Method for making plant tissue culture medium using agar production byproducts and tissue culture process

Abstract

The application provides a method for preparing plant tissue culture medium by using agar production by-products, agar production by-products are unloaded from a filter plate, the by-products comprise perlite 40-55%, agar 10-15%, fiber 10-20% and water 10-40%, hot water is added to soak the agar production by-products so that the agar production by-products have a certain flow state; a beating machine is used to beat and uniformly disperse the clumped agar production by-products, and a homogenizer is used to uniformly mix the various components of the agar production by-products; cellulase is added to hydrolyze, and after hydrolysis, the homogenizer is used to uniformly mix to obtain slurry; the slurry is pumped into a roller for drying, crushing and sieving to obtain finished modified agar production by-products; the modified agar production by-products are weighed and poured into a culture medium mother liquor, and the addition proportion of the modified agar production by-products is 5-20% of the total culture medium solution. The method has the advantages that the modified agar production by-products are used to replace agar to prepare the culture medium, the modified agar production by-products can be used as a coagulating agent in the plant tissue culture medium, the preparation cost is greatly reduced, and the economic benefit is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant culture medium preparation, in particular to a method for preparing a plant tissue culture medium by using agar production byproducts and a tissue culture process. BACKGROUND

[0002] The culture medium used for plant tissue culture is generally a solid culture medium using agar as a coagulant. Therefore, in factory seedling raising, the demand for agar is large, and the price is high, which seriously affects the economic benefits of seedling raising. In addition, the common problems of conventional plant tissue culture are as follows:

[0003] In plant tissue culture, when the explant is cut, the cells at the wound are damaged, the separation is broken, and in the presence of oxygen, polyphenol oxidase (PPO) oxidizes phenolic compounds into brown quinone compounds, and then under the action of enzymes such as tyrosinase, the proteins in the explant cells are polymerized, and then the cell growth is arrested or even dies. Therefore, the existing plant culture medium has a high browning rate, which may lead to tissue culture failure. In addition, the soluble protein and total sugar content of the tissue culture plant is generally lower than that of the wild plant, and the root system of the tissue culture plant directly affects the strength of the plant, but the root system of the tissue culture plant often has a poor development probability.

[0004] In view of this, the present inventors have conducted in-depth research on the above problems, and thus the present application is produced. SUMMARY

[0005] The present application solves the technical problems of high cultivation cost and high browning rate during tissue culture of the existing plant tissue culture medium, and provides a method for preparing a plant tissue culture medium by using agar production byproducts and a tissue culture process.

[0006] The present application is implemented as follows: a method for preparing a plant tissue culture medium by using agar production byproducts, comprising the following steps:

[0007] Step 1: preparing a culture medium mother liquor;

[0008] Step 2: preparing a modified agar production byproduct: removing the agar production byproduct from the filter plate, adding boiling water to soak the agar production byproduct to have a certain flow state; using a beater to beat and evenly distribute some still clumped agar production byproducts, and then using a homogenizer to mix the various components of the agar production byproduct evenly; adding cellulase for hydrolysis, and then mixing evenly with the homogenizer after hydrolysis to obtain a slurry; pumping the slurry into a roller for drying, crushing, and sieving to obtain a finished product of the modified agar production byproduct;

[0009] Step three: plant tissue culture medium preparation: weigh the modified agar production by-products, pour into the culture medium mother liquor, the addition of modified agar production by-products is 5-20% of the total culture medium solution, 100 ℃ high temperature heating and stirring, then the medium is hot packed in tissue culture bottles;

[0010] Step four: finished product: cover the tissue culture bottle with a lid, autoclave at 110-125 ℃ for 30 minutes, and finally cool to room temperature to obtain the plant tissue culture medium.

[0011] Further, the components of the agar production by-products include:

[0012] perlite 40-55%

[0013] agar 10-15%

[0014] fiber 10-20%

[0015] water 10-40%.

[0016] Further, in step two, the temperature is controlled at 90-100 ℃ during pulping.

[0017] Further, in step two, the mass fraction of cellulase is 0.1-0.6%, and the hydrolysis time is 6-8 hours.

[0018] Further, in step three, the addition of modified agar production by-products is 8-14% of the total culture medium solution.

[0019] Further, in step four, the sterilization temperature is 121 ℃.

[0020] Further, in step one, the preparation method of the culture medium mother liquor includes the following steps:

[0021] Step 1.1: according to the preparation method of MS, White, B5, SH, N6, NN, DKW and other plant culture medium mother liquor, mother liquor A is prepared;

[0022] Step 1.2: weigh 100 mg of plant growth regulator, heat in water bath, and finally dilute to 100 mL to obtain 1 mg / mL of mother liquor B;

[0023] Step 1.3: weigh mother liquor A into a beaker, add the required concentration of mother liquor B, shake evenly, add 2%-4% sucrose to the prepared solution, and stir to dissolve;

[0024] Step 1.4: measure the pH value of the solution with a pH meter or pH test paper, and adjust the pH value with sodium hydroxide or hydrochloric acid until the pH of the culture medium solution is 6-8. The required culture medium mother liquor is obtained.

[0025] Further, in step 1.2, the plant growth regulator includes one or both of naphthalene acetic acid and 6-benzylaminopurine; the naphthalene acetic acid is dissolved in a small amount of ethanol when heated in a water bath, and the 6-benzylaminopurine is dissolved in a small amount of NaOH solution when heated in a water bath.

[0026] In another aspect, a tissue culture process uses the plant tissue culture medium prepared by the method for preparing a plant tissue culture medium using agar production by-products; then inoculation, culture, and transplanting.

[0027] The present application has the advantages of:

[0028] 1. The present application uses modified agar production by-products to replace agar to prepare culture medium, which can be used as a coagulant in MS, White, B5, SH, N6, NN, DKW, and other plant tissue culture media, greatly reducing the cost of preparation and improving economic efficiency.

[0029] 2. The modified agar production by-products of the present application contain perlite and modified fibers, which can be used as adsorbents to adsorb quinone compounds produced by browning, and harmful substances produced by browning in the culture medium environment can be quickly transferred away, reducing the toxic effect and further preventing browning; thereby reducing the browning rate of tissue culture. The higher the concentration of modified agar production by-products, the better the anti-browning effect.

[0030] 3. The modified agar production by-products of the present application have a similar effect to humus, which can adsorb plant growth regulators and other substances that are beneficial to root growth, thus promoting the rooting of tissue culture seedlings.

[0031] 4. Since the modified agar production by-products of the present application can promote root formation and growth, they also have strong ability to absorb water and inorganic salts and other nutrients, providing abundant raw materials for the biosynthesis of proteins and carbohydrates; at the same time, the fibers after enzymatic hydrolysis also provide abundant raw materials; thereby significantly increasing the content of soluble protein and total sugar in the body of tissue culture plants. BRIEF DESCRIPTION OF DRAWINGS

[0032] The present application will be further described below with reference to the accompanying drawings and examples.

[0033] Figure 1 is a process flow chart of the method for preparing a plant tissue culture medium using agar production by-products in Example 1.

[0034] Figure 2 is a preparation flow chart of the modified agar production by-products in Example 1.

[0035] Figure 3 is a preparation flow chart of the culture medium mother liquor in Example 1.

[0036] Figure 4 is a flow chart of the tissue culture process in Example Two.

DETAILED DESCRIPTION

[0037] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0038] Example One

[0039] Referring to the drawings shown in the accompanying drawings, Figures 1-3 the present embodiment provides a method for preparing plant tissue culture medium by using agar production by-products, comprising the following steps:

[0040] Step One: Preparation of culture medium mother liquor.

[0041] Step Two: Preparation of modified agar production by-products: the temperature for removing agar production by-products from the filter plate is generally between 55-100°C, and hot water is added for soaking so that the agar production by-products have a certain flow state; a beater is used to beat and evenly disperse some agar production by-products that are still in clumps, and the temperature is controlled at 90-100°C; a homogenizer is then used to mix the various components of the agar production by-products evenly; cellulase is added for hydrolysis, and after hydrolysis is completed, the homogenizer is used again to mix evenly to obtain slurry; the slurry is pumped into a roller for drying, crushing and sieving to obtain the finished modified agar production by-products.

[0042] Step Three: Preparation of plant tissue culture medium: the modified agar production by-products are weighed and poured into the culture medium mother liquor, and the addition ratio of the modified agar production by-products is 5-20% of the total culture medium solution; high-temperature heating and stirring are performed at 100°C, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0043] Step Four: Finished product: the tissue culture bottles are capped, high-pressure sterilized at a temperature of 110-125°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0044] The method for preparing plant tissue culture medium by using agar production by-products provided in the present embodiment has the following advantages:

[0045] 1. Existing plant tissue culture media generally use agar as a solid medium. Therefore, in factory-scale seedling production, the demand for agar is large. However, the high price of agar leads to high media preparation costs, severely impacting the economic benefits of seedling production. To reduce costs and increase revenue, this embodiment uses agar production byproducts as the main raw material to prepare modified agar production byproducts to replace agar as the solidifying agent for plant tissue culture media. Furthermore, the proportion of modified agar production byproducts added is only 5-20% of the total media solution, significantly reducing the production cost and improving economic efficiency.

[0046] 2. Byproducts of modified agar production include perlite and modified fibers, which can be used as adsorbents to adsorb quinone compounds produced by browning. At the same time, harmful substances produced by browning in the culture medium environment can be quickly transferred away, reducing toxicity and further preventing browning, thereby reducing the browning rate of tissue culture.

[0047] 3. Modified agar production byproducts can adsorb plant growth regulators and other substances that promote rooting, thus promoting rooting of tissue culture seedlings.

[0048] 4. Because modified agar production byproducts are added to the culture medium, they can promote root formation and growth, and also develop a strong ability to absorb nutrients such as water and inorganic salts, providing abundant raw materials for the biosynthesis of proteins and sugars; at the same time, a large amount of fiber is produced during the enzymatic hydrolysis of agar production byproducts, which also provides abundant raw materials for the growth of tissue culture seedlings.

[0049] In step two, the components of the agar production byproduct that has just been unloaded include: 40-55% perlite, 10-15% agar, 10-20% fiber, and 10-40% water. After enzymatic hydrolysis, the agar production byproduct generates modified fiber, providing abundant raw materials for plant root growth.

[0050] In step two, the mass fraction of cellulase is 0.1–0.6%, and the hydrolysis time is 6–8 hours. The optimal cellulase ratio is 0.1–0.6%; below this ratio, degradation efficiency is low, while above it, cost is high, and the degradation effect is not optimal. The modified agar production byproduct obtained after 6–8 hours of hydrolysis is best suited for tissue culture. Excessive degradation time reduces gelation efficiency, while insufficient time results in inadequate modification of the agar production byproduct.

[0051] In step three, preferably, the proportion of the modified agar production by-product added is 8-14% of the total culture medium solution.

[0052] In step four, the preferred sterilization temperature is 121°C. The medium contains the necessary carbon source, nitrogen source and inorganic salts for microbial growth, and is usually sterilized by moist heat autoclaving at 121°C for 15 minutes to ensure that both bacteria and their spores are inactivated. Higher temperatures and atmospheric pressure sterilization can be better, but can affect the ingredients in the medium.

[0053] In step one, the method for preparing the medium stock solution includes the following steps:

[0054] Step 1.1: Prepare stock solution A according to the preparation method of MS, White, B5, SH, N6, NN, DKW, etc. plant medium stock solution.

[0055] Step 1.2: Weigh 100 mg of plant growth regulator, heat in water bath, and finally dilute to 100 mL to obtain a stock solution B of 1 mg / mL. The plant growth regulator includes one or both of naphthalene acetic acid (NAA) and 6-benzylaminopurine (6-BA); a small amount of ethanol is used to dissolve naphthalene acetic acid when heated in a water bath, and a small amount of NaOH solution is used to dissolve 6-benzylaminopurine when heated in a water bath.

[0056] Step 1.3: Weigh stock solution A into a beaker, add the required concentration of stock solution B, shake well, and add 2% to 4% sucrose to the prepared solution and stir to dissolve.

[0057] Step 1.4: Measure the pH value of the solution with a pH meter or pH paper, and adjust the pH value with sodium hydroxide or hydrochloric acid until the pH of the medium solution is 6 to 8; the required medium stock solution is obtained.

[0058] Example Two

[0059] Referring to the accompanying Figure 4 The present embodiment provides a tissue culture process using the plant tissue culture medium prepared by the method of Example One; then inoculation, culture, and transplantation; finally, detecting the browning rate of the tissue culture seedlings, observing the rooting of the tissue culture seedlings, and measuring the content of soluble protein and total sugar in the tissue culture seedlings.

[0060] Next, a plurality of experiments are performed using the tissue culture process described in the present embodiment, and the following selected test groups and control groups are used to further illustrate the beneficial effects of the tissue culture process.

[0061] Test Group One

[0062] (1) Prepare the medium stock solution.

[0063] (2) The temperature for removing the agar production by-products from the filter plate is generally between 55-100°C, the agar production by-products include 40% perlite, agar 10%, fiber 10%, and water 40%; hot water is added to soak the agar production by-products to make them have a certain flow state; a beater is used to beat and evenly disperse some of the agar production by-products that are still in clumps, at this time the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the agar production by-products evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, after hydrolysis is completed, the homogenizer is used to mix evenly to obtain a slurry; the slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-products.

[0064] (3) The modified agar production by-products are weighed and poured into the culture medium mother liquor, the addition ratio of the modified agar production by-products is 5% of the total culture medium solution, high temperature heating and stirring at 100°C, then the culture medium is hotly divided and packed into tissue culture bottles.

[0065] (4) The tissue culture bottles are capped, high pressure sterilization at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0066] (5) Inoculation, culture, and transplanting; finally, the browning rate of the tissue culture is detected, the rooting condition of the cultured seedlings is observed, and the content of soluble protein and total sugar in the tissue culture plants is measured.

[0067] Test group two

[0068] (1) Preparation of the culture medium mother liquor.

[0069] (2) The temperature for removing the agar production by-products from the filter plate is generally between 55-100°C, the agar production by-products include 40% perlite, agar 10%, fiber 10%, and water 40%; hot water is added to soak the agar production by-products to make them have a certain flow state; a beater is used to beat and evenly disperse some of the agar production by-products that are still in clumps, at this time the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the agar production by-products evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, after hydrolysis is completed, the homogenizer is used to mix evenly to obtain a slurry; the slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-products.

[0070] (3) The modified agar production by-products are weighed and poured into the culture medium mother liquor, the addition ratio of the modified agar production by-products is 5% of the total culture medium solution, high temperature heating and stirring at 100°C, then the culture medium is hotly divided and packed into tissue culture bottles.

[0071] (4) The tissue culture bottles are capped, high pressure sterilization at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0072] (5) Inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the seedlings, and measuring the soluble protein and total sugar contents in the tissue culture plants.

[0073] Test Group Three

[0074] (1) Preparation of the medium mother liquor.

[0075] (2) The temperature for removing the agar production by-product from the filter plate is generally between 55-100°C, and the agar production by-product includes 55% perlite, 15% agar, 20% fiber, and 10% water. Hot water is added to soak the agar production by-product so that it has a certain flow state. A beater is used to beat and evenly distribute some still clumped agar production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the agar production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours. After hydrolysis is complete, the homogenizer is used again to mix evenly to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0076] (3) The modified agar production by-product is weighed and poured into the medium mother liquor. The addition ratio of the modified agar production by-product is 5% of the total medium solution. High-temperature heating and stirring are performed at 100°C, after which the medium is hotly divided and packaged in tissue culture bottles.

[0077] (4) The tissue culture bottles are capped, high-pressure sterilized at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0078] (5) Inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the seedlings, and measuring the soluble protein and total sugar contents in the tissue culture plants.

[0079] Test Group Four

[0080] (1) Preparation of the medium mother liquor.

[0081] (2) The temperature for removing the agar production by-product from the filter plate is generally between 55-100°C, and the agar production by-product includes 55% perlite, 15% agar, 20% fiber, and 10% water. Hot water is added to soak the agar production by-product so that it has a certain flow state. A beater is used to beat and evenly distribute some still clumped agar production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the agar production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours. After hydrolysis is complete, the homogenizer is used again to mix evenly to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0082] (3) Take the modified agar production by-products, pour into the culture medium mother liquor, the modified agar production by-products of the total medium solution is added to the proportion of 8%, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0083] (4) Put the tissue culture bottle cover, high pressure sterilization at 121 ℃ for 30 minutes, finally cooled to room temperature, the plant tissue culture medium is obtained.

[0084] (5) Inoculation, culture, transplanting; finally test the browning rate of tissue culture, observe the rooting of the seedlings, and measure the content of soluble protein and total sugar in the tissue culture plant.

[0085] Test group five

[0086] (1) Preparation of culture medium mother liquor.

[0087] (2) The temperature of the agar production by-products from the filter plate is generally between 55-100 ℃, the agar production by-products include 52% perlite, agar 13%, fiber 15%, water 20%; add hot water to soak so that the agar production by-products have a certain flow state; use the beater to beat some still clumps of agar production by-products evenly, the temperature is controlled at 90-100 ℃; then use the homogenizer to mix the various components of the agar production by-products evenly; add 0.1-0.6% cellulase for hydrolysis for 6-8 hours, after hydrolysis, mix evenly with the homogenizer to obtain the slurry; the slurry is pumped into the roller for drying, crushing and sieving to obtain the finished modified agar production by-products.

[0088] (3) Take the modified agar production by-products, pour into the culture medium mother liquor, the modified agar production by-products of the total medium solution is added to the proportion of 8%, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0089] (4) Put the tissue culture bottle cover, high pressure sterilization at 121 ℃ for 30 minutes, finally cooled to room temperature, the plant tissue culture medium is obtained.

[0090] (5) Inoculation, culture, transplanting; finally test the browning rate of tissue culture, observe the rooting of the seedlings, and measure the content of soluble protein and total sugar in the tissue culture plant.

[0091] Test group six

[0092] (1) Preparation of culture medium mother liquor.

[0093] (2) The temperature for removing the agar production by-products from the filter plate is generally between 55-100°C, the agar production by-products include 52% perlite, 13% agar, 15% fiber, and 20% water; hot water is added to soak the agar production by-products to make them have a certain flow state; a beater is used to beat and evenly disperse some of the agar production by-products that are still in clumps, at this time the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the agar production by-products evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, after hydrolysis is completed, the homogenizer is used to mix evenly to obtain a slurry; the slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-products.

[0094] (3) The modified agar production by-products are weighed and poured into the culture medium mother liquor, the addition ratio of the modified agar production by-products is 14% of the total culture medium solution, high-temperature heating and stirring are carried out at 100°C, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0095] (4) The tissue culture bottles are capped, high-pressure sterilization is carried out at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0096] (5) Inoculation, culture, and transplanting; finally, the browning rate of the tissue culture is detected, the rooting condition of the seedlings is observed, and the content of soluble protein and total sugar in the tissue culture plant is measured.

[0097] Test group seven

[0098] (1) Preparation of the culture medium mother liquor.

[0099] (2) The temperature for removing the agar production by-products from the filter plate is generally between 55-100°C, the agar production by-products include 52% perlite, 13% agar, 15% fiber, and 20% water; hot water is added to soak the agar production by-products to make them have a certain flow state; a beater is used to beat and evenly disperse some of the agar production by-products that are still in clumps, at this time the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the agar production by-products evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, after hydrolysis is completed, the homogenizer is used to mix evenly to obtain a slurry; the slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-products.

[0100] (3) The modified agar production by-products are weighed and poured into the culture medium mother liquor, the addition ratio of the modified agar production by-products is 14% of the total culture medium solution, high-temperature heating and stirring are carried out at 100°C, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0101] (4) The tissue culture bottles are capped, high-pressure sterilization is carried out at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0102] (5) Inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the seedlings, and measuring the content of soluble protein and total sugar in the tissue culture plant.

[0103] Comparative Group One

[0104] (1) Preparation of the medium mother liquor.

[0105] (2) The temperature for removing the agar production by-product from the filter plate is generally between 55-100℃, and the agar production by-product includes 52% perlite, 13% agar, 15% fiber, and 20% water. Hot water is added for soaking so that the agar production by-product has a certain flow state. A beater is used to beat and evenly distribute some still clumped agar production by-products, with the temperature controlled at 90-100℃. A homogenizer is then used to mix the various components of the agar production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours. After hydrolysis is completed, the homogenizer is used again to mix evenly to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0106] (3) The modified agar production by-product is weighed and poured into the medium mother liquor. The addition proportion of the modified agar production by-product is 4% of the total medium solution. High-temperature heating and stirring are performed at 100℃, after which the medium is hotly divided and packaged into tissue culture bottles.

[0107] (4) The tissue culture bottles are capped, high-pressure sterilized at 121℃ for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0108] (5) Inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the seedlings, and measuring the content of soluble protein and total sugar in the tissue culture plant.

[0109] Comparative Group Two

[0110] (1) Preparation of the medium mother liquor.

[0111] (2) The temperature for removing the agar production by-product from the filter plate is generally between 55-100℃, and the agar production by-product includes 52% perlite, 13% agar, 15% fiber, and 20% water. Hot water is added for soaking so that the agar production by-product has a certain flow state. A beater is used to beat and evenly distribute some still clumped agar production by-products, with the temperature controlled at 90-100℃. A homogenizer is then used to mix the various components of the agar production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours. After hydrolysis is completed, the homogenizer is used again to mix evenly to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0112] (3) Take the modified agar production by-products, pour into the culture medium mother liquor, the modified agar production by-products of the total medium solution is added to the proportion of 22%, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0113] (4) Put the tissue culture bottle cover, high pressure sterilization at 121 ℃ for 30 minutes, finally cooled to room temperature, the plant tissue culture medium is obtained.

[0114] (5) Inoculation, culture, transplanting; finally detect the browning rate of tissue culture, observe the rooting of the seedlings, and measure the content of soluble protein and total sugar in the tissue culture plant.

[0115] Comparison group three

[0116] (1) Preparation of medium mother liquor.

[0117] (2) The temperature of the agar production by-products from the filter plate is generally between 55-100 ℃, the agar production by-products include 52% perlite, agar 20%, fiber 10%, water 18%; add hot water to soak so that the agar production by-products have a certain flow state; use the beater to beat some still clumps of agar production by-products evenly, the temperature is controlled at 90-100 ℃; then use the homogenizer to mix the various components of the agar production by-products evenly; add 0.1-0.6% cellulase to hydrolyze for 6-8 hours, after hydrolysis, mix evenly with the homogenizer to obtain the slurry; the slurry is pumped into the roller for drying, crushing and sieving to obtain the finished modified agar production by-products.

[0118] (3) Take the modified agar production by-products, pour into the culture medium mother liquor, the modified agar production by-products of the total medium solution is added to the proportion of 22%, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0119] (4) Put the tissue culture bottle cover, high pressure sterilization at 121 ℃ for 30 minutes, finally cooled to room temperature, the plant tissue culture medium is obtained.

[0120] (5) Inoculation, culture, transplanting; finally detect the browning rate of tissue culture, observe the rooting of the seedlings, and measure the content of soluble protein and total sugar in the tissue culture plant.

[0121] Comparison group four

[0122] (1) Preparation of medium mother liquor.

[0123] (2) The temperature of the agar production by-product removed from the filter plate is generally between 55-100°C, and the agar production by-product includes 52% perlite, 25% agar, 10% fiber, and 13% water. The agar production by-product is soaked in boiling water to have a certain flow state. Some still clumped agar production by-products are uniformly dispersed by using a beater, and the temperature is controlled at 90-100°C. The various components of the agar production by-product are uniformly mixed by using a homogenizer. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours, and the hydrolysis is uniformly mixed by using a homogenizer to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0124] (3) The modified agar production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified agar production by-product is 11% of the total culture medium solution. High-temperature heating and stirring are performed at 100°C, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0125] (4) The tissue culture bottle is covered with a lid, high-pressure sterilization is performed at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0126] (5) Inoculation, culture, and transplantation; finally, the browning rate of the tissue culture is detected, the rooting condition of the cultured seedlings is observed, and the content of soluble protein and total sugar in the tissue culture plant is measured.

[0127] Comparative Group Five

[0128] (1) The culture medium mother liquor is prepared.

[0129] (2) The temperature of the agar production by-product removed from the filter plate is generally between 55-100°C, and the agar production by-product includes 52% perlite, 25% agar, 10% fiber, and 13% water. The agar production by-product is soaked in boiling water to have a certain flow state. Some still clumped agar production by-products are uniformly dispersed by using a beater, and the temperature is controlled at 90-100°C. The various components of the agar production by-product are uniformly mixed by using a homogenizer. Cellulase with a mass fraction of 0.1-0.6% is added for hydrolysis for 6-8 hours, and the hydrolysis is uniformly mixed by using a homogenizer to obtain a slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product.

[0130] (3) The modified agar production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified agar production by-product is 11% of the total culture medium solution. High-temperature heating and stirring are performed at 100°C, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0131] (4) The tissue culture bottle is covered with a lid, high-pressure sterilization is performed at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0132] (5) Inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the seedlings, and measuring the content of soluble protein and total sugar in the tissue culture plants.

[0133] According to the above-mentioned multiple experiments, the relevant experimental data are listed, including the following:

[0134] Table 1 Specific components of test groups 1 to 7 and comparison groups 1 to 5

[0135]

[0136] Table 2 Comparison of detection results of each test group and comparison group

[0137]

[0138]

[0139] Then according to the experimental results in Table 2, it can be seen that:

[0140] 1. Higher perlite content is beneficial to reduce the browning rate, but too high or too low addition of modified agar production by-products will also affect the browning rate, which may be due to the hardness or softness of the gel effect, which is not conducive to plant growth and increases the browning rate. Modified agar production by-products contain perlite and modified fiber, which can be used as an adsorbent to adsorb quinone compounds produced by browning, and harmful substances produced by browning in the culture medium environment can be quickly transferred away.

[0141] 2. Higher cellulose content is more conducive to improving the rooting rate of plants; but too high or too low addition of modified agar production by-products will affect the rooting rate, which may be due to the hardness or softness of the gel effect, which is not conducive to rooting. Therefore, modified agar production by-products can adsorb plant growth regulators and other substances conducive to rooting, promoting the rooting of tissue culture seedlings.

[0142] 3. Due to the addition of modified agar production by-products in the culture medium, it can promote root formation and growth, and also form a strong ability to absorb water and inorganic salts and other nutrients, providing abundant raw materials for protein and sugar biosynthesis; at the same time, a large amount of fiber is produced during the enzymatic hydrolysis of agar production by-products, which also provides abundant raw materials for the growth of tissue culture seedlings. Therefore, the content of soluble protein and total sugar in the tissue culture plants is related to the growth of root system, and the better the root system grows, the more conducive to improving the content of soluble protein and total sugar.

[0143] While the foregoing description has described specific embodiments of the application, one ordinary skill in the art will appreciate that various modifications and changes can be made thereto without departing from the spirit and scope of the application, as set forth in the appended claims.

Claims

1. A method for making plant tissue culture media using agar production byproducts, the method comprising: It comprises the following steps: ​ Step one: preparing the medium mother liquor; the preparation method of the medium mother liquor comprises the following steps: Step 1.1: according to the preparation method of MS, White, B5, SH, N6, NN, DKW plant medium mother liquor, mother liquor A is prepared; Step 1.2: weigh 100 mg of plant growth regulator, heat in water bath, and finally make up to 100 mL, i.e. 1 mg / mL of mother liquor B; the plant growth regulator comprises one or both of naphthalene acetic acid and 6-benzylaminopurine; a small amount of ethanol is used to dissolve naphthalene acetic acid when heated in water bath, and a small amount of NaOH solution is used to dissolve 6-benzylaminopurine when heated in water bath; Step 1.3: weigh mother liquor A into a beaker, add the required concentration of mother liquor B into the beaker, shake uniformly, and add 2%-4% sucrose to the prepared solution and stir to dissolve; Step 1.4: measure the pH value of the solution with a pH meter or pH test paper, and adjust the pH value with sodium hydroxide or hydrochloric acid until the pH of the medium solution is 6-8; i.e. the required medium mother liquor is obtained; Step two: preparing modified agar production by-product: remove the agar production by-product from the filter plate, soak it in boiling water to make it have a certain flow state; use a beater to beat some of the still clumped agar production by-products uniformly; when beating, the temperature is controlled at 90-100°C; then use a homogenizer to mix the various components of the agar production by-product uniformly; add cellulase for hydrolysis, and then mix uniformly with the homogenizer after hydrolysis to obtain slurry; Draw the slurry into a roller for drying, crushing, and sieving to obtain the finished modified agar production by-product; The components of the agar production by-product include: 40-55% perlite, 10-15% agar, 10-20% fiber, and 10-40% water; the mass fraction of cellulase is 0.1-0.6%; the hydrolysis time is 6-8 hours; Step three: plant tissue culture medium preparation: weigh the modified agar production by-product, pour it into the medium mother liquor, the addition ratio of the modified agar production by-product is 8-14% of the total medium solution, heat and stir at 100°C, and then the medium is hotly divided into tissue culture bottles; Step four: finished product: cover the tissue culture bottles with lids, autoclave at 110-125°C for 30 minutes, and then cool to room temperature to obtain the plant tissue culture medium.

2. The method for producing a plant tissue culture medium using agar production by-products according to claim 1, characterized by: In step four, the sterilization temperature is 121°C.

3. A tissue culture process, characterized by: Use the plant tissue culture medium prepared by the method for preparing plant tissue culture medium using agar production by-product according to any one of claims 1-2; then inoculate, culture, and transplant.

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