Method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings

Abstract

The present invention provides a method for producing a snow cane that is free of sugarcane mosaic virus and a virus-free seedling. The method mainly comprises the following steps that are performed in sequence: (1) preparation of qualified single bud seed stems; (2) disinfection; (3) planting; (4) preheating treatment; (5) double high temperature change detoxification treatment; (6) stem tip culture; (7) obtaining virus-free seedlings. The invention overcomes the disadvantage that the existing sugarcane detoxification methods are not applicable to the snow cane seed stems and axillary buds due to their high water content. The obtained detoxified seedlings are tested by RT-PCR molecular detection of sugarcane mosaic virus, and the detoxification rate reaches 100%. The present invention provides a method for producing a snow cane that is free of sugarcane mosaic virus and a virus-free seedling. The detoxification technical method of preparing qualified single bud seed stems and performing preheating treatment on them, double high temperature change combined with alternating light / dark cycle treatment, and stripping a larger (8-10 mm) stem tip for culture is the core innovation of the present invention, which is simple to operate, less polluting, and has a high survival rate, thereby improving the detoxification efficiency of snow cane.

Description

Technical Field

[0001] The invention relates to a method for producing a snow cane detoxified with sugarcane mosaic virus and virus-free seedlings, and is applied in the technical field of snow cane virus-free seedling production. Background Art

[0002] Snow cane is a high-quality variety of sugar cane (fruit cane) with Fujian local characteristics. It has the advantages of thick stems, thin skin, rich and soft juice, sweet and refreshing taste, and less residue and clumping. It is favored by consumers. Sugarcane farmers can earn 8,000 to 10,000 yuan per mu of snow cane, which is the main source of income for local farmers. The healthy development of the snow cane industry is conducive to the development of local agricultural specialization, scale and industrialization.

[0003] It has been found that snow cane is susceptible to viral diseases, and the main infection is Sugarcane mosaic virus (SCMV), which can be quickly spread through seedlings, production tools, aphids, etc. The main symptoms of infected plants are mottled stem bark, short stems, few tillers, difficult leaf peeling, and low sucrose content, resulting in a 20-40% drop in yield, poor quality of commercial sugarcane, and low selling price, which seriously affects the production and benefits of sugarcane farmers and has become a major problem plaguing snow cane production and industrial development. At present, sugarcane mosaic virus is difficult to control with chemicals. The most effective solution is to plant snow cane virus-free seedlings, which can increase the yield per mu by about 20% and the sugar content by about 0.5%.

[0004] There are many methods for plant detoxification, such as heat treatment detoxification, callus and shoot tip culture detoxification, chemical agent detoxification, ultra-low temperature detoxification, shoot tip micro-grafting detoxification and combined methods, etc. At present, there are two main effective methods for sugarcane virus removal: one is warm water detoxification technology (see patent CN 101637087A), which uses the biological characteristics of virus inactivation by heat, and treats sugarcane seed stems with 50°C constant temperature hot water for 120 minutes to quickly inactivate the virus and obtain attenuated sugarcane seedlings, but this method cannot completely remove the virus from sugarcane seedlings. The other is a heat treatment combined with stem tip culture detoxification technology, that is, the sugarcane seed stems are first treated with 50-55°C constant temperature hot water for 30-120 minutes, and then grown into sugarcane buds after being cultured at a high temperature of 35-45°C for 1-4 weeks, and the stem tips of about 1.0-5.0 mm in length at the top of the sugarcane buds are cut for in vitro culture, and finally detoxified seedlings are obtained, and the detoxification rate can reach 60-100% (see patents CN101361458A, CN102090340A, CN107494271A and CN107646238A); however, these methods still have many disadvantages, such as high requirements for equipment, great difficulty in operation, many and complicated steps, high contamination rate, low survival rate if the stem tip is too small, reduced detoxification rate if it is too large, and damaged sugarcane buds are easily killed by heat, etc., resulting in low detoxification efficiency. The applicant has found through a large number of experiments that the above two methods are not feasible for snow cane virus removal, because the snow cane seed stems and buds have a high water content (about 80%), and after being treated with 50-55°C warm water for 30-120 minutes, all of the buds die and do not germinate. Therefore, it is important to provide a method for removing sugarcane mosaic virus from snow cane and virus-free seedlings that is suitable for heat treatment of snow cane, simple to operate, and has high virus removal efficiency, in order to promote the healthy development of the snow cane industry. Summary of the invention

[0005] In order to overcome the disadvantage that the existing sugarcane detoxification methods are not applicable to the snowcane seed stems and axillary buds due to their high water content, the present invention provides a method for producing snowcane detoxified sugarcane mosaic virus and detoxified seedlings, which comprises the following steps: firstly preparing a single bud seed stem and performing a preheating treatment on it, then adopting a double high temperature change combined with an alternating light / dark cycle treatment to ensure that the snowcane buds have a high germination rate and the snowcane bud seedlings have a good growth potential; then peeling off the larger stem tip (8-10 mm) of the snowcane bud seedlings for in vitro culture, and cultivating the snowcane detoxified seedlings free of sugarcane mosaic virus.

[0006] The present invention is implemented by the following technical solutions:

[0007] A method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings, mainly comprising the following steps performed in sequence:

[0008] (1) Preparation of qualified single bud seed stems: Using snow cane stems from the elongation stage to the early maturity stage as the material, cut them into multiple single bud seed stems with a single sugarcane bud using a sharp knife for later use, and the internode length of the sugarcane bud from the upper and lower ends of the single bud seed stem is 3-4 cm;

[0009] (2) Disinfection: Soak the single bud seed stem obtained in step (1) in a disinfectant for 15-20 minutes, then take it out and dry the cut for later use;

[0010] (3) Planting: First, lay each single bud seed stem treated in step (2) flat on a substrate with a thickness of ≥6 cm, and place each sugarcane bud sideways in the same direction; then, cover each single bud seed stem with the substrate until the substrate just covers the single bud seed stem; finally, sprinkle the substrate with warm water at 50-52°C;

[0011] (4) Preheating treatment: the single bud seed stem after planting in step (3) is placed in a dark culture at a temperature of 38±2°C and a relative humidity of ≥80% for 24 hours;

[0012] (5) Double high temperature detoxification treatment: the single bud seed stems preheated in step (4) are first treated in the dark at 46-48°C for 3-6 hours, and then treated at 36-40°C with 12000Lx light for 3-8 hours, and then this alternating treatment is continued for 12 days; after each sugarcane bud sprouts, the substrate is irrigated with warm water at 50-52°C once every 3 days until each single bud seed stem grows into a sugarcane bud seedling with 3-5 fully expanded true leaves;

[0013] (6) Shoot tip culture detoxification: remove the top leaves of the sugarcane bud seedlings obtained in step (5), then disinfect the surface of the sugarcane bud seedlings with medical alcohol for 30 seconds, then place them in a 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinse them with sterile water for 3 times; peel off 8-10 mm shoot tips under sterile conditions and inoculate them on adventitious bud induction liquid culture medium and culture them for 3 weeks to induce adventitious buds, during which the adventitious bud induction liquid culture medium is replaced every 7 days;

[0014] (7) Obtaining virus-free seedlings: inoculating the adventitious buds obtained in step (6) into a proliferation liquid culture medium and culturing for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds; selecting clustered buds that are negative for sugarcane mosaic virus for subculture and proliferation, subculturing once every 3 weeks, and controlling the number of subcultures within 10 times; finally, inoculating the proliferated clustered buds into a rooting liquid culture medium and culturing for 3 weeks to obtain snow cane virus-free seedlings free of sugarcane mosaic virus.

[0015] In the step (5), the single bud seed stems preheated in the step (4) are first treated in the dark at 47° C. for 5 hours, and then treated at 38° C. for 7 hours under the condition of 12000 Lx light, and then the treatment is repeated for 12 days.

[0016] The preferred double-high variable temperature detoxification treatment parameters not only achieve a detoxification rate of 100.00%, but also have better germination rate, height of sugarcane sprouts and health index.

[0017] The disinfectant in step (2) is a 1000-fold dilution of 50% carbendazim wettable powder.

[0018] The preferred disinfectant can achieve the best disinfection effect while ensuring that the single bud seed stems are not damaged.

[0019] The substrate in step (3) is a mixture prepared by mixing peat soil, coconut husk and vermiculite in a volume ratio of 1:1:1.

[0020] The matrix of the preferred components and proportions can achieve long-lasting water retention and air permeability to the greatest extent.

[0021] The adventitious bud induction liquid culture medium described in step (6) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 2.0 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.5 mg / L, sucrose with a mass concentration of 20 g / L, and activated carbon with a mass concentration of 0.2 g / L.

[0022] The preferred adventitious bud induction liquid culture medium has a better effect of inducing adventitious buds from the shoot tips.

[0023] The proliferation liquid culture medium described in step (7) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 0.5 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.2 mg / L, and sucrose with a mass concentration of 30 g / L.

[0024] The preferred proliferation liquid culture medium makes the proliferation effect of adventitious buds and clustered buds better.

[0025] The rooting liquid culture medium described in step (7) is: 1 / 2MS culture medium with a pH value of 6.0, which also contains 1-naphthylacetic acid with a mass concentration of 3.0 mg / L, sucrose with a mass concentration of 50 g / L, and activated carbon with a mass concentration of 1.0 g / L.

[0026] The preferred rooting liquid medium has a higher rooting rate.

[0027] The core innovation of the invention is the production method of removing sugarcane mosaic virus from snow cane and virus-free seedlings, which adopts the following methods: preparing qualified single bud seed stems and preheating them, double high temperature change combined with alternating light / dark cycle treatment, and stripping larger (8-10 mm) stem tips for cultivation:

[0028] (1) Preparing single-bud seed stems and preheating them: The single-bud seed stem preparation technology is used to cut qualified seed stem segments with a single sugarcane bud, which has a higher germination rate than the traditional double-bud seed stem segments, and the seedlings emerge in an orderly manner; and the germination rate is further improved by controlling the distance between the incision and the sugarcane bud (3-4 cm), thereby avoiding the problem that the incision is too close to the sugarcane bud when the seed stem is prepared, which makes it easier for fungi and viruses to invade and harm the sugarcane bud (see Appendix). Figure 2 ), or the distance between the incision and the sugarcane bud is too large, leaving a lot of stem tissue and increasing the virus load;

[0029] (2) Adopting double high temperature change and combining light / dark cycle alternating treatment: The step of constant temperature hot water soup treatment of sugarcane buds in the existing detoxification method of sugarcane is omitted, and two high temperature sections (46-48°C and 36-40°C) are directly used in combination with the alternating temperature change of light / dark cycle for 11-13 days. The snow cane buds not only have a high survival rate and good growth, but also the sugarcane mosaic virus is well passivated and inhibited, thereby expanding the virus-free area of ​​the apical meristem, making up for the shortcomings of low germination rate and low detoxification efficiency of the existing constant temperature hot water soup detoxification treatment;

[0030] (3) After preheating and double high temperature treatment combined with alternating light / dark cycles, the snow cane buds can be peeled to a stem tip of 8-10 mm (see Appendix Figure 6 ), compared with the length of the stem tip commonly obtained by microscopic dissection during the existing sugarcane detoxification (mostly 1.0-5.0 mm) (see the background technology section for details), the stripping process does not require the aid of a body dissecting microscope, the difficulty of stripping the stem tip is greatly reduced, and the survival rate of the stem tip can be significantly improved, making the operation faster and the contamination rate lower. The detoxification rate can reach 100.00%, greatly improving the efficiency of detoxification.

[0031] It can be seen that the technology of single bud seed stem preparation, double high temperature detoxification treatment and light / dark treatment combined not only has little effect on the normal growth of snow cane buds, but also ensures the good effect of virus passivation and inhibition after the detoxification of snow cane seed stems and buds; the stem tip peeled during the stem tip culture is large (8-10mm), has a high survival rate and detoxification rate, has the advantages of simple operation, strong practicality and high detoxification efficiency, provides technical support for further mass production of snow cane detoxified seedlings, and solves the problem that sugarcane mosaic virus disease seriously affects the quality and yield in the existing snow cane planting process. Among them, the single bud seed stem obtained in step (1) has a good germination rate and sugarcane bud height, which can not only improve the utilization rate of the seed stem, but also help to reduce the virus dose carried in the seed stem.

[0032] In order to verify the relevant technical effects of this application, the applicant conducted the following scientific experiments:

[0033] 1 Test materials

[0034] 1.1 Snow cane seed stems: The materials are the snow cane stems from the elongation stage to the early maturity stage (the virus-free test specially selected the stems of the diseased snow cane plants infected with sugarcane mosaic virus)

[0035] 1.2 Substrate: A mixture of equal volumes of peat soil, coconut husk and vermiculite

[0036] 1.3 Disinfectant: 1000 times dilution of 50% carbendazim wettable powder

[0037] 1.4 Culture medium:

[0038] Adventitious bud induction liquid medium (Y): MS + 6-BA (2.0 mg / L) + NAA (0.5 mg / L) + sucrose (20 g / L) + activated carbon (0.2 g / L), pH 6.0;

[0039] Proliferation liquid medium (Z): MS + 6-BA (0.5 mg / L) + NAA (0.2 mg / L) + sucrose (30 g / L), pH 6.0;

[0040] Rooting liquid medium (G): 1 / 2MS + NAA (3.0 mg / L) + sucrose (50 g / L) + activated carbon (1.0 g / L), pH 6.0.

[0041] 2 Test methods and results

[0042] 2.1 Comparative test of germination between single-bud seed stem and double-bud seed stem

[0043] 2.1.1 Experimental treatment

[0044] Cut the middle of the internode of the snow cane stem with a sharp knife and cut it into two kinds of stems with a single bud and a double bud respectively (two treatments), 20 buds in each treatment, and three replicates for each treatment. Soak the stems of each treatment in disinfectant for 20 minutes, then plant them in a substrate, water them and put them in an artificial climate box for germination culture, water them once every 3 days, and the culture conditions are set as follows: the optimum temperature is 28°C, the humidity is 80%, and the light is 14 hours per day; after 30 days, the germination rate and height of the sugarcane bud seedlings of each treatment are counted.

[0045] The number of sugarcane sprouts with a height of more than 5 mm was counted, i.e., the number of sprouts, and the percentage of the number of sprouts in the total number of sprouts in the treated samples was calculated, i.e., the germination rate (%); the height from the substrate surface to the bifurcation of the leaves at the top of the sugarcane sprouts was measured with a ruler (0.1 cm), i.e., the height of the sugarcane sprouts.

[0046] 2.1.2 Test results (see Table 1)

[0047] Table 1 Comparison of germination between single-bud seed stem and double-bud seed stem

[0048]

[0049] Note: The numbers in the table are mean ± standard error. Lowercase letters indicate significant differences at the level of P < 0.05, and uppercase letters indicate significant differences at the level of P < 0.01, the same below.

[0050] As shown in Table 1, the germination rate of single-bud seed stems (88.33%) is significantly higher than that of double-bud seed stems (63.33%). Although the height of sugarcane sprouts of single-bud seed stems (9.66 cm) is slightly lower than that of double-bud seed stems (10.49 cm), the difference between the two is not significant.

[0051] 2.2 Experiment on the effect of incision position on the germination of single bud seed stem

[0052] 2.2.1 Experimental treatment

[0053] The snow cane stems were cut into single bud seed stems with a sharp knife, and the internode lengths of the sugarcane buds from the upper and lower ends of the single bud seed stems were controlled at 1 cm, 2 cm, 3 cm, 4 cm and ≥ 5 cm respectively. Each treatment had 20 buds and each treatment was repeated three times. The seed stems of each treatment were soaked in disinfectant for 20 minutes, then planted in a substrate, watered and placed in an artificial climate box for germination culture. Water was poured every 3 days. The culture conditions were set as follows: the optimum temperature was 28°C, the humidity was 80%, and the light was 14 hours per day. After 30 days, the germination rate and height of sugarcane bud seedlings of each treatment were counted.

[0054] 2.2.2 Test results (see Table 2)

[0055] Table 2 Effect of incision position on sugarcane bud germination

[0056]

[0057] As shown in Table 2, the different distances between the incision and the sugarcane bud greatly affect the germination rate of the single bud seed stem and the height of the sugarcane bud seedling. The germination rate and height of the single bud seed stem are significantly reduced when the incision is less than 3 cm away from the sugarcane bud. The reason is that fungal viruses are more likely to invade through the incision and harm the sugarcane bud (see Appendix Figure 2 ); while the germination rate and height of sugarcane buds between single bud seed stems with the incision 3 cm or more away from the sugarcane buds were higher, and the difference was not significant.

[0058] 2.3 Double high temperature detoxification treatment test

[0059] 2.3.1 Test materials

[0060] The infected snow cane seed stems confirmed by molecular detection were cut into qualified single bud seed stems, that is, the internode length of the sugarcane buds from the upper and lower ends of the single bud seed stems was controlled to be 3-4 cm. The single bud seed stems were soaked in disinfectant for 20 minutes and then planted with a substrate.

[0061] 2.3.2 Drugs and reagents

[0062] TransScript One-Step RT-PCR SuperMix kit, TransZol UpPlus RNA Kit, Tap enzyme, etc. from Trans Company, and other drugs were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0063] 2.3.3 Main instruments

[0064] Zhejiang Ningbo Safe Intelligent Artificial Climate Chamber PRX-450C, Hangzhou Bioer Technology Co., Ltd.'s PCR amplifier, etc.

[0065] 2.3.4 Uniform design to optimize double high temperature detoxification treatment conditions

[0066] The planted single bud seed stems were preheated for 24 hours under the following conditions: temperature 38±2℃, relative humidity ≥80%, dark culture. After preheating, double high temperature detoxification treatment was carried out for 12 days according to the uniform design scheme (see Table 3), after which the germination rate and the height of the sugarcane bud seedlings were counted. Ten single bud seed stems were treated for each treatment, repeated 3 times, and the single bud seed stems treated at room temperature (28±1℃) were used as the control (CK). And +1 leaf (the leaf with the highest visible hypertrophy at the top of the seedling) was sampled for SCMV virus molecular detection.

[0067] Table 3U 6 (3 2 ×2 1 ) Uniform design experimental plan

[0068]

[0069] Note: D1-42-2-6: double high temperature treatment conditions are 42℃2h dark / and38℃6h light; D4-42-4-3 is 42℃4h dark / 38℃3h light; D5-45-6-6 is 45℃6h dark / 38℃6h light; D2-45-2-3 is 45℃2h dark / 38℃3h light; D6-48-4-6 is 48℃4h dark / 38℃6h light; D3-48-6-3 is 48℃6h dark / 38℃3h light; CK is the control, 28±1℃12h dark / 28±1℃12h light; the light intensity of the above treatments is 12000Lx.

[0070] 2.3.5 Molecular detection of Sugarcane mosaic virus (SCMV)

[0071] The RNA of sugarcane seedlings +1 leaf was extracted using TransZol Up Plus RNA Kit (TRANS), and the purity and concentration of RNA were detected by a micro-ultraviolet-visible spectrophotometer (Thermo NanoDrop One). The RT-PCR detection of SCMV was performed using the TransScript One-Step RT-PCR SuperMix Kit of TRANS. The primer sequences used were F: 5`-GAAGAXGTYTTCCAYCAAFCXGGAAC-3`, R: 5`-AGCTGTGTGTCTCTCTGTATTCTC-3`, and the target amplification product was 906 bp. The amplification system was 20.00 μL: 2×TS One-Step Reaction Mix 10.00 μL, total RNA template 2.00 μL, RNase-free water 6.80 μL, TransScript One-Step Enzyme mix 0.40 μL, and 0.40 μL of upstream and downstream primers respectively. The amplification program was as follows: 45°C, 30 min; 94°C pre-denaturation for 3 min; 95°C denaturation for 30 s; 55°C annealing for 30 s; 72°C

[0072] Extension for 1 min, 35 cycles; extension at 72°C for 10 min. Electrophoresis was performed using 1% agarose gel at 190 V and 300 mA. The detoxification rate was calculated according to formula (1).

[0073] Virus-free rate % = number of SCMV positive samples / total number of samples × 100% (1)

[0074] 2.3.6 Data Statistics and Analysis

[0075] Excel 2020 was used for data statistics, SPSS 21 for variance analysis, Photoshop for electrophoresis image editing, and Minitab software for mathematical modeling.

[0076] 2.3.7 Verification test

[0077] The uniform design test results were mathematically modeled, and the optimal double-high temperature variable temperature detoxification treatment test plan was analyzed and inferred, and a verification test was conducted. The growth of sugarcane seedlings and mosaic symptoms of different leaf positions in the verification test were observed and analyzed, as well as the sugarcane mosaic virus molecular detection, to determine whether the verification test achieved the best detoxification effect.

[0078] 2.3.8 Test results (see Table 4)

[0079] Table 4 Effects of double high temperature treatment on growth indexes of snow cane

[0080]

[0081] Note: The numbers of germination rate and seedling height represent mean ± standard deviation, lowercase letters represent significant differences at P < 0.05, and uppercase letters represent significant differences at P < 0.01, the same below. In the health index, "3" means very healthy, with green leaves, "2" means healthy, with some yellow leaves, "1" means basically healthy, with yellow leaves, and "0" means unhealthy, with yellow buds and no leaves.

[0082] As shown in Table 4, the germination rate of each group treated with double high temperature and detoxification (hereinafter referred to as double high temperature) was higher than that of the untreated control group, but the difference was not significant. The double high temperature treatment had a significant effect on the seedling height and seedling growth (health index). The seedling height of the D1-42-2-6 and D2-45-2-3 treatment groups was significantly higher than that of the control group and other treatment groups (P <

[0083] 0.01), the health index was the same as the control, and the leaves were green and growing well. The seedling height and health index of the other four treatment groups were lower than the control, and the leaves were yellow, especially the D3-48-6-3 treatment group, which had the lowest health index, and the growth of the seedlings was severely hindered, and most of the leaves did not grow (see attached Figure 3 ).

[0084] It can be seen that short-term (no more than 2 hours) high temperature treatment will increase the germination rate and the health index of axillary bud seedlings, such as D1-42-2-6 and D2-45-2-32, and the 45℃ treatment is better than the 42℃ treatment; long-term high temperature treatment (more than 2 hours) has little effect on the germination rate, but inhibits the growth of sugarcane seedlings, making the seedling height and health index lower than the control.

[0085] Table 5 Comparison of the effects of double high temperature detoxification treatment on SCMV removal

[0086]

[0087] SCMV virus molecular detection (see attached Figure 8 ——Lane N in the figure is a blank control for monitoring the environment during the test), the SCMV detoxification rate of the control group is 0%, the detoxification rates of the double high temperature treatment groups D6-48-4-6 and D3-48-6-3 are both 100%, the detoxification rate of the D5-45-6-6 group is better, reaching 77.6%, the detoxification rate of the D4-42-4-3 group is not high, only 55.2%, and the remaining two groups (D1-42-2-6, D2-45-2-3) have the worst detoxification effect. Minitab software was used to perform regression analysis on the detoxification rates in Table 5 above, and the regression analysis results are shown in Table 6.

[0088] Table 6 Variance analysis table of detoxification rate of double high temperature detoxification treatment

[0089]

[0090] The results of variance analysis of the virus-free rate showed that the P value of the selected model was less than 0.0001, indicating that the model had high accuracy and proved that the selected model was reasonable and accurate. The value of the virus-free rate model coefficient R2 was 0.9978, which was very close to 1, showing good correlation. The adjusted R2 and predicted R2 were 0.9969 and 0.9950, respectively, and the values ​​of the two were close to 1, and the difference between the two was less than 0.02, indicating that the model had high prediction accuracy.

[0091] As shown in Table 6, the detoxification rate is significantly affected by the high temperature factor X1, the high temperature treatment time factor X2, and the 38°C treatment time factor X3. The quadratic term of the high temperature factor X1 and the quadratic term of the high temperature treatment time factor X2 have significant effects on the detoxification rate. By analyzing the relationship between the parameters and the detoxification rate, the mathematical models of the two can be obtained. The empirical model is shown in formula (2):

[0092] Y=940-53.4*X1+71.01*X2+1.337*X3+0.669*X1*X1-6.900*X2*X2 (2)

[0093] According to the mathematical model of formula (2), the virus-free rates of D6-48-4-6 and D3-48-6-3 in Table 2-5 were calculated to be 99.838% and 99.847% respectively. The actual virus-free rates of D6-48-4-6 and D3-48-6-3 were both 100%, with high actual and predicted accuracy, but the health status of D6-48-4-6 seedlings was better than that of D3-48-6-3. Compared with D3-48-6-3, D6-48-4-6 had a short high-temperature treatment time and a long 38° treatment time. When the virus-free rates were equal, the health index was higher. Considering the two indicators of virus-free rate and health index, the double high temperature treatment method with a virus-free rate greater than 99% and a high health index was the best choice. This choice can ensure a high virus-free rate to obtain healthy seedlings on the one hand, and minimize the damage to the seedlings caused by the double high temperature treatment to obtain strong sugarcane seedlings on the other hand. According to the mathematical model, the theoretical optimal combination of double high temperature treatment conditions was obtained as 47℃ for 5h and 38℃ for 7h, and the expected detoxification rate was 99.93%.

[0094] Through the verification test, it was found that after the single bud seed stem of snow cane infected with SCMV was treated with a double high temperature change mode of 47℃ dark treatment for 5h and 38℃ light treatment for 7h for 12 days, its germination rate was as high as 96.67%, the height of the sugarcane bud seedling was 8.93cm, and the virus-free rate reached 100.00% (see attached Figure 8 ), the health index is 2 (see attached Figure 4 The leaves of the sugarcane seedlings in the validation test showed no mosaic symptoms, while the leaves of the control treatment showed obvious mosaic symptoms (see Appendix Figure 5). The results of the verification test were compared with the optimal solution D6-48-4-6 in Table 5. The virus-free rate was the same, but the germination rate, height of sugarcane sprouts and health index were all improved.

[0095] 2.4 Effect of stem tip size on survival rate and virus-free rate

[0096] 2.4.1 Experimental treatment

[0097] After the single bud seed stem of snow cane infected with SCMV was treated with double high temperature alternation (high temperature 47°C dark treatment for 5h and high temperature 38°C light treatment for 7h) for 12 days, the base of the sugarcane bud seedling was cut from the connection of the seed stem, the top leaves were removed, the surface of the bud seedling was sprayed with medical alcohol for disinfection for 30 seconds, and then placed in 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinsed with sterile water for 3 times. Under sterile conditions, five groups of stem tips of different sizes, such as 0.5mm, 1.0mm, 5.0mm, 10.0mm and 15.0mm, were peeled off and inoculated into adventitious bud induction liquid culture medium (Y) for in vitro culture for 3 weeks to induce adventitious buds. During this period, the adventitious bud induction liquid culture medium (Y) was replaced every 7 days, and the survival rate of each group was counted; then the adventitious buds were inoculated into the proliferation liquid culture medium (Z) for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds (see Appendix Figure 7 ,in, Figure 7 A: in vitro culture of shoot tips; B: adventitious buds induced from shoot tips; C: induced clustered buds; D: rooted snow cane virus-free seedlings), leaves were cut and tested for sugarcane mosaic virus RT-PCR molecular detection, and the virus-free rate of each group was counted. Each group of the experiment treated 15 shoot tips once, repeated three times, and the mean of each group was used for one-way ANOVA.

[0098] 2.4.2 Test results

[0099] Table 7 Effect of stem tip size on survival rate and virus-free rate

[0100]

[0101] As shown in Table 7, the size of the stem tip has a great influence on the survival rate and virus-free rate of the stem tip after double high temperature detoxification treatment, and the difference is extremely significant. The smaller the stem tip, the lower the survival rate. When the stem tip size is 0.5mm, the survival rate of the stem tip is only 17.78%, while when the stem tip is larger (≥10.0mm), the survival rate of the stem tip is higher, reaching more than 97.78%. Although the survival rate of the 10.0mm stem tip is less than that of the 15.0mm stem tip, the difference is not significant. When the stem tip size is controlled within 10.0mm, the virus-free rate of the stem tip can reach 100.00%, but as the stem tip increases to 15.0mm, the virus-free rate drops significantly to 75.56%.

[0102] 3 Experimental summary

[0103] 3.1 Single-bud seed stems have a higher germination rate than double-bud seed stems, and have no obvious effect on the growth of sugarcane seedlings.

[0104] 3.2 When the distance between the cut of the single-bud seed stem and the sugarcane bud is ≥3cm, the germination rate of the seed stem and the height of the sugarcane bud seedling are both good.

[0105] 3.3 Double high temperature detoxification treatment can improve the germination rate of single bud seed stems. The detoxification rate after double high temperature detoxification treatment is significantly affected by high temperature factor X1, high temperature treatment time factor X2 and 38℃ high temperature treatment time factor X3. Among them, the high temperature (factor X1) and the length of high temperature treatment time (factor X2) have the greatest impact on the detoxification rate. When it reaches above 45℃ and the treatment time is longer than 2 hours, there is a higher detoxification rate. The extension of 38℃ treatment time (factor X3) is beneficial to improve the health index of sugarcane sprouts.

[0106] 3.4 After the double high temperature treatment, the size of the stem tip had a great influence on the survival rate and virus-free rate of the stem tip, and the difference was extremely significant. The smaller the stem tip, the lower the survival rate, and the virus-free rate reached 100.00%; when the stem tip was larger (10.0mm), the survival rate of the stem tip was high, and the virus-free rate still reached 100.00%; but as the stem tip increased to 15.0mm, the virus-free rate decreased significantly.

[0107] 4 Experimental conclusion

[0108] 4.1 The use of single-bud seed stems as detoxification materials has a higher germination rate than double-bud seed stems, which is more conducive to improving the utilization rate of seed stems.

[0109] 4.2 The single bud seed stem with the distance between the incision and the sugarcane bud controlled at 3-4cm is the most suitable and qualified, which can not only improve the germination rate of the sugarcane bud and the utilization rate of the seed stem, but also help to reduce the dose of virus carried in the seed stem.

[0110] 4.3 According to the mathematical model calculation and experimental verification, the best conditions for double high temperature treatment are 47℃ dark treatment for 5h and 38℃ light treatment for 7h. After 12 days of alternating treatment, the detoxification rate can reach 100.00%.

[0111] 4.4 After the double high temperature detoxification treatment, when the size of the stem tip is controlled at 10.0 mm, the survival rate is very high.

[0112] The detoxification rate can also reach 100.00%, with the highest detoxification efficiency.

[0113] Compared with the prior art, the present invention has the following advantages:

[0114] (1) The present invention adopts a single-bud seed stem preparation technology. By cutting qualified seed stem segments into single buds, the germination rate is higher than that of the traditional double-bud seed stem segments, and the seedlings emerge in an orderly manner. The germination rate is further improved by controlling the distance between the incision and the sugarcane bud (3-4 cm).

[0115] (2) The present invention adopts a double high variable temperature detoxification treatment technology, which eliminates the step of treating the sugarcane buds with constant temperature hot water in the existing sugarcane detoxification method, and changes the method of constant temperature detoxification treatment of the sugarcane buds. It directly adopts two high temperature periods (46-48°C and 36-40°C) and combines the alternating temperature treatment of light / dark cycle for 12 days. The snow cane buds not only have a high survival rate and good growth, but also the sugarcane mosaic virus is well inhibited, which makes up for the shortcoming of the existing constant temperature hot water detoxification treatment that the snow cane buds do not germinate.

[0116] (3) The snow cane buds treated by the present invention can be peeled off with a stem tip of up to 10 mm. The peeling process does not require the use of a body dissecting microscope, which greatly reduces the difficulty of peeling the stem tip. It can also significantly improve the survival rate of the stem tip, making the operation faster, and the detoxification rate reaches 100.00%, greatly improving the efficiency of detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 A schematic photo of a single bud seed stem obtained in step (1) of the method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings of the present invention;

[0118] Figure 2 Schematic photo of fungal viruses invading the snow cane seed stem through the cut and harming the cane buds;

[0119] Figure 3 Photos showing the effects of each treatment on the growth of snow cane seedlings in the double high temperature detoxification test of the method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings of the present invention;

[0120] Figure 4 Growth comparison photos of the T-47-5-7 sample treated with double high temperature detoxification and the control sugarcane sprout seedlings in the method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings of the present invention;

[0121] Figure 5 Comparison photos of leaf mosaic disease characteristics of T-47-5-7 sample treated with double high temperature change and control sugarcane seedlings in the method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings of the present invention;

[0122] Figure 6 A schematic photograph of a larger snow cane stem tip (10 mm) peeled off in the stem tip culture and virus removal step of the method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings of the present invention;

[0123] Figure 7Schematic photo of the process of in vitro induction and cultivation of virus-free seedlings of the snow cane for removing sugarcane mosaic virus and producing virus-free seedlings of the present invention;

[0124] Figure 8 Electrophoretic diagram of SCMV molecule detection of each detoxification treatment in the method for removing sugarcane mosaic virus from snow cane and producing detoxified seedlings of the present invention. DETAILED DESCRIPTION

[0125] The technical solution of the present invention is described in detail below in conjunction with the embodiments of the specification.

[0126] Example 1

[0127] A method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings, mainly comprising the following steps performed in sequence:

[0128] (1) Preparation of qualified single bud seed stems: Using snow sugarcane stems from the elongation stage to the early maturity stage as the material, cut them into multiple single bud seed stems with a single sugarcane bud using a sharp knife for later use, and the internode length of the sugarcane bud from the upper and lower ends of the single bud seed stem is 3.5 cm;

[0129] (2) Disinfection: Soak the single bud seed stem obtained in step (1) in a disinfectant for 15 minutes, then take it out and dry the cut for later use;

[0130] (3) Planting: First, lay the single bud seed stems treated in step (2) flat on a substrate with a thickness of 7 cm, and place the sugarcane buds sideways in the same direction; then, cover the single bud seed stems with the substrate until the substrate just covers the single bud seed stems; finally, sprinkle the substrate with warm water at 51°C;

[0131] (4) Preheating treatment: the single bud seed stem after planting in step (3) is placed in a dark culture at a temperature of 36° C. and a relative humidity of 90% for 24 hours;

[0132] (5) Double high temperature detoxification treatment: the single bud seed stems preheated in step (4) were first treated in the dark at 46°C for 6 hours, and then treated at 40°C for 12000Lx for 3 hours, and then this alternating treatment was continued for 12 days; after each sugarcane bud sprouted, the substrate was irrigated with 50°C warm water once every 3 days until each single bud seed stem grew into a sugarcane bud seedling with 4 fully expanded true leaves; (6) Shoot tip culture detoxification: the top leaves of the sugarcane bud seedlings obtained in step (5) were removed, and then the surface of the sugarcane bud seedlings was disinfected with medical alcohol for 30 seconds, and then placed in 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinsed with sterile water for 3 times; 9mm shoot tips were peeled under sterile conditions and inoculated into adventitious bud induction liquid culture medium and cultured for 3 weeks to induce adventitious buds, during which the adventitious bud induction liquid culture medium was replaced every 7 days;

[0133] (7) Obtaining virus-free seedlings: the adventitious buds obtained in step (6) are inoculated into a proliferation liquid culture medium and cultured for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds; the clustered buds that are negative for sugarcane mosaic virus are selected for subculture and proliferation, and the subculture is performed once every 3 weeks, and the number of subcultures is 9 times; finally, the proliferated clustered buds are inoculated into a rooting liquid culture medium and cultured for 3 weeks to obtain the snow cane virus-free seedlings free of sugarcane mosaic virus.

[0134] The disinfectant in step (2) is a 1000-fold dilution of 50% carbendazim wettable powder.

[0135] The substrate in step (3) is a mixture prepared by mixing peat soil, coconut husk and vermiculite in a volume ratio of 1:1:0.5.

[0136] The adventitious bud induction liquid culture medium described in step (6) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 2.0 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.5 mg / L, sucrose with a mass concentration of 20 g / L, and activated carbon with a mass concentration of 0.2 g / L.

[0137] The proliferation liquid culture medium described in step (7) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 0.5 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.2 mg / L, and sucrose with a mass concentration of 30 g / L.

[0138] The rooting liquid culture medium described in step (7) is: 1 / 2MS culture medium with a pH value of 6.0, which also contains 1-naphthylacetic acid with a mass concentration of 3.0 mg / L, sucrose with a mass concentration of 50 g / L, and activated carbon with a mass concentration of 1.0 g / L.

[0139] Example 2

[0140] A method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings, mainly comprising the following steps performed in sequence:

[0141] (1) Preparation of qualified single-bud seed stems: Using snow sugarcane stems from the elongation stage to the early maturity stage as the material, cut them into multiple single-bud seed stems with a single sugarcane bud using a sharp knife for later use, and the internode length of the sugarcane bud from the upper and lower ends of the single-bud seed stem is 4 cm;

[0142] (2) Disinfection: Soak the single bud seed stem obtained in step (1) in a disinfectant for 18 minutes, then take it out and dry the cut for later use;

[0143] (3) Planting: First, lay the single bud seed stems treated in step (2) flat on a substrate with a thickness of 6 cm, and place the sugarcane buds sideways in the same direction; then, cover the single bud seed stems with the substrate until the substrate just covers the single bud seed stems; finally, sprinkle the substrate with warm water at 50°C;

[0144] (4) Preheating treatment: the single bud seed stems planted in step (3) are placed in a dark culture at a temperature of 38° C. and a relative humidity of 80% for 24 hours;

[0145] (5) Double high temperature detoxification treatment: the single bud seed stems preheated in step (4) were first treated in the dark at 47°C for 5 hours, and then treated at 38°C for 7 hours under the condition of 12000Lx light, and then this alternating treatment was continued for 12 days; after each sugarcane bud sprouted, the substrate was irrigated with 52°C warm water once every 3 days until each single bud seed stem grew into a sugarcane bud seedling with 3 fully expanded true leaves; (6) Shoot tip culture detoxification: the top leaves of the sugarcane bud seedlings obtained in step (5) were removed, and then the surface of the sugarcane bud seedlings was disinfected with medical alcohol for 30 seconds, and then placed in 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinsed with sterile water for 3 times; 10mm shoot tips were peeled under sterile conditions and inoculated into adventitious bud induction liquid culture medium and cultured for 3 weeks to induce adventitious buds, during which the adventitious bud induction liquid culture medium was replaced every 7 days;

[0146] (7) Obtaining virus-free seedlings: inoculating the adventitious buds obtained in step (6) into a proliferation liquid culture medium and culturing for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds; selecting clustered buds that are negative for sugarcane mosaic virus for subculture and proliferation, subculturing once every 3 weeks, and subculturing 10 times; finally, inoculating the proliferated clustered buds into a rooting liquid culture medium and culturing for 3 weeks to obtain snow cane virus-free seedlings free of sugarcane mosaic virus.

[0147] The disinfectant in step (2) is a 1000-fold dilution of 50% carbendazim wettable powder.

[0148] The substrate in step (3) is a mixture prepared by mixing peat soil, coconut husk and vermiculite in a volume ratio of 1:1:1.

[0149] The adventitious bud induction liquid culture medium described in step (6) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 2.0 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.5 mg / L, sucrose with a mass concentration of 20 g / L, and activated carbon with a mass concentration of 0.2 g / L.

[0150] The proliferation liquid culture medium described in step (7) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 0.5 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.2 mg / L, and sucrose with a mass concentration of 30 g / L.

[0151] The rooting liquid culture medium described in step (7) is: 1 / 2MS culture medium with a pH value of 6.0, which also contains 1-naphthylacetic acid with a mass concentration of 3.0 mg / L, sucrose with a mass concentration of 50 g / L, and activated carbon with a mass concentration of 1.0 g / L.

[0152] Example 3

[0153] A method for removing sugarcane mosaic virus from snow cane and producing virus-free seedlings, mainly comprising the following steps performed in sequence:

[0154] (1) Preparation of qualified single-bud seed stems: Using snow sugarcane stems from the elongation stage to the early maturity stage as the material, cut them into multiple single-bud seed stems with a single sugarcane bud using a sharp knife for later use, and the internode length of the sugarcane bud from the upper and lower ends of the single-bud seed stem is 3 cm;

[0155] (2) Disinfection: Soak the single bud seed stem obtained in step (1) in a disinfectant for 20 minutes, then take it out and dry the cut for later use;

[0156] (3) Planting: First, lay the single bud seed stems treated in step (2) flat on a substrate with a thickness of 8 cm, and place the sugarcane buds sideways in the same direction; then, cover the single bud seed stems with the substrate until the substrate just covers the single bud seed stems; finally, sprinkle the substrate with warm water at 52°C;

[0157] (4) Preheating treatment: the single bud seed stems planted in step (3) are placed in a dark culture at a temperature of 40° C. and a relative humidity of 100% for 24 hours;

[0158] (5) Double high temperature detoxification treatment: the single bud seed stems preheated in step (4) were first treated in the dark at 48°C for 3 hours, and then treated at 36°C for 8 hours under the condition of 12000Lx light, and then this alternating treatment was continued for 12 days; after each sugarcane bud sprouted, the substrate was irrigated with warm water at 51°C once every 3 days until each single bud seed stem grew into a sugarcane bud seedling with 5 fully expanded true leaves; (6) Shoot tip culture detoxification: the top leaves of the sugarcane bud seedlings obtained in step (5) were removed, and then the surface of the sugarcane bud seedlings was disinfected with medical alcohol for 30 seconds, and then placed in 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinsed with sterile water for 3 times; 8mm shoot tips were peeled under sterile conditions and inoculated into adventitious bud induction liquid culture medium and cultured for 3 weeks to induce adventitious buds, during which the adventitious bud induction liquid culture medium was replaced every 7 days;

[0159] (7) Obtaining virus-free seedlings: inoculating the adventitious buds obtained in step (6) into a proliferation liquid culture medium and culturing them for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds; selecting clustered buds that are negative for sugarcane mosaic virus for subculture and proliferation, subculturing once every 3 weeks, and subculturing 8 times; finally, inoculating the proliferated clustered buds into a rooting liquid culture medium and culturing them for 3 weeks to obtain snow cane virus-free seedlings free of sugarcane mosaic virus.

[0160] The disinfectant in step (2) is a 1000-fold dilution of 50% carbendazim wettable powder.

[0161] The substrate in step (3) is a mixture prepared by mixing peat soil, coconut husk and vermiculite in a volume ratio of 1:1:1.5.

[0162] The adventitious bud induction liquid culture medium described in step (6) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 2.0 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.5 mg / L, sucrose with a mass concentration of 20 g / L, and activated carbon with a mass concentration of 0.2 g / L.

[0163] The proliferation liquid culture medium described in step (7) is: MS culture medium with a pH value of 6.0, which also contains 6-benzylaminopurine with a mass concentration of 0.5 mg / L, 1-naphthaleneacetic acid with a mass concentration of 0.2 mg / L, and sucrose with a mass concentration of 30 g / L.

[0164] The rooting liquid culture medium described in step (7) is: 1 / 2MS culture medium with a pH value of 6.0, which also contains 1-naphthylacetic acid with a mass concentration of 3.0 mg / L, sucrose with a mass concentration of 50 g / L, and activated carbon with a mass concentration of 1.0 g / L.

Claims

1. A method for producing snow cane seedlings free of sugarcane mosaic virus and virus-free seedlings, Features: It mainly includes the following steps in order: (1) Preparation of qualified single-bud seed stems: Using snow cane stems from the elongation stage to the early maturity stage as the material, cut them into multiple single-bud seed stems with a single sugarcane bud using a sharp knife for later use, and the internode length of the sugarcane bud from the upper and lower ends of the single-bud seed stem is 3-4 cm; (2) Disinfection: Soak the single bud seed stem obtained in step (1) in disinfectant for 15-20 minutes, then take out and dry the cut for later use; (3) Planting: First, lay the single bud seed stems treated in step (2) flat on a substrate with a thickness of ≥ 6 cm, and place the sugarcane buds sideways in the same direction; then, cover the single bud seed stems with the substrate until the substrate just covers the single bud seed stems; finally, sprinkle the substrate with warm water at 50-52°C; (4) Preheating treatment: the single bud seed stems planted in step (3) are placed in a dark incubator at a temperature of 38±2°C and a relative humidity of ≥80% for 24 hours; (5) Double high temperature detoxification treatment: the single bud seed stems preheated in step (4) are first treated in the dark at 46-48°C for 3-6 h, and then treated at 36-40°C with 12000 Lx light for 3-8 h, and then this alternating treatment is continued for 12 days; after each sugarcane bud sprouts, the substrate is irrigated with warm water at 50-52°C once every 3 days until each single bud seed stem grows into a sugarcane bud seedling with 3-5 fully expanded true leaves; (6) Shoot tip culture detoxification: remove the top leaves of the sugarcane bud seedlings obtained in step (5), then disinfect the surface of the sugarcane bud seedlings with medical alcohol for 30 seconds, then place them in a 0.1% mercuric chloride solution for disinfection for 20 minutes, and finally rinse them with sterile water for 3 times; peel off 8-10 mm shoot tips under sterile conditions and inoculate them into adventitious bud induction liquid culture medium and culture them for 3 weeks to induce adventitious buds. During this period, the adventitious bud induction liquid culture medium was replaced every 7 days; (7) Obtaining virus-free seedlings: inoculating the adventitious buds obtained in step (6) into a proliferation liquid culture medium and culturing for 3 weeks to induce the adventitious buds to proliferate and grow into clustered buds; selecting clustered buds that are negative for sugarcane mosaic virus for subculture and proliferation, subculturing once every 3 weeks, and controlling the number of subcultures within 10 times; finally, inoculating the proliferated clustered buds into a rooting liquid culture medium and culturing for 3 weeks to obtain snow cane virus-free seedlings free of sugarcane mosaic virus; The adventitious bud induction liquid culture medium described in step (6) is: MS culture medium with a pH value of 6.0, to which 6-benzylaminopurine with a mass concentration of 2.0 mg / L, 1-naphthylacetic acid with a mass concentration of 0.5 mg / L, sucrose with a mass concentration of 20 g / L, and activated carbon with a mass concentration of 0.2 g / L are added; The proliferation liquid culture medium in step (7) is: MS culture medium with a pH value of 6.0, to which 6-benzylaminopurine at a mass concentration of 0.5 mg / L, naphthylacetic acid at a mass concentration of 0.2 mg / L, and sucrose at a mass concentration of 30 g / L are added; The rooting liquid culture medium described in step (7) is: 1 / 2MS culture medium with a pH value of 6.0, to which 1-naphthylacetic acid with a mass concentration of 3.0 mg / L, sucrose with a mass concentration of 50 g / L, and activated carbon with a mass concentration of 1.0 g / L are added.

2. The method for producing snow cane seedlings free of sugarcane mosaic virus according to claim 1, Features: In the step (5), the single bud seed stems preheated in the step (4) are first treated in the dark at 47°C for 5 hours, and then treated at 38°C for 7 hours under the condition of 12000 Lx light, and then the alternating treatment is repeated for 12 days.

3. The method for producing snow cane seedlings free of sugarcane mosaic virus according to claim 1, Features: The disinfectant in step (2) is a 1000-fold dilution of 50% carbendazim wettable powder.

4. The method for producing snow cane seedlings free of sugarcane mosaic virus according to claim 1, Features: The substrate in step (3) is a mixture prepared by mixing peat soil, coconut husk and vermiculite in a volume ratio of 1:1:1.

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