Cultivation method of virus-free tissue culture seedlings of trichosanthes kirilowii maxim and virus-free tissue culture seedlings obtained by method

Abstract

The invention belongs to the technical field of plant tissue culture, and discloses a method for cultivating detoxified tissue culture seedlings of trichosanthes kirilowii maxim and the detoxified tissue culture seedlings obtained by the method. The method comprises the following steps: taking a terminal bud or an axillary bud of a trichosanthes kirilowii Maxim plant, performing surface disinfection, and stripping a 0.3-0.5 mm stem tip under a sterile condition to serve as an explant; inoculating the stem tips into a primary culture medium for induction culture, wherein the primary culture medium is an MS culture medium added with 1.0 mg / L 6-BA and 0.2 mg / L NAA; after the adventitious buds germinate, sequentially performing multiplication culture, subculture and rooting culture to obtain complete tissue culture seedlings; and finally carrying out acclimatization and transplantation. According to the method, a systematic and efficient stem tip detoxification culture system is established for trichosanthes kirilowii maxim for the first time, and by optimizing explant selection, disinfection procedures, a key culture medium formula and culture conditions, rapid propagation is achieved, and meanwhile the survival rate of the trichosanthes kirilowii maxim is increased. Various viruses such as cucurbit light mottle mosaic virus (CuMMV) and zucchini green mottle mosaic virus (ZGMMV) carried in a trichosanthes kirilowii Maxim plant can be efficiently removed, and the detoxification rate can reach 96% or above.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and relates to a method for stem tip tissue culture of Trichosanthes kirilowii (Trichosanthes tangutorum). In particular, it relates to a rapid propagation method of Trichosanthes kirilowii tissue culture that can efficiently remove viruses and obtain virus-free seedlings, as well as the virus-free tissue culture seedlings obtained by this method. Background Technology

[0002] Trichosanthes kirilowii Maxim., also known as medicinal melon, wild melon, and hanging melon, is a perennial twining vine belonging to the genus Trichosanthes in the Cucurbitaceae family. It is a major traditional Chinese medicinal herb, with its roots, fruits, and seeds all used medicinally. Previously, domestic research on Trichosanthes kirilowii largely focused on its medicinal components and value. In the 1950s, in the Dabie Mountains region, Trichosanthes kirilowii seeds were used as a side dish for entertaining guests, gradually forming an industry development model primarily focused on food while also considering medicinal uses. In recent years, research on Trichosanthes kirilowii has not only expanded its traditional medicinal value but has also continuously discovered its edible and ornamental value. Trichosanthes kirilowii is one of the specialty economic crops with great potential in the current agricultural restructuring, and its planting scale is constantly expanding, leading to an increasing demand for high-quality seedlings.

[0003] Traditional methods of propagating Trichosanthes kirilowii seedlings often involve direct sowing from seeds or germinating seedlings from small sections of underground tubers (Trichosanthes kirilowii rootlets). Because Trichosanthes kirilowii is dioecious, seed sowing often results in a severe imbalance in the male-to-female ratio, producing a large number of ineffective male seedlings and causing waste. Cultivating seedlings from tubers requires a large number of underground tubers, leading to low propagation efficiency, and the quality of seedlings is affected by the quality of the tubers, making them susceptible to viral accumulation leading to genetic degeneration and the spread of root-knot nematodes. Tissue culture technology can achieve rapid and large-scale propagation of Trichosanthes kirilowii, suitable for the large-scale promotion of superior varieties, plant sex control, and germplasm preservation.

[0004] Current research on Trichosanthes kirilowii tissue culture mainly focuses on using explants such as cotyledons, leaf segments, petioles, tendrils, stem segments, root segments, and terminal or axillary buds of seed tubers. By adding different hormones to induce the development of adventitious buds and roots, complete plants can be cultured, achieving the goal of rapid propagation. However, while tissue culture using these explants can achieve rapid propagation, these conventional explants often carry viruses from the parent plant, resulting in tissue culture seedlings that still carry viruses. This fails to fundamentally solve the problems of virus-carrying seedlings and virus accumulation and degeneration.

[0005] Shoot tip culture is one of the most effective methods for plant virus elimination. Its principle is based on the characteristics of vigorous cell division and extremely low or even virus-free virus content in the meristematic tissue of plant shoot tips. Virus-free plants are obtained by culturing tiny shoot tips. Currently, there are no publicly reported studies on the successful application of shoot tip virus elimination technology to *Trichosanthes kirilowii*, along with the establishment of a complete, efficient, and stable culture system from virus elimination and rapid propagation to seedling formation. Furthermore, existing technology lacks optimized solutions specifically for the characteristics of *Trichosanthes kirilowii* shoot tips (such as difficulty in sterilization, high browning rate, and low induction rate), and there is no experimental data to confirm its virus elimination effect. Summary of the Invention

[0006] The primary objective of this invention is to overcome the shortcomings of existing rapid propagation techniques for Trichosanthes kirilowii tissue culture, which cannot remove viruses, and to provide an efficient and stable method for virus-free culture of Trichosanthes kirilowii stem tips.

[0007] Another objective of this invention is to provide a virus-free tissue culture seedling of Trichosanthes kirilowii that is not carrying major viruses (such as CuMMV and ZGMMV) obtained by the above method.

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

[0009] The first aspect of this invention is to provide a method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii, comprising the following steps:

[0010] S1. Explant preparation and disinfection: Select the terminal or axillary buds of healthy Trichosanthes kirilowii plants, wash them with running water, and then disinfect them by soaking them in 75% alcohol for a short time combined with soaking them in 0.1% mercuric chloride solution for 5 minutes. This effectively reduces the contamination rate and ensures the viability of the explants.

[0011] S2. Shoot tip explant: Under a clean bench and dissecting microscope, aseptically explant approximately 0.3-0.5 mm long shoot tip meristems with 1-2 leaf primordia as explants.

[0012] S3, Primary culture: Shoot tips were inoculated into optimized primary culture medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA) and cultured under suitable light and temperature conditions to induce adventitious bud germination;

[0013] S4. Proliferation Culture: The germinating adventitious shoots were transferred to proliferation medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L KT) for efficient propagation;

[0014] S5. Subculture: The proliferated shoot clusters were separated into individual plants and cultured in subculture medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA) to further strengthen the seedlings;

[0015] S6. Rooting culture: Transfer robust rootless seedlings to rooting medium (MS + 0.6 mg / LIBA) to induce the formation of a well-developed root system;

[0016] S7. Hardening and Transplanting: After hardening, the complete tissue culture seedlings are transplanted into a substrate with a specific ratio (peat soil: perlite: vermiculite = 2:1:1) to complete the transition from test-tube seedlings to field seedlings.

[0017] Preferably, in step S1, the specific method for surface disinfection is as follows: first, soak in 75% alcohol by volume for 30 seconds, then soak in 0.1% mercuric chloride solution by mass for 5 minutes, and finally rinse with sterile water 5 times.

[0018] Preferably, in steps S3, S4, S5, and S6, the pH value of each culture medium is 5.8, and the culture conditions are: temperature 26-28℃, humidity 50-60%, light intensity 2500-3000 Lux, and photoperiod 16 hours / day.

[0019] Preferably, in step S4, the proliferation medium is MS medium, and is supplemented with 1.0 mg / L 6-BA, 0.1 mg / L NAA and 0.1 mg / L KT.

[0020] Preferably, in step S5, the subculture medium is MS medium, with 1.0 mg / L 6-BA and 0.1 mg / L NAA added.

[0021] Preferably, in step S6, the rooting medium is MS medium with 0.6 mg / L IBA added.

[0022] Preferably, in step S7, the hardening-off time is 3-5 days; the cultivation substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:1, and after transplanting, the temperature is maintained at 25-28℃ and the humidity at 80-90%.

[0023] A second aspect of the present invention is to provide a virus-free tissue culture seedling of Trichosanthes kirilowii obtained by any of the methods described above, wherein the virus-free tissue culture seedling does not carry at least one of Cucurbita Mottle Mosaic Virus (CuMMV) and Zucchini Green Mottle Mosaic Virus (ZGMMV).

[0024] Preferably, the virus-free tissue culture seedlings are of the varieties Wanlou No. 9, Wanlou No. 17, or Wanlou No. 20.

[0025] A third aspect of the present invention is to provide the application of the virus-free tissue culture seedlings of Trichosanthes kirilowii as described above in the cultivation of virus-free seedlings for production.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0027] (1) Highly efficient virus removal effect: This invention is the first to apply the shoot tip culture technology system to the virus removal of Trichosanthes kirilowii, and the results have been verified by RT-PCR detection. Experiments have shown that this method has extremely high removal efficiency against Cucurbita Mottle Mosaic Virus (CuMMV) and Zucchini Green Mottle Mosaic Virus (ZGMMV), which are common and serious pests in Trichosanthes kirilowii fields. The virus removal rate can reach more than 96%, which fundamentally solves the problem of virus transmission in seedlings.

[0028] (2) Optimized technical system: Through comparative experiments, this invention has determined the most suitable disinfection method (0.1% mercuric chloride treatment for 5 minutes) and basic culture medium (MS medium) for the stem tips of Trichosanthes kirilowii, which reduces the contamination rate of the first generation culture to 0% and the survival rate to 93.3%. At the same time, the key hormone ratios of each stage from the first generation induction to rooting and seedling growth have been optimized, forming a complete, efficient and stable culture process.

[0029] (3) Rapid propagation capacity: The proliferation medium provided by this invention can enable the average bud proliferation coefficient of Trichosanthes kirilowii tissue culture seedlings to reach 7.97, and the propagation speed is fast, which can meet the requirements of industrial seedling production for the propagation coefficient.

[0030] (4) Wide applicability: The method of this invention has been successfully applied to multiple Trichosanthes kirilowii varieties such as Wanlou No. 9, Wanlou No. 17, and Wanlou No. 20, indicating that the technology system has good universality and is easy to promote.

[0031] (5) High quality of seedlings: The virus-free tissue culture seedlings obtained by this method are robust, have well-developed root systems, have a high survival rate after transplanting, and are free from the virus problem. Their growth potential and yield potential are significantly better than those of virus-infected seedlings. Attached Figure Description

[0032] Figure 1 and Figure 2 The image shows the microstem tips at the axillary buds of Trichosanthes kirilowii in Example 1 and the cut microstem tips (×40x magnification).

[0033] Figure 3 , Figure 4 and Figure 5 The growth status of Trichosanthes kirilowii stem tips after 7, 14, and 30 days of initial culture in Example 2 is shown.

[0034] Figure 6 and Figure 7 The images show the growth status of the first and second transplantation of Trichosanthes kirilowii explants in Example 3.

[0035] Figure 8 and Figure 9 The image shows the rooting of Trichosanthes kirilowii tissue culture seedlings in Example 4 at concentrations of 0.05 mg / L NAA, 0.01 mg / L NAA, and 0.2 mg / L NAA.

[0036] Figure 10 and Figure 11 The growth chart for Trichosanthes kirilowii tissue culture seedlings in Example 4 is shown at concentrations of 0.05 mg / L NAA, 0.01 mg / L NAA, and 0.2 mg / L NAA.

[0037] Figure 12 and Figure 13 The growth chart for Trichosanthes kirilowii tissue culture seedlings in Example 4 is shown at concentrations of 0.2 mg / LIBA, 0.4 mg / LIBA, and 0.6 mg / LIBA.

[0038] Figure 14 and Figure 15 The image shows the rooting of Trichosanthes kirilowii tissue culture seedlings in Example 4 at concentrations of 0.2 mg / LIBA, 0.4 mg / LIBA, and 0.6 mg / LIBA.

[0039] Figure 16 This is the growth status of Trichosanthes kirilowii tissue culture seedlings after hardening and transplanting in Example 4.

[0040] Figure 17 This is an RNA gel electrophoresis image from Example 5, showing the RT-PCR detection of nine common viruses, including Potato Virus Y (PVY).

[0041] Figure 18 This is an RNA gel electrophoresis image of Comparative Example 1 for RT-PCR detection of gourd mild mottle mosaic virus.

[0042] Figure 19 This is an RNA gel electrophoresis image of RT-PCR detection of zucchini green mottle mosaic virus, which is the result of Comparative Example 1. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and do not constitute a limitation thereof.

[0044] Example 1: Effects of different sterilization methods and basal culture media on primary culture of Trichosanthes kirilowii shoot tips

[0045] The peeled stem tip of Trichosanthes kirilowii is like Figure 1 and Figure 2 As shown. By Figure 1 As can be seen, the stem tip at the axillary bud is significantly lighter in color than the stem segment, slightly translucent, and two leaf primordia are faintly visible. Microscopically, the tip measures approximately 3 millimeters in size. The carefully peeled and cut micro-stem tip... Figure 2 As shown in Table 1, the statistical results of primary cultures of shoot tips using different sterilization methods on different basic cultures are presented.

[0046] Table 1. Effects of different sterilization methods and basal culture media on the primary culture of Trichosanthes kirilowii shoot tips (30 days)

[0047] Note: +: The growth rate of the microstem tip is slow, most of the buds are slightly yellow, and some show signs of vitrification. ++: The growth rate of the microstem tip is slightly slower, and the color of the buds is slightly yellowish. +++: The microstem tip is growing well, the buds are growing rapidly, the color is bright green, and the shape is normal.

[0048] Observing the data in Table 1 and comparing the disinfection methods, the contamination rate after disinfection with 0.1% mercuric chloride solution was lower than that after disinfection with 4% sodium hypochlorite. After disinfection with 0.1% mercuric chloride solution, the contamination rate of samples inoculated into MS medium was as low as 0.0%; while after disinfection with 4% sodium hypochlorite, the contamination rate of samples inoculated into MS medium was 16.7%. Regarding survival rate, the survival rate of samples inoculated into MS medium after disinfection with 0.1% mercuric chloride solution was the highest, reaching 93.3%, which is significantly higher than the survival rate of 10.0% after disinfection with 4% sodium hypochlorite. Therefore, it is evident that 0.1% mercuric chloride solution has the most ideal disinfection effect.

[0049] Compared with the basal culture medium, experimental data after sterilization with 0.1% mercuric chloride solution were used as the basis. The shoot tips inoculated on MS medium had the highest survival rate, reaching 93.3%, and the micro-shoot tip growth status was +++; the shoot tips inoculated on 1 / 2 MS medium had a survival rate of 63.3%, and the micro-shoot tip growth status was ++; the shoot tips inoculated on B5 medium had a survival rate of 56.7%, and the micro-shoot tip growth status was +.

[0050] The results showed that: (1) the contamination rate of disinfection method ② was 0% and the survival rate was significantly higher than that of method ①, making it the better disinfection method among the two; (2) by comparing the growth status of microstem tips, the effect of basal medium MS was better than that of 1 / 2MS and B5.

[0051] Example 2: Effects of different hormone ratios on primary culture of Trichosanthes kirilowii shoot tips

[0052] The effects of different hormone concentration combinations on the primary culture of Trichosanthes kirilowii shoot tips were observed and statistically analyzed. The data obtained are shown in Table 2 below. The culture conditions are as follows: Figure 3 , Figure 4 and Figure 5 As shown.

[0053] Table 2. Results of primary culture of Trichosanthes kirilowii shoot tips under different hormone ratios (30 days)

[0054] The data in Table 2 clearly show that different combinations of hormone concentrations have a significant impact on various indicators of the primary culture of Trichosanthes kirilowii shoot tips.

[0055] Bud height: In all groups: (1) The contamination rate of disinfection method ② was 0%, and the survival rate was significantly higher than that of method ①, which is the better disinfection method among the two; (2) By comparing the growth status of microstem buds, the effect of basal medium MS was better than 1 / 2MS and B5.

[0056] Seedling thickness: The seedlings in group ④ were the thickest, measuring 1.8±0.4 mm. The seedlings in groups ① and ⑤ were relatively thick, measuring 1.5±0.4 mm and 1.6±0.3 mm respectively, while the seedlings in group ③ were the thinnest, measuring 1.0±0.2 mm. This indicates that when 6-BA and NAA are combined at 1.0 mg / L and 0.2 mg / L, respectively, they effectively promote lateral growth of the stem tips, resulting in stronger stem tips.

[0057] Leaf count: Group ④ had the most leaves, with 4.0 ± 0.6 leaves. Groups ① and ⑤ had relatively more leaves, with 3.5 ± 0.5 and 3.8 ± 0.5 leaves respectively. Group ② had fewer leaves, with only 2.8 ± 0.5 leaves, and Group ③ had the fewest leaves, with only 2.0 ± 0.3 leaves. This indicates that the combination of 6-BA and NAA at 1.0 mg / L and 0.2 mg / L has a positive effect on leaf differentiation and growth.

[0058] Color and growth vigor: The stem tips of groups ①, ④, and ⑤ are bright green and exhibit vigorous or relatively vigorous growth, indicating that the hormone combinations in these three groups create a favorable environment for stem tip growth and promote healthy stem tip development. Group ② is light green and exhibits average growth vigor; Group ③ is yellowish-green and exhibits weak growth vigor, indicating that the hormone combinations in these two groups have a relatively limited effect on promoting stem tip growth.

[0059] Based on the above indicators, the hormone combination of ④MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA performed the best in the primary culture of Trichosanthes kirilowii shoot tips, significantly promoting shoot tip growth and development and greatly improving the effectiveness of primary culture.

[0060] Example 3: Subculture of Trichosanthes kirilowii shoot tips

[0061] During the subculture of Trichosanthes kirilowii shoot tips, detailed observations and data recording were conducted on the number of inoculated shoots, total number of shoots after proliferation, shoot height, color, number of new shoots, and shoot proliferation coefficient of the first and second subcultures. The results are shown in Table 3 below. The growth status of the Trichosanthes kirilowii explants after the first and second subcultures is as follows. Figure 6 and Figure 7 As shown.

[0062] Table 3. Growth of explants at different stages (15 days)

[0063] The results showed that: (1) This experiment was a two-parallel replicate experiment of subculture proliferation culture. The average bud proliferation coefficient was the final conclusion of the data: (7.17+8.76)÷2=7.97. On this proliferation medium, the average bud proliferation coefficient of Trichosanthes kirilowii tissue culture seedlings was 7.97, and the buds after proliferation were bright green, increased in height, and had good physiological condition. (2) The high average bud proliferation coefficient indicates that the proliferation capacity of the culture system designed in this experiment is strong, and the culture conditions have a good promoting effect on bud proliferation.

[0064] By comparing the bud growth status under different subculture cycles in Table 3, we can more intuitively understand the changes in bud growth. After the first subculture, the bud height is relatively low, and the number of new buds is also relatively small. As the number of subculture cycles increases, the buds gradually grow taller, new buds sprout one after another, the clustered buds become more and more dense, and the overall growth is good. This is consistent with the data results presented in Table 3, fully demonstrating that the MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L KT subculture proliferation medium and the corresponding culture conditions can fully meet the various requirements of Trichosanthes kirilowii shoot tip subculture proliferation culture, effectively improve the proliferation coefficient of Trichosanthes kirilowii shoot tips, and ensure growth quality.

[0065] Example 4: Screening of culture medium for rooting and strengthening Trichosanthes kirilowii test-tube seedlings

[0066] The growth of explants in different rooting and seedling strengthening media was observed and statistically analyzed. The data obtained are shown in Table 4 below. The plant growth and rooting status are as follows: Figures 8 to 16 As shown.

[0067] Table 4. Experimental results of rooting and seedling vigor development of Trichosanthes kirilowii plantlets in different culture media (15 days)

[0068] The results showed that: (1) Compared with ①②③, all rooting indicators gradually increased with the increase of NAA. The increase of NAA concentration is beneficial to promoting the rooting of Trichosanthes kirilowii tissue culture seedlings. (2) Compared with ④⑤⑥, the conclusions of all statistical indicators are the same as those in (1). (3) Trichosanthes kirilowii test-tube seedlings can achieve good rooting and seedling strengthening effects in MS + 0.6 mg / LIBA medium. After 15 days of culture, the rooting rate can reach 86.7%, the total number of roots can reach 11.7, the seedling height increases by 0.8 cm, the seedling diameter increases by 0.4 mm, and 1.6 new leaves are added.

[0069] Example 5: Method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii

[0070] This embodiment uses Wanlou No. 9, No. 17, and No. 20 as materials, and the specific steps are as follows:

[0071] S1. Obtaining test materials

[0072] Select thick, large tuberous roots from healthy Trichosanthes kirilowii plants and cultivate them in loamy soil containing a certain amount of river sand at room temperature and natural light. When the plant produces terminal and axillary buds, cut them off from the plant, wash them with running water to remove surface dust and impurities, and store them in a clean glass bottle for later use.

[0073] S2. Surface disinfection of test materials

[0074] The young apical and axillary buds of Trichosanthes kirilowii collected in step S1 are transferred to a clean bench. They are first soaked in 75% alcohol for 30 seconds, then transferred to a sterile bottle and disinfected with 0.1% mercuric chloride solution for 5 minutes. After disinfection, they are rinsed 5 times with sterile water for 1 minute each time.

[0075] S3, stem tip stripping

[0076] After wiping the surface moisture of the explants disinfected in step S2 with filter paper, carefully peel off the outer young leaves and leaf primordia using a sterile dissecting microscope (40x) in a clean bench under strict aseptic conditions, and cut off the stem tip containing meristems, which is about 0.3-0.5 mm long.

[0077] S4, primary shoot tip culture

[0078] The shoot tips obtained in step S3 were inoculated into MS medium containing 1.0 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, 6.2 g / L agar, and pH 5.8 using a sterile dissecting needle. The culture conditions were: 26℃-28℃, humidity 50%-60%, light intensity 2500 Lx-3000 Lx, and photoperiod 16 h / d. After 30 days of culture, the shoot tip survival rate reached 93.3%, and the emerging adventitious buds were bright green and grew vigorously.

[0079] S5, adventitious bud proliferation

[0080] After one month of shoot tip culture, vigorous, bright green adventitious shoots (approximately 1-2 cm in height) obtained in step S4 were selected and transferred to proliferation medium for further culture. The proliferation medium consisted of MS medium containing 1.0 mg / L 6-BA, 0.1 mg / L NAA, 0.1 mg / L KT, 30 g / L sucrose, 6.2 g / L agar, and pH 5.8. The culture conditions were the same as in step S4. Under these conditions, dense clusters of shoots formed after approximately 15 days. The average shoot proliferation coefficient was measured to be 7.97.

[0081] S6, Plant Subculture

[0082] Once the adventitious buds obtained in step S5 have grown to 2-3 cm in height, they are subcultured. In a clean bench, using a sterile scalpel, the adventitious bud clusters are separated into individual plants. Using forceps, they are inoculated onto the subculture medium following their natural growth direction. Subculture is performed every 30 days to expand propagation and promote seedling growth. The subculture medium formula is: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6.2 g / L agar + pH 5.8. The culture conditions are the same as in step S4.

[0083] S7, Plant Rooting

[0084] After subculturing in step S6, when the tissue culture seedlings reach a height of 4-5 cm and have 3-4 stem nodes, they are transferred to rooting medium for rooting culture. The rooting medium formula is: MS + 0.6 mg / L IBA + 30 g / L sucrose + 6.2 g / L agar + pH 5.8. The culture conditions are the same as in step S4. After 15 days of culture, the rooting rate reaches 86.7%, with an average of 11.7 roots per seedling, and the plants are growing vigorously.

[0085] S8, Seedling Hardening and Transplanting

[0086] After rooting culture in step S7, when the roots of the tissue culture seedlings reach 2-3 cm in length, open the culture bottle and harden the seedlings under flash light for 3-5 days. Remove the seedlings, wash off the agar from the roots, and transplant them into a sterilized substrate of peat moss:perlite:vermiculite = 2:1:1, maintaining a temperature of 25℃-28℃ and humidity of 80%-90%. After 20-30 days of growth in the substrate, when new leaves appear, transplant them to the field according to climatic conditions.

[0087] Example 6: Virus Detection in Trichosanthes kirilowii Seedlings

[0088] To verify whether the Trichosanthes seedlings cultivated in this invention carry multiple viruses such as Potato Virus Y, virus detection was performed on the virus-free tissue culture seedlings of Wanlou No. 9, No. 17, and No. 20 cultivated by the method in Example 5 above.

[0089] Test samples: 12 samples of Trichosanthes kirilowii seedlings cultured in Example 5 above, namely 17-1, 17-2, 17-3, 20-1, 20-2, 20-3, 9, 9-1, 9-2, 9-3, 17, and 20.

[0090] Testing Unit: Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences

[0091] Testing method: RT-PCR detection was performed using specific primers to detect nine common viruses, including Potato Virus Y (PVY).

[0092] Test results:

[0093] For the 12 *Trichosanthes kirilowii* seedlings submitted for testing, nine detection targets were identified. The samples were processed according to the methods specified in GB / T 28071-201, "Quarantine Identification Method for Cucumber Green Mottle Mosaic Virus," to extract total RNA, synthesize cDNA via reverse transcription, and perform RT-PCR detection using the cDNA as a template. For CGMMV, specific primers were designed according to the national standard; for the other detection targets, specific primers were designed based on relevant standards and literature. The test results are as follows: Figure 17 As shown.

[0094] Depend on Figure 17 The shown RNA gel electrophoresis images indicate the following lane order: For non-virus-free vaccines, lanes 1:17-1, 2:17-2, 3:17-3, 4:20-1, 5:20-2, 6:20-3, 8:9-1, 9:9-2, and 10:9-3 are lanes. For virus-free vaccines, lanes 7:9, 11:17, and 12:20 are shown in the red box.

[0095] The results showed that none of the samples carried PVY, PVX, TMV, or TSWV. Sample 9-3 carried CMV; samples 20-1, 20-2, 9-1, 9-2, and 9-3 carried ZGMMV; and samples 17-1, 17-2, 17-3, 20-1, 20-2, 9, 9-2, 9-3, 17, and 20 carried CuMMV. Samples 17-1, 17-2, 17-3, 20-1, 20-2, 20-3, 9, 9-1, 9-2, 9-3, and 20 carried TrMMV. None of the samples carried CGMMV. CuMMV was detected in sample 17, and both CuMMV and TrMMV were detected in samples 9 and 20.

[0096] Comparative Example 1: Detoxification Effect Test

[0097] To verify the detoxification effect of the method of the present invention, the detoxification rate was tested on two detoxified tissue culture seedlings (Wanlou No. 9 and Wanlou No. 17) and their corresponding original field seedlings, which were cultivated by the method of Example 5 above, for two viruses (CuMMV and ZGMMV) that caused severe disease in Trichosanthes kirilowii in the field.

[0098] Test samples: The virus-free seedlings of Wanlou No. 9 and Wanlou No. 17 cultivated in Example 5 above were used as experimental groups, and the corresponding field original seedlings of Wanlou No. 9 and Wanlou No. 17 were used as control groups.

[0099] Testing Unit: Institute of Plant Protection and Agricultural Product Quality and Safety, Anhui Academy of Agricultural Sciences

[0100] Detection method: RT-PCR was used, focusing on detecting Cucurbita Mild Mottle Mosaic Virus (CuMMV) and Zucchini Green Mottle Mosaic Virus (ZGMMV), which caused severe disease outbreaks in the field. RT-PCR results are shown below. Figure 18 and Figure 19 As shown in Table 5 below, the detoxification rate test data are presented.

[0101] Table 5. Detection of CuMMV and ZGMMV virus-carrying rates in field seedlings and virus-free seedlings.

[0102] The results showed that the field virus carriage rates of CuMMV for the two varieties, Wanlou 9 and Wanlou 17, were 61% and 76%, respectively, and after detoxification through shoot tip culture, the virus carriage rates of both varieties were 0%. Similarly, the field virus carriage rates of ZGMMV for the two varieties, Wanlou 9 and Wanlou 17, were 88% and 87%, respectively, and after detoxification through shoot tip culture, the virus carriage rates of both varieties were 0% and 4%. These results indicate that the shoot tip culture detoxification method for Trichosanthes kirilowii was highly effective, achieving a detoxification rate of over 96%.

[0103] Conclusion: The shoot tip culture method described in this invention has an extremely significant effect on eliminating the main viruses CuMMV and ZGMMV in Trichosanthes kirilowii, with a comprehensive virus elimination rate of over 96%. This fully demonstrates that this invention is not only a rapid propagation technique, but also an effective virus elimination technique capable of producing high-quality virus-free Trichosanthes kirilowii seedlings.

[0104] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii, characterized in that, Includes the following steps: S1. Explant preparation and disinfection: Take the terminal bud or axillary bud of the Trichosanthes kirilowii plant and disinfect its surface. S2. Stem tip removal: Under aseptic conditions, remove stem tips of 0.3-0.5 mm in size from the material sterilized in step S1; S3. Primary culture: The shoot tips obtained in step S2 are inoculated into the primary culture medium for induction culture. The primary culture medium is MS medium supplemented with 1.0 mg / L 6-BA and 0.2 mg / L NAA. S4. Proliferation culture: The adventitious shoots obtained in step S3 are transferred to the proliferation culture medium for propagation. S5. Subculture: The clustered shoots obtained from step S4 are separated into individual plants and transferred to a subculture medium for culture. S6. Rooting culture: Transfer the robust tissue culture seedlings obtained in step S5 to the rooting culture medium to induce rooting. S7. Hardening and Transplanting: After hardening the complete tissue culture seedlings obtained in step S6, transplant them into the cultivation substrate.

2. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In step S1, the specific method for surface disinfection is as follows: first, soak in 75% alcohol for 30 seconds, then soak in 0.1% mercuric chloride solution for 5 minutes, and finally rinse with sterile water 5 times.

3. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In steps S3, S4, S5, and S6, the pH value of each culture medium was 5.8, and the culture conditions were: temperature 26-28℃, humidity 50-60%, light intensity 2500-3000 Lux, and photoperiod 16 hours / day.

4. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In step S4, the proliferation medium is MS medium, and 1.0 mg / L 6-BA, 0.1 mg / L NAA and 0.1 mg / L KT are added.

5. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In step S5, the subculture medium is MS medium, with 1.0 mg / L 6-BA and 0.1 mg / L NAA added.

6. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In step S6, the rooting medium is MS medium with 0.6 mg / L IBA added.

7. The method for cultivating virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 1, characterized in that, In step S7, the hardening-off time is 3-5 days; the cultivation substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:1, and after transplanting, the temperature is maintained at 25-28℃ and the humidity at 80-90%.

8. A virus-free tissue culture seedling of Trichosanthes kirilowii obtained by the method according to any one of claims 1-7, characterized in that, The virus-free tissue culture seedlings do not carry at least one of Cucurbita Mottle Mosaic Virus (CuMMV) and Zucchini Green Mottle Mosaic Virus (ZGMMV).

9. The virus-free tissue culture seedlings of Trichosanthes kirilowii according to claim 8, characterized in that, The virus-free tissue culture seedlings are of varieties such as Wanlou No. 9, Wanlou No. 17, or Wanlou No.

20.

10. The application of the virus-free tissue culture seedlings of Trichosanthes kirilowii as described in claim 8 or 9 in the cultivation of virus-free seedlings for production.

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