A tissue culture method of sugarcane

Abstract

The application provides a sugarcane tissue culture method, and belongs to the technical field of sugarcane tissue culture, and the tissue culture method comprises the following steps: (1) taking sugarcane tender stems as explants, alcohol disinfection, and obtaining pretreated explants; (2) placing the pretreated explants in an induction culture medium, inducing culture, and obtaining callus; (3) placing the callus in a proliferation culture medium, proliferating culture, and obtaining adventitious buds; (4) placing the adventitious buds in a rooting culture medium, rooting culture, and obtaining tissue culture seedlings; (5) placing the tissue culture seedlings in a seedling raising substrate, seedling raising, and obtaining seedlings; the seedling raising substrate in step (5) comprises the following raw material components in mass parts: 10-20 parts of sugarcane residues, 40-60 parts of peat soil, 10-15 parts of sheep manure, and 8-10 parts of rice husk charcoal. The tissue culture method provided by the application can improve the rooting rate and transplanting survival rate of sugarcane tissue culture seedlings, and the growth traits of the tissue culture seedlings can also be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of sugarcane tissue culture technology, and particularly relates to a method for tissue culture of sugarcane. Background Technology

[0002] Sugarcane is a temperate and tropical crop, used as a raw material for sugar production, and can also be used to extract ethanol as an energy substitute. Sugarcane is rich in sugar and water, as well as various vitamins, fats, proteins, organic acids, calcium, iron, and other substances that are very beneficial to human metabolism.

[0003] Sugarcane tissue culture is a highly efficient propagation technique that can accelerate the rapid propagation of superior sugarcane varieties, solving the problem of consuming large quantities of raw sugarcane due to the large amount of seed required. Furthermore, healthy seedlings obtained through tissue culture can improve sugarcane quality. However, current production capacity for healthy sugarcane seedlings is far from meeting production needs, and the rapid propagation technology for sugarcane seedlings is not yet fully mature. Problems often arise during the cultivation process, such as high contamination rates, poor plant growth, slow growth, low rooting rates, and high mortality rates during the acclimatization period. Moreover, subculture requires a significant amount of labor, significantly increasing the production cost of rapid sugarcane seedling propagation.

[0004] Therefore, establishing an efficient rapid propagation system for healthy sugarcane seedlings, reducing production costs, and providing high-quality healthy sugarcane seedlings to sugarcane-growing areas is of great significance for promoting the development of the sugarcane industry. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a tissue culture method for sugarcane, which can obtain sugarcane seedlings with high rooting rate and transplant survival rate and excellent growth traits.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for tissue culture of sugarcane, comprising the following steps:

[0008] (1) Sugarcane tender stems were used as explants and disinfected with alcohol to obtain pretreated explants;

[0009] (2) Place the pretreated explants in an induction medium and induce culture to obtain callus tissue;

[0010] (3) Place the callus tissue in a proliferation culture medium and proliferate to obtain adventitious shoots;

[0011] (4) Place the adventitious buds in a rooting medium and root them to obtain tissue culture seedlings;

[0012] (5) Place the tissue culture seedlings in the hardening substrate to harden them and obtain seedlings;

[0013] The seedling substrate described in step (5) comprises the following raw material components in parts by weight: 10-20 parts sugarcane bagasse, 40-60 parts peat moss, 10-15 parts sheep manure, and 8-10 parts rice husk charcoal.

[0014] Preferably, the sugarcane stalks in step (1) are obtained by cutting from newly grown stalks that have grown to 5-8cm, and the length of the sugarcane stalks is 2-3cm.

[0015] Preferably, the volume fraction of alcohol in step (1) is 70% to 80%, the alcohol disinfection time is 15 to 20 seconds per time, and the alcohol disinfection is performed 1 to 2 times.

[0016] Preferably, the induction culture medium in step (2) comprises: MS + 2.2-2.5 mg / L 2,4-D + 5-10 g / L agar + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the induction culture temperature is 25-28°C, and the induction culture time is 15-20 days.

[0017] Preferably, the proliferation culture medium in step (3) comprises: MS + 2.0-2.2 mg / L 6-BA + 0.10-0.15 mg / L NAA + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the proliferation culture temperature is 25-30℃, and the proliferation culture time is 10-15 days.

[0018] Preferably, the rooting culture medium in step (4) comprises: MS + 1.3-1.4 g / L NH4H2PO4 + 0.5-0.6 mg / L NAA + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the temperature of the rooting culture is 25-28℃, and the rooting culture time is 15-20 days.

[0019] Preferably, the temperature for hardening the seedlings in step (5) is 28-31°C, and the hardening time is 8-10 days.

[0020] Preferably, the antibacterial solution uses water as a solvent and is composed of garlic juice and sodium hypochlorite.

[0021] Preferably, the mass ratio of the garlic juice to sodium hypochlorite is 1-2:15-20.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a method for sugarcane tissue culture. By limiting the composition of the culture medium, especially the rooting medium and hardening substrate, this method yields sugarcane seedlings with high rooting rates, high transplant survival rates, and excellent growth traits. This invention inhibits the growth of exogenous bacteria by adding antibacterial agents to the induction medium, proliferation medium, and rooting medium, reducing the degree of explant contamination during culture. Simultaneously, the antibacterial agents promote the rooting of adventitious buds, providing a favorable foundation for the vigorous growth of sugarcane tissue-cultured seedlings. Furthermore, by limiting the raw material components in the hardening substrate, this invention provides high-quality nutrients to the sugarcane tissue-cultured seedlings, improves their growth performance, and shortens their recovery time.

[0024] The tissue culture method provided by this invention can lay the foundation for obtaining a new type of high-quality sugarcane seedling that can grow healthily, thereby promoting the sound development of the sugarcane industry. Detailed Implementation

[0025] This invention provides a method for tissue culture of sugarcane, comprising the following steps:

[0026] (1) Sugarcane tender stems were used as explants and disinfected with alcohol to obtain pretreated explants;

[0027] (2) Place the pretreated explants in an induction medium and induce culture to obtain callus tissue;

[0028] (3) Place the callus tissue in a proliferation culture medium and proliferate to obtain adventitious shoots;

[0029] (4) Place the adventitious buds in a rooting medium and root them to obtain tissue culture seedlings;

[0030] (5) Place the tissue culture seedlings in the hardening substrate to harden them and obtain seedlings;

[0031] In this invention, sugarcane stalks are used as explants, which are disinfected with alcohol to obtain pretreated explants.

[0032] In this invention, the sugarcane stalks are preferably obtained from newly grown stalks that have grown to 5-8 cm, more preferably 6-7 cm, and even more preferably 6.5 cm; the length of the sugarcane stalks is preferably 2-3 cm, more preferably 2.2-2.8 cm, even more preferably 2.4-2.6 cm, and even more preferably 2.5 cm; the volume fraction of the alcohol is preferably 70%-80%, more preferably 72%-78%, even more preferably 74%-76%, and even more preferably 75%; the alcohol disinfection time is preferably 15-20 s / time, more preferably 16-18 s / time, and even more preferably 17 s / time; the number of alcohol disinfection cycles is preferably 1-2 times.

[0033] In this invention, pretreated explants are placed in an induction culture medium and induced to obtain callus tissue.

[0034] In this invention, the induction culture medium preferably comprises: MS + 2.2–2.5 mg / L 2,4-D + 5–10 g / L agar + 28–30 g / L sucrose + 5–10 g / L antibacterial solution; more preferably, it comprises: MS + 2.3–2.4 mg / L 2,4-D + 6–8 g / L agar + 28.5–29.5 g / L sucrose + 6–8 g / L antibacterial solution; and even more preferably, it comprises: MS + 2.35 mg / L 2,4-D + 7 g / L agar + 29 g / L sucrose + 7 g / L antibacterial solution. The induction culture temperature is preferably 25–28°C, more preferably 26–27°C, and even more preferably 26.5°C. The induction culture time is preferably 15–20 days, more preferably 16–18 days, and even more preferably 17 days.

[0035] In this invention, callus tissue is placed in a proliferation culture medium and cultured to obtain adventitious shoots.

[0036] In this invention, the proliferation culture medium preferably comprises: MS + 2.0–2.2 mg / L 6-BA + 0.10–0.15 mg / L NAA + 28–30 g / L sucrose + 5–10 g / L antibacterial solution; more preferably, it comprises: MS + 2.1 mg / L 6-BA + 0.12–0.14 mg / L NAA + 29 g / L sucrose + 6–8 g / L antibacterial solution; the proliferation culture temperature is preferably 25–30°C, more preferably 26–28°C, and even more preferably 27°C; the proliferation culture time is preferably 10–15 days, more preferably 12–14 days, and even more preferably 13 days.

[0037] In this invention, adventitious buds are placed in a rooting culture medium and rooted to obtain tissue culture seedlings.

[0038] In this invention, the rooting culture medium preferably comprises: MS + 1.3–1.4 g / L NH4H2PO4 + 0.5–0.6 mg / L NAA + 28–30 g / L sucrose + 5–10 g / L antibacterial solution; more preferably, it comprises: MS + 1.32–1.38 g / L NH4H2PO4 + 0.52–0.58 mg / L NAA + 28.5–29.5 g / L sucrose + 6–8 g / L antibacterial solution; and even more preferably, it comprises: MS + 1.34–1.36 g / L NH4H2PO4 + 0.54–0.56 mg / L NAA. NAA + 29 g / L sucrose + 7 g / L antibacterial solution; the rooting culture temperature is preferably 25-28℃, more preferably 26-27℃, and even more preferably 26.5℃; the rooting culture time is preferably 15-20 days, more preferably 16-18 days, and even more preferably 17 days.

[0039] In this invention, the antibacterial solution in the induction medium, proliferation medium, and rooting medium is preferably water-based and composed of garlic juice and sodium hypochlorite; the mass ratio of garlic juice to sodium hypochlorite is preferably 1-2:15-20, more preferably 1.2-1.8:16-18, and even more preferably 1.4-1.6:17; the mass concentration of the antibacterial solution is preferably 0.5%-1%, more preferably 0.6%-0.8%, and even more preferably 0.7%; the garlic juice, used as an antibacterial agent, can effectively inhibit the contamination of the culture medium, improve the survival rate and proliferation coefficient of explants, and improve the rooting rate of sugarcane; the use of garlic juice and sodium hypochlorite can reduce production costs and shorten tissue culture time.

[0040] In this invention, tissue culture seedlings are placed in a hardening substrate to harden the seedlings and obtain seedlings.

[0041] In this invention, the preferred temperature for hardening off seedlings is 28–31°C, more preferably 29–30°C, and even more preferably 29.5°C; the preferred hardening time is 8–10 days, more preferably 8.5–9.5 days, and even more preferably 9 days; the preferred hardening substrate comprises the following raw material components by weight: 10–20 parts sugarcane bagasse, 40–60 parts peat moss, 10–15 parts sheep manure, and 8–10 parts rice husk charcoal; more preferably, it comprises the following raw material components by weight: 12–18 parts sugarcane bagasse, 45–55 parts peat moss, 12–14 parts sheep manure, and 8.5–9.5 parts rice husk charcoal; and even more preferably, it comprises the following... The raw material components by weight are: 14-16 parts sugarcane bagasse, 50 parts peat moss, 13 parts sheep manure, and 9 parts rice husk charcoal. The sugarcane bagasse contains a large number of beneficial bacteria and active substances, including water-soluble humic acid ≥14% and crude protein ≥6.5%. When used to cultivate sugarcane tissue culture seedlings, it can improve the transplant survival rate of sugarcane seedlings. The water-soluble humic acid it contains can improve the soil, stimulate crop growth, and improve the quality of agricultural products. The crude protein can provide certain nutrients for sugarcane tissue culture seedlings and promote sugarcane growth. The rice husk charcoal can improve the growth characteristics of tissue culture seedlings, increase the plant height and stem diameter, and promote the root growth of sugarcane seedlings.

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A method for tissue culture of sugarcane, the steps of which are as follows:

[0045] (1) Cut a 2cm long sugarcane stem from a newly grown 5cm stem and use it as an explant. Disinfect it twice with 70% alcohol for 20s each time to obtain a pretreated explant.

[0046] (2) The pretreated explants were placed in an induction medium and induced at 25°C for 15 days to obtain callus tissue;

[0047] The induction medium composition is as follows: MS + 2.2 mg / L 2,4-D + 5 g / L agar + 28 g / L sucrose + 5 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.5%).

[0048] (3) Place the callus tissue in the proliferation medium and culture it at 25℃ for 15 days to obtain adventitious shoots;

[0049] The proliferation medium composition is as follows: MS + 2.0 mg / L 6-BA + 0.10 mg / L NAA + 28 g / L sucrose + 5 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.5%);

[0050] (4) Place the adventitious buds in a rooting medium and root them at 25°C for 15 days to obtain tissue culture seedlings;

[0051] The rooting medium composition is as follows: MS + 1.3 g / L NH4H2PO4 + 0.5 mg / L NAA + 28 g / L sucrose + 5-10 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.5%).

[0052] (5) Place the tissue culture seedlings in a hardening substrate containing 10 parts sugarcane bagasse, 60 parts peat moss, 10 parts sheep manure and 8 parts rice husk charcoal, harden the seedlings at 28℃ for 10 days to obtain seedlings.

[0053] The antibacterial solution uses water as a solvent and is composed of garlic juice and sodium hypochlorite in a mass ratio of 1:15.

[0054] Example 2

[0055] A method for tissue culture of sugarcane, the steps of which are as follows:

[0056] (1) Cut a 3cm long sugarcane stem from the new shoots that have grown to 8cm and use it as an explant. Disinfect it once with 80% alcohol for 15s each time to obtain pretreated explants.

[0057] (2) Pretreated explants were placed in induction medium and induced at 28°C for 20 days to obtain callus tissue;

[0058] The induction medium composition is as follows: MS + 2.5 mg / L 2,4-D + 10 g / L agar + 30 g / L sucrose + 10 g / L antibacterial solution (the mass concentration of the antibacterial solution is 1%).

[0059] (3) Place the callus tissue in the proliferation medium and culture it at 30℃ for 10 days to obtain adventitious shoots;

[0060] The proliferation medium composition is as follows: MS + 2.2 mg / L 6-BA + 0.15 mg / L NAA + 30 g / L sucrose + 10 g / L antibacterial solution (the mass concentration of the antibacterial solution is 1%);

[0061] (4) Place the adventitious buds in a rooting medium and root them at 28℃ for 20 days to obtain tissue culture seedlings;

[0062] The rooting medium composition is as follows: MS + 1.4 g / L NH4H2PO4 + 0.6 mg / L NAA + 30 g / L sucrose + 10 g / L antibacterial solution (the mass concentration of the antibacterial solution is 1%).

[0063] (5) Place the tissue culture seedlings in a hardening substrate containing 20 parts sugarcane bagasse, 40 parts peat moss, 15 parts sheep manure and 10 parts rice husk charcoal, harden the seedlings at 31℃ for 8 days to obtain seedlings.

[0064] The antibacterial solution uses water as a solvent and is composed of garlic juice and sodium hypochlorite in a mass ratio of 2:20.

[0065] Example 3

[0066] A method for tissue culture of sugarcane, the steps of which are as follows:

[0067] (1) Cut a 2.5cm long sugarcane stalk from a new stalk that has grown to 7cm and use it as an explant. Disinfect it twice with 75% alcohol for 17s each time to obtain pretreated explants.

[0068] (2) Pretreated explants were placed in induction medium and induced at 27°C for 18 days to obtain callus tissue;

[0069] The induction medium composition is as follows: MS + 2.3 mg / L 2,4-D + 7 g / L agar + 29 g / L sucrose + 7 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.7%).

[0070] (3) The callus tissue was placed in the proliferation medium and cultured at 27°C for 13 days to obtain adventitious shoots;

[0071] The proliferation medium composition is as follows: MS + 2.1 mg / L 6-BA + 0.13 mg / L NAA + 29 g / L sucrose + 7 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.7%);

[0072] (4) Place the adventitious buds in a rooting medium and root them at 26℃ for 17 days to obtain tissue culture seedlings;

[0073] The rooting medium composition is as follows: MS + 1.35 g / L NH4H2PO4 + 0.55 mg / L NAA + 29 g / L sucrose + 7 g / L antibacterial solution (the mass concentration of the antibacterial solution is 0.7%);

[0074] (5) Place the tissue culture seedlings in a hardening substrate containing 15 parts sugarcane bagasse, 50 parts peat moss, 13 parts sheep manure and 9 parts rice husk charcoal, harden the seedlings at 29℃ for 9 days to obtain seedlings.

[0075] The antibacterial solution uses water as a solvent and is composed of garlic juice and sodium hypochlorite in a mass ratio of 1.5:17.

[0076] Comparative Example 1

[0077] Sugarcane seedlings were cultured according to the method in Example 1, with the only difference being that the antibacterial solution in the rooting medium was discarded.

[0078] Comparative Example 2

[0079] Sugarcane seedlings were cultivated according to the method in Example 1, except that the rice husk charcoal in the hardening substrate was replaced with wheat bran.

[0080] Comparative Example 3

[0081] Sugarcane seedlings were cultivated according to the method in Example 1, with the only difference being that the sugarcane bagasse in the hardening substrate was discarded.

[0082] Experimental Example 1

[0083] Sugarcane tissue culture seedlings were cultured according to the methods in Examples 1-3 and Comparative Example 1. Before rooting culture, 150 adventitious buds with basically uniform growth were randomly selected from each group and randomly divided into 3 replicate treatment groups of 50 seedlings each for rooting culture. After the culture was completed and before being transferred to the hardening substrate, the rooting rate of the sugarcane tissue culture seedlings was counted. The rooting rate was calculated using the formula: Rooting rate = Number of rooted seedlings / Number of inoculated seedlings × 100%. The results are shown in Table 1.

[0084] Table 1. Effects of different rooting media on the rooting rate of sugarcane tissue culture seedlings

[0085] Group Rooting rate / % Example 1 100.0 Example 2 99.3 Example 3 99.3 Comparative Example 1 81.3

[0086] As shown in Table 1, the present invention improves the rooting rate of sugarcane tissue culture seedlings by adding antibacterial solution to the rooting medium.

[0087] Experimental Example 2

[0088] Sugarcane seedlings were cultivated according to the methods described in Examples 1-3 and Comparative Examples 2-3. Before hardening off, 60 uniformly growing sugarcane tissue culture seedlings were randomly selected from each group. These 60 seedlings were then randomly divided into three replicate treatment groups of 20 seedlings each. After hardening off, the seedlings were transplanted into a plastic greenhouse. The transplant survival rate was calculated after 30 days. The formula for calculating the transplant survival rate was: (Number of surviving seedlings after transplanting / Number of transplanted seedlings) × 100%. The results are shown in Table 2.

[0089] Table 2. Transplant survival rate of sugarcane seedlings in different treatment groups.

[0090] Group Transplant survival rate / % Example 1 100.0 Example 2 98.3 Example 3 96.7 Comparative Example 2 85.2 Comparative Example 3 84.3

[0091] As shown in Table 2, using a hardening substrate containing sugarcane bagasse and rice husk charcoal can improve the transplant survival rate of sugarcane seedlings.

[0092] Experimental Example 3

[0093] Sugarcane seedlings were cultivated according to the methods in Examples 1-3 and Comparative Examples 1-3. 60 sugarcane seedlings with uniform growth were randomly selected from each group and randomly divided into 3 groups of 20 seedlings each. The seedlings were placed in a greenhouse with a day / night temperature of 30℃ / 25℃ (day / night) for 45 days. After cultivation, the growth of sugarcane seedlings in different treatment groups was observed, and the plant height, root length, and stem diameter of the sugarcane seedlings were recorded.

[0094] Table 3 Growth traits of sugarcane seedlings in different treatment groups

[0095] Group Plant height / cm Root length / cm Stem diameter / cm Example 1 62.51 26.71 2.25 Example 2 61.73 26.56 2.31 Example 3 62.28 26.77 2.18 Comparative Example 1 51.60 15.16 1.77 Comparative Example 2 55.49 16.22 1.84 Comparative Example 3 54.71 16.07 1.88

[0096] As shown in Table 3, the growth traits of sugarcane seedlings in Examples 1-3, such as plant height, root length, and stem diameter, were significantly improved. This result indicates that the tissue culture method provided by the present invention can improve the growth traits of sugarcane and further obtain a high-quality healthy sugarcane seedling.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for tissue culture of sugarcane, characterized in that, Includes the following steps: (1) Sugarcane tender stems were used as explants and disinfected with alcohol to obtain pretreated explants; (2) Place the pretreated explants in an induction medium and induce culture to obtain callus tissue; (3) Place the callus tissue in a proliferation culture medium and proliferate to obtain adventitious shoots; (4) Place the adventitious buds in a rooting medium and root them to obtain tissue culture seedlings; (5) Place the tissue culture seedlings in the hardening substrate to harden them and obtain seedlings; The seedling substrate described in step (5) comprises the following raw material components in parts by weight: 10-20 parts sugarcane bagasse, 40-60 parts peat moss, 10-15 parts sheep manure, and 8-10 parts rice husk charcoal.

2. The tissue culture method according to claim 1, characterized in that, The sugarcane stalks mentioned in step (1) are obtained by cutting from newly grown stalks that have grown to 5-8cm, and the length of the sugarcane stalks is 2-3cm.

3. The tissue culture method according to claim 1, characterized in that, The volume fraction of alcohol in step (1) is 70-80%, the alcohol disinfection time is 15-20 seconds per time, and the alcohol disinfection is performed 1-2 times.

4. The tissue culture method according to claim 1, characterized in that, The induction culture medium in step (2) includes: MS + 2.2-2.5 mg / L 2,4-D + 5-10 g / L agar + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the induction culture temperature is 25-28℃, and the induction culture time is 15-20 days.

5. The tissue culture method according to claim 1, characterized in that, The proliferation culture medium in step (3) includes: MS + 2.0-2.2 mg / L 6-BA + 0.10-0.15 mg / L NAA + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the proliferation culture temperature is 25-30℃, and the proliferation culture time is 10-15 days.

6. The tissue culture method according to claim 1, characterized in that, The rooting culture medium in step (4) includes: MS + 1.3-1.4 g / L NH4H2PO4 + 0.5-0.6 mg / L NAA + 28-30 g / L sucrose + 5-10 g / L antibacterial solution; the rooting culture temperature is 25-28℃, and the rooting culture time is 15-20 days.

7. The tissue culture method according to claim 1, characterized in that, The temperature for hardening the seedlings in step (5) is 28-31℃, and the hardening time is 8-10 days.

8. The tissue culture method according to any one of claims 4 to 6, characterized in that, The antibacterial solution uses water as a solvent and is composed of garlic juice and sodium hypochlorite.

9. The tissue culture method according to claim 8, characterized in that, The mass ratio of the garlic juice to sodium hypochlorite is 1-2:15-20.