Method for producing plant culture medium by using carrageenan production by-product and tissue culture process

Abstract

The application provides a method for preparing plant tissue culture medium by using carrageenan production by-products, and the carrageenan production by-products are unloaded from a filter plate, the by-products include perlite 40-55%, carrageenan 10-15%, fiber 10-20% and water 10-40%, and boiled water is added for soaking so that the carrageenan production by-products have a certain flow state; a beating machine is used to beat and uniformly disperse the clumped carrageenan production by-products, and a homogenizer is used to uniformly mix the various components of the carrageenan production by-products; cellulase is added for hydrolysis, and after the hydrolysis is completed, 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 carrageenan production by-products; the modified carrageenan production by-products are weighed and poured into a culture medium mother liquor, and the addition proportion of the modified carrageenan production by-products is 5-20% of the total culture medium solution. The method has the advantages that the modified carrageenan production by-products are used to replace carrageenan to prepare the culture medium, the modified carrageenan production by-products can be used as a coagulant in the plant tissue culture medium, the production 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 culture medium from carrageenan production by-products and a tissue culture process. BACKGROUND

[0002] The culture medium used in plant tissue culture generally adopts a solid culture medium with carrageenan as a coagulant. Therefore, in factory seedling production, a large amount of carrageenan is required, and the cost is high due to the high price of carrageenan, which seriously affects the economic benefits of seedling production. 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 site 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 polymerize, and then cause cell growth arrest or even death. 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 contents in the tissue culture plant are generally lower than those in the wild plant, and the root system of the tissue culture plant directly affects the strength of the plant, but the probability of poor development of the root system of the tissue culture plant often occurs.

[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 culture medium from carrageenan production by-products and a tissue culture process.

[0006] The present application is implemented as follows: a method for preparing a plant culture medium from carrageenan production by-products, comprising the following steps:

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

[0008] Step 2: preparing a modified carrageenan production by-product: removing the carrageenan production by-product from the filter plate, adding boiling water to soak the carrageenan production by-product to have a certain flow state; using a beater to beat and evenly distribute some still clumped carrageenan production by-products, and then using a homogenizer to mix the various components of the carrageenan production by-product 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 modified carrageenan production by-product;

[0009] Step three: plant tissue culture medium preparation: weigh the modified carrageenan production by-products, pour into the culture medium mother liquor, the addition of modified carrageenan 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 121℃ for 30 minutes, and finally cool to room temperature to obtain the plant tissue culture medium.

[0011] Further, in step two, the components of the carrageenan production by-products include:

[0012]

[0013] Further, in step two, the temperature during beating is controlled at 90-100℃.

[0014] Further, in step two, the mass fraction of cellulase is 0.1-0.6%, and the hydrolysis time is 6-8 hours.

[0015] Further, in step three, the addition of modified carrageenan production by-products is 8-14% of the total culture medium solution.

[0016] Further, in step four, the sterilization temperature is 121℃.

[0017] Further, in step one, the preparation method of the culture medium mother liquor includes the following steps:

[0018] Step 1.1: prepare mother liquor A according to the preparation method of MS, White, B5, SH, N6, NN, DKW, etc. plant culture medium mother liquor;

[0019] 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;

[0020] 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;

[0021] 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.

[0022] Further, in step 1.2, the plant growth regulator includes one or both of naphthalene acetic acid and 6-benzylaminopurine; a small amount of ethanol is used to dissolve naphthalene acetic acid during water bath heating, and a small amount of NaOH solution is used to dissolve 6-benzylaminopurine during water bath heating.

[0023] In another aspect, a tissue culture process using the plant culture medium prepared by the method for preparing plant culture medium using carrageenan production by-products.

[0024] The present application has the following advantages:

[0025] 1. The present application uses modified carrageenan production by-products to replace carrageenan 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 preparing plant tissue culture media and improving economic efficiency.

[0026] 2. The modified carrageenan 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.

[0027] 3. The modified carrageenan production by-products of the present application have a similar effect to humus soil, which can adsorb plant growth regulators and other substances that are beneficial to root growth, thus promoting the rooting of tissue culture seedlings.

[0028] 4. Since the modified carrageenan 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

[0029] The present application will be further described below with reference to the accompanying drawings and examples.

[0030] Figure 1 is a process flow chart of the method for preparing plant tissue culture medium using carrageenan production by-products in Example 1.

[0031] Figure 2 is a preparation flow chart of the modified carrageenan production by-products in Example 1.

[0032] Figure 3 is a preparation flow chart of the culture medium mother liquor in Example 1.

[0033] Figure 4 is a process flow chart of the tissue culture process in Example 2. DETAILED DESCRIPTION

[0034] In order to make the purposes, 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 part 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 the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection 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. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0035] Embodiment one

[0036] Referring to the drawings, the present embodiment provides a method for preparing plant tissue culture medium by using carrageenan production by-products, comprising the following steps: Figures 1-3

[0037] Step one: preparing a culture medium mother liquor.

[0038] Step two: preparing modified carrageenan production by-products: the temperature of the carrageenan production by-products removed from the filter plate is generally between 55-100℃, and hot water is added for soaking so that the carrageenan production by-products have a certain flow state; a beater is used to beat and evenly disperse some still clumped carrageenan production by-products, and the temperature is controlled at 90-100℃ at this time; a homogenizer is used to further mix the various components of the carrageenan production by-products evenly; cellulase is added for hydrolysis, and 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 carrageenan production by-products.

[0039] Step three: preparing plant tissue culture medium: the modified carrageenan production by-products are weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-products is 5-20% of the total culture medium solution; high-temperature heating and stirring are performed at 100℃, and then the culture medium is hotly divided and packaged into tissue culture bottles.

[0040] Step four: finished product: the tissue culture bottles are capped, high-pressure sterilized at a temperature of 110-125℃ for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0041] The method for preparing plant tissue culture medium by using carrageenan production by-products provided in the present embodiment has the following advantages:

[0042] ​1. Existing plant tissue culture media generally use solid media with carrageenan as a solidifying agent. Therefore, in factory-scale seedling production, the demand for carrageenan is relatively large. However, due to the high price of carrageenan, the preparation cost of the media is high, which seriously affects the economic benefits of seedling production. In order to reduce costs and increase revenue, this embodiment uses carrageenan production by-products as the main raw material to prepare modified carrageenan production by-products to replace carrageenan for the solidifying agent required for plant tissue culture media. Furthermore, the addition ratio of modified carrageenan production by-products is only 5-20% of the total culture medium solution, which greatly reduces the production cost of the culture medium and improves economic benefits.

[0043] 2. Byproducts of modified carrageenan 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.

[0044] 3. Modified carrageenan production byproducts can adsorb plant growth regulators and other substances that promote rooting, thus promoting rooting of tissue culture seedlings.

[0045] 4. Because modified carrageenan production byproducts are added to the culture medium, it can promote root formation and growth, and also form 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, the enzymatic hydrolysis of carrageenan production byproducts produces a large amount of fiber, which also provides abundant raw materials for the growth of tissue culture seedlings.

[0046] In step two, the components of the carrageenan production byproduct that has just been unloaded include: 40-55% perlite, 10-15% carrageenan, 10-20% fiber, and 10-40% water. After enzymatic hydrolysis, the carrageenan production byproduct generates modified fiber, providing abundant raw materials for plant root growth.

[0047] In step two, the mass fraction of cellulase is 0.1–0.6%, and the hydrolysis time is 6–8 hours. A cellulase ratio of 0.1–0.6% is optimal; below this level, degradation efficiency is low, while above it, cost is high, and the degradation effect is not optimal. Hydrolysis for 6–8 hours yields the best modified carrageenan production byproducts for tissue culture. Excessive degradation time reduces gelation efficiency, while insufficient time results in inadequate modification of the carrageenan production byproducts.

[0048] In step three, preferably, the proportion of the modified carrageenan production by-product added is 8-14% of the total culture medium solution.

[0049] In step four, the sterilization temperature is preferably 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.

[0050] In step one, the method for preparing the medium stock solution includes the following steps:

[0051] 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.

[0052] 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 water bath, and a small amount of NaOH solution is used to dissolve 6-benzylaminopurine when heated in water bath.

[0053] 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.

[0054] 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.

[0055] Example Two

[0056] 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 using carrageenan production by-products to produce plant tissue culture medium described in Example One; then inoculation, culture, transplanting; 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.

[0057] 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 described in the present embodiment.

[0058] Test Group One

[0059] (1) Prepare the medium stock solution.

[0060] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 40% perlite, 10% carrageenan, 10% fiber, and 40% water. Hot water is added to soak the carrageenan production by-product to make it have a certain flow state. A beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added to hydrolyze for 6-8 hours, and after hydrolysis is complete, 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 carrageenan production by-product.

[0061] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 5% 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.

[0062] (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.

[0063] (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 plant is measured.

[0064] Test Group Two

[0065] (1) Preparation of the culture medium mother liquor.

[0066] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 40% perlite, 10% carrageenan, 10% fiber, and 40% water. Hot water is added to soak the carrageenan production by-product to make it have a certain flow state. A beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added to hydrolyze for 6-8 hours, and after hydrolysis is complete, 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 carrageenan production by-product.

[0067] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 5% 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.

[0068] (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.

[0069] (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.

[0070] Test Group Three

[0071] (1) Preparation of the medium mother liquor.

[0072] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 55% perlite, 15% carrageenan, 20% fiber, and 10% water. Hot water is added to soak the carrageenan production by-product to have a certain flow state. A beater is used to beat and evenly distribute some still clumped carrageenan production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan 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 the slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified carrageenan production by-product.

[0073] (3) The modified carrageenan production by-product is weighed and poured into the medium mother liquor. The addition ratio of the modified carrageenan 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.

[0074] (4) The tissue culture bottles are capped and autoclaved at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0075] (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.

[0076] Test Group Four

[0077] (1) Preparation of the medium mother liquor.

[0078] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 55% perlite, 15% carrageenan, 20% fiber, and 10% water. Hot water is added to soak the carrageenan production by-product to have a certain flow state. A beater is used to beat and evenly distribute some still clumped carrageenan production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan 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 the slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified carrageenan production by-product.

[0079] (3) Take the modified carrageenan production by-products, pour into the culture medium mother liquor, the addition of modified carrageenan production by-products ratio is 8% of the total medium solution, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0080] (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.

[0081] (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.

[0082] Test group five

[0083] (1) Preparation of medium mother liquor.

[0084] (2) The temperature of carrageenan production by-products from the filter plate is generally between 55-100 ℃, and the carrageenan production by-products include 52% perlite, 13% carrageenan, 15% fiber and 20% water. Add boiling water to make the carrageenan production by-products have a certain flow state; use the beater to beat some still clumps of carrageenan production by-products evenly, the temperature is controlled at 90-100 ℃; then use the homogenizer to mix the various components of the carrageenan production by-products evenly; add 0.1-0.6% cellulase for hydrolysis for 6-8 hours, then 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 carrageenan production by-products.

[0085] (3) Take the modified carrageenan production by-products, pour into the culture medium mother liquor, the addition of modified carrageenan production by-products ratio is 8% of the total medium solution, 100 ℃ high temperature heating and stirring, then the medium while hot into the tissue culture bottle.

[0086] (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.

[0087] (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.

[0088] Test group six

[0089] (1) Preparation of medium mother liquor.

[0090] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 13% carrageenan, 15% fiber, and 20% water. Hot water is added to soak the carrageenan production by-product to make it have a certain flow state. A beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added to hydrolyze for 6-8 hours, and after hydrolysis is complete, 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 carrageenan production by-product.

[0091] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 14% 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.

[0092] (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.

[0093] (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 plant is measured.

[0094] Test group seven

[0095] (1) The culture medium mother liquor is prepared.

[0096] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 13% carrageenan, 15% fiber, and 20% water. Hot water is added to soak the carrageenan production by-product to make it have a certain flow state. A beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan production by-product evenly. Cellulase with a mass fraction of 0.1-0.6% is added to hydrolyze for 6-8 hours, and after hydrolysis is complete, 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 carrageenan production by-product.

[0097] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 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.

[0098] (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.

[0099] (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.

[0100] Comparative Group One

[0101] (1) Preparation of the medium mother liquor.

[0102] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 13% carrageenan, 15% fiber, and 20% water. Hot water is added for soaking to make the carrageenan production by-product have a certain flow state. A beater is used to beat and evenly distribute some still clumped carrageenan production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan 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 slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified carrageenan production by-product.

[0103] (3) The modified carrageenan production by-product is weighed and poured into the medium mother liquor. The addition ratio of the modified carrageenan production by-product is 4% 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.

[0104] (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.

[0105] (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.

[0106] Comparative Group Two

[0107] (1) Preparation of the medium mother liquor.

[0108] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 13% carrageenan, 15% fiber, and 20% water. Hot water is added for soaking to make the carrageenan production by-product have a certain flow state. A beater is used to beat and evenly distribute some still clumped carrageenan production by-products, with the temperature controlled at 90-100°C. A homogenizer is then used to mix the various components of the carrageenan 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 slurry. The slurry is pumped into a roller for drying, crushing, and sieving to obtain the finished modified carrageenan production by-product.

[0109] (3) Weigh the modified carrageenan production by-product, pour it into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 22% of the total culture medium solution. High-temperature heating and stirring are performed at 100°C, and then the culture medium is hotly divided into tissue culture bottles.

[0110] (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.

[0111] (5) Inoculation, culture, and transplantation; finally, the browning rate of 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.

[0112] Comparison group three

[0113] (1) Preparation of culture medium mother liquor.

[0114] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 20% carrageenan, 10% fiber, and 18% water. Hot water is added for soaking to make the carrageenan production by-product have a certain flow state. The beating machine is used to beat and evenly distribute some still clumped carrageenan production by-products, and the temperature is controlled at 90-100°C. The homogenizer is used to mix the various components of the carrageenan production by-product evenly. 0.1-0.6% cellulase is added for hydrolysis for 6-8 hours, and then the homogenizer is used for mixing to obtain the slurry. The slurry is pumped into the roller for drying, crushing, and sieving to obtain the finished modified carrageenan production by-product.

[0115] (3) Weigh the modified carrageenan production by-product, pour it into the culture medium mother liquor, and the addition ratio of the modified carrageenan production by-product is 22% of the total culture medium solution. High-temperature heating and stirring are performed at 100°C, and then the culture medium is hotly divided into tissue culture bottles.

[0116] (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.

[0117] (5) Inoculation, culture, and transplantation; finally, the browning rate of 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.

[0118] Comparison group four

[0119] (1) Preparation of culture medium mother liquor.

[0120] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 25% carrageenan, 10% fiber, and 13% water; hot water is added to soak the carrageenan production by-product to make it have a certain flow state; a beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the carrageenan production by-product evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, and 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 carrageenan production by-product.

[0121] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan 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.

[0122] (4) The tissue culture bottle is capped, high-pressure sterilized at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0123] (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 plant is measured.

[0124] Comparative Group Five

[0125] (1) The culture medium mother liquor is prepared.

[0126] (2) The temperature of the carrageenan production by-product removed from the filter plate is generally between 55-100°C, and the carrageenan production by-product includes 52% perlite, 25% carrageenan, 10% fiber, and 13% water; hot water is added to soak the carrageenan production by-product to make it have a certain flow state; a beater is used to beat and evenly disperse some of the still clumped carrageenan production by-product, and the temperature is controlled at 90-100°C; a homogenizer is used to mix the various components of the carrageenan production by-product evenly; 0.1-0.6% cellulase by mass fraction is added to hydrolyze for 6-8 hours, and 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 carrageenan production by-product.

[0127] (3) The modified carrageenan production by-product is weighed and poured into the culture medium mother liquor, and the addition ratio of the modified carrageenan 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.

[0128] (4) The tissue culture bottle is capped, high-pressure sterilized at 121°C for 30 minutes, and finally cooled to room temperature to obtain the plant tissue culture medium.

[0129] (5) inoculation, culture, transplanting; finally, detecting the browning rate of the tissue culture, observing the rooting condition of the tissue culture seedlings, and measuring the content of soluble protein and total sugar in the tissue culture plant.

[0130] Table 1: specific components of test groups 1 to 7 and comparison groups 1 to 5

[0131]

[0132] Table 2: comparison of detection results of each test group and comparison group

[0133]

[0134]

[0135] Then according to the experimental results in Table 2, it can be seen that:

[0136] 1. The higher the content of perlite is, the more conducive to reducing the browning rate, but adding too much or too little of the modified carrageenan production by-product will also affect the browning rate, which may be due to the fact that the gel effect is too hard or too soft, which is not conducive to plant growth and increases the browning rate. The modified carrageenan production by-product contains 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.

[0137] 2. The higher the content of cellulose is, the more conducive to improving the rooting rate of plants; but adding too much or too little of the modified carrageenan production by-product will also affect the rooting rate, which may be due to the fact that the gel effect is too hard or too soft, which is not conducive to rooting. Therefore, the modified carrageenan production by-product can adsorb plant growth regulating substances and other substances conducive to rooting, and promote the rooting of tissue culture seedlings.

[0138] 3. Because the modified carrageenan production by-product is added to the culture medium, it can promote the formation and growth of roots, and also form a strong ability to absorb water and inorganic salts and other nutrients, 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 the carrageenan production by-product, 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 plant is related to the growth of root system, and the better the growth of root system is, the more conducive to improving the content of soluble protein and total sugar.

[0139] Although the specific embodiments of the present application are described above, those skilled in the art should understand that the specific examples described are only illustrative, and are not intended to limit the scope of the present application, and equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for preparing plant culture medium using carrageenan production byproducts, characterized in that: Includes the following steps: Step 1: Preparation of culture medium stock solution; the preparation method of the culture medium stock solution includes the following steps: Step 1.1: Prepare stock solution A according to the preparation methods of MS, White, B5, SH, N6, NN, and DKW plant culture medium stock solutions; Step 1.2: Weigh 100 mg of plant growth regulator, heat in a water bath, and finally bring the volume to 100 mL to obtain a 1 mg / mL mother liquor B. The plant growth regulator includes one or both of naphthaleneacetic acid and 6-benzylaminopurine. When heating naphthaleneacetic acid in a water bath, a small amount of ethanol is used to dissolve it, and when heating 6-benzylaminopurine in a water bath, a small amount of NaOH solution is used to dissolve it. Step 1.3: Weigh mother liquor A into a beaker, measure the required concentration of mother liquor B into the beaker, shake well, add 2%~4% sucrose to the prepared solution, and stir to dissolve; Step 1.4: Measure the pH value of the solution using 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; this yields the required culture medium stock solution. Step 2: Preparation of modified carrageenan production by-products: Remove the carrageenan production by-products from the filter plate and soak them in boiling water to make them flow. Use a pulper to break up any remaining clumps of carrageenan production by-products evenly. During pulping, control the temperature at 90~100℃. Then use a homogenizer to mix all the components of the carrageenan production by-products evenly. Add cellulase for hydrolysis. After hydrolysis, use a homogenizer to mix evenly to obtain a slurry. The slurry is pumped into a drum for drying, crushing, and sieving to obtain the finished modified carrageenan production byproduct. The composition of the carrageenan production by-product includes: 40-55% perlite, 10-15% carrageenan, 10-20% fiber, and 10-40% water; The cellulase has a mass fraction of 0.1-0.6% and a hydrolysis time of 6-8 hours; Step 3: Preparation of plant tissue culture medium: Weigh the modified carrageenan production by-product and pour it into the culture medium stock solution. The addition ratio of the modified carrageenan production by-product is 8-14% of the total culture medium solution. Heat and stir at 100℃, and then dispense the culture medium into tissue culture bottles while it is still hot. Step 4: Finished product: Cover the tissue culture bottle and autoclave at 121℃ for 30 minutes. Finally, cool to room temperature to obtain the plant tissue culture medium.

2. A tissue culture process, characterized in that: The plant culture medium is prepared using the method for producing plant culture medium from carrageenan production by-products as described in claim 1; then, the plant is inoculated, cultured, and transplanted.

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