Sterile seedling culture medium capable of increasing survival rate of rhizoma bletillae seedlings and preparation method of sterile seedling culture medium

Abstract

The invention relates to the technical field of trichosanthes kirilowii resistant new variety cultivation, and discloses a sterile seedling culture medium for improving the survival rate of bletilla striata seedlings and a preparation method thereof, the sterile seedling culture medium comprises the following components: a basic culture medium, a plant growth regulator, an organic additive, an anti-browning agent, a carbon source, a curing agent and a pH regulator; according to the culture medium, the proportion of nutritional ingredients and a plant growth regulator is optimized, the survival rate of rhizoma bletillae seedlings can be remarkably increased, tissue browning caused by oxidation in the culture process is remarkably reduced through combination of activated carbon, vitamin C and other anti-browning agents, the success rate of tissue culture is increased, and the survival rate of rhizoma bletillae seedlings is increased. The optimal proportion of NAA and 6-BA can effectively promote root growth and improve the transplanting survival rate of seedlings, coconut water and potato juice are added to provide natural auxin, polysaccharide and vitamins and enhance the stress resistance and growth vigor of the seedlings, and through accurate pH adjustment, sterilization treatment and sterile operation, the stability and pollution-free property of the culture medium are guaranteed, and the survival rate of the seedlings is increased. And the repeatability and operability of the experiment are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and specifically relates to a special culture medium formula for aseptic seedling cultivation of Bletilla striata seedlings and a preparation method thereof, which is particularly suitable for improving the germination rate of seeds or protocorms, inhibiting browning and optimizing the seedling rate. Background Art

[0002] Bletilla striata is an important traditional Chinese medicinal plant. Its rhizome is rich in polysaccharide compounds and has significant medicinal value. However, due to the lack of endosperm in its seeds and low natural germination rate, traditional seed propagation methods are difficult to meet the needs of large-scale seedling cultivation. Therefore, the rapid propagation of Bletilla striata using sterile tissue culture technology has become an important means of seedling cultivation.

[0003] The existing tissue culture method of Bletilla striata is mainly based on MS (Murashige & Skoog) medium, but in actual application, the survival rate of seedlings is low, due to unreasonable medium formula, severe browning phenomenon, poor root growth, etc. Therefore, optimizing the medium formula and improving the survival rate of Bletilla striata seedlings are of great significance to improving the production efficiency of Bletilla striata. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies in the prior art, the present invention provides a sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings and a preparation method thereof. The culture medium improves the survival rate, growth quality and stress resistance of Bletilla striata seedlings by adjusting basic nutrients, adding anti-browning agents, optimizing the proportion of growth regulators and supplementing organic additives.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings, comprising the following components and their mass volume ratios:

[0008] Basal culture medium: MS culture medium mother solution, with a concentration of 0.8-1.2× the stock solution concentration, wherein the MS mother solution contains 1650 mg / L ammonium nitrate (NH4NO3), 1900 mg / L potassium nitrate (KNO3), 170 mg / L potassium dihydrogen phosphate (KH2PO4), 370 mg / L magnesium sulfate (MgSO4·7H2O), 440 mg / L calcium chloride (CaCl2·2H2O), 27.8 mg / L iron salt (Fe-EDTA), and no inositol is added;

[0009] Plant growth regulators: naphthaleneacetic acid (NAA) 0.1-0.5 mg / L, 6-benzylaminopurine (6-BA) 0.2-1.0 mg / L, wherein the molar concentration ratio of NAA to 6-BA is 1:2 to 1:5;

[0010] Organic additives: coconut water 50-100 mL / L (taken from mature coconuts, filtered after centrifugation at 5000 rpm for 10 minutes to remove suspended matter), potato juice 20-50 mL / L (made from peeled potatoes, sliced, boiled for 30 minutes, and the squeezed juice filtered through double-layer gauze);

[0011] Anti-browning agent: a complex of activated carbon 0.5-2.0g / L and vitamin C 0.05-0.2g / L;

[0012] Carbon source: sucrose 20-40 g / L (purity ≥ 99%);

[0013] Solidifying agent: agar 6-10g / L (gel strength ≥900g / cm 2 );

[0014] pH regulator: Use 0.1 mol / L NaOH or HCl to adjust the pH of the culture medium to 5.8-6.2;

[0015] Auxiliary ingredients: Vitamin B1 0.1mg / L, niacin 0.5mg / L.

[0016] The concentration of the MS medium mother solution is 1.0× the original solution concentration, and magnesium sulfate (MgSO4·7H2O) is adjusted to 400 mg / L and calcium chloride (CaCl2·2H2O) is adjusted to 460 mg / L to enhance ion balance.

[0017] The invention is characterized in that the ratio of the plant growth regulator is NAA 0.3 mg / L and 6-BA 0.5 mg / L, and the two are dissolved in sterile water by 40 kHz ultrasonic treatment for 10 minutes at 25°C, and then added to the culture medium after forming a homogeneous solution.

[0018] The coconut water needs to be sterilized by high pressure (121° C., 15 minutes) and then cooled to 25° C. before use. 0.05% (w / v) papain is added to the potato juice and enzymolyzed at 45° C. for 1 hour to release soluble polysaccharides.

[0019] The anti-browning agent is a ternary complex of 1.0 g / L activated carbon, 0.1 g / L vitamin C and 0.05 g / L cysteine ​​hydrochloride, and the three need to be pre-mixed and ground to a particle size of ≤50 μm.

[0020] Preferably, the method for preparing the sterile seedling culture medium comprises the following steps:

[0021] (1) Weigh MS mother solution, sucrose and agar according to the ratio, add deionized water to make up to 70% of the total volume, heat to 80°C and stir until the agar is completely dissolved;

[0022] (2) adding plant growth regulator, organic additive and anti-browning agent in sequence, and mixing for 20 minutes under a magnetic stirrer (speed of 500 rpm);

[0023] (3) Use a pH meter to monitor in real time, adjust the pH to 5.8-6.2 with 0.1 mol / L NaOH or HCl, and add deionized water to the target volume;

[0024] (4) Dispense into transparent glass culture bottles (100 mL capacity, 40 mL liquid volume / bottle), seal and sterilize at 121°C for 20 min, and shake the bottle when cooled to 45°C to ensure uniform distribution of the ingredients;

[0025] (5) The sterilized culture medium was placed in a sterile operating table for 24 hours and used after the temperature dropped to 25°C.

[0026] In step (2), the organic additives must be sterilized by two-stage filtration through 0.45 μm and 0.22 μm filter membranes in sequence, and aseptically injected into the clean bench when the culture medium is cooled to 50°C.

[0027] In the step (5), the static environment is light-proof and the temperature is controlled at 20-25° C. After static, a uniform gel layer is formed on the surface of the culture medium with a thickness of 2-3 mm.

[0028] Preferably, the sterile seedling culture medium is used for the sterile sowing of Bletilla striata seeds or protocorms. Before sowing, the seeds or protocorms need to be soaked in 0.1% HgCl2 solution for 8-10 minutes and rinsed with sterile water 3-5 times; the culture conditions are: light intensity 1000-2000Lux, 12-16 hours of light per day, culture temperature 22±2°C, humidity 60-80%, and culture period of 30-40 days.

[0029] (III) Beneficial technical effects

[0030] (1) Improving the survival rate: The culture medium of the present invention optimizes the ratio of nutrients and plant growth regulators, and can significantly improve the survival rate of Bletilla striata seedlings.

[0031] (2) Inhibition of browning: Through the combination of activated carbon, vitamin C and other anti-browning agents, the browning of tissues caused by oxidation during the culture process can be significantly reduced, thereby improving the success rate of tissue culture.

[0032] (3) Promote root development: The optimized ratio of NAA and 6-BA can effectively promote root growth and improve the survival rate of seedlings after transplanting.

[0033] (4) Enhanced nutrient supply: Adding coconut water and potato juice provides natural growth hormones, polysaccharides and vitamins to enhance the stress resistance and growth potential of seedlings.

[0034] (5) Optimize the preparation method: Through precise pH adjustment, sterilization and aseptic operation, ensure the stability and contamination-free nature of the culture medium and improve the repeatability and operability of the experiment. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below.

[0036] Example 1

[0037] The embodiment of the present invention provides a sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings, comprising the following components and their mass volume ratios:

[0038] Basal culture medium: MS culture medium mother solution, with a concentration of 0.8-1.2× the stock solution concentration, wherein the MS mother solution contains 1650 mg / L ammonium nitrate (NH4NO3), 1900 mg / L potassium nitrate (KNO3), 170 mg / L potassium dihydrogen phosphate (KH2PO4), 370 mg / L magnesium sulfate (MgSO4·7H2O), 440 mg / L calcium chloride (CaCl2·2H2O), 27.8 mg / L iron salt (Fe-EDTA), and no inositol is added;

[0039] Plant growth regulators: naphthaleneacetic acid (NAA) 0.1-0.5 mg / L, 6-benzylaminopurine (6-BA) 0.2-1.0 mg / L, wherein the molar concentration ratio of NAA to 6-BA is 1:2 to 1:5;

[0040] Organic additives: coconut water 50-100 mL / L (taken from mature coconuts, filtered after centrifugation at 5000 rpm for 10 minutes to remove suspended matter), potato juice 20-50 mL / L (made from peeled potatoes, sliced, boiled for 30 minutes, and the squeezed juice filtered through double-layer gauze);

[0041] Anti-browning agent: a complex of activated carbon 0.5-2.0g / L and vitamin C 0.05-0.2g / L;

[0042] Carbon source: sucrose 20-40 g / L (purity ≥ 99%);

[0043] Solidifying agent: agar 6-10g / L (gel strength ≥900g / cm 2 );

[0044] pH regulator: Use 0.1 mol / L NaOH or HCl to adjust the pH of the culture medium to 5.8-6.2;

[0045] Auxiliary ingredients: Vitamin B1 0.1mg / L, niacin 0.5mg / L.

[0046] The concentration of the MS medium mother solution is 1.0× the original solution concentration, and magnesium sulfate (MgSO4·7H2O) is adjusted to 400 mg / L and calcium chloride (CaCl2·2H2O) is adjusted to 460 mg / L to enhance ion balance.

[0047] The invention is characterized in that the ratio of the plant growth regulator is NAA 0.3 mg / L and 6-BA 0.5 mg / L, and the two are dissolved in sterile water by 40 kHz ultrasonic treatment for 10 minutes at 25°C, and then added to the culture medium after forming a homogeneous solution.

[0048] The coconut water needs to be sterilized under high pressure (121° C., 15 minutes) and then cooled to 25° C. before use. 0.05% (w / v) papain is added to the potato juice and enzymolyzed at 45° C. for 1 hour to release soluble polysaccharides.

[0049] The anti-browning agent is a ternary complex of 1.0 g / L activated carbon, 0.1 g / L vitamin C and 0.05 g / L cysteine ​​hydrochloride, and the three need to be pre-mixed and ground to a particle size of ≤50 μm.

[0050] Another embodiment of the present invention is a method for preparing the sterile seedling culture medium, comprising the following steps:

[0051] (1) Weigh MS mother solution, sucrose and agar according to the ratio, add deionized water to make up to 70% of the total volume, heat to 80°C and stir until the agar is completely dissolved;

[0052] (2) adding plant growth regulator, organic additive and anti-browning agent in sequence, and mixing for 20 minutes under a magnetic stirrer (speed of 500 rpm);

[0053] (3) Use a pH meter to monitor in real time, adjust the pH to 5.8-6.2 with 0.1 mol / L NaOH or HCl, and add deionized water to the target volume;

[0054] (4) Dispense into transparent glass culture bottles (100 mL capacity, 40 mL liquid volume / bottle), seal and sterilize at 121°C for 20 min, and shake the bottle when cooled to 45°C to ensure uniform distribution of the ingredients;

[0055] (5) The sterilized culture medium was placed in a sterile operating table for 24 hours and used after the temperature dropped to 25°C.

[0056] In step (2), the organic additives must be sterilized by two-stage filtration through 0.45 μm and 0.22 μm filter membranes in sequence, and aseptically injected into the clean bench when the culture medium is cooled to 50°C.

[0057] In the step (5), the static environment is light-proof and the temperature is controlled at 20-25° C. After static, a uniform gel layer is formed on the surface of the culture medium with a thickness of 2-3 mm.

[0058] Furthermore, the sterile seedling culture medium is used for sterile sowing of Bletilla striata seeds or protocorms. Before sowing, the seeds or protocorms need to be soaked in 0.1% HgCl2 solution for 8-10 minutes and rinsed with sterile water 3-5 times; the culture conditions are: light intensity 1000-2000Lux, daily light 12-16 hours, culture temperature 22±2°C, humidity 60-80%, and culture period of 30-40 days.

[0059] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and describes these embodiments in detail to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is only limited by the claims and their full scope and equivalents. The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and describes these embodiments in detail to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings, characterized in that: Includes the following ingredients and their mass volume ratios: Basal culture medium: MS culture medium mother solution, with a concentration of 0.8-1.2× the stock solution concentration, wherein the MS mother solution contains 1650 mg / L ammonium nitrate (NH4NO3), 1900 mg / L potassium nitrate (KNO3), 170 mg / L potassium dihydrogen phosphate (KH2PO4), 370 mg / L magnesium sulfate (MgSO4·7H2O), 440 mg / L calcium chloride (CaCl2·2H2O), 27.8 mg / L iron salt (Fe-EDTA), and no inositol is added; Plant growth regulators: naphthaleneacetic acid (NAA) 0.1-0.5 mg / L, 6-benzylaminopurine (6-BA) 0.2-1.0 mg / L, wherein the molar concentration ratio of NAA to 6-BA is 1:2 to 1:5; Organic additives: coconut water 50-100 mL / L (taken from mature coconuts, filtered after centrifugation at 5000 rpm for 10 minutes to remove suspended matter), potato juice 20-50 mL / L (made from peeled potatoes, sliced, boiled for 30 minutes, and the squeezed juice filtered through double-layer gauze); Anti-browning agent: a complex of activated carbon 0.5-2.0g / L and vitamin C 0.05-0.2g / L; Carbon source: sucrose 20-40 g / L (purity ≥ 99%); Solidifying agent: agar 6-10g / L (gel strength ≥900g / cm 2 ); pH regulator: Use 0.1 mol / L NaOH or HCl to adjust the pH of the culture medium to 5.8-6.2; Auxiliary ingredients: Vitamin B1 0.1mg / L, niacin 0.5mg / L.

2. The sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings according to claim 1, characterized in that: The concentration of the MS medium mother solution is 1.0× the original solution concentration, and magnesium sulfate (MgSO4·7H2O) is adjusted to 400 mg / L and calcium chloride (CaCl2·2H2O) is adjusted to 460 mg / L to enhance ion balance.

3. The sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings according to claim 1, characterized in that: The plant growth regulator has a ratio of NAA 0.3 mg / L and 6-BA 0.5 mg / L, and both are dissolved in sterile water at 25° C. by 40 kHz ultrasonic treatment for 10 minutes, and then added to the culture medium after forming a homogeneous solution.

4. The sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings according to claim 1, characterized in that: The coconut water needs to be sterilized by high pressure (121° C., 15 minutes) and then cooled to 25° C. before use. 0.05% (w / v) papain is added to the potato juice and enzymolyzed at 45° C. for 1 hour to release soluble polysaccharides.

5. The sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings according to claim 1, characterized in that: The anti-browning agent is a ternary complex of 1.0 g / L activated carbon, 0.1 g / L vitamin C and 0.05 g / L cysteine ​​hydrochloride, and the three need to be pre-mixed and ground to a particle size of ≤50 μm.

6. A method for preparing the sterile seedling culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Weigh MS mother solution, sucrose and agar according to the ratio, add deionized water to make up to 70% of the total volume, heat to 80°C and stir until the agar is completely dissolved; (2) adding plant growth regulator, organic additive and anti-browning agent in sequence, and mixing for 20 minutes under a magnetic stirrer (speed of 500 rpm); (3) Use a pH meter to monitor in real time, adjust the pH to 5.8-6.2 with 0.1 mol / L NaOH or HCl, and add deionized water to the target volume; (4) Dispense into transparent glass culture bottles (100 mL capacity, 40 mL liquid volume / bottle), seal and sterilize at 121°C for 20 min, and shake the bottle when cooled to 45°C to ensure uniform distribution of the ingredients; (5) The sterilized culture medium was placed in a sterile operating table for 24 hours and used after the temperature dropped to 25°C.

7. The sterile seedling culture medium and preparation method thereof according to claim 6, characterized in that: In step (2), the organic additives must be sterilized by two-stage filtration through 0.45 μm and 0.22 μm filter membranes in sequence, and aseptically injected into the clean bench when the culture medium is cooled to 50°C.

8. The sterile seedling culture medium and preparation method thereof according to claim 6, characterized in that: In the step (5), the static environment is light-proof and the temperature is controlled at 20-25° C. After static, a uniform gel layer is formed on the surface of the culture medium with a thickness of 2-3 mm.

9. The sterile seedling culture medium for improving the survival rate of Bletilla striata seedlings according to claim 1, characterized in that: The sterile seedling culture medium is used for the sterile sowing of Bletilla striata seeds or protocorms. Before sowing, the seeds or protocorms need to be soaked in a 0.1% HgCl2 solution for 8-10 minutes and rinsed with sterile water for 3-5 times. The culture conditions are: light intensity of 1000-2000 Lux, daily light for 12-16 hours, culture temperature of 22±2° C., humidity of 60-80%, and culture period of 30-40 days.