A tissue culture method for improving sugarcane proliferation coefficient

Abstract

The invention discloses a tissue culture method for improving the proliferation coefficient of sugarcane, and the steps include disinfecting healthy axillary buds; inoculating axillary buds into an induction medium and culturing for 15-20 days, inducing new buds, and establishing a sterile system; cutting new buds, transferring to a proliferation medium and culturing for 20-25 days, and then transferring once every 20 days or so to obtain clustered buds; transferring clustered buds to a rooting medium, culturing for 10 days so that the clustered buds grow new roots, and waiting for the root system to be complete; taking out the clustered buds with new roots from the culture bottle, and hedging on the seedbed, after 30 days of hardening, the clustered sugarcane seedlings are divided into individual plants, transplanted into a light matrix material cup, and after 45-60 days of hardening management, the sugarcane seedlings grow to 30-40cm, and can be sold out of the field or moved to field planting. The cultivation method of the present invention effectively improves the proliferation coefficient of sugarcane, reduces the generation of invalid buds, reduces the risk of infection, improves the quality of seedlings, and reduces production costs.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a tissue culture method for improving the proliferation coefficient of sugarcane. Background Art

[0002] Sugarcane (Saccharum offici narum L.) is a perennial tall solid herb with a strong and well-developed rhizome. In sugarcane production, the long-term use of asexual reproduction and seed retention has led to a series of problems, such as variety degeneration, reduced yield, and reduced quality. In addition, this planting method also consumes a large amount of raw sugarcane as planting material.

[0003] In order to solve these problems, sugarcane tissue culture technology came into being. This technology accelerates the rapid reproduction of sugarcane varieties. The healthy seedlings obtained through tissue rapid propagation technology can improve the quality of sugarcane. More importantly, this method can effectively eliminate viral diseases such as mosaic disease and perennial dwarfing disease, prevent the degeneration of varieties, purify and rejuvenate, and increase the yield and sucrose content of sugarcane.

[0004] However, the current ability to produce healthy sugarcane seedlings is far from meeting production needs, and the rapid propagation technology of sugarcane seedlings is not yet fully mature. Problems such as high contamination rate, poor plant growth, slow growth, low rooting rate, and high mortality rate during the domestication period often occur during the cultivation process, which seriously restrict the widespread application of sugarcane tissue culture technology.

[0005] The existing sugarcane tissue culture method usually adopts the following steps: first, the axillary buds of sugarcane are used, washed with clean water and disinfected, and then inoculated into the induction medium. Under appropriate light and temperature conditions, they are cultured for about 30 days to induce sterile buds. Then, the sterile buds are cut and transferred to the proliferation medium, cultured for 20-30 days, and the sterile buds grow into sterile cluster buds. Transfer and proliferation are carried out every 20 days or so, and each cluster bud can proliferate about 2 times. Finally, the cluster buds are transferred to the rooting medium, and after 20-25 days of culture, a complete plant with a well-developed root system can be obtained.

[0006] However, these methods also have some obvious defects: First, the proliferation coefficient of sugarcane tissue culture buds is basically less than 3, which means low reproduction efficiency. Second, due to the high nitrogen content in the culture medium and the single cytokinin, there are many ineffective buds in the cultured sugarcane buds, and few effective buds that can be used for rooting. In addition, during the propagation of sugarcane tissue culture seedlings, the risk of infection with pathogens is extremely high, which not only affects the quality of the seedlings, but also greatly increases the production cost.

[0007] The present invention is proposed in view of the above problems. Summary of the invention

[0008] In order to overcome the above-mentioned defects of the prior art, the present invention provides a tissue culture method for improving the proliferation coefficient of sugarcane, which has the advantages of improving the proliferation coefficient of sugarcane, reducing ineffective buds, reducing infection risks, improving seedling quality and reducing production costs, so as to solve the problems raised in the above-mentioned background technology.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A tissue culture method for improving the proliferation coefficient of sugarcane comprises the following steps:

[0011] (1) Disinfect healthy axillary buds;

[0012] (2) inoculating the axillary buds into an induction medium and culturing them for 15-20 days to induce new buds and establish a sterile system;

[0013] (3) cutting the new shoots obtained in step (2), transferring them to a proliferation medium and culturing them for 20-25 days, and then transferring them every 20 days or so to obtain clustered shoots;

[0014] (4) transferring the clustered buds to a rooting medium and culturing them for 10 days to allow the clustered buds to grow new roots until the root system is complete;

[0015] (5) taking the clustered buds with new roots grown in step (4) out of the culture bottle, transplanting them on a seedling bed, and hardening them for 30 days, then dividing the clustered sugarcane seedlings into individual plants and transplanting them into cups of light matrix materials. After 45-60 days of hardening management, when the sugarcane seedlings grow to 30-40 cm, they can be sold or transplanted to the field for planting;

[0016] The induction medium is MS+2.0-3.0mg / LBA+0.1-0.5mg / LNAA+2.0-4.0mg / LPVP+30-40g / L sucrose+5-10g / L agar;

[0017] The proliferation medium is improved MS+1.0-2.0mg / LBA+0.1-0.5mg / LZT+0.1-0.5mg / LNAA+30-40g / L sucrose+5-10g / L agar;

[0018] The rooting medium is 1 / 2MS+2.0-3.0mg / LNAA+0.5-1.0mg / LI BA+30-50g / L sucrose+5-8g / L agar.

[0019] Further, in step (1), healthy and pest-free sugarcane axillary buds are selected, rinsed with clean water, soaked in 75% alcohol for 40-50 seconds, then transferred to 0.1% mercuric chloride solution for 15-20 minutes, and finally rinsed with sterile water for 4-5 times. Selecting healthy and pest-free sugarcane axillary buds is a prerequisite for ensuring the effect of the entire disinfection treatment. The axillary buds are rinsed with clean water to remove the dirt and impurities on the surface. Then soaked in 75% alcohol for 40-50 seconds, mainly to kill bacteria and fungi on the surface. Transferred to 0.1% mercuric chloride solution for 15-20 minutes, this step is mainly to further kill pathogenic microorganisms that may exist on the surface and inside of the axillary buds. Finally, rinsed with sterile water several times to remove residual mercuric chloride to avoid adverse effects on subsequent culture. The alcohol soaking time can be adjusted between 40-50 seconds according to the specific situation to achieve the best disinfection effect.

[0020] Furthermore, in step (2), the pH of the induction medium is 5.8-6.0, the light intensity of the culture is 1800-2000 Lux, the temperature is 22-25°C, and the culture is 15-20 days. The pH value range of the induction medium is 5.8-6.0, which helps to provide an acid-base environment suitable for the growth of sugarcane sprouts. The light intensity is set to 1800-2000 Lux, which ensures the effective photosynthesis and promotes the healthy growth of the sprouts. The temperature is controlled at 22-25°C, which provides suitable thermal conditions for the growth of sugarcane sprouts and helps to improve the growth rate and quality. The culture time is 15-20 days to ensure that the sprouts can fully develop during this period to form a healthy sterile system.

[0021] Furthermore, in step (3), the pH of the proliferation medium is 5.8-6.0, the illumination of the culture is 2000-2200 Lux, the temperature is 23-25°C, and the culture is 20-25 days. The pH value of the proliferation medium is set between 5.8-6.0, which helps to maintain the stability of the culture medium and the appropriate pH value; the illumination intensity of the culture is set at 2000-2200 Lux to ensure that the sugarcane obtains sufficient light energy during the proliferation process; the temperature is controlled at 23-25°C to provide suitable temperature conditions for the growth of sugarcane. Through the mutual coordination of these technical features, the problems of regulating the pH value, illumination and temperature of the culture medium during the proliferation of sugarcane can be effectively solved, and the proliferation efficiency and health of sugarcane can be improved.

[0022] Furthermore, in step (4), the pH of the rooting medium is 5.8-6.0, the light intensity of the culture is 2200-2500 Lux, the temperature is 23-25° C., and the culture is carried out for 25 days until the root system is fully developed. The pH value is controlled between 5.8-6.0 to ensure that the pH of the culture medium is suitable for root growth; the light intensity is controlled at 2200-2500 Lux, providing sufficient energy to promote photosynthesis and root growth; the temperature is controlled at 23-25° C., providing suitable temperature conditions for root growth; and extending the culture time to 25 days ensures that the root system has enough time to mature, ultimately achieving the purpose of a fully developed root system.

[0023] Further, in step (5), the matrix of the seedbed includes the following raw material components in parts by weight: 20-30 parts of bagasse, 100-150 parts of soil, 30-40 parts of straw, and 30-50 parts of peat soil. As an important component of the matrix, bagasse has good air permeability and water retention, which is beneficial to the respiration and water absorption of the roots of the sugarcane seedlings. As the basic component of the matrix, soil provides necessary minerals and trace elements to support the normal growth of sugarcane seedlings. The addition of straw not only improves the air permeability of the matrix, but also increases the organic matter content, improving the structure and fertility of the matrix. As a supplementary component of the matrix, peat soil has high water retention and nutrient content, which helps to keep the matrix moist and provide a continuous supply of nutrients.

[0024] Furthermore, 30-50 ml of biological agent is sprayed per kilogram of the matrix of the seedbed, and the concentration of the biological agent is 100 times diluted with water per gram, and the biological agent is Bacillus licheniformis, Bacillus subtilis and Pseudomonas. These biological agents can effectively inhibit the growth of pathogens, thereby reducing the pathogen infection in the sugarcane seedbed matrix. Through the above scheme, the present application solves the problem of pathogen infection in the sugarcane seedbed matrix. The use of biological agents can not only inhibit the growth of harmful pathogens, but also promote the reproduction of beneficial microorganisms, thereby improving the microbial environment of the seedbed matrix and ensuring the healthy growth of sugarcane seedlings.

[0025] Furthermore, the biological bacterial agent also includes EM bacteria, and the mass ratio of Bacillus licheniformis, Bacillus subtilis and Pseudomonas is 1:1:2; the mass ratio of Bacillus licheniformis, Bacillus subtilis, Pseudomonas and EM bacteria is 2:2:4:1.

[0026] Furthermore, the induction medium, proliferation medium and rooting medium also include an antibacterial agent, the concentration of the antibacterial agent is 6-15g / L, and the antibacterial agent is a mixture of extracts of mint, cactus, scutellaria and ginger.

[0027] Furthermore, the mass ratio of the extracts of mint, cactus, scutellaria and ginger is 2:1:1:3.

[0028] The beneficial effects of the present invention relative to the prior art are:

[0029] (1) The present invention is modified on the basis of MS culture medium, which is simple, easy and easy to operate, and has strong operability. The tissue culture method has a culture cycle of 25 days. Each bottle of subculture seedlings can multiply 5.5 bottles in each cycle, while the existing technology can only multiply 2 bottles in each cycle. Therefore, the proliferation coefficient of the new method and new technology is 2 to 3 times that of the existing technology. The application of the new method in production reduces production costs, improves production efficiency, and greatly increases the output of factory-based seedling cultivation.

[0030] (2) The sugarcane tissue culture seedlings produced by the present invention have strong plants, developed root systems, strong stress resistance, and restore the excellent properties of sugarcane itself. When planted in the field, the sugarcane yield can be increased by 30-50% and the sucrose content can be increased by 0.2-0.5%.

[0031] (3) The present invention adds biological microbial agents to the matrix of the seedbed, which not only promotes the increase of organic matter in the matrix, but also produces a synergistic effect between the biological bacteria and the microorganisms in the matrix, balances the bacterial flora, promotes the absorption of multiple nutrients by the cluster buds, and thus promotes the growth of young plants.

[0032] (4) The present invention adds antibacterial agents to the induction medium, proliferation medium and rooting medium. The active ingredients of the antibacterial liquid disinfect the medium and resist bacterial infection, effectively preventing the adverse effects of pathogens in the environment on the reproduction of sugarcane tissue culture seedlings. The inoculation, culture and other operating environment conditions have low requirements and strong adaptability, which simplifies the production operation process of tissue culture seedlings, greatly improves work efficiency and reduces production costs.

[0033] From the above, it can be seen that the tissue culture method for improving the proliferation coefficient of sugarcane provided in the present application includes the steps of disinfection, induction culture, proliferation culture, rooting culture and seedling hardening. By optimizing the culture medium formula and culture conditions, and adding antibacterial agents and biological agents, the proliferation coefficient of sugarcane is effectively improved, the production of invalid buds is reduced, the risk of infection is reduced, the quality of seedlings is improved, and the production cost is reduced. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the 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.

[0035] The components, concentrations and parameters of the three types of culture media used in the present invention are listed below:

[0036] The induction medium was MS+2.0mg / LBA+0.1mg / LNAA+2.0mg / LPVP+30g / L sucrose+5g / L agar.

[0037] The induction medium consisted of the following components:

[0038] KNO3: 1900mg / L;

[0039] MH4NO3: 1650mg / L;

[0040] KH2PO4: 170mg / L;

[0041] MgSO4·7H2O: 370mg / L;

[0042] CaCl2·I H2O: 440mg / L;

[0043] KI = 0.62 mg / L;

[0044] H3BO3: 4.6mg / L;

[0045] MnSO4·4H2O: 25.2mg / L;

[0046] ZnS04·7H2O: 7.1mg / L;

[0047] Na2M0O4·2H20: 0.21mg / L;

[0048] CuSO4·5H2O: 0.025mg / L;

[0049] CoCl2·6H20: 0.025mg / L;

[0050] Na2EDTA: 37.2mg / L;

[0051] FeSO4·7H20: 27.8mg / L;

[0052] Inositol: 100 mg / L;

[0053] Glycine: 2 mg / L;

[0054] Thiamine hydrochloride: 0.1 mg / L;

[0055] Pyridoxine hydrochloride: 0.5 mg / L;

[0056] Niacin: 0.5 mg / L;

[0057] Sucrose: 30g / L;

[0058] Agar: 4.0 g / L;

[0059] The solvent is deionized water.

[0060] The proliferation medium was modified MS + 1.0 mg / L BA + 0.1 mg / L ZT + 0.1 mg / L NAA + 30 g / L sucrose + 5 g / L agar;

[0061] KNO3: 1900mg / L;

[0062] NH4NO3: 800mg / L;

[0063] KH2PO4: 160mg / L;

[0064] MgSO4·7H2O: 370mg / L;

[0065] CaCl2·2H2O: 440mg / L;

[0066] KI: 0.62mg / L;

[0067] H3BO3: 4.6mg / L;

[0068] MnSO4·4H20: 25.2mg / L;

[0069] ZnS04·7H2O: 7.1mg / L;

[0070] Na2MO4·2H2O: 0.21mg / L;

[0071] CuSO4·5H2O: 0.025mg / L;

[0072] Coc2·6H2O: 0.025mg / L;

[0073] Na2EDTA: 37.2mg / L;

[0074] FeSO4·7H20: 27.8mg / L;

[0075] Inositol: 10 mg / L;

[0076] Glycine: 2 mg / L;

[0077] Thiocolloid hydrochloride = 0.1 mg / L;

[0078] Pyridoxine hydrochloride: 0.5 mg / L;

[0079] Niacin: 0.5 mg / L;

[0080] Sucrose: 30g / L;

[0081] Agar: 5.0 g / L;

[0082] The solvent is deionized water.

[0083] The rooting medium is 1 / 2MS+2.0mg / LNAA+0.5mg / LI BA+30g / L sucrose+5g / L agar.

[0084] KNO3: 950mg / L;

[0085] MH4NO3: 800mg / L;

[0086] KH2PO4: 170mg / L;

[0087] MgS04·7H2O: 370mg / L;

[0088] CaC2·2H2O: 440mg / L;

[0089] KI: 0.b2mg / L;

[0090] H3BO3: 4.6mg / L;

[0091] MnS04·4H2O: 25.2mg / L;

[0092] ZnS04·7H2O: 7.1mg / L;

[0093] Na2MO4·2H2O: 0.21mg / L;

[0094] CuO4·5H2O: 0.025mg / L;

[0095] COCl2·6H2O: 0.025mg / L;

[0096] Na2EDTA: 37.2mg / L;

[0097] FeS04·7H2O: 27.8mg / L;

[0098] Inositol: 10 mg / L;

[0099] Glycine: 2 mg / L;

[0100] Thiocolloid hydrochloride: 0.1mg / L;

[0101] Pyridoxine hydrochloride: 0.5 mg / L;

[0102] Niacin: 0.5 mg / L;

[0103] Sucrose: 30g / L;

[0104] Agar: 5.0 g / L;

[0105] The solvent is deionized water.

[0106] In the above three culture media, KNO3, MH4NO3, KH2PO4, MgSO4·7H2O, CaCl 2·2H2O, KI, H3BO3, MnSO4·4H2O, ZnSO4·7H2O, Na2M0O4·2H2O, CuS04·5H2O, C0Cl 2·6H2O, Na2EDTA, FeS047H2O, etc. are all analytically pure, i.e. AR grade, and are produced by Sichuan Xilong Science Co., Ltd. Inositol, glycine, thiamine hydrochloride, pyridoxine hydrochloride, niacin, BA, NAA, I BA, etc. are all produced by Sinopharm Chemical Reagent Co., Ltd.

[0107] Example 1

[0108] A tissue culture method for improving the proliferation coefficient of sugarcane comprises the following steps:

[0109] (1) Select healthy and pest-free sugarcane axillary buds, rinse them with clean water, soak them in 75% alcohol for 40-50 seconds, then soak them in 0.1% mercuric chloride solution for 15-20 minutes, and finally rinse them with sterile water; this step can effectively disinfect the sugarcane axillary buds, remove the pathogens on the surface and inside, and ensure a sterile environment in the subsequent cultivation process.

[0110] (2) Inoculate the axillary buds into an induction medium to induce new buds and establish a sterile system; the induction medium is MS+2.0 mg / L BA+0.1 mg / L NAA+2.0 mg / LPVP+30 g / L sucrose+5 g / L agar; the pH of the induction medium is 5.8-6.0, the light intensity during culture is 1800-2000 Lux, the temperature is 22-25° C., and the culture is carried out for 15-20 days.

[0111] (3) Cut the new shoots from step (2), transfer them to a proliferation medium and culture them for 20-25 days, and then transfer them every 20 days or so to obtain clustered shoots; the proliferation medium is modified MS + 1.0 mg / L BA + 0.1 mg / L ZT + 0.1 mg / L NAA + 30 g / L sucrose + 5 g / L agar; the pH value of the proliferation medium is 5.8-6.0, the light intensity of the culture is 2000-2200 Lux, the temperature is controlled at 23-25° C., and the culture is carried out for 20-25 days.

[0112] (4) The clustered buds were transferred to a rooting medium and cultured for 10 days to allow the clustered buds to grow new roots and wait for the root system to be complete; the rooting medium was 1 / 2MS + 2.0 mg / L NAA + 0.5 mg / LI BA + 30 g / L sucrose + 5 g / L agar; the pH value of the root culture medium was controlled between 5.8 and 6.0, the light intensity of the culture was 2200-2500 Lux, the temperature was controlled at 23-25 ​​° C, and the culture time was extended to 25 days to promote the complete development of the root system.

[0113] (5) taking the clustered buds with new roots grown in step (4) out of the culture bottle, and transplanting them on a common substrate in a seedling bed. After 30 days of hardening, the clustered sugarcane seedlings are divided into individual plants and transplanted into a cup of light substrate material. After 45-60 days of hardening, when the sugarcane seedlings grow to 30-40 cm, they can be sold or transplanted to the field for planting.

[0114] Example 2

[0115] The other steps are the same as those of Example 1, except that in step (5), the matrix of the seedbed includes the following raw material components in parts by weight: 20-30 parts of bagasse, 100-150 parts of soil, 30-40 parts of straw, and 30-50 parts of peat soil.

[0116] Example 3

[0117] The other steps are the same as those in Example 1. In step (5), 30-50 ml of the biological agent is sprayed per kilogram of the substrate of the seedbed. The concentration of the biological agent is 100 times diluted with water per gram. The biological agents are Bacillus licheniformis, Bacillus subtilis and Pseudomonas. The mass ratio of Bacillus licheniformis, Bacillus subtilis and Pseudomonas is 1:1:2.

[0118] Example 4

[0119] The other steps are the same as those in Example 1. In step (5), 30-50 ml of the biological agent is sprayed per kilogram of the substrate of the seedbed, and the concentration of the biological agent is 100 times diluted with water per gram; the biological agent is Bacillus licheniformis, Bacillus subtilis, Pseudomonas and EM bacteria in a mass ratio of 2:2:4:1.

[0120] Example 5

[0121] The other steps are the same as those in Example 1, except that the induction medium, the proliferation medium and the rooting medium also include an antibacterial agent, the concentration of the antibacterial agent is 6-15 g / L, the antibacterial agent is a mixture of extracts of mint, cactus, scutellaria and ginger, and the mass ratio of the extracts of mint, cactus, scutellaria and ginger is 2:1:1:3.

[0122] Comparative Example 1

[0123] (1) Rinse the axillary buds of sugarcane with clean water, disinfect them with 75% alcohol and 0.05-0.1% mercuric chloride, and then inoculate them into the induction culture medium. Adjust the light and temperature and culture them for about 30 days to induce sterile buds.

[0124] (2) Cut the sterile buds and transfer them to the proliferation medium: MS+6-BA (1.0-2.0)+NAA (0.1-0.5) for 20-30 days. The sterile buds will grow into sterile clustered buds. They will be transferred and multiplied every 20 days or so. Each time, the buds can multiply by about 2 times. Finally, the buds will be transferred to the rooting medium: 1 / 2MS+NAA (2.0-3.0)+I BA (1.0). After 20-25 days of culture, they will grow into complete plants with well-developed root systems.

[0125] The proliferation and rooting growth of tissue culture seedlings in different implementation modes are shown in Table 1. It can be seen from Table 1 that except for the contamination rate of 3.8% in Comparative Example 1, the other tissue culture seedling propagation methods are not contaminated. The cultivation method provided in the present invention, combined with the formulation of the culture medium, the matrix of the seedbed and the antibacterial agent, is effective in preventing the sugarcane tissue culture seedling propagation and cultivation process from being contaminated by pathogens.

[0126] As shown in Table 1, after 25 days of tissue culture seedling proliferation, the proliferation times of sugarcane tissue culture seedlings in Examples 1 to 5 were between 5.5 and 6.6, the proliferation times of Comparative Example 1 was 2, and the proliferation coefficients of Examples 1 to 5 were 2 to 3 times that of Comparative Example 1. It can be seen that the formula of the growth medium, the matrix of the seedbed and the antibacterial agent regulate the reproduction of sugarcane tissue culture seedlings and synergistically promote the proliferation of sugarcane tissue culture seedlings.

[0127] As can be seen from Table 1, the rooting rates of Examples 1-5 all reached 100%, and the transplanting survival rates were all above 99%. The sugarcane tissue culture seedling proliferation method of the present invention has obvious advantages.

[0128] Table 1

[0129]

[0130] The present application effectively solves the problem of low sugarcane proliferation coefficient through the above combination. Compared with the prior art, the improved combination of the biological agent and culture medium of the present application can significantly increase the proliferation coefficient of sugarcane, reduce pollution problems, and improve the quality and growth rate of sugarcane seedlings. Therefore, the present application has significant advantages in increasing the proliferation coefficient of sugarcane and can provide an efficient and stable method for the production of sugarcane seedlings.

[0131] Through the synergistic effect of the above steps and the culture medium, the proliferation coefficient of sugarcane is significantly improved, and the problems of low proliferation coefficient, many invalid buds, and high risk of infection with pathogens in the prior art are solved. Compared with the prior art, the method provided by the present application has a low contamination rate during the culture process, good plant growth, fast growth rate, high rooting rate, and low mortality rate during the acclimation period, which significantly improves the proliferation coefficient and production efficiency of sugarcane.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tissue culture method for improving the proliferation coefficient of sugarcane, characterized in that: The following steps are involved: (1) Disinfect healthy axillary buds; (2) inoculating the axillary buds into an induction medium and culturing them for 15-20 days to induce new buds and establish a sterile system; (3) cutting the new shoots obtained in step (2), transferring them to a proliferation medium and culturing them for 20-25 days, and then transferring them every 20 days or so to obtain clustered shoots; (4) transferring the clustered buds to a rooting medium and culturing them for 10 days to allow the clustered buds to grow new roots until the root system is complete; (5) taking the clustered buds with new roots grown in step (4) out of the culture bottle, transplanting them on a seedling bed, and hardening them for 30 days, then dividing the clustered sugarcane seedlings into individual plants and transplanting them into cups of light matrix materials. After 45-60 days of hardening management, when the sugarcane seedlings grow to 30-40 cm, they can be sold or transplanted to the field for planting; The induction medium is MS+2.0-3.0mg / LBA+0.1-0.5mg / LNAA+2.0-4.0mg / LPVP+30-40g / L sucrose+5-10g / L agar; The proliferation medium is improved MS+1.0-2.0mg / LBA+0.1-0.5mg / LZT+0.1-0.5mg / LNAA+30-40g / L sucrose+5-10g / L agar; The rooting medium is 1 / 2MS+2.0-3.0mg / LNAA+0.5-1.0mg / LIBA+30-50g / L sucrose+5-8g / L agar.

2. A tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 1, characterized in that: In step (1), healthy sugarcane axillary buds free of disease and insect pests are selected, rinsed with clean water, soaked in 75% alcohol for 40-50 seconds, then transferred to 0.1% mercuric chloride solution and soaked for 15-20 minutes, and finally rinsed with sterile water.

3. A tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 1, characterized in that: In step (2), the pH of the induction medium is 5.8-6.0, the illumination of the culture is 1800-2000 Lux, the temperature is 22-25° C., and the culture is carried out for 15-20 days.

4. The tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 1, characterized in that: In step (3), the pH of the proliferation medium is 5.8-6.0, the illumination of the culture is 2000-2200 Lux, the temperature is 23-25° C., and the culture is carried out for 20-25 days.

5. The tissue culture method for improving the proliferation coefficient of sugarcane according to claim 1, characterized in that: In step (4), the pH of the rooting medium is 5.8-6.0, the light intensity is 2200-2500 Lux, the temperature is 23-25° C., and the culture is carried out for 25 days until the root system is fully developed.

6. The tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 1, characterized in that: In step (5), the matrix of the seedbed includes the following raw material components in parts by weight: 20-30 parts of bagasse, 100-150 parts of soil, 30-40 parts of straw, and 30-50 parts of peat soil.

7. A tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 6, characterized in that: Spray 30-50 ml of biological agent per kilogram of the substrate of the seedbed, the concentration of the biological agent is 100 times diluted with water per gram, and the biological agent is Bacillus licheniformis, Bacillus subtilis and Pseudomonas.

8. A tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 7, characterized in that: The biological bacterial agent also includes EM bacteria, and the mass ratio of Bacillus licheniformis, Bacillus subtilis and Pseudomonas is 1:1:2; the mass ratio of Bacillus licheniformis, Bacillus subtilis, Pseudomonas and EM bacteria is 2:2:4:

1.

9. The tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 1, characterized in that: The induction medium, proliferation medium and rooting medium also include an antibacterial agent, the concentration of the antibacterial agent is 6-15g / L, and the antibacterial agent is a mixed solution of extracts of mint, cactus, scutellaria and ginger.

10. A tissue culture method for increasing the proliferation coefficient of sugarcane according to claim 9, characterized in that: The mass ratio of the extracts of mint, cactus, scutellaria and ginger is 2:1:1:3.

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