Tissue culture method for reducing browning of cercis chinensis

Abstract

The invention discloses a tissue culture method for reducing browning of cercis chinensis, which comprises the following steps: S1, cleaning and disinfecting stems with axillary buds of cercis chinensis, and taking the axillary buds from a sodium sulfite solution; s2, carrying out primary culture on the axillary buds to obtain aseptic seedlings, and carrying out anti-browning culture and growth promoting culture on the aseptic seedlings to obtain cercis chinensis tissue culture seedlings; a culture medium used in the anti-browning culture comprises the following components: improved MS, IBA, 2, 4-D, rutin and sodium sulfite. Compared with the prior art, the method has the advantages that the aseptic seedling obtaining rate is increased by 81.67% by improving the disinfection method, the transplanting survival rate of the cercis chinensis aseptic seedlings obtained through anti-browning growth-promoting culture is 88.33%, and the surviving cercis chinensis aseptic seedlings have dark green or cyan leaves and more robust stems.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a tissue culture method for reducing browning in Bauhinia purpurea. Background Technology

[0003] Although nature reserves were established to protect Bauhinia trees a long time ago, the wild resources of Bauhinia trees are still declining rapidly. Therefore, it is very necessary to preserve the germplasm resources of Bauhinia trees through plant tissue culture.

[0004] To protect the germplasm resources of *Bauhinia purpurea*, the inventors of this invention have focused on obtaining *Bauhinia purpurea* tissue culture seedlings through plant tissue culture. However, during the tissue culture process, the inventors discovered the following difficulties in *Bauhinia purpurea* tissue culture: (1) The axillary buds of Bauhinia purpurea showed severe contamination and browning in the initial culture stage, resulting in a low rate of obtaining sterile seedlings; (2) During the cultivation process, the culture medium of the inoculated axillary buds or stem segments of Cercis chinensis was stained light yellow or brown with the inoculation point as the center, and the inoculated material also showed different degrees of browning. Light browning would affect growth, and severe browning would cause the plant to wither and die. (3) The slow growth of the stem nodes of the Bauhinia stem segment results in a long cultivation time and makes it difficult to sprout larger leaves.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The main objective of this invention is to provide a tissue culture method that reduces browning in Bauhinia purpurea seedlings, aiming to address the problem of decreasing wild Bauhinia purpurea resources and potential lack of Bauhinia purpurea germplasm resources, and to overcome the difficulties in the tissue culture process of Bauhinia purpurea as described in the background art.

[0007] To achieve the above objectives, the present invention provides a tissue culture method for reducing browning in Bauhinia purpurea, the tissue culture method comprising: S1. Clean and disinfect the stem segments of Bauhinia purpurea with axillary buds, and take the axillary buds in sodium sulfite solution; S2, the axillary buds are initially cultured to obtain sterile seedlings. After the sterile seedlings are subjected to anti-browning and growth-promoting culture, the stem segments of the sterile seedlings are taken for subculture. The culture medium used for the anti-browning culture includes: modified MS, IBA, 2,4-D, L-cysteine, rutin, and sodium sulfite.

[0008] Preferably, in the above technical solution, the culture medium used for the anti-browning culture consists of modified MS + 1-2 mg / L IBA + 2-4 mg / L 2,4-D + 1-2 mg / L L-cysteine ​​+ 1-2 mg / L rutin + 1-2 mg / L sodium sulfite.

[0009] Preferably, in the above technical solution, the culture conditions for the anti-browning and growth-promoting culture are as follows: cultured at a culture temperature of 25±2℃, a light intensity of 3000-4000Lx, and a light duration of 12-14 h / d until the sterile seedlings reach a height of 5-7cm, and then subcultured.

[0010] Preferably, in the above technical solution, the specific steps for cleaning and disinfecting the Bauhinia stem segment with axillary buds and extracting the axillary buds in sodium sulfite solution in step S1 are as follows: S11, take branches of Bauhinia purpurea containing 2-3cm axillary buds; S12, soak the Bauhinia branches in alcohol for 30-120 seconds and then rinse with distilled water, and soak them in H2O2 for 5-8 minutes and then rinse with distilled water. S13, in sodium sulfite solution, remove 2-3 cm axillary buds from the branches of the Bauhinia tree to obtain axillary buds.

[0011] Preferably, in the above technical solution, the length of the Bauhinia branch is 3-5 cm between adjacent nodes of the stem node containing the 2-3 cm axillary bud.

[0012] According to the above technical solution, the present invention protects the axillary bud by retaining 3-5 cm of the internode between the adjacent nodes of the stem node where the axillary bud is located, thereby avoiding damage to the cell membrane of cells near the cut caused by disinfectant during the disinfection process of the Bauhinia explant, and reducing explant death caused by axillary bud cell damage.

[0013] Preferably, in the above technical solution, the H2O2 concentration is 10-12%.

[0014] Preferably, in the above technical solution, the sodium sulfite solution is obtained by adding 5-7 µL of 1 mg / L sodium sulfite to 20-40 mL of distilled water.

[0015] Preferably, in the above technical solution, the culture medium used for the initial culture consists of WPM + 1-2 mg / L sodium sulfite.

[0016] Preferably, in the above technical solution, the initial culture conditions are: culture at a temperature of 18±2℃ and a light intensity of 500-1000Lx, with a light duration of 6-8 h / d for 4-5 days.

[0017] Preferably, in the above technical solution, in the step of the sterile seedling undergoing anti-browning and growth-promoting culture, the sterile seedling is transplanted as a whole plant.

[0018] Compared with the prior art, the present invention has the following beneficial effects: After disinfection, the axillary buds of *Bauhinia purpurea* were taken from a sodium sulfite solution. This served two purposes: first, to further kill microorganisms on the surface of the axillary buds, reducing contamination to only 5.00%; and second, to inhibit polyphenol oxidase activity in conjunction with WPM and sodium sulfite culture medium, thus reducing browning of the axillary buds in the initial culture stage, with a browning rate of only 13.33% and a sterile seedling acquisition rate of 81.67%. A culture medium that reduces browning and promotes vegetative growth in Bauhinia purpurea was selected, namely, modified MS, IBA, 2,4-D, L-cysteine, rutin, and sodium sulfite. The transplant survival rate was 88.33%, and the surviving sterile Bauhinia purpurea seedlings had dark green or blue leaves and stronger stems. Attached Figure Description

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0020] Figure 1 The images show varying degrees of browning in the axillary buds of Bauhinia purpurea during the initial cultivation stage. Figure 2 This is a diagram of the axillary buds of *Bauhinia purpurea* during the initial cultivation stage of Embodiment 1 of the present invention; Figure 3 This is a diagram showing the severe browning of the axillary buds of *Cercis chinensis* in Comparison 4 during the anti-browning and growth-promoting stage. Figure 4 This is a diagram showing the browning of axillary buds of *Cercis chinensis* in Comparison 5 during the browning resistance and growth promotion stage; Figure 5 This is a diagram of axillary bud germination in Bauhinia purpurea during the browning resistance and growth promotion stage, as shown in Comparison 6. Figure 6 This is a diagram of axillary bud germination in Bauhinia purpurea during the browning resistance and growth promotion stage, as shown in Figure 7. Figure 7 This is a diagram of axillary bud germination in Bauhinia purpurea during the browning resistance and growth promotion stage (Figure 8). Figure 8 This is a diagram of axillary bud germination in *Bauhinia purpurea* during the anti-browning and growth-promoting stage of Example 1; Figure 9 This is a diagram of axillary bud germination in a Bauhinia purpurea during the anti-browning and growth-promoting stage of a particular embodiment. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention have been clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Figure 1 During the initial culture stage of *Bauhinia purpurea*, the inventors discovered that browning of the *Bauhinia purpurea* explants caused the culture medium to turn dark brown or light yellow centered on the explants, and as the browning deepened, the inoculated material eventually lost its viability. Therefore, without improving the culture medium and addressing the browning problem of *Bauhinia purpurea*, the rate of obtaining sterile seedlings of *Bauhinia purpurea* cannot be increased, severely restricting the preservation of *Bauhinia purpurea* germplasm resources through tissue culture. Example 1

[0023] A tissue culture method for reducing browning in Bauhinia purpurea, the method comprising: S1. Clean and disinfect the stem segments of Bauhinia purpurea with axillary buds, and extract the axillary buds from the sodium sulfite solution. Specific steps include: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 30 seconds, rinse twice with distilled water, soak in 10% H2O2 for 5 minutes, and rinse twice with distilled water. S13, in a sodium sulfite solution, cut off 2-3 cm axillary buds from the branches of the Bauhinia tree to obtain axillary buds; wherein, the sodium sulfite solution is 20 mL of distilled water with 5 µL of 1 mg / L sodium sulfite added.

[0024] S2, Initial Culture: The axillary buds from step S13 were inoculated into a culture medium of WPM + 1 mg / L sodium sulfite. After 10 days of culture at a temperature of 18±2℃, a light intensity of 500 Lx, and a light duration of 6 h / d, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0025] Anti-browning and growth-promoting culture: The sterile seedlings were transferred to a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 2 mg / L L-cysteine, 2 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were cultured at 25 ± 2 °C with a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning until they reached a height of 5-7 cm or had sprouted 4-5 leaves. Subculture was then carried out.

[0026] Successive generations of training: In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture were removed, and the seedlings were cut into stem segments retaining 1-2 nodes. These stem segments were inoculated into a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 2 mg / L L-cysteine, 2 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were first cultured at 18±2℃ with a light intensity of 500 Lx and a photoperiod of 6 h / d for 5 days, and then transferred to a culture temperature of 25±2℃ with a light intensity of 4000 Lx and a photoperiod of 12 h / d. The sodium sulfite solution was prepared by adding 5 µL of 1 mg / L sodium sulfite to 20 mL of distilled water.

[0027] The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0028] Example 2 A tissue culture method for reducing browning in Bauhinia purpurea, the method comprising: S1. Clean and disinfect the stem segments of Bauhinia purpurea with axillary buds, and extract the axillary buds from the sodium sulfite solution. Specific steps include: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5-7 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 60 seconds, rinse twice with distilled water, soak in 12% H2O2 for 5 minutes, and rinse twice with distilled water. S13, in a sodium sulfite solution, 2-3 cm axillary buds are cut off from the branches of the Bauhinia tree to obtain axillary buds; wherein, the sodium sulfite solution is prepared by adding 5 µL of 1 mg / L sodium sulfite to 20 mL of distilled water.

[0029] S2, Initial Culture: The axillary buds from step S13 were inoculated into a culture medium of WPM + 1.5 mg / L sodium sulfite. After culturing for 5 days at a temperature of 18±2℃, a light intensity of 800 Lx, and a light duration of 8 h / d, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0030] Anti-browning and growth-promoting culture: The sterile seedlings were transferred to a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 2 mg / L L-cysteine, 2 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were cultured at 25 ± 2 °C with a light intensity of 3000 Lx and a photoperiod of 14 h / d to promote growth and prevent browning until they reached a height of 5-7 cm or had sprouted 4-5 leaves. Subculture was then carried out.

[0031] Successive generations of training: In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture were removed, and stem segments were cut, retaining 1-2 nodes. These stem segments were inoculated into a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 1-2 mg / L L-cysteine, 2 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were first cultured at 18±2℃ with a light intensity of 8000 Lx and a photoperiod of 8 h / d for 5 days, and then transferred to a culture temperature of 25±2℃ with a light intensity of 3000 Lx and a photoperiod of 14 h / d. The sodium sulfite solution was prepared by adding 5 µL of 1 mg / L sodium sulfite to 20 mL of distilled water.

[0032] The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0033] Example 3 A tissue culture method for reducing browning in Bauhinia purpurea, the method comprising: S1. Clean and disinfect the stem segments of Bauhinia purpurea with axillary buds, and extract the axillary buds from the sodium sulfite solution. Specific steps include: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 30 seconds, rinse twice with distilled water, soak in 12% H2O2 for 5 minutes, and rinse twice with distilled water. S13, in a sodium sulfite solution, 2-3 cm axillary buds are cut off from the branches of the Bauhinia tree to obtain axillary buds; wherein, the sodium sulfite solution is prepared by adding 7 µL of 1 mg / L sodium sulfite to 40 mL of distilled water.

[0034] S2, Initial Culture: The axillary buds from step S13 were inoculated into a culture medium of WPM + 2 mg / L sodium sulfite. After 10 days of culture at a temperature of 18±2℃, a light intensity of 1000 Lx, and a light duration of 6 h / d, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0035] Anti-browning and growth-promoting culture: The sterile seedlings were transferred to a modified MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 1 mg / L L-cysteine, 1 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were cultured at 25 ± 2 °C with a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning until they reached a height of 5-7 cm or had sprouted 4-5 leaves. Subculture was then carried out.

[0036] Successive generations of training: In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture were removed, and the seedlings were cut into stem segments retaining 1-2 nodes. These stem segments were inoculated into a modified MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 1 mg / L L-cysteine, 1 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were first cultured at 18±2℃ with a light intensity of 1000 Lx and a photoperiod of 6 h / d for 5 days, and then transferred to a culture temperature of 25±2℃ with a light intensity of 3500 Lx and a photoperiod of 14 h / d. The sodium sulfite solution was prepared by adding 7 µL of 1 mg / L sodium sulfite to 40 mL of distilled water.

[0037] The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0038] Example 4 A tissue culture method for reducing browning in Bauhinia purpurea, the method comprising: S1. Clean and disinfect the stem segments of Bauhinia purpurea with axillary buds, and extract the axillary buds from the sodium sulfite solution. Specific steps include: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 30 seconds, rinse twice with distilled water, soak in 12% H2O2 for 5 minutes, and rinse twice with distilled water. S13, in a sodium sulfite solution, cut off 2-3 cm axillary buds from the branches of the Bauhinia tree to obtain axillary buds; wherein, the sodium sulfite solution is a sodium sulfite solution in which 7 µL of 1 mg / L sodium sulfite is added to 40 mL of distilled water.

[0039] S2, Initial Culture: The axillary buds from step S13 were inoculated into a culture medium of WPM + 2 mg / L sodium sulfite. After 10 days of culture at a temperature of 18±2℃, a light intensity of 800 Lx, and a light duration of 6 h / d, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0040] Anti-browning and growth-promoting culture: The sterile seedlings were transferred to a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 2 mg / L L-cysteine, 2 mg / L rutin, 2 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were cultured at 25 ± 2 °C with a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning until they reached a height of 5-7 cm or had sprouted 4-5 leaves. Subculture was then carried out.

[0041] Successive generations of training: In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture were removed, and stem segments were cut, retaining 1-2 nodes. These stem segments were inoculated into a modified MS medium containing 2 mg / L IBA, 4 mg / L 2,4-D, 2 mg / L L-cysteine, 2 mg / L rutin, 2 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The seedlings were first cultured at 18±2℃ with a light intensity of 800 Lx and a photoperiod of 6 h / d for 5 days, and then transferred to a culture temperature of 25±2℃ with a light intensity of 3500 Lx and a photoperiod of 14 h / d. The sodium sulfite solution was prepared by adding 7 µL of 1 mg / L sodium sulfite to 40 mL of distilled water.

[0042] The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0043] Comparative Example 1 A method for disinfection and initial cultivation of Bauhinia purpurea, the method comprising: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, soak in 0.3% sodium hypochlorite for 5 minutes, rinse twice with distilled water, and then cut off the 2-3cm axillary bud on the Bauhinia branch to obtain the axillary bud; S13, the axillary buds were inoculated into WPM medium, a total of 60 bottles were inoculated, one Bauhinia axillary bud in each bottle, and they were placed in a culture environment of 25±2℃, light intensity of 500Lx and light duration of 6 h / d. After 10 days of culture, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0044] Comparative Example 2 A method for disinfection and initial cultivation of Bauhinia purpurea, comprising: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 30 seconds, rinse twice with distilled water, soak in 10% H2O2 for 5 minutes, rinse twice with distilled water, then cut off the 2-3cm axillary bud on the Bauhinia branch to obtain the axillary bud; S13, the axillary buds were inoculated into WPM medium, a total of 60 bottles were inoculated, one Bauhinia axillary bud in each bottle, and they were placed in a culture environment of 25±2℃, light intensity of 500Lx and light duration of 6 h / d. After 10 days of culture, sterile seedlings were obtained. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded daily, and contaminated tissue culture bottles were removed in a timely manner.

[0045] Comparative Example 3 A method for disinfection and initial cultivation of Bauhinia purpurea, the method comprising: S11, the collection target is a healthy and vigorous single Bauhinia tree with a tree age of 5 years or more, free from pests and diseases, and the branches with 2-3cm axillary buds are taken. S12, retain 3-5cm between the two ends of the stem node where the axillary bud is located, soak in saturated detergent water for 5 minutes, then gently brush with a soft brush, rinse 3 times with clean water, transfer to the inoculation room, soak in 70% alcohol for 30 seconds, rinse twice with distilled water, soak in 10% H2O2 for 5 minutes, rinse twice with distilled water, cut off the 2-3cm axillary bud on the Bauhinia branch to obtain the axillary bud; S13, Inoculate the axillary buds from step S12 into the culture medium: WPM + 1 mg / L sodium sulfite, for a total of 60 bottles, with one Bauhinia axillary bud inoculated in each bottle. Place them in the culture conditions of 25±2℃, light intensity of 500 Lx and light duration of 6 h / d, and obtain sterile seedlings after 10 days of culture. During the culture process, observe and record the contamination and browning of the tissue culture seedlings at regular intervals every day, and remove the contaminated tissue culture bottles in a timely manner.

[0046] Contamination rate (%) = (Number of contaminated strains / Total number of inoculated strains) × 100%; Browning rate (%) = (Number of browned plants / Total number of inoculated plants) × 100%; Sterile vaccine acquisition rate (%) = (Uncontaminated and non-browned / Total number of inoculated vaccines) × 100%. Table 1. Contamination rate and browning rate of initial culture name Pollution rate / % Browning rate / % Sterile vaccine acquisition rate / % Culture medium color Example 1 Initial Culture 5.00 13.33 81.67 Transparent, white Comparative Example 1 18.33 38.33 43.34 pale yellow Comparative Example 2 11.67 33.33 55.00 pale yellow Comparative Example 3 6.67 15.00 78.33 Transparent, white Table 1 shows that after the culture was completed, the use of alcohol and hydrogen peroxide for disinfection, the addition of sodium sulfite to the WPM medium, and the extraction of axillary buds from the sodium sulfite solution all had a significant impact on the contamination rate, browning rate, and sterile seedling acquisition rate of Bauhinia purpurea during the initial culture process. Appropriate disinfection and culture methods resulted in a contamination rate of less than 10%, a browning rate of less than 15%, and a sterile seedling acquisition rate of 81.67% for the initial culture of Bauhinia purpurea. In contrast, the axillary buds of Bauhinia purpurea in Comparative Example 1, which were disinfected with sodium hypochlorite and inoculated into WPM medium, had a high browning rate and a sterile seedling acquisition rate of less than 50%.

[0047] Comparative Example 4 Culture medium preparation: Modified MS medium + 1 mg / L IBA + 2 mg / L NAA + 12.5 g / L sucrose + 7.5 g / L agar, 60 bottles in total; The sterile seedlings were transferred one seedling per bottle to modified MS medium containing 1 mg / L IBA, 2 mg / L NAA, 12.5 g sucrose, and 7.5 g agar. The culture was then incubated at 25 ± 2 °C under a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning. Figure 3 ) The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0048] Comparative Example 5 Culture medium preparation: Modified MS medium + 1 mg / L IBA + 2 mg / L 2,4-D + 12.5 g / L sucrose + 7.5 g / L agar, 60 bottles in total; The sterile seedlings were transferred one seedling per bottle to modified MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 12.5 g / L sucrose, and 7.5 g / L agar. The culture was then incubated at 18 ± 2 °C under a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning. Figure 4 ) The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0049] Comparative Example 6 Culture medium preparation: Modified MS + 1 mg / L IBA + 2 mg / L 2,4-D + 2 mg / L L-cysteine ​​+ 1 mg / L sodium sulfite + 12.5 g / L sucrose + 7.5 g / L agar, 60 bottles in total; The sterile seedlings were transferred, one seedling per bottle, to a modified MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 2 mg / L rutin, 1 mg / L sodium sulfite, 12.5 g sucrose, and 7.5 g agar. The culture was then incubated at 18 ± 2℃ with a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning. Figure 5 ) The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0050] Comparative Example 7 Culture medium preparation: Modified MS + 1 mg / L IBA + 2 mg / L 2,4-D + 2 mg / L VC + 1 mg / L sodium sulfite + 12.5 g / L sucrose + 7.5 g / L agar, 60 bottles in total; The sterile seedlings were transferred, one seedling per bottle, to a modified MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 2 mg / L L-cysteine, 1 mg / L sodium sulfite, 12.5 g / L sucrose, and 7.5 g / L agar. The culture was then incubated at 25 ± 2 °C under a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning. Figure 6 ) The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0051] Comparative Example 8 Culture medium preparation: Modified MS + 1 mg / L IBA + 2 mg / L 2,4-D + 1 mg / L PVP + 1 mg / L sodium sulfite + 12.5 g / L sucrose + 7.5 g / L agar, 60 bottles in total; The sterile seedlings were transferred one seedling per bottle to MS medium containing 1 mg / L IBA, 2 mg / L 2,4-D, 1 mg / L PVP, and 1 mg / L sodium sulfite. The culture was then incubated at 25 ± 2 °C under a light intensity of 4000 Lx and a photoperiod of 12 h / d to promote growth and prevent browning. Figure 7 ) The specific components of the modified MS medium in this embodiment are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

[0052] The transplant survival rate and browning rate of each group after the anti-browning and growth-promoting culture stage.

[0053] Table 2. Transplant survival and browning status in each group during the anti-browning and growth-promoting culture stage. name Transplant survival rate / % Browning rate / % Example 1: Anti-browning and growth-promoting culture 88.33 11.67 Comparative Example 4 55.00 45.00 Comparative Example 5 63.33 36.67 Comparative Example 6 80.00 20.00 Comparative Example 7 73.33 26.67 Comparative Example 8 71.67 28.33 As shown in Table 2, Example 1, using modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine, and rutin, demonstrated a more significant effect in preventing browning in sterile Bauhinia seedlings. Compared to the addition of adsorbents PVP and sodium sulfite, the transplant survival rate was higher in the culture medium containing L-cysteine ​​and sodium sulfite, indicating that the addition of adsorbents and sodium sulfite was ineffective in preventing browning in sterile Bauhinia seedlings. Comparing Comparative Examples 6 and 7, it was shown that the anti-browning effect (80.00%) of the culture medium containing modified MS, IBA, 2,4-D, sodium sulfite, and L-cysteine ​​was superior to the anti-browning effect (73.33%) of the culture medium containing modified MS, IBA, 2,4-D, sodium sulfite, and VC. Using modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine, and rutin as transfer media for Bauhinia purpurea aseptic seedlings can improve the browning resistance and transplant survival rate of Bauhinia purpurea aseptic seedlings, providing convenient aseptic seedlings for subsequent Bauhinia purpurea tissue culture research.

[0054] In Example 1 of this invention, the aseptic seedlings of *Cercis chinensis* obtained through anti-browning and growth-promoting culture had large leaves that were dark green or bluish-green, and thick stems, such as... Figure 7 As shown. After the completion of the anti-browning and growth-promoting culture phase, the physiological indicators in the leaves of each group were measured, such as malondialdehyde content, chlorophyll a content, and chlorophyll b content.

[0055] Table 3 Physiological indices of leaves after browning resistance and growth promotion culture in each group name MDA (µmol / g) Chlorophyll a (mg / g) Chlorophyll b (mg / g) Example 1: Anti-browning and growth-promoting culture 0.43 0.25 0.16 Comparative Example 4 0.66 0.16 0.08 Comparative Example 5 0.57 0.19 0.10 Comparative Example 6 0.45 0.22 0.12 Comparative Example 7 0.50 0.19 0.11 Comparative Example 8 0.52 0.19 0.10 Table 3 shows that the modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine, and rutin culture medium used in this invention to promote growth and prevent browning has a more significant effect on the physiological indicators of leaves of sterile Bauhinia seedlings. In particular, the malondialdehyde content is lower, indicating that the cell membrane damage of the sterile Bauhinia seedlings in Example 1 is very low, and the activity of free radical scavenging enzymes in the body is stable and normal. At the same time, the chlorophyll a and chlorophyll b contents are higher, indicating that the combination of sodium sulfite, L-cysteine, and rutin is more effective than sodium sulfite with PVP or VC in maintaining the stable and normal activity of free radical scavenging enzymes in sterile Bauhinia seedlings, which is more conducive to the growth of sterile Bauhinia seedlings.

[0056] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A tissue culture method for reducing browning in Bauhinia purpurea, characterized in that, The tissue culture method includes: S1. Clean and disinfect the Bauhinia stem segments with axillary buds. The specific steps for extracting the axillary buds in sodium sulfite solution are as follows: S11, take branches of Bauhinia purpurea containing 2-3cm axillary buds; S12, soak the Bauhinia branches in alcohol for 30-120 seconds and then rinse with distilled water, and soak them in H2O2 for 5-8 minutes and then rinse with distilled water. S13, in a sodium sulfite solution, remove 2-3 cm axillary buds from the branches of the Bauhinia tree to obtain axillary buds, wherein the sodium sulfite solution is obtained by adding 5-7 µL of 1 mg / L sodium sulfite to 20-40 mL of distilled water; S2, the axillary buds are initially cultured to obtain sterile seedlings. After the sterile seedlings are subjected to anti-browning and growth-promoting culture, the stem segments of the sterile seedlings are taken for subculture. The culture medium used for the anti-browning culture consists of modified MS + 1-2 mg / L IBA + 2-4 mg / L 2,4-D + 1-2 mg / L L-cysteine ​​+ 1-2 mg / L rutin + 1-2 mg / L sodium sulfite. The culture medium used for the initial culture consists of WPM + 1-2 mg / L sodium sulfite.

2. The tissue culture method for reducing browning in Bauhinia purpurea as described in claim 1, characterized in that, The specific components of the modified MS medium are as follows: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, 0.025 mg / L cobalt chloride, 37.3 mg / L disodium EDTA, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid.

3. The tissue culture method for reducing browning in Bauhinia purpurea as described in claim 1, characterized in that, The culture conditions for the anti-browning and growth-promoting culture are as follows: cultured at a temperature of 25±2℃, a light intensity of 3000-4000Lx, and a light duration of 12-14 h / d until the sterile seedlings reach a height of 5-7cm, and then subcultured.

4. The tissue culture method for reducing browning in Bauhinia purpurea as described in claim 1, characterized in that, The length of the Bauhinia branch is 3-5 cm between the adjacent nodes of the stem node containing the 2-3 cm axillary bud.

5. The tissue culture method for reducing browning in Bauhinia purpurea as described in claim 1, characterized in that, The H2O2 concentration is 10-12%.

6. The tissue culture method for reducing browning in Bauhinia purpurea as described in claim 1, characterized in that, The initial culture conditions are as follows: culture temperature 18±2℃, light intensity 500-1000Lx, and light duration 6-8 h / d for 4-5 days.

7. The tissue culture method for reducing browning of Bauhinia purpurea as described in claim 1, characterized in that, In the step of culturing sterile seedlings to resist browning and promote growth, the sterile seedlings are transplanted as a whole plant.

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