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] The invention relates to the technical field of plant tissue culture, and in particular to a tissue culture method for reducing the browning of Cercis chinensis. Background Art

[0002] Bauhinia (Madhucapasquieri), also known as wood peanut and star wood, is an evergreen tree of the genus Bauhinia in the family Sapotaceae. It is one of the main precious tree species in Guangxi and one of the "five major hardwoods in Guangxi". It is listed as a national key protected wild plant (level II). It is also a wild plant with extremely small populations, a national key plant protection object and a key protected plant in Guangxi.

[0003] Although nature reserves were established long ago to protect Bauhinia, the wild resources of Bauhinia are still decreasing rapidly. It is very necessary to preserve the germplasm resources of Bauhinia through plant tissue culture.

[0004] In order to protect the germplasm resources of Bauhinia chinensis, the inventors of the present invention have devoted themselves to obtaining Bauhinia chinensis tissue culture seedlings through plant tissue culture. However, during the tissue culture of Bauhinia chinensis, the inventors found that the difficulties of Bauhinia chinensis tissue culture are as follows:

[0005] (1) The axillary buds of Cercis chinensis were seriously contaminated and browned during the initial culture stage, resulting in a low rate of obtaining sterile seedlings;

[0006] (2) During the culture process, the culture medium of the inoculated axillary buds or stem segments of Cercis chinensis was dyed light yellow or brown with the inoculation point as a dot, and the inoculated materials also showed varying degrees of browning. The browning affected growth in mild cases, and withered and died in severe cases;

[0007] (3) The slow growth of the nodes of the stem segments of Cercis chinensis results in a long cultivation time and difficulty in producing larger leaves.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0009] The main purpose of the present invention is to provide a tissue culture method for reducing the browning of Bauhinia chinensis test tube seedlings, aiming to solve the problem of the decreasing wild resources of Bauhinia chinensis and the possible lack of Bauhinia chinensis germplasm resources, and overcome the difficulties in the tissue culture process of Bauhinia chinensis plants described in the background technology.

[0010] To achieve the above object, the present invention provides a tissue culture method for reducing browning of Cercis chinensis, the tissue culture method comprising:

[0011] S1, the stem segment of Cercis australis with axillary buds was cleaned and disinfected, and the axillary buds were taken in sodium sulfite solution;

[0012] S2, the axillary buds are initially cultured to obtain sterile seedlings, and after the sterile seedlings are cultured for anti-browning and growth promotion, the stem segments of the sterile seedlings are taken for subculture; the culture medium components used in the anti-browning culture include: modified MS, IBA, 2,4-D, L-cysteine, rutin, and sodium sulfite.

[0013] Preferably, in the above technical solution, the culture medium components used in the anti-browning culture are 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.

[0014] Preferably, in the above technical scheme, the culture conditions of the anti-browning growth-promoting culture are: culture at a culture temperature of 25±2°C, a light intensity of 3000-4000Lx, and a lighting time of 12-14h / d until the sterile seedlings are 5-7cm high and then subculture is carried out.

[0015] Preferably, in the above technical solution, in step S1, the specific steps of washing and disinfecting the Cercis chinensis stem segment with axillary buds and taking the axillary buds in a sodium sulfite solution are as follows:

[0016] S11, take branches of Cercis australis containing 2-3 cm axillary buds;

[0017] S12, soaking the Cercis chinensis branches in alcohol for 30-120 seconds and then rinsing with distilled water, soaking them in H2O2 for 5-8 minutes and then rinsing with distilled water;

[0018] S13, in sodium sulfite solution, remove the 2-3 cm axillary buds on the branches of Cercis grandiflora to obtain axillary buds.

[0019] Preferably, in the above technical solution, the length of the Cercis chinensis branch is 3-5 cm of the adjacent internodes of the stem node where the 2-3 cm axillary buds are located.

[0020] According to the above technical scheme, the present invention protects the axillary buds by retaining the 3-5 cm adjacent internodes of the stem node where the 2-3 cm axillary buds are located, thereby preventing the disinfectant from causing damage to the cell membranes of cells near the incision of the Bauhinia explant during the disinfection process, thereby reducing the death of the explant caused by axillary bud cell damage.

[0021] Preferably, in the above technical solution, the H2O2 concentration is 10-12%.

[0022] 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.

[0023] Preferably, in the above technical solution, the culture medium components used in the initial culture consist of WPM + 1-2 mg / L sodium sulfite.

[0024] Preferably, in the above technical solution, the culture conditions of the initial culture are: culture temperature of 18±2°C, light intensity of 500-1000Lx, and light time of 6-8h / d for 4-5 days.

[0025] Preferably, in the above technical solution, in the step after the sterile seedlings are subjected to anti-browning and growth-promoting culture, the sterile seedlings are transplanted as whole plants.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) After disinfection, the axillary buds of Bauhinia were removed in a sodium sulfite solution. First, the microorganisms on the surface of the axillary buds were further killed to reduce the contamination of the axillary buds of Bauhinia, and the contamination rate was only 5.00%; second, the polyphenol oxidase activity was inhibited together with WPM and sodium sulfite culture medium to reduce the browning of the axillary buds of Bauhinia in the initial culture stage, and the browning rate was only 13.33%, and the sterile seedling acquisition rate was 81.67%;

[0028] (2) A culture medium was screened out that reduces the browning of Bauhinia chinensis and promotes its nutritional growth, namely, modified MS, IBA, 2,4-D, L-cysteine, rutin and sodium sulfite. The transplant survival rate was 88.33%, and the leaves of the surviving Bauhinia chinensis sterile seedlings were dark green or blue, and the stems were stronger. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings.

[0030] Figure 1 This is a picture of the axillary buds of Cercis australis showing different degrees of browning during the initial culture stage;

[0031] Figure 2 is a picture of axillary buds of Cercis australis in the initial culture stage according to Example 1 of the present invention;

[0032] Figure 3 is a picture showing severe browning of axillary buds of Cercis australis in comparative example 4 at the stage of anti-browning and growth promotion;

[0033] Figure 4 is a graph of browning of axillary buds of Cercis australis in comparative example 5 at the stage of anti-browning and growth promotion;

[0034] Figure 5 is a diagram of the axillary bud germination of Cercis australis in comparative example 6 at the stage of anti-browning and growth promotion;

[0035] Figure 6 is a diagram of the axillary bud germination of Cercis australis in comparative example 7 at the stage of anti-browning and growth promotion;

[0036] Figure 7 is a diagram of the axillary bud germination of Cercis australis in comparative example 8 at the stage of anti-browning and growth promotion;

[0037] Figure 8 is a diagram of axillary bud germination of Cercis australis in the anti-browning and growth-promoting stage of Example 1;

[0038] Fig. 9 The figure is a diagram showing the axillary bud germination of Cercis chinensis in the anti-browning and growth-promoting stage of a certain embodiment. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely. 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.

[0040] Figure 1 The inventor discovered during the initial culture stage of Bauhinia wood that due to the browning of Bauhinia wood explants, the culture medium showed dark brown or light yellow around the Bauhinia wood explants, and as the degree of browning deepened, the inoculated material eventually lost its vitality. Therefore, if the problem of culture medium and Bauhinia wood browning is not improved, the rate of obtaining sterile seedlings of Bauhinia wood cannot be improved, which seriously restricts the preservation of Bauhinia wood germplasm resources by tissue culture.

[0041] Example 1

[0042] A tissue culture method for reducing browning of Cercis chinensis, the method comprising:

[0043] S1, cleaning and disinfecting the stem segment of Cercis australis with axillary buds, and taking the axillary buds in a sodium sulfite solution, the specific steps include:

[0044] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0045] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary bud is located, soak it in saturated detergent water for 5 minutes, brush it gently with a soft brush, rinse it with clean water 3 times, transfer it to the inoculation room, soak it in 70% alcohol for 30 seconds, rinse it with distilled water twice, soak it in 10% H2O2 for 5 minutes, and rinse it with distilled water twice;

[0046] S13, cutting off 2-3 cm axillary buds on branches of Cercis chinensis in a sodium sulfite solution 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.

[0047] S2, initial cultivation:

[0048] The axillary buds in step S13 were inoculated into the culture medium: WPM + 1 mg / L sodium sulfite. Under the conditions of culture temperature of 18 ± 2 ° C, light intensity of 500Lx, and light time of 6h / d, sterile seedlings were obtained after 10 days of culture. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded regularly every day, and the contaminated tissue culture bottles were removed in time.

[0049] Anti-browning and growth-promoting culture:

[0050] The sterile seedlings are transferred to the improved culture medium MS + 2mg / LIBA + 4mg / L 2,4-D + 2mg / L L-cysteine ​​+ 2mg / L rutin + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, and anti-browning growth-promoting culture is carried out at a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light time of 12h / d until the sterile seedlings are 5-7cm tall or 4-5 leaves have sprouted, and then subculture is carried out.

[0051] Subculture:

[0052] In the sodium sulfite solution, all the leaves of the sterile seedlings after anti-browning and growth-promoting culture were cut off and cut into stem segments with 1-2 stem nodes, and the stem segments were inoculated into the improved culture medium MS + 2mg / L IBA + 4mg / L 2,4-D + 2mg / LL-cysteine ​​+ 2mg / L rutin + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, first cultured for 5 days at a culture temperature of 18±2°C, a light intensity of 500Lx, and a light duration of 6h / d, and then transferred to a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light duration of 12h / d for culture, wherein the sodium sulfite solution is prepared by adding 5μL 1mg / L sodium sulfite to 20mL distilled water.

[0053] Example 2

[0054] A tissue culture method for reducing browning of Cercis chinensis, the method comprising:

[0055] S1, cleaning and disinfecting the stem segment of Cercis australis with axillary buds, and taking the axillary buds in a sodium sulfite solution, the specific steps include:

[0056] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0057] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary bud is located, soak it in saturated detergent water for 5-7min, brush it lightly with a soft brush, rinse it with clean water 3 times, transfer it to the inoculation room, soak it in 70% alcohol for 30-120s, rinse it with distilled water 2 times, soak it in 12% H2O2 for 5min, and rinse it with distilled water 2 times;

[0058] S13, cutting off 2-3 cm axillary buds on branches of Cercis grandiflora in a sodium sulfite solution to obtain axillary buds; wherein the sodium sulfite solution is prepared by adding 5 μL of 1 mg / L sodium sulfite to 20-40 mL of distilled water.

[0059] S2, initial cultivation:

[0060] The axillary buds in step S13 were inoculated into the culture medium: WPM + 1.5 mg / L sodium sulfite. Under the conditions of culture temperature of 18 ± 2 ° C, light intensity of 800Lx, and light time of 8h / d, sterile seedlings were obtained after 5 days of culture. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded regularly every day, and the contaminated tissue culture bottles were removed in time.

[0061] Anti-browning and growth-promoting culture:

[0062] The sterile seedlings are transferred to the improved culture medium MS + 2mg / L IBA + 4mg / L 2,4-D + 2mg / L L-cysteine ​​+ 2mg / L rutin + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, and anti-browning and growth-promoting culture is carried out at a culture temperature of 25±2°C, a light intensity of 3000Lx, and a light time of 14h / d until the sterile seedlings are 5-7cm tall or 4-5 leaves have sprouted, and then subculture is carried out.

[0063] Subculture:

[0064] In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture are cut off and cut into stem segments with 1-2 stem nodes, and the stem segments are inoculated into a modified culture medium of MS+2mg / LIBA+4mg / L 2,4-D+1-2mg / LL-cysteine+2mg / L rutin+1mg / L sodium sulfite+12.5g / L sucrose+7.5g / L agar, first cultured at a culture temperature of 18±2°C, a light intensity of 8000Lx, and a light duration of 8h / d for 4-5 days, and then transferred to a culture temperature of 25±2°C, a light intensity of 3000Lx, and a light duration of 14h / d for culture; the sodium sulfite solution is prepared by adding 5μL1mg / L sodium sulfite to 20-40mL distilled water.

[0065] Example 3

[0066] A tissue culture method for reducing browning of Cercis chinensis, the method comprising:

[0067] S1, cleaning and disinfecting the stem segment of Cercis australis with axillary buds, and taking the axillary buds in a sodium sulfite solution, the specific steps include:

[0068] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0069] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary bud is located, soak it in saturated detergent water for 5 minutes, brush it gently with a soft brush, rinse it with clean water 3 times, transfer it to the inoculation room, soak it in 70% alcohol for 30 seconds, rinse it with distilled water twice, soak it in 12% H2O2 for 5 minutes, and rinse it with distilled water twice;

[0070] S13, cutting off 2-3 cm axillary buds on branches of Cercis grandiflora in a sodium sulfite solution 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.

[0071] S2, initial cultivation:

[0072] The axillary buds in step S13 were inoculated into the culture medium: WPM + 2 mg / L sodium sulfite. Under the conditions of culture temperature of 18 ± 2 ° C, light intensity of 1000Lx, and light time of 6h / d, sterile seedlings were obtained after culturing for 10 days. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded regularly every day, and the contaminated tissue culture bottles were removed in time.

[0073] Anti-browning and growth-promoting culture:

[0074] The sterile seedlings are transferred to the improved culture medium MS + 1mg / LIBA + 2mg / L 2,4-D + 1mg / LL-cysteine ​​+ 1mg / L rutin + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, and anti-browning and growth-promoting culture is carried out at a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light time of 12h / d until the sterile seedlings are 5-7cm tall or 4-5 leaves have sprouted, and then subculture is carried out.

[0075] Subculture:

[0076] In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture are cut off and cut into stem segments with 1-2 stem nodes, and the stem segments are inoculated into a modified culture medium of MS + 1mg / L IBA + 2mg / L 2,4-D + 1mg / L L-cysteine ​​+ 1mg / L rutin + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, first cultured for 5 days at a culture temperature of 18±2°C, a light intensity of 1000Lx, and a light duration of 6h / d, and then transferred to a culture temperature of 25±2°C, a light intensity of 3500Lx, and a light duration of 14h / d for culture; the sodium sulfite solution is prepared by adding 7μL 1mg / L sodium sulfite to 40mL distilled water.

[0077] Example 4

[0078] A tissue culture method for reducing browning of Cercis chinensis, the method comprising:

[0079] S1, cleaning and disinfecting the stem segment of Cercis australis with axillary buds, and taking the axillary buds in a sodium sulfite solution, the specific steps include:

[0080] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0081] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary bud is located, soak it in saturated detergent water for 5 minutes, brush it gently with a soft brush, rinse it with clean water 3 times, transfer it to the inoculation room, soak it in 70% alcohol for 30 seconds, rinse it with distilled water twice, soak it in 12% H2O2 for 5 minutes, and rinse it with distilled water twice;

[0082] S13, cutting off 2-3 cm axillary buds on branches of Cercis chinensis in a sodium sulfite solution to obtain axillary buds; wherein the sodium sulfite solution is a sodium sulfite solution prepared by adding 7 μL of 1 mg / L sodium sulfite to 40 mL of distilled water.

[0083] S2, initial cultivation:

[0084] The axillary buds in step S13 were inoculated into the culture medium: WPM + 2 mg / L sodium sulfite. Under the conditions of culture temperature of 18 ± 2 ° C, light intensity of 800Lx, and light time of 6h / d, sterile seedlings were obtained after 10 days of culture. During the culture process, the contamination and browning of the tissue culture seedlings were observed and recorded regularly every day, and the contaminated tissue culture bottles were removed in time.

[0085] Anti-browning and growth-promoting culture:

[0086] The sterile seedlings are transferred to the improved culture medium MS + 2mg / LIBA + 4mg / L 2,4-D + 2mg / L L-cysteine ​​+ 2mg / L rutin + 2mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, and anti-browning growth-promoting culture is carried out at a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light time of 12h / d until the sterile seedlings are 5-7cm tall or 4-5 leaves have sprouted, and then subculture is carried out.

[0087] Subculture:

[0088] In a sodium sulfite solution, all leaves of sterile seedlings after anti-browning and growth-promoting culture are cut off and cut into stem segments with 1-2 stem nodes, and the stem segments are inoculated into a modified culture medium of MS + 2mg / L IBA + 4mg / L 2,4-D + 2mg / L L-cysteine ​​+ 2mg / L rutin + 2mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, first cultured for 5 days at a culture temperature of 18±2°C, a light intensity of 800Lx, and a light duration of 6h / d, and then transferred to a culture temperature of 25±2°C, a light intensity of 3500Lx, and a light duration of 14h / d for culture; the sodium sulfite solution is prepared by adding 7μL 1mg / L sodium sulfite to 40mL distilled water.

[0089] Comparative Example 1

[0090] A method for disinfecting and initially cultivating Cercis chinensis, comprising:

[0091] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0092] S12, retain 3-5 cm of the internodes at both ends of the stem node where the axillary buds are located, soak them in saturated detergent water for 5-7 min, brush them lightly with a soft brush, rinse them with clean water 3 times, soak them in 0.3% sodium hypochlorite for 5 min, rinse them with distilled water 2 times, cut off the 2-3 cm axillary buds on the branches of Cercis chinensis, and obtain the axillary buds;

[0093] S13, inoculating the axillary buds into WPS culture medium, inoculating 60 bottles in total, inoculating one axillary bud of Cercis chinensis in each bottle, placing the bottles under the conditions of a culture temperature of 25±2°C, a light intensity of 500-1000Lx, and a light duration of 6-8h / d, and obtaining sterile seedlings after culturing for 10 days, observing and recording the contamination and browning of the tissue culture seedlings on a daily basis during the culture process, and removing the contaminated tissue culture bottles in a timely manner.

[0094] Comparative Example 2

[0095] A method for disinfecting and initially cultivating redbud wood comprises:

[0096] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0097] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary buds are located, soak them in saturated detergent water for 5-7min, brush them lightly with a soft brush, rinse them with clean water 3 times, transfer them to the inoculation room, soak them in 70% alcohol for 30-120s, rinse them with distilled water 2 times, soak them in 10% H2O2 for 5min, rinse them with distilled water 2 times, and then cut off the 2-3cm axillary buds on the branches of Cercis chinensis to obtain the axillary buds;

[0098] S13, inoculating the axillary buds into WPS culture medium, inoculating 60 bottles in total, inoculating one axillary bud of Cercis chinensis in each bottle, placing the bottles under the conditions of a culture temperature of 25±2°C, a light intensity of 500-1000Lx, and a light duration of 6-8h / d, and obtaining sterile seedlings after culturing for 10 days, observing and recording the contamination and browning of the tissue culture seedlings on a daily basis during the culture process, and removing the contaminated tissue culture bottles in a timely manner.

[0099] Comparative Example 3

[0100] A method for disinfecting and initially cultivating Cercis chinensis, comprising:

[0101] S11, select excellent single trees of Cercis chinensis that are over 5 years old, lush, healthy and free of diseases and insect pests as the collection objects, and take branches with axillary buds of 2-3 cm;

[0102] S12, retain 3-5cm of the internodes at both ends of the stem node where the axillary buds are located, soak them in saturated detergent water for 5-7min, brush them lightly with a soft brush, rinse them with clean water 3 times, transfer them to the inoculation room, soak them in 70% alcohol for 30-120s, rinse them with distilled water 2 times, soak them in 10% H2O2 for 5min, rinse them with distilled water 2 times, cut off the 2-3cm axillary buds on the branches of Cercis chinensis, and obtain the axillary buds;

[0103] S13, inoculate the axillary buds in step S12 into the culture medium: WPM+1-2mg / L sodium sulfite, inoculate 60 bottles in total, inoculate one axillary bud of Cercis chinensis in each bottle, place them under the conditions of culture temperature of 25±2°C, light intensity of 500-1000Lx, and light time of 6-8h / 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 regularly every day, and remove the contaminated tissue culture bottles in time.

[0104] Contamination rate (%) = (number of contaminated strains / total number of inoculated strains) × 100%;

[0105] Browning rate (%) = (number of browning plants / total number of inoculations) × 100%;

[0106] The rate of sterile seedlings obtained (%) = (uncontaminated and unbrown / total number of inoculations) × 100%.

[0107] Table 1 Contamination rate and browning rate of primary culture

[0108] name Pollution rate / % Browning rate / % Sterile seedling 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 Light yellow Comparative Example 2 11.67 33.33 55.00 Light yellow Comparative Example 3 6.67 15.00 78.33 Transparent, white

[0109] It can be concluded from Table 1 that after the culture, the use of alcohol and hydrogen peroxide for disinfection, the addition of sodium sulfite to the WPM medium and the collection of axillary buds in the sodium sulfite solution all have a great influence on the contamination rate, browning rate and sterile seedling acquisition rate during the initial culture of Cercis wood. Appropriate disinfection and culture methods make the contamination rate of the initial culture of Cercis wood lower than 10%, the browning rate lower than 15%, and the sterile seedling acquisition rate of 81.67%; while in Comparative Example 1, the axillary buds of Cercis wood disinfected with sodium hypochlorite and inoculated into the WPS medium for culture had a high browning rate and a sterile seedling acquisition rate of less than 50%.

[0110] Comparative Example 4

[0111] Prepare the culture medium: MS+1mg / L IBA+2mg / L NAA+12.5g / L sucrose+7.5g / L agar, 60 bottles in total;

[0112] The sterile seedlings were transferred to the modified medium MS+1mg / L IBA+2mg / L NAA+12.5g sucrose+7.5g agar at one bottle per plant, and the anti-browning growth-promoting culture was carried out at a culture temperature of 25±2°C, a light intensity of 3000-4000Lx, and a light time of 12h / d. Figure 3 )

[0113] Comparative Example 5

[0114] Prepare the culture medium: modified MS + 1mg / L IBA + 2mg / L 2,4-D + 12.5g / L sucrose + 7.5g / L agar, 60 bottles in total;

[0115] The sterile seedlings were transferred to the modified medium MS+1mg / L IBA+2mg / L2,4-D+12.5g / L sucrose+7.5g / L agar at one bottle per plant, and the anti-browning growth-promoting culture was carried out at a culture temperature of 18±2°C, a light intensity of 4000Lx, and a light time of 12h / d. Figure 4 )

[0116] Comparative Example 6

[0117] Prepare the culture medium: modified MS + 1mg / L IBA + 2mg / L 2,4-D + 2mg / L L-cysteine ​​+ 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, 60 bottles in total;

[0118] The sterile seedlings were transferred to the modified medium MS+1mg / L IBA+2mg / L2,4-D+2mg / L rutin+1mg / L sodium sulfite+12.5g sucrose+7.5g agar at one bottle per plant, and the anti-browning growth-promoting culture was carried out at a culture temperature of 18±2°C, a light intensity of 4000Lx, and a light time of 12h / d. Figure 5 )

[0119] Comparative Example 7

[0120] Prepare the culture medium: modified MS + 1mg / L IBA + 2mg / L 2,4-D + 2mg / LVC + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, 60 bottles in total;

[0121] The sterile seedlings were transferred one bottle at a time to the improved culture medium MS + 1mg / L IBA + 2mg / L2,4-D + 2mg / L L-cysteine ​​+ 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, and anti-browning and growth-promoting culture was carried out at a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light time of 12h / d.

[0122] ( Figure 6 )

[0123] Comparative Example 8

[0124] Prepare the culture medium: modified MS + 1mg / L IBA + 2mg / L 2,4-D + 1mg / LPVP + 1mg / L sodium sulfite + 12.5g / L sucrose + 7.5g / L agar, 60 bottles in total;

[0125] The sterile seedlings were transferred to the culture medium MS+1mg / L IBA+2mg / L2,4-D+1mg / LPVP+1mg / L sodium sulfite according to one bottle, and the anti-browning growth-promoting culture was carried out at a culture temperature of 25±2°C, a light intensity of 4000Lx, and a light time of 12h / d. Figure 7 )

[0126] Transplant survival rate and browning rate during the anti-browning and growth-promoting culture stage after anti-browning and growth-promoting culture in each group.

[0127] Table 2 Transplant survival and browning in each group during the anti-browning and growth-promoting culture stage

[0128]

[0129] As can be seen from Table 2, Example 1 of the present invention using modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine ​​and rutin is more effective in preventing the anti-browning of sterile seedlings of Bauhinia wood. Compared with the addition of adsorbent PVP and sodium sulfite, the transplant survival rate of the culture medium with the addition of L-cysteine ​​and sodium sulfite is higher, indicating that the anti-browning effect of sterile seedlings of Bauhinia wood by adding adsorbents and sodium sulfite is not good. Comparative Example 6 and Comparative Example 7 are compared, and the anti-browning effect of adding modified MS, IBA, 2,4-D, sodium sulfite and L-cysteine ​​as a culture medium is 80.00%, which is better than the anti-browning effect of using modified MS, IBA, 2,4-D, sodium sulfite and VC as a culture medium of 73.33%. Using modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine ​​and rutin as the transfer culture medium for Bauhinia sterile seedlings can improve the anti-browning ability of Bauhinia sterile seedlings, increase the transplantation survival rate of Bauhinia sterile seedlings, and provide convenient sterile seedlings for subsequent Bauhinia tissue culture research.

[0130] The leaves of the sterile seedlings of Bauhinia obtained by the anti-browning and growth-promoting culture in Example 1 of the present invention are relatively large and dark green or cyan, and the stems are thick, such as Figure 7 After the anti-browning and growth-promoting cultivation phases were completed, the physiological indices in the leaves of each group were tested, such as the malondialdehyde content, chlorophyll a and chlorophyll b content.

[0131] Table 3 Physiological indexes of leaves after anti-browning and growth-promoting culture in each group

[0132]

[0133] It can be concluded from Table 3 that the use of modified MS, IBA, 2,4-D, sodium sulfite, L-cysteine ​​and rutin as anti-browning growth-promoting culture medium in the present invention has a more significant effect on the physiological indicators of the leaves of the sterile seedlings of Bauhinia wood, especially the lower content of malondialdehyde, indicating that the degree of damage to the cell membrane of the sterile seedlings of Bauhinia wood in Example 1 is very low, the activity of free radical scavenging enzymes in the body is stable and normal, and the content of chlorophyll a and chlorophyll b is higher, indicating that the combination of sodium sulfite, L-cysteine ​​and rutin can maintain the stable and normal activity of free radical scavenging enzymes of the sterile seedlings of Bauhinia wood better than sodium sulfite and PVP or VC, and is more conducive to the growth of sterile seedlings of Bauhinia wood.

[0134] The present invention can be implemented in various ways and is not limited to the embodiments described. A person skilled in the art can understand that the present invention can be implemented in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary and are not intended to limit the present invention.

Claims

1. A tissue culture method for reducing browning of Cercis chinensis, characterized in that: The tissue culture method comprises: S1, the stem segment of Cercis australis with axillary buds was cleaned and disinfected, and the axillary buds were taken in sodium sulfite solution; S2, the axillary buds are initially cultured to obtain sterile seedlings, and after the sterile seedlings are cultured for anti-browning and growth promotion, the stem segments of the sterile seedlings are taken for subculture; the culture medium components used in the anti-browning culture include: modified MS, IBA, 2,4-D, L-cysteine, rutin, and sodium sulfite.

2. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 1, characterized in that: The culture medium components used in the anti-browning culture are 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.

3. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 1, characterized in that: The culture conditions of the anti-browning growth-promoting culture are: culture at a culture temperature of 25±2° C., a light intensity of 3000-4000Lx, and a light duration of 12-14h / d until the sterile seedlings are 5-7cm high, and then subculture is performed.

4. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 1, characterized in that: In step S1, the Cercis chinensis stem segment with axillary buds is cleaned and disinfected, and the specific steps of taking the axillary buds in a sodium sulfite solution are as follows: S11, take branches of Cercis australis containing 2-3 cm axillary buds; S12, soaking the Cercis chinensis branches in alcohol for 30-120 seconds and then rinsing with distilled water, soaking them in H2O2 for 5-8 minutes and then rinsing with distilled water; S13, in sodium sulfite solution, remove the 2-3 cm axillary buds on the branches of Cercis grandiflora to obtain axillary buds.

5. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 4, characterized in that: The length of the Cercis chinensis branch is 3-5 cm of the adjacent internodes of the stem node where the 2-3 cm axillary buds are located.

6. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 4, characterized in that: The H2O2 concentration is 10-12%.

7. The tissue culture method for reducing browning of Cercis aurantium as claimed in claim 4, characterized in that: The sodium sulfite solution is obtained by adding 5-7 μL of 1 mg / L sodium sulfite to 20-40 mL of distilled water.

8. The tissue culture method for reducing browning of Cercis aurantium as claimed in claim 1, characterized in that: The culture medium components used in the initial culture consist of WPM+1-2 mg / L sodium sulfite.

9. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 1, characterized in that: The initial culture conditions are as follows: culture temperature of 18±2° C., light intensity of 500-1000 Lx, and light duration of 6-8 h / d for 4-5 days.

10. The tissue culture method for reducing browning of Cercis aurantii as claimed in claim 1, characterized in that: In the step after the sterile seedlings are subjected to anti-browning and growth-promoting culture, the sterile seedlings are transplanted as a whole plant.

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