCl treatment for 0.5 h + 10% Na3PO4 treatment for 3 h: Soak the poisonous tomato seeds in 5 times the volume of boiling water, and quickly stir and cool to 40-45 ° C. Then soak the seeds in 2% HCl solution for 30 min, and then wash them with sterile water for 3-5 times. Then soak the seeds in 10% Na3PO4 solution for 3 h, and then filter the solution. Wash the seeds with sterile water for 3-5 times. After filtering the aqueous solution, place the seeds on a rack and place them in an oven at 40 ° C for 72 h to prevent the seeds from mildew and germination.

[0020] Treatment 11: Moist heat treatment + 2% HCl treatment for 1 hour + 10% Na3PO4 treatment for 3 hours: Soak the poisonous tomato seeds in 5 times the volume of boiling water, and quickly stir and cool to 40-45°C. Then soak the seeds in 2% HCl solution for 60 minutes, and then wash them with sterile water for 3-5 times. Then soak the seeds in 10% Na3PO4 solution for 3 hours, and then filter the solution. Wash the seeds with sterile water for 3-5 times. After filtering the aqueous solution, place the seeds on a wire rack and place them in an oven at 40°C for 72 hours to prevent the seeds from mildew and germination.

[0021] Treatment 12: Control (CK): Tomato seeds that were not detoxified were used as the control.

[0022] Virus Detection: (1) Total RNA extraction from tomato seeds Virus-free and non-virus-free seeds were immersed in 10 mL of 0.1 M phosphate buffer (Na2HPO4 / KH2PO4, pH 7.2) and incubated overnight at approximately 4°C. The seeds were then homogenized using a FastPrep homogenizer at 5 rpm for 40 seconds. After centrifugation at 10,000 g for 10 minutes at 4°C, the supernatant was used for RNA extraction using the RNeasy Plant Mini Kit (Qiagen).

[0023] (2) One-step real-time RT-PCR The primer and probe sequences used for ToBRFV are: ToBRFV-F: 5'-CAATCAGAGCACATTTGAAAGTGCA-3'; ToBRFV-R:5'-CAGACACAATCTGTTATTTAAGCATC-3'; ToBRFV-Probe:5'-6FAM-ACAATGGTCCTCTGCACCTG-BHQ1-3'; The reaction detection system is shown in Table 1.

[0024] Table 1 Reaction detection system

[0025] Real-time fluorescence quantitative RT-PCR cycling conditions: reverse transcription at 48°C for 15 min, denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, 40 cycles, and annealing and extension at 60°C for 1 min. After the reaction, the PCR amplification curve Ct value of each reaction sample was recorded. The critical value was 35; values ​​below 35 were considered positive, and values ​​above 35 were considered negative.

[0026] Results and Analysis: Real-time fluorescence quantitative PCR test results are shown in Figure 1 As shown in Table 2, the nucleic acid detection Ct values ​​of tomato seeds treated with moist heat treatment + 10% Na3PO4 treatment for 3 h, 2% HCl treatment for 0.5 h + 10% Na3PO4 treatment for 3 h, 2% HCl treatment for 1 h + 10% Na3PO4 treatment for 3 h, moist heat treatment + 2% HCl treatment for 0.5 h + 10% Na3PO4 treatment for 3 h, and moist heat treatment + 2% HCl treatment for 1 h + 10% Na3PO4 treatment for 3 h were all greater than 35 or there was no amplification curve, indicating that the treatment results of the five treatment measures were all negative and did not contain viruses.

[0027] Table 2 Nucleic acid detection results of different treatments

[0028] Example 2: Fifty tomato seeds from each of the virus-free and non-virus-free treatments in Example 1 were randomly selected, soaked in cold water for 4 h, and placed in a germination box. The filter paper constant temperature germination method was used to conduct a seed germination test in a constant temperature incubator at 28°C. Starting from the third day after treatment, the germination status was observed and recorded daily, and the germination rate and germination potential of each group were calculated. The germination potential was determined on the day with the largest number of germinations in each virus-free treatment, and the germination test was ended on the 10th day.

[0029] Germination rate = (number of germinated seeds / total number of test seeds) × 100%; Germination potential = number of seeds germinated within the specified number of days at the beginning of the germination test / number of test seeds × 100%.

[0030] The results of the seed germination test are shown in Table 3. As can be seen from Table 3, the germination potential and germination rate of the control seeds were 38.1% and 95.24%, respectively. Among them, the treatment method with the greatest impact on germination potential and germination rate was the moist heat treatment + 2% HCl treatment for 1 hour + 10% Na3PO4 treatment for 3 hours. The germination potential and germination rate of the pepper seeds treated with this method decreased by about 24% and 70%, respectively, compared with the control. Except for the moist heat treatment + 10% Na3PO4 treatment for 3 hours, the germination potential and germination rate of the pepper seeds treated with other detoxification methods were lower than those of the control. The germination potential and germination rate of the pepper seeds treated with the moist heat treatment + 10% Na3PO4 treatment for 3 hours were 45.45% and 100%, respectively, which were higher than those of the control.

[0031] Table 3 Effects of different virus removal methods on tomato seed germination rate and germination potential

[0032] In summary, the method of the present invention can not only completely remove tomato brown wrinkled fruit virus from tomato seeds, but also has no effect on the germination rate and germination potential of tomato seeds, which is of great significance for blocking and controlling the spread and harm of tomato brown wrinkled fruit virus.

[0033] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for removing tomato brown fruit virus from tomato seeds, characterized by: The steps include: S1. Moist heat treatment: Soak the infected tomato seeds in 5 times the volume of boiling water, quickly stir and cool to 40-45°C, and then filter the aqueous solution; S2. Chemical treatment and seed filtration: soak the tomato seeds from which the aqueous solution has been drained in 5 volumes of 10% Na3PO4 solution, filter, and obtain the tomato seeds; S3. Rinse: Rinse the tomato seeds 3-5 times with sterile water and drain the sterile water; S4, drying: placing the tomato seeds obtained in step S3 on a wire rack in an oven at 40° C. for 72 h to prevent the seeds from germinating and becoming moldy.

2. The method for removing tomato brown fruit virus from tomato seeds as claimed in claim 1, wherein: In the step S1, the stirring frequency is >200 r / min.

3. The method for removing tomato brown fruit virus from tomato seeds as claimed in claim 1, wherein: In step S2, the tomato seeds from which the aqueous solution has been filtered are soaked in 5 times the volume of 10% Na3PO4 solution for 3 h and then filtered.

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