A method for removing hop dwarfing viroids by combining sRNA with stem tip ultra-low temperature therapy

Abstract

The present invention discloses a method for removing hop dwarf viroid by combining sRNA with ultra-low temperature therapy of stem apex, and belongs to the technical field of detoxified improved varieties production. The sRNA includes a sense chain as shown in SEQ ID NO: 5 and an antisense chain as described in SEQ ID NO: 6. The method includes the following steps: treating tomato stems infected with hop dwarf viroid with sRNA to obtain a sample in which hop dwarf viroid is suppressed; obtaining a stem apex after disinfecting the sample in which viroid is suppressed, and then pre-culturing and loading the stem apex; treating the loaded stem apex with cryoprotectant PVS2, and then performing ultra-low temperature treatment, unloading and post-culturing to obtain a detoxified sample. The method can remove viroids that are generally impossible to remove on plants, and does not require a long period of low temperature passivation treatment of viroids. The present invention is suitable for quickly obtaining detoxified seedlings free of hop dwarf viroids on tomatoes.

Description

Technical Field

[0001] The invention relates to the technical field of virus-free improved variety production, and in particular to a method for removing hop dwarfing viroids by combining sRNA with stem tip ultra-low temperature therapy. Background Art

[0002] Tomato is one of the most widely grown and consumed vegetables in the world. The stable and safe commercial production of tomatoes is threatened by viroid infection. Viroids are a class of single-stranded closed RNA molecules with a genome of approximately 240-430bp. They are the smallest known specialized parasitic pathogens. Viroids are important quarantine diseases and seed-borne pathogens. When tomatoes are infected with viroids, they can cause virus-like symptoms and host nutritional and reproductive growth disorders. In addition, as a global quarantine disease, the circulation of seeds and seedling germplasm resources carrying viroids is restricted. It is of great significance to develop effective inhibition and viroid elimination technologies. However, because most viral diseases are not transmitted through seeds and it is difficult to remove seed-borne viruses and viroids, detoxification technology is often applied to vegetatively propagated crops. Therefore, the development of viroid detoxification technology in seed-propagated crops is often overlooked.

[0003] 20-23nt long sRNAs are involved in post-transcriptional gene expression regulation in all organisms and play a key role in various important physiological processes. sRNA initiates targeted RNA interference and is recognized and cleaved by the Dicer protein in RNAs of about 20nt in length. One strand of the duplex is loaded into a ribonucleoprotein complex, called the RNA-induced silencing complex, which consists of at least one ARGONAUTE protein and one sRNA. The expression of any RNA with a complementary sequence to the sRNA will be specifically silenced at the transcriptional or post-transcriptional level. Studies have found that the regulation of sRNA is not limited to endogenous, it can act as a signaling molecule in heterologous cells, deliver different organisms while maintaining biological activity, and regulate gene expression in target cells. Recently, gene expression regulation can be achieved in different types of organisms by exogenously applying natural sRNAs or artificially synthesized sRNAs homologous to the target gene sequence. At present, exogenous targeted sRNAs have been applied to inhibit diseases caused by fungi, oomycetes, viruses and pests on plants. In the research related to viroids, exogenous sRNA can induce resistance to potato spindle tuber viroids, citrus exoviruses and chrysanthemum chlorotic mottle viroids in plants. In previous studies, long-term low temperature treatment is usually used to inhibit the movement and replication of viroids in plants, but the low temperature treatment method is not suitable for thermophilic crops such as tomatoes. Therefore, exogenous application of targeted sRNA can be used as an effective method to inhibit viroids in tomatoes.

[0004] Detoxification therapy is a method based on the principle of uneven distribution of pathogens in the host, by obtaining tissues that are not infected with pathogens (mainly the apical meristem of the stem tip) and cultivating them into complete plants. Unlike most viruses that cannot attack the cells of the apical meristem of the stem tip, viroids can infect all cells of the plant and will not leave viroid-free areas or tissues in the diseased plants, so they are difficult to remove. Ultra-low temperature therapy is the latest developed detoxification technology. Based on the ultra-low temperature's efficient freezing and killing ability of mature and pathogen-carrying cells, and the ability to accurately screen the topmost, most vigorous, less or no-toxin cells at the stem tip, the production efficiency of detoxified seedlings is significantly higher than that of various detoxification techniques based on stem tip culture. However, there is currently a lack of efficient and rapid methods for removing plant viroids in tomato tissue culture. Summary of the invention

[0005] The purpose of the present invention is to provide a method for removing hop dwarf viroids by combining sRNA with shoot tip ultra-low temperature therapy to solve the problems existing in the above-mentioned prior art. The method has a high detoxification rate, and the detoxified plants grow more vigorously than the infected plants. The method is suitable for quickly obtaining hop dwarf viroid-free seedlings on tomatoes.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a targeted sRNA for inactivating hop stunt viroid in tomatoes. The sRNA comprises a sense strand as shown in SEQ ID NO: 5 and an antisense strand as shown in SEQ ID NO: 6.

[0008] The present invention also provides a method for removing hop dwarf viroids in tomatoes by using the sRNA combined with the stem tip ultra-low temperature therapy, comprising the following steps:

[0009] Tomato stems infected with hop dwarf viroid were treated with sRNA to obtain samples in which the viroid was suppressed;

[0010] Disinfecting the sample with suppressed viroids to obtain the shoot tip, and then pre-culturing and loading the shoot tip;

[0011] The loaded shoot tips are treated with cryoprotectant PVS2, followed by ultra-low temperature treatment, unloading and post-cultivation to obtain virus-free plants.

[0012] Preferably, the viroid-inhibited sample is obtained by immersing the base of a tomato stem with an apical bud or an axillary bud into a 20-30 μM sRNA solution and culturing at room temperature for 36 hours under light.

[0013] Preferably, the shoot apex is a shoot apex containing 3-4 leaf primordia and apical meristem, and has a length of 1.8-2.0 mm.

[0014] Preferably, the pre-culture conditions are: using MS liquid culture medium containing 0.4 M sucrose, pre-culture at 22-28° C. in the dark for 16 h.

[0015] Preferably, the loading condition is: loading the pre-cultured shoot tips with a loading solution at room temperature for 30 minutes, wherein the loading solution is an MS liquid culture medium containing 0.1 M sucrose and 2 M glycerol.

[0016] Preferably, the loaded stem tip is treated with cryoprotectant PVS2 for 15-20 min;

[0017] The cryoprotectant PVS2 comprises the following components: MS liquid culture medium + 0.4M sucrose + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide.

[0018] Preferably, the ultra-low temperature treatment includes the step of ultra-low temperature freezing the shoot tip treated with cryoprotectant PVS2 in liquid nitrogen for 1-2 hours.

[0019] Preferably, the unloading condition is: treating the ultra-low temperature frozen shoot tip in an unloading solution for 20 minutes for unloading and rewarming, and the unloading solution is an MS liquid culture medium containing 1.2 M sucrose.

[0020] Preferably, the post-culture conditions are: culturing with post-culture medium for 8 weeks, wherein the first 3 days are cultured under dark conditions, and light culture is started after 3 days; the post-culture medium comprises components in the following concentrations: MS liquid culture medium + 0.25 mg / L NAA + 0.25 mg / L TDZ + 0.16 uM glutathione.

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

[0022] The present invention combines exogenous application of viroid-targeted sRNA with ultra-low temperature therapy of the shoot tip to detoxify tomato plants infected with hop dwarf viroid. The results show that the detoxification method provided by the present invention can remove viroids that are generally indestructible on plants, and does not require a long period of low-temperature passivation treatment of viroids. The detoxification rate is high, and the survival rate and regeneration rate of detoxified tomato seedlings are also high. The present invention is suitable for quickly obtaining detoxified seedlings of hop dwarf viroid on tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The secondary structure and partitioning of hop dwarf viroid; the yellow blocks indicate the regions where various sRNAs target the hop dwarf viroid genome;

[0025] Figure 2 Symptoms of tomatoes infected with hop stunt viroid; 1 and 2: healthy plants; 3: healthy leaves; 4 and 5: hop stunt viroid infected plants; 6: infected leaves;

[0026] Figure 3 is the distribution of exogenous sRNA in tomato plants;

[0027] Figure 4 is the distribution of exogenous sRNA in tomato stem apex;

[0028] Figure 5 The results of screening of different exogenous sRNA efficiencies; the inhibitory effect of sRNA on the titer of hop dwarf viroid was determined by detecting the expression of HSV genome by RT-qPCR; the data are expressed as mean ± standard error; differences with different lowercase letters indicate statistically significant differences (P<0.05);

[0029] Figure 6 Comparison results of growth indicators among healthy tomato plants, plants infected with hop dwarf viroid and virus-free plants; data are expressed as mean ± standard error; differences with different lowercase letters indicate statistically significant differences (P<0.05). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental reagents and consumables used are purchased from conventional biochemical reagent stores unless otherwise specified. The experimental instruments can be replaced by other brands. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0036] Example 1 Inoculation of tomatoes with hop stunt viroid

[0037] 1. Plant materials

[0038] The tomato variety chosen was 'Ailsa Craig'.

[0039] 2. In vitro transcription of hop dwarf viroid

[0040] The pGEM-T plasmid containing the hop dwarf viroid dimer cDNA (accession number: MH537075.1297nt) was transcribed in vitro with T7 RNA polymerase at 37°C, and the obtained dimer RNA was dissolved in 1% K 2 HPO 4 Hop stunt viroid-K 2 HPO 4 Inoculation buffer.

[0041] 3. Inoculation of hop dwarfing virus

[0042] Hop stunt viroid-K 2 HPO 4The inoculation buffer was mixed with a small amount of quartz sand and mechanically rubbed onto the second expanded leaf of a tomato plant cultured for 40 days to obtain tomato seedlings inoculated with hop dwarf viroid.

[0043] 4. Cultivation of inoculated seedlings

[0044] Seedlings inoculated with hop dwarf viroid were cultured in a 3:1 peat / perlite mixture and incubated in a growth chamber at 25°C under a 16h light / 8h dark photoperiod and kept moist. 40 days after inoculation, newly developed tomato leaves were collected and their total RNA was extracted, and RT-PCR was used to verify whether the hop dwarf viroid was successfully inoculated.

[0045] 5. RT-PCR detection of hop dwarf viroid

[0046] Total RNA was extracted from leaves using the Trizol kit (Invitrogen, Carlsbad, USA). After reverse transcription and first-strand cDNA synthesis were completed using SuperScript IV (thermofisher, USA), PCR amplification was performed in a PCR instrument (C1000TM Thermal Cycler, BIO-RAD, Singapore). The hop dwarf viroid forward primer (5′-CTGGGAATTCTCGAGT-3′) and reverse primer (5′-AGGGTCAAGAGAGGAT-3′) were used to amplify the hop dwarf viroid genome band. The PCR program was an initial denaturation step of 3 min at 94°C, 35 cycles at 94°C for 30 s, 56°C for 20 s, and 72°C for 30 s, followed by a final extension step of 10 min at 72°C. A 1.5% agarose gel was prepared and specific bands were read by agarose gel electrophoresis.

[0047] 6. Results and Analysis

[0048] 6.1 Analysis of the secondary structure of hop dwarf viroid

[0049] Mfold was used to analyze the secondary structure of hop dwarf viroid (http: / / unafold.rna.albany.edu / ?q=mfold). According to previous reports, the genome of hop dwarf viroid is divided into five regions: the terminal left region, the pathogenic region, the central conserved region, the variable region and the terminal right region. Figure 1 shown.

[0050] 6.2 Hop dwarf viroid tomato disease

[0051] Compared with healthy seedlings, hop dwarf viroid infected plants have severely curled and slightly yellowed leaves and lower plant height, such as Figure 2 shown.

[0052] Example 2: Verification of the penetration of hop dwarf viroid-targeted sRNA in tomato plants

[0053] 1. Design and synthesis of sRNA

[0054] 21nt long double-stranded sRNAs were randomly selected from the left end, pathogenic region, central conserved region, variable region and right end of the hop stunt viroid genome, and the sequences were:

[0055] ds-sRNA-H1: the sense strand is 5'-CUCGAGUCGCCGCAAAAGGCA-3' (SEQ ID NO: 1), and the antisense strand is 3'-UGCCUUUUGCGGCGACUCGAG-5' (SEQ ID NO: 2);

[0056] ds-sRNA-H2: the sense strand is 5'-AACUAGGCAGGGAGGCGCUUA-3' (SEQ ID NO: 3), and the antisense strand is 3'-UAAGCGCCUCCCUGCCUAGUU-5' (SEQ ID NO: 4);

[0057] ds-sRNA-H3: the sense strand is 5'-CUGAGAAAGGAGCCCCGGGGC-3' (SEQ ID NO: 5), and the antisense strand is 3'-GCCCCGGGGCUCCUUUCUCAG-5' (SEQ ID NO: 6);

[0058] ds-sRNA-H4: the sense strand is 5'-AGAAUCCAGCGAGAGGCGUGG-3' (SEQ ID NO: 7); the antisense strand is 3'-CCACGCCUCUCGCUGGAUUCU-5' (SEQ ID NO: 8);

[0059] ds-sRNA-H5: the sense strand is 5'-GCGGUGCUCUGGAGUAGAGGC-3' (SEQ ID NO: 9); the antisense strand is 3'-GCCUCUACUCCAGAGCACCGC-5' (SEQ ID NO: 10).

[0060] The above double-stranded sRNA was synthesized by Nanjing GenScript Company.

[0061] 2. sRNA FAM fluorescent labeling

[0062] Use FAM labeled sRNA labeling kit (Silencer TM, AM1634, Thermo Fisher, USA) was used to label the synthesized ds-sRNA with FAM green fluorescence.

[0063] 3. sRNA exogenous treatment

[0064] 100 μL of FAM-labeled ds-sRNA (20 μM) was applied to the main stem of tomato plants cultured for 40 days. After incubation for 2 h, the samples were washed two to three times with distilled water to prevent the labeled sRNA from adhering to the outer surface of the plant material, and paraffin sections were prepared using the petiole and blade of the second unfolded leaf and the stem tip as samples.

[0065] 4. sRNA permeability detection

[0066] Tomato seedlings were treated with FAM fluorescently labeled sRNA for 2 h, and the distribution of FAM-labeled sRNA in plants or shoot tips was observed using a confocal laser scanning microscope (Zeiss, LSM780, Germany). FAM-sRNA fluorescence was excited by 495 nm laser and detected at 520 nm. The results showed that the results observed in ds-sRNA-H1 to ds-sRNA-H5 treatments were consistent, and fluorescence could be detected in all cells. sRNA can penetrate into all cells in the sample, including vascular cells, parenchyma cells, and apical meristem cells of the shoot tip, such as Figure 3 and Figure 4 shown.

[0067] Example 3 Detection of the inhibitory effect of sRNA on the genome of hop dwarf viroid in tomato

[0068] 1. Exogenous treatment of sRNA in the stem tip of hop dwarf viroid

[0069] From tomato plants infected with hop stunt viroid, stem segments with one young leaf and 1 cm long stem tip were harvested, and the wound at the end of the stem was immersed in exogenous ds-sRNA solution. The treatment was carried out in PCR tubes containing 100 μL of various ds-sRNA (20 μM), with one sample per tube. 2 O was used as a control instead of ds-sRNA. The titer of hop dwarf viroid of each treatment was analyzed by RT-qPCR at 0, 24, 48 and 72 h after ds-sRNA treatment.

[0070] 2. RT-qPCR detection

[0071] Total RNA was extracted from leaves using the Trizol kit (Invitrogen, Carlsbad, USA). Reverse transcription was performed by the ReverTra-Ace qPCR RT kit (Toyobo, Osaka, Japan). qPCR analysis was performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche, Basel, Switzerland) on a FQD-96 Real-Time PCR Detection System (Roche, Basel, Switzerland). The primers used were hop stunt viroid qF: 5′-CAACTCTCAGAATCCAGCGA-3′ and hop stunt viroid qR: 5′-TCGAGTGTTTCGAAGGCA-3′. Three independent biological replicates were used for each assay, each with at least three technical replicates. The Acting gene (NM_001330119.1) was used as a housekeeping gene. 2 -ΔΔCT The method was used to calculate relative gene expression.

[0072] 3. Inhibitory effects of different sRNAs on the titer of hop dwarf viroid

[0073] The titers of hop stunt viroid were significantly inhibited by ds-sRNA-H2, ds-sRNA-H3, ds-sRNA-H4 and ds-sRNA-H5 after 24h, 36h and 48h treatment. The titers of hop stunt viroid in tomato were also significantly inhibited by ds-sRNA-H1 at 36h and 48h treatment, while the titers of hop stunt viroid remained at the control level when ds-sRNA-H1 was treated for 24h. The concentrations of hop stunt viroid were higher when tomato stems were exogenously applied with ds-sRNA-H1, ds-sRNA-H2, ds-sRNA-H4 and ds-sRNA-H5 for 72h compared with the treatment for 24-48h, and only H3 showed hop stunt viroid inhibition. The ds-sRNA-H3 treatment for 36 h had the best effect. Therefore, exogenous application of sRNA targeting the central conserved region of hop dwarf viroid had the best effect, and ds-sRNA-H3 treatment for 36 h was selected for subsequent experiments, such as Figure 5 shown.

[0074] Example 4 Ultra-low temperature therapy of shoot apex treated with ds-sRNA-H3

[0075] 1. Surface disinfection of stem segments

[0076] The tomato stem segments infected with hop dwarf viroid treated with ds-sRNA-H3 solution for 36 hours were used as materials. After being rinsed under running water for 2 hours, the surface was washed in 75% (v / v) ethanol for 30 seconds in an ultra-clean workbench (SJ-CJ-1F, Zhengde Intelligence, Suzhou), and disinfected in 5% (v / v) hypochlorite solution for 5 minutes. The stem tips of plants infected with hop dwarf viroid were surface disinfected and then repeatedly washed with sterile water.

[0077] 2. Acquisition of the stem apex

[0078] After surface disinfection, 2 The shoot tips (1.8-2.0 mm) containing 3-4 leaf primordia and apical meristem were cut from the stem segments treated with O (control material) and ds-sRNA-H3 for 36 h.

[0079] 3. Shoot tip pre-cultivation and loading

[0080] The shoot tips were inoculated in MS liquid medium (pH=5.8) containing 0.4 M sucrose and pre-cultured in the dark at 25° C. for 16 h. The pre-cultured shoot tips were loaded with loading solution [MS liquid medium (pH=5.8,) containing 0.1 M sucrose and 2 M glycerol] at room temperature for 30 min.

[0081] 4. PVS2 solution treatment

[0082] The loaded shoot tips were treated in PVS2 solution [MS liquid culture medium (pH=5.8) + 0.4 M sucrose + 30% (w / v) glycerol + 15% (w / v) ethylene glycol + 15% (w / v) dimethyl sulfoxide] for 5, 10, 15 and 20 min respectively.

[0083] 5. Ultra-low temperature treatment

[0084] Each sprout was transferred to an aluminum foil strip (0.5 cm × 2 cm) carrying a 2.0 μL PVS2 droplet, with one sprout placed in each droplet, and then directly immersed in liquid nitrogen for 10 min, and then transferred to a 2 mL cryovial previously pre-frozen in liquid nitrogen and ultra-low-temperature frozen for 1 h.

[0085] 6. Unloading of the shoot apex

[0086] The aluminum foil strips with the shoot tips were quickly taken out of liquid nitrogen and quickly placed in an unloading solution [composed of MS liquid medium (pH=5.8) containing 1.2 M sucrose] for 20 min for unloading and rewarming.

[0087] 7. Post-cultivation of the shoot tip

[0088] The buds were placed on sterilized filter paper for 2 seconds to remove excess unloading solution. They were then cultured for 8 weeks in MS medium (pH = 5.8) supplemented with 0.25 mg / L NAA, 0.25 mg / L TDDZ and 0.16 uM glutathione. The culture conditions were dark culture in a normal tissue culture environment for the first 3 days and light culture after 3 days.

[0089] 8. Testing of survival rate and regeneration rate

[0090] The shoot survival rate was defined as the percentage of shoots showing green fresh tissues after 8 weeks of culture to the total number of shoots in the treatment. The regeneration rate was defined as the percentage of shoots regenerated into normal shoots (0.5 cm in length) after 8 weeks of culture to the total number of shoots in the treatment.

[0091] 9. Detection of virus-free rate

[0092] Total RNA was extracted from leaves using the Trizol kit (Invitrogen, Carlsbad, USA). After reverse transcription and first-strand cDNA synthesis were completed using SuperScript IV (thermofisher, USA), PCR amplification was performed in a PCR instrument (C1000TM Thermal Cycler, BIO-RAD, Singapore). The hop dwarf viroid forward primer (5′-CTGGGAATTCTCGAGT-3′) and reverse primer (5′-AGGGTCAAGAGAGGAT-3′) were used to amplify the hop dwarf viroid genome band. The PCR program was an initial denaturation step at 94°C for 3 minutes, 35 cycles at 94°C for 30s, 56°C for 20s, and 72°C for 30s, followed by a final extension step at 72°C for 10min. A 1.5% agarose gel was prepared and specific bands were read by agarose gel electrophoresis. The virus-free rate was defined as the percentage of buds without hop dwarf viroid-specific bands detected by RT-PCR and agarose gel electrophoresis to the total number of treated buds after 8 weeks of post-culture.

[0093] 10. Survival rate, regeneration rate and virus-free rate of shoot tip cryotherapy

[0094] The samples treated with ds-sRNA-H3 obtained a 6.7%-10.0% hop dwarf viroid-free rate after 15 and 20 min of PVS2 treatment. The proportion of shoot tips regenerating into complete plants was 13.3-20.0%, and exogenous ds-sRNA treatment did not reduce the survival and regeneration of meristems. Regardless of the duration of PSV2 treatment, ddH 2 The control materials treated with O were unable to obtain the above-mentioned hop dwarf viroid detoxification rate, as shown in Table 1.

[0095] Table 1 Screening results of ultra-low temperature therapy detoxification system

[0096]

[0097] The data are expressed as mean ± standard error. The differences with different lowercase letters indicate statistically significant differences (P<0.05).

[0098] Example 5 Comparison of growth indicators of healthy tomatoes, tomatoes infected with hop dwarf viroid and free of virus

[0099] 1. Materials

[0100] After acclimation of the regenerated tissue culture seedlings for 1 week, the HVSd virus-free seedlings were transplanted into a 3:1 peat / perlite mixed substrate and cultured in a plant growth room at 25°C under a photoperiod of 16 h light / 8 h dark for 80 days. Main stems with a length of 10 cm were cut from healthy controls, hop dwarf viroid-infected tomatoes and virus-free tomatoes and cultured again under the same culture conditions for 80 days as test materials.

[0101] 2. Growth index determination

[0102] The vegetative growth of healthy tomatoes, hop dwarf viroid-infected tomatoes, and virus-free tomatoes was evaluated by measuring the average plant height, stem diameter, number of leaves, root length, and fresh root weight using an electronic vernier caliper (Xibei Qingliang, Qinghai) and counting method. Leaf area and root area were measured using a leaf area and plant root scanner (12000XL, Expression, Tokyo, Japan).

[0103] 3. Comparison results

[0104] The virus-free seedlings regenerated from the ultra-low temperature detoxification of the shoot tips showed similar plant growth status compared with the healthy control. There were no significant differences in stem length, leaf number, root length, root fresh weight, leaf area and root area, while stem length, stem diameter, leaf number, root length, root fresh weight, leaf area and root area were significantly higher than those of tomatoes infected with hop dwarf viroid. Figure 6 shown.

[0105] The above results showed that the tomato stem tips treated with ds-sRNA-H3 for 36h were virus-free samples, and after being pretreated with ultra-low temperature therapy for 16h, loaded for 30min, treated with PVS2 for 15min and 20min, unloaded for 20min, and cultured on MS medium (pH=5.8) supplemented with 0.25mg / L NAA, 0.25mg / LTDZ and 0.16uM glutathione, 6.7% and 10.0% virus-free rates of hop dwarf viroid were obtained. Compared with virus-infected tomato plants, virus-free plants grew more vigorously.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A targeted sRNA for inactivating hop dwarf viroid in tomatoes, characterized in that: The sRNA includes a sense strand as shown in SEQ ID NO:5 and an antisense strand as shown in SEQ ID NO:

6.

2. A method for removing hop dwarf viroid from tomatoes by using the sRNA combined with the stem tip ultra-low temperature therapy according to claim 1, characterized in that: The following steps are involved: Tomato stems infected with hop dwarf viroid were treated with sRNA to obtain samples in which the viroid was suppressed; Disinfecting the sample with suppressed viroids to obtain the stem tip, and then pre-culturing and loading the stem tip; The loaded stem tip is treated with cryoprotectant PVS2, and then subjected to ultra-low temperature treatment, unloading and post-cultivation to obtain virus-free plants; The ultra-low temperature treatment comprises the step of ultra-low temperature freezing the shoot tip treated with cryoprotectant PVS2 in liquid nitrogen for 1-2 hours.

3. The method according to claim 2, characterized in that The sample in which the viroid is inhibited is obtained by immersing the base of a tomato stem with an apical bud or an axillary bud into a 20-30 μM sRNA solution and culturing it under light at room temperature for 36 hours.

4. The method according to claim 2, characterized in that The shoot apex is a shoot apex containing 3-4 leaf primordia and apical meristem, and has a length of 1.8-2.0 mm.

5. The method according to claim 2, characterized in that The pre-culture conditions are: using MS liquid culture medium containing 0.4 M sucrose, pre-culture at 22-28° C. in the dark for 16 hours.

6. The method according to claim 2, characterized in that The loading condition is: the pre-cultured shoot tips are loaded with a loading solution at room temperature for 30 minutes, and the loading solution is an MS liquid culture medium containing 0.1 M sucrose and 2 M glycerol.

7. The method according to claim 2, characterized in that The loaded stem tip is treated with cryoprotectant PVS2 for 15-20 min; The cryoprotectant PVS2 comprises the following components: MS liquid culture medium + 0.4M sucrose + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide.

8. The method according to claim 2, characterized in that The unloading condition is as follows: the shoot tip that has been subjected to ultra-low temperature freezing is treated in an unloading solution for 20 minutes for unloading and rewarming, and the unloading solution is an MS liquid culture medium containing 1.2 M sucrose.

9. The method according to claim 2, characterized in that The post-culture conditions are: culture with post-culture medium for 8 weeks, wherein the first 3 days are cultured under dark conditions, and light culture is started after 3 days; the post-culture medium includes components of the following concentrations: MS liquid culture medium + 0.25 mg / L NAA + 0.25 mg / L TDZ + 0.16 μM glutathione.

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