Efficient sugarcane detoxification rapid propagation method

Abstract

The invention provides an efficient sugarcane detoxification rapid propagation method, which belongs to the technical field of plant propagation, and comprises the following steps: S1, sugarcane plant top explant preparation and callus induction, S2, callus warm bath, S3, callus differentiation culture, S4, seedling multiplication culture, S5, seedling rooting culture, and S6, seedling transplanting. The invention relates to an efficient healthy sugarcane seedling breeding technology for removing viral diseases and bacterial diseases by combining somatic embryo regeneration with warm bath treatment. By utilizing the technology, more than 3000 virus-free healthy seedlings can be bred by a single sugarcane top explant, and the time consumption from cutting the sugarcane top explant to transplanting the healthy seedlings to a sand bed is about 3 months. Therefore, the method is an efficient, rapid and low-cost healthy sugarcane seedling breeding technology.

Description

Technical Field

[0001] The invention relates to a plant rapid propagation method, in particular to a sugarcane detoxification rapid propagation method, and belongs to the technical field of plant propagation. Background Art

[0002] Sugarcane is an important sugar crop and an asexually propagated crop. Years of asexual propagation have led to the accumulation of various diseases in sugarcane cultivars, resulting in serious variety degeneration, which seriously affects the growth and yield of sugarcane.

[0003] Sugarcane mosaic disease is a viral disease that is common in my country's sugarcane growing areas. The new leaves of the infected sugarcane show light yellow mottled spots or slight chlorotic stripes, and the old leaves show long chlorotic stripes or mosaic. The incidence rate is very high, resulting in a decrease in sugar content and yield, and the decline of the variety, affecting the quality.

[0004] Sugarcane perennial root stunting disease is an important bacterial disease of sugarcane, which is also common in major sugarcane production areas in my country. The main symptoms of sugarcane perennial root stunting disease are that the infected sugarcane plants become shorter, the sugarcane stems become thinner, the growth is sluggish, and the perennial roots are less. Summary of the invention

[0005] In view of the above problems, the present invention provides an efficient sugarcane detoxification and rapid propagation method to remove sugarcane viral diseases and bacterial diseases and efficiently obtain healthy sugarcane seedlings.

[0006] To achieve the above object, the technical solution of the present invention is: An efficient sugarcane detoxification and rapid propagation method comprises the following steps: S1 Preparation of sugarcane plant apical explants and callus induction Taking the tail of the sugarcane plant and obtaining the tightly curled cylindrical young white core leaf tissue at the top growth point as the explant, the explant is prepared into a sterile explant after pretreatment; The sterile explants were cut into thin slices, transferred into a container containing callus induction medium, and cultured in a constant temperature dark environment to obtain callus tissue; S2 Callus Warming Bath The callus tissue is warmed in a preheated warm bath medium for a predetermined time to obtain a callus tissue, wherein the warm bath medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 2,4-D, and pH 5.8; S3 Callus Differentiation Culture The callus tissue after warm bath is inoculated into differentiation medium, first cultured in dark at constant temperature, then cultured in light, and callus tissue with young shoots can be differentiated; S4 seedling proliferation culture The callus tissue that differentiates into young shoots is cut into small pieces, and re-inoculated into differentiation medium for secondary differentiation culture to obtain clustered seedlings; S5 seedling rooting culture The clustered seedlings obtained by secondary differentiation are cut into small pieces, inoculated into rooting medium, and subjected to light rooting culture; S6 seedling transplanting After the rooting culture is completed, the seedlings are washed, sterilized, and then moved to a sand bed for growth to obtain healthy sugarcane seedlings.

[0007] Furthermore, in step S1, the explants are pretreated by washing with sterile water, disinfecting, and then washing with sterile water again, wherein the disinfection is disinfecting with 75% alcohol by volume for 30 seconds, and then transferring to 0.1% mercuric chloride or 5% sodium hypochlorite solution by volume for disinfection for 10 minutes; The sterile explants were cut into 1 mm thick slices, and the moisture on the surface of the sterile explants was dried before slicing; the callus induction medium was replaced every 14-20 days, and callus was obtained after 1-3 changes; the temperature of the constant temperature dark culture was 25-28°C; The callus induction medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 2,4-D, 5-9 g / L plant gel / agar, and pH 5.8.

[0008] Furthermore, the callus tissue is air-dried before the warm bath in step S2, and the warm bath medium is preheated to 40-50° C., and the warm bath time is 5-10 minutes.

[0009] Furthermore, in step S3, the constant temperature dark culture temperature is 28°C for 7 days; the light culture is cultured in a light incubator for 14-20 days, the light conditions are 1500-2100 lux for 14 hours a day, 30-35°C during the day, and 28-30°C at night; The differentiation medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 6-BA, 5-9 g / L plant gel / agar, and pH 5.8.

[0010] Furthermore, the time of the secondary differentiation culture in step S4 is 14-20 days.

[0011] Furthermore, in step S5, the time for light rooting culture is 20-30 days, the lighting conditions are 1500-2100 lux for 14 hours a day, 30-35°C during the day and 28-30°C at night; the rooting culture medium is B5, N6 or MS culture medium as the basic culture medium, with 20-30g / L sucrose, 0.01-0.1g / L activated carbon, 0-3mg / L NAA, 5-9g / L plant gel / agar and other culture medium solids added, and pH 5.8.

[0012] Furthermore, in step S6, the seedlings are cleaned by washing away the rooting medium on the seedlings with clean water, and the seedlings are disinfected by soaking them in carbendazim disinfectant solution.

[0013] Furthermore, after step S6, a test of the virus-free rate of the plants needs to be performed.

[0014] Furthermore, the virus-free rate detection includes randomly collecting leaf and stem samples of the sugarcane plants obtained in step S6, extracting RNA from the leaves for detecting viral diseases such as sugarcane mosaic disease or yellow leaf disease; and extracting DNA from the stems for detecting bacterial diseases such as perennial dwarfing disease or white stripe disease.

[0015] Furthermore, the detection rates of viral diseases and bacterial diseases of sugarcane plants obtained in step S6 are both lower than 10%.

[0016] The beneficial effects of the efficient sugarcane detoxification and rapid propagation method of the present invention are: The present invention is an efficient sugarcane healthy seedling breeding technology that utilizes somatic embryo callus regeneration combined with warm bath treatment to remove viral diseases and bacterial diseases. Using this technology, a single sugarcane top explant can breed more than 3,000 virus-free healthy seedlings, and it takes about 3 months from cutting the sugarcane top explant to transplanting healthy seedlings into a sand bed. Therefore, this method is an efficient, fast and low-cost sugarcane healthy seedling breeding technology.

[0017] The method of the invention has a high virus-free rate for seedlings, and the detection rates of viral diseases and bacterial diseases of the seedlings are both lower than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a diagram showing the detection results of sugarcane streak mosaic virus in explants in Example 1 of the present invention, wherein M: DNA Markerladder, CK+: positive control sample, CK-: H2O, 1: plant sample; Figure 2This is a diagram of the detection result of sugarcane ratoon dwarfing disease on explants in Example 1 of the present invention, wherein M: DNA Markerladder, CK+: positive control sample, CK-: H2O, 1: plant sample; Figure 3 This is a diagram of callus induction results in Example 1 of the present invention; Figure 4 This is a diagram showing the callus warm bath results in Example 1 of the present invention; Figure 5 This is a diagram of the callus differentiation culture results in Example 1 of the present invention; Figure 6 This is a diagram showing the results of seedling rooting culture and transplanting in Example 1 of the present invention; Figure 7 7-A is a graph showing the results of the virus-free rate detection of sugarcane stripe mosaic virus in virus-free plants, M: DNA Marker ladder; 1-24: virus-free seedling samples; 7-B is a graph showing the results of the virus-free rate detection of sugarcane ratoon dwarf disease in virus-free plants, M: DNA Marker ladder; 1-24: virus-free seedling samples. DETAILED DESCRIPTION

[0020] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0021] An efficient sugarcane detoxification and rapid propagation method, the specific implementation steps are as follows: 1. Preparation of sugarcane plant apical explants and callus induction: Cut the tail of the sugarcane plant planted in the sugarcane field, peel off the green leaves and leaf sheaths layer by layer, and obtain the tightly curled cylindrical tender white core leaf tissue (about 8 cm) at the top growth point as the explant. After the explant is washed with sterile water, it is transferred to 75% alcohol for disinfection for 30 seconds and quickly transferred to 0.1% mercuric chloride or 5% sodium hypochlorite solution for disinfection for 10 minutes. Take it out and soak it in sterile water again to successfully prepare sterile explants.

[0022] Remove the explant from the sterile water, dry it with sterile filter paper, cut it into slices of about 1 mm with a sterile surgical blade, transfer it to a culture dish containing callus induction medium, and place it in an incubator at 25-28°C in the dark. Replace the callus induction medium every 14-20 days, and obtain callus tissue after 1-3 changes. The callus induction medium is based on B5, N6 or MS culture medium, and 20-30g / L sucrose, 0.01-0.1g / L activated carbon, 0-3mg / L 2,4-D, 5-9g / L plant gel / agar and other culture medium solids are added according to the variety, and the pH is 5.8 (Table 1).

[0023] 2. Warm callus bath Collect the induced callus tissue in a culture dish with filter paper and place it in a clean bench to slightly air dry for 10-30 minutes, collect it in a conical bottle and warm it in a preheated 45-50℃ warm bath medium for 5-10 minutes (preferably 45℃ warm bath for 10 minutes). The warm bath medium is based on B5, N6 or MS culture medium, and 20-30g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 2,4-D are added according to the variety, pH 5.8. After warm bath, filter out the callus tissue, dry the liquid culture medium with sterile filter paper, and slightly dry the callus tissue in the clean bench.

[0024] 3. Callus differentiation culture The callus tissue after warm bath was inoculated into differentiation medium, and then cultured in dark at 25-28℃ for 7 days, and then transferred to the light incubator for light culture for 14-20 days. The light conditions were 1500-2100lux for 14 hours a day, 30-35℃ during the day, and 28-30℃ at night. The callus differentiation medium was based on B5, N6 or MS culture medium, and 20-30g / L sucrose, 0.01-0.1g / L activated carbon, 0-3mg / L 6-BA, 5-9g / L plant gel / agar and other culture medium solids were added according to the variety, and the pH was 5.8 (Table 1).

[0025] 4. Seedling proliferation culture The callus tissue that has differentiated into young shoots is cut into small pieces and re-inoculated into differentiation medium for secondary light differentiation culture for 14-20 days. The culture medium is the same as step 3.

[0026] 5. Rooting and cultivation of seedlings The clustered seedlings obtained by secondary differentiation were cut into small pieces and inoculated into rooting medium for light rooting culture for 20-30 days. The light culture conditions were 1500-2100 lux for 14 hours a day, 30-35℃ during the day, and 28-30℃ at night. The plant rooting medium was based on B5, N6 or MS culture medium, and 20-30g / L sucrose, 0.01-0.1g / L activated carbon, 0-3mg / L NAA, 5-9g / L plant gel / agar and other culture medium solids were added according to the variety, and the pH was 5.8 (Table 1).

[0027] 6. Transplanting seedlings After the rooting culture is completed, the sugarcane plants are taken out from the rooting medium, the rooting medium is washed off with clean water, and after being soaked in disinfectant such as carbendazim, they are transplanted to a sand bed for growth to obtain healthy sugarcane seedlings.

[0028] 7. Plant virus-free rate detection Leaf and stem samples were randomly collected from the plants, and RNA was extracted from the leaves for the detection of viral diseases such as sugarcane mosaic disease and yellow leaf disease, while DNA was extracted from the stems for the detection of bacterial diseases such as perennial dwarfing disease and white stripe disease.

[0029] Table 1: Culture medium formulation Example 1

[0030] An efficient method for rapid propagation of sugarcane virus-free (efficient rapid propagation of diseased sugarcane plants) Material: The sugarcane cultivar ROC22 with the sugarcane streak mosaic virus (SCSMV) and the fruit cane cultivar Cheriben with the ratoon stunting disease (RSD) have been planted in the germplasm resource nursery for a long time.

[0031] Experimental methods: 1. Pathogen detection of sugarcane materials Cut the leaf tissue of the sugarcane cultivar ROC22 planted in the resource garden with typical symptoms of sugarcane mosaic disease, extract total RNA using the Trizol method, and detect the pathogen of sugarcane mosaic disease by RT-PCR. Cut the stems of the fruit cane cultivar ROC22 with short internodes and obvious perennial root dwarfing disease symptoms, peel them, cut them into thin slices, grind them into powder with liquid nitrogen, extract DNA using the CTAB method, and detect perennial root dwarfing disease by PCR. (Detection method reference: Li Wenfeng, 2016, Molecular detection technology of important sugarcane diseases) The results of explant disease detection are as follows: like Figure 1As shown in the figure, the leaf tissue of the sugarcane cultivar ROC22 with typical sugarcane mosaic disease symptoms planted in the resource garden was cut. Figure 1 -A, after total RNA was extracted by Trizol method, the results of RT-PCR detection of sugarcane mosaic disease pathogen were as follows Figure 1 -B, the sample amplified band was consistent with the expected band size (939 bp), indicating that the sugarcane cultivar ROC22 was infected with stripe mosaic virus (SCSMV).

[0032] like Figure 2 As shown in the figure, the stems of the fruit cane cultivar Cheliben with short internodes and obvious symptoms of ratoon dwarfing disease were cut. Figure 2 -A, DNA was extracted by CTAB method and PCR test for perennial root dwarfing disease was performed. The results were as follows Figure 2 -B, the sample amplified band is consistent with the expected band size (438 bp), indicating that the fruit cane cultivar C. schrenkiana is infected with ratoon stunting disease (RSD).

[0033] 2. Preparation of sugarcane plant apical explants and callus induction Cut the tail tips of sugarcane ROC22 and fruit cane stripe caribbean plants infected with sugarcane mosaic disease and perennial root dwarfing disease, peel off the green leaves and leaf sheaths, and obtain the tightly curled cylindrical young white core leaf tissue (about 8 cm) at the top growth point as explants. After the explants are washed with sterile water, transfer to 75% alcohol for 30 seconds and quickly transfer to 0.1% mercuric chloride for 10 minutes, take out and soak and wash again with sterile water to obtain sterile explants.

[0034] Remove the explants from the sterile water, dry them with sterile filter paper, cut them into slices of about 1 mm with a sterile surgical blade, transfer them into a culture dish containing callus induction medium, and place them in an incubator at 28°C in the dark. After 14 days, replace the callus induction medium with fresh one, and culture under the same conditions for another 14 days to obtain callus.

[0035] like Figure 3 As shown in the figure, the tail tips of sugarcane ROC22 and fruit cane striped caribbean plants infected with sugarcane mosaic disease and ratoon dwarf disease were cut, the green leaves and leaf sheaths were peeled off, and the tightly curled cylindrical young white core leaf tissue (about 8 cm) at the top growth point was obtained as explants, as shown in the figure. Figure 3 -A. Cut into slices of about 1 mm and place on callus induction medium, such as Figure 3 -B. After 14 days of dark culture Figure 3 -C, secondary callus subculture for 14 days Figure 4 -D.

[0036] 3. Callus warming and differentiation culture The callus tissues induced for more than 28 days from the above-mentioned infected sugarcane plants were used as the warm bath objects, and the warm bath medium was preheated to 40, 45 and 50°C for 5, 10 and 15 minutes respectively. In this example, the induced callus tissues were collected in a culture dish with filter paper and placed in a clean bench for 10 minutes under breeze. Figure 4 -A, transfer to a tissue culture bottle and incubate in preheated 45°C warm bath medium for 10 minutes. Figure 4 -B.

[0037] The callus tissue after warm bathing in the warm bath medium was transferred to the differentiation medium, and cultured in the dark at 28°C for 7 days, and then transferred to the light incubator for light culture for 14 days. Adventitious buds were differentiated on the surface of the callus tissue. Figure 5 -A, the lighting conditions are 1500-2100 lux 14 hours a day, 30-35℃ during the day, and 28-30℃ at night.

[0038] 4. Adventitious bud proliferation (seedling proliferation culture) The callus tissue that differentiated into adventitious buds was cut into small pieces and re-inoculated into differentiation medium for secondary differentiation culture for 14 days. Figure 5 -B, clustered seedlings were obtained. The light conditions were 1500-2100 lux for 14 hours per day, 30-35°C during the day, and 28-30°C at night.

[0039] 5. Rooting and cultivation of seedlings The clustered seedlings obtained by secondary differentiation were cut into small pieces, inoculated into rooting medium, and cultured for rooting under light for 20 days. Figure 6 -A, a sugarcane seedling plant with a well-developed root system was obtained. The light conditions were 1500-2100 lux for 14 hours a day, 30-35°C during the day, and 28-30°C at night.

[0040] 6. Transplanting seedlings After the rooting culture is completed, take out the sugarcane plants from the rooting medium and rinse off the rooting medium with clean water. Figure 6 -B; after soaking with disinfectant such as carbendazim, transplant to the sand bed for growth, such as Figure 6 -C, healthy sugarcane seedlings were obtained and the regeneration rate was calculated, regeneration rate = number of explants / number of transplanted seedlings.

[0041] 7. Plant virus-free rate detection Leaf and stem samples of virus-free seedlings ROC22 and striped sedge were randomly collected. RNA was extracted from the leaves of virus-free seedlings ROC22 for the detection of viral diseases such as sugarcane mosaic disease and yellow leaf disease. DNA was extracted from the stems of striped sedge plants for the detection of perennial root dwarfing disease. (Detection method reference: Li Wenfeng, 2016, Molecular detection technology of important sugarcane diseases) Specifically, 24 leaves of ROC22 virus-free seedlings were randomly collected to extract RNA and tested for stripe mosaic virus (SCSMV). Figure 7 -A, only 2 samples (No. 4 and No. 18) among the 24 samples were tested positive for stripe mosaic virus, indicating that the virus-free rate of sugarcane stripe mosaic virus in this experiment was 91.7%.

[0042] 24 samples of R. striata stems were randomly collected to extract DNA and tested for RSD. The results were as follows: Figure 7 -B, only one sample (No. 6) among the 24 samples was detected with perennial root dwarfing disease, indicating that the perennial root dwarfing disease-free rate of the present invention is 95.8%.

[0043] Comparative Example 1 Comparative Example 1 is a screening comparison test of the temperature and time of the callus warm bath treatment. The test conditions are to use the preheated warm bath medium at 40, 45 and 50°C for 5, 10 and 15 minutes respectively. In Example 1, the preheated warm bath medium at 45°C was used for 10 minutes. The results are shown in Table 2: Table 2 Callus warm bath regeneration and detoxification

[0044] The effects of warm bath on the callus detoxification rate and regeneration rate are shown in Table 2. With the increase of warm bath temperature and warm bath time, the detoxification rate of viral disease SCSMV increased slightly, with the lowest detoxification rate of 92.5% at 40℃ for 5 minutes and the highest detoxification rate of 96.7% at 50℃ for 15 minutes. The detoxification rate of callus regeneration plants in the control group without warm bath treatment also reached 93.6%, indicating that the detoxification process of the virus is mainly the regeneration process of somatic embryo callus, and the temperature and duration of warm bath have little effect on the detoxification effect of viral diseases. For the detoxification rate of bacterial diseases, with the increase of warm bath temperature and warm bath time, the detoxification rate increased significantly. The detoxification rate was only 43.2% at 40℃ for 5 minutes, and the highest detoxification rate was 94.1% at 50℃ for 15 minutes, while the RSD detection rate of regeneration plants in the control group without warm bath treatment reached 88.8%.

[0045] The data in Table 2 also show that the temperature and time of warm bathing have a significant effect on callus regeneration. The average regeneration rate of the explants of the two varieties that were not warm bathed was 1:3306, and the 40℃ warm bath had little effect on callus regeneration, with an average regeneration rate of 1:3229. The 45℃ warm bath had a significant effect on callus regeneration, with an average regeneration rate of 1:3258.5 for 5 minutes at 45℃ and an average regeneration rate of 2850 for 15 minutes at 45℃. The 50℃ warm bath had a greater effect on callus regeneration, with an average regeneration rate of 1:2487 for 5 minutes at 50℃ and a minimum average regeneration rate of 1:546 for 15 minutes at 50℃.

[0046] The results showed that the dedifferentiation and somatic embryo regeneration pathways of sugarcane explants can effectively remove viral diseases. The virus removal rate was higher than 90% without warm bath treatment, and warm bath callus would further increase the virus removal rate. The regeneration of somatic embryo callus combined with warm bath treatment can effectively remove bacterial diseases. After warm bathing at 45℃ for 10 minutes, the regeneration rate was 1:3005, and the bacterial disease removal rate was 86.3%. After warm bathing at 50℃ for 5 minutes, the regeneration rate dropped to 1:2431, and the bacterial disease removal rate was 90.5%. Therefore, if the explant material is only infected with viral diseases, virus-free healthy seedlings can be directly obtained through somatic embryo callus regeneration (the virus-free rate will be further improved after warm bathing). If infected with bacterial diseases, virus-free healthy seedlings need to be obtained through somatic embryo callus warm bath regeneration. The higher the temperature and the longer the warm bath time, the more favorable the bacterial disease removal. However, the appropriate warm bath temperature and time need to be determined by comprehensively considering the regeneration rate.

[0047] Comparative Example 2 The steps are basically the same as those in Example 1, except that the object of the warm bath is different. Specifically, the object of the warm bath treatment in step 3 is the young core leaf explants of the infected sugarcane plants before slicing in step 2, and the warm bath medium is preheated at 40, 45 and 50°C for 5, 10 and 15 minutes respectively. After the warm bath, callus induction is performed in step 2, callus differentiation is performed in step 3, adventitious bud proliferation is performed in step 4, rooting culture is performed in step 5, seedling transplantation is performed in step 6 and the regeneration rate is calculated, and finally the virus-free rate is calculated in step 7. The results are shown in Table 3: Table 3 Results of warm bath treatment of core leaf explants

[0048] As can be seen from Table 3, the detoxification efficiency of RSD by direct warming of explants was significantly lower than that of warming of callus tissue.

[0049] Comparative Example 3 The steps are basically the same as those in Example 1, except that the warm bath object is different. Specifically, step 3 does not warm bath the callus tissue, but the callus tissue that differentiates into adventitious buds after differentiation culture is used as the warm bath object, and the preheated warm bath medium at 40, 45 and 50°C is warm bathed for 5, 10 and 15 minutes respectively. After the warm bath, the adventitious buds are multiplied in step 4, the rooting culture is carried out in step 5, the seedlings are transplanted and the regeneration rate is calculated in step 6, and the virus-free rate is calculated in step 7. The results are shown in Table 4: Table 4 Results of warm-bath treatment of callus that differentiated into adventitious buds

[0050] It can be seen from Table 4 that the detoxification effect of adventitious buds differentiated by warm bath is much worse than that of warm bathing callus tissue, and the regeneration rate is more affected.

[0051] Comparative Example 4 The steps are basically the same as those in Example 1, except that the solvent used for the warm bath is different. In Comparative Example 4, instead of using the warm bath medium for the warm bath, sterile water is used for the warm bath. The other steps and conditions are the same as those in Example 1. The results are shown in Table 5: Table 5 Results of callus tissue sterile water temperature bath treatment

[0052] It can be seen from Table 5 that the detoxification rate of callus tissue in sterile water warm bath is worse than that in warm bath medium, and sterile water warm bath has a greater impact on the regeneration rate.

[0053] Comparative Example 5 The steps are basically the same as those in Example 1, except that the solvent used for the warm bath is different. In Comparative Example 5, the warm bath medium is not used for the warm bath, but the sucrose in the warm bath medium is removed and used as the warm bath solvent. The other steps and conditions are the same as those in Example 1. The results are shown in Table 6: Table 6 Callus warm bath treatment results

[0054] It can be seen from Table 6 that the detoxification rate of warm bath callus tissue by changing the formula of warm bath medium is worse than that by using warm bath medium, and warm bathing with the changed warm bath medium has a greater impact on the regeneration rate.

[0055] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. An efficient sugarcane detoxification and rapid propagation method, characterized in that: The steps include: S1 Preparation of sugarcane plant apical explants and callus induction Taking the tail of the sugarcane plant and obtaining the tightly curled cylindrical young white core leaf tissue at the top growth point as the explant, the explant is pretreated to prepare a sterile explant; The sterile explants were cut into thin slices, transferred into a container containing callus induction medium, and cultured in a constant temperature dark environment to obtain callus tissue; S2 Callus Warming Bath The callus tissue is warmed for a predetermined time in a preheated warm bath medium to obtain a warm bathed callus tissue, wherein the warm bath medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 2,4-D, and pH 5.8; S3 Callus Differentiation Culture The callus tissue after warm bath is inoculated into differentiation medium, first cultured in dark at constant temperature, then cultured in light, and callus tissue with young shoots can be differentiated; S4 seedling proliferation culture The callus tissue that differentiates into young shoots is cut into small pieces, and re-inoculated into differentiation medium for secondary differentiation culture to obtain clustered seedlings; S5 seedling rooting culture The clustered seedlings obtained by secondary differentiation are cut into small pieces, inoculated into rooting medium, and subjected to light rooting culture; S6 seedling transplanting After the rooting culture is completed, the seedlings are washed, sterilized, and then moved to a sand bed for growth to obtain healthy sugarcane seedlings.

2. An efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: In step S1, the explants are pretreated by washing with sterile water, disinfecting, and then washing with sterile water again, wherein the disinfection is disinfecting with 75% alcohol by volume for 30 seconds, and then transferring to 0.1% mercuric chloride or 5% sodium hypochlorite solution by volume for disinfection for 10 minutes; The sterile explants were cut into 1 mm thick slices, and the moisture on the surface of the sterile explants was dried before slicing; the callus induction medium was replaced every 14-20 days, and callus was obtained after 1-3 changes; the temperature of the constant temperature dark culture was 25-28°C; The callus induction medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 2,4-D, 5-9 g / L plant gel / agar, and pH 5.

8.

3. An efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: In step S2, the callus tissue is air-dried before the warm bath, and the warm bath medium is preheated to 40-50° C. The warm bath time is 5-10 minutes.

4. The efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: In step S3, the constant temperature dark culture temperature is 25-28°C for 7 days; the light culture is cultured in a light incubator for 14-20 days, the light conditions are 1500-2100 lux for 14 hours a day, 30-35°C during the day, and 28-30°C at night; The differentiation medium is based on B5, N6 or MS medium, supplemented with 20-30 g / L sucrose, 0.01-0.1 g / L activated carbon, 0-3 mg / L 6-BA, 5-9 g / L plant gel / agar, and pH 5.

8.

5. The efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: The duration of the secondary differentiation culture in step S4 is 14-20 days.

6. The efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: The time of light rooting culture in step S5 is 20-30 days, the light conditions are 1500-2100 lux 14 hours a day, 30-35°C during the day and 28-30°C at night; the rooting medium is B5, N6 or MS medium as the basic medium, supplemented with 20-30g / L sucrose, 0.01-0.1g / L activated carbon, 0-3mg / L NAA, 5-9g / L plant gel / agar, pH5.

8.

7. The efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: In step S6, the seedlings are cleaned by washing away the rooting medium on the seedlings with clean water, and the seedlings are disinfected by soaking them in carbendazim disinfectant solution.

8. The efficient sugarcane detoxification and rapid propagation method according to claim 1, characterized in that: After step S6, the virus-free rate of the plants needs to be tested.

9. An efficient sugarcane detoxification and rapid propagation method according to claim 8, characterized in that: The virus-free rate detection includes randomly collecting leaf and stem samples of the sugarcane plants obtained in step S6, extracting RNA from the leaves for viral disease detection of sugarcane mosaic disease and yellow leaf disease, and extracting DNA from the stems for bacterial disease detection of perennial root dwarfing disease and white stripe disease.

10. An efficient sugarcane detoxification and rapid propagation method according to any one of claims 1 to 9, characterized in that: The detection rates of viral diseases and bacterial diseases of sugarcane plants obtained in step S6 are both lower than 10%.

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