Culture medium composition for garlic gene editing breeding and preparation method thereof

Abstract

The invention discloses a culture medium composition for garlic gene editing breeding and a preparation method of the culture medium composition, and belongs to the technical field of tissue culture media. Copper peroxide is generated on the surface of modified rice hull biochar, and double bonds of acrylate are attacked by using hydrogen atoms with high nucleophilic property of thiol, so that the garlic gene editing breeding efficiency is improved. Polythioether-acrylate containing thioether bonds is formed, polythioether-acrylate and 2-hydroxyethyl acrylate are copolymerized under the action of an initiator, the ROS-responsive porous rice husk biochar adsorbent containing thioether bonds is obtained, the thioether bonds are broken in a high-reactive oxygen species (ROS) environment, internal supported modified rice husk biochar is exposed, and the adsorbent is used for adsorbing the ROS-responsive porous rice husk biochar adsorbent containing the thioether bonds and the ROS-responsive porous rice husk biochar adsorbent containing the thioether bonds and the ROS-responsive porous rice husk biochar adsorbent containing the thioether bonds and the ROS-responsive porous rice husk biochar. When the antibacterial agent is added into a culture medium, the culture medium has a good growth environment, garlic growth can be promoted, harmful substances such as phenols released in the garlic growth process can be reduced, and bacterium breeding can be avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue culture media, and specifically relates to a culture medium composition for garlic gene editing breeding and a preparation method thereof. Background Art

[0002] Garlic is an annual plant of the genus Allium in the Liliaceae family. Most cultivated varieties of garlic cannot form seeds and are usually propagated asexually. This restricts the biodiversity and genetic characteristics of garlic, causing serious degeneration of varietal characteristics and seriously affecting the yield and quality of garlic. Long-term asexual propagation also leads to the continuous accumulation of garlic viruses, seriously affecting the growth of garlic plants and the commercial value of bulbs. Currently, the application of technologies such as plant tissue culture, artificial mutagenesis, genetic engineering, and garlic polyploid breeding has greatly improved the breeding work of excellent garlic varieties. By optimizing the garlic genetic transformation system and constructing gene editing vectors, precise editing of the garlic FT homologous gene has been successfully achieved, and multiple new germplasms with early maturity, no bolt, and improved bulb traits have been obtained, providing technical support for the upgrading of the garlic industry.

[0003] A Chinese patent with the publication number CN106386500B discloses a culture medium for garlic virus-free tissue culture seedlings. Adding a certain concentration of activated carbon to this culture medium can effectively reduce the discoloration of garlic tissues and the culture medium, effectively adsorb harmful substances and impurities in the culture medium, and also play an adsorption role on the harmful metabolites generated during the tissue culture process. However, in this scheme, activated carbon mainly captures substances through physical adsorption and pore filling, and its adsorption range covers small molecule organic substances. Therefore, while adsorbing harmful substances, activated carbon can also adsorb beneficial substances such as growth regulators, vitamin B6, folic acid, and niacin, thus affecting the growth of garlic. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture medium composition for garlic gene editing breeding and a preparation method thereof. Copper peroxide is generated on the surface of modified rice husk biochar, and by utilizing the highly nucleophilic hydrogen atoms of thiol to attack the double bond of acrylate, a polythioether-acrylate containing a thioether bond is formed. The polythioether-acrylate and 2-hydroxyethyl acrylate copolymerize under the action of an initiator to obtain a ROS-responsive porous rice husk biochar adsorbent containing a thioether bond. In a high reactive oxygen species (ROS) environment, the thioether bond breaks, exposing the internal supported modified rice husk biochar, which is added to the culture medium to enable the culture medium to have a good growth environment, promote the growth of garlic, reduce harmful substances such as phenols released during the growth of garlic, and avoid the growth of bacteria.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A preparation method of a culture medium composition for garlic gene editing breeding, which is prepared by the following steps:

[0007] Generate a ROS-responsive porous rice husk biochar adsorbent containing thioether bonds on the surface of the supported modified rice husk biochar; stir and dissolve sucrose, plant gel, 2,4-dichlorophenoxyacetic acid, indoleacetic acid, ticarcillin sodium clavulanate (Timentin), kanamycin sulfate and the porous rice husk biochar adsorbent evenly in the MS medium to obtain a culture medium composition for garlic gene editing breeding.

[0008] Furthermore, the dosage ratio of sucrose, plant gel, 2,4-dichlorophenoxyacetic acid, indoleacetic acid, Timentin, kanamycin sulfate and the porous rice husk biochar adsorbent is 20-30 g: 2-3 g: 1-2 mg: 1-2 mg: 0.2-0.3 g: 0.1-0.2 g: 5-6 g.

[0009] Furthermore, the porous rice husk biochar adsorbent is specifically prepared by the following steps:

[0010] Add polythioether-acrylate, 2-hydroxyethyl acrylate and dimethyl sulfoxide into the reaction kettle, stir at 40-55 °C and 400-500 r / min for 1-2 h, then add 1-2 g of sodium dodecyl sulfate and deionized water, heat to 40-50 °C, continue to stir for 20-30 min, introduce ammonia gas at a flow rate of 10-12 min / L for 30-40 min, heat to 70-80 °C, add ammonium persulfate, continue to stir and react in a nitrogen atmosphere for 6-7 h, then add the supported modified rice husk biochar into the reaction kettle, continue to stir for 3-4 h, filter, wash the filter cake with deionized water and absolute ethanol respectively for 2-3 times, and vacuum dry at 60-80 °C for 1-2 h to obtain the porous rice husk biochar adsorbent.

[0011] Furthermore, the dosage ratio of polythioether-acrylate, 2-hydroxyethyl acrylate, dimethyl sulfoxide, sodium dodecyl sulfate, deionized water, ammonium persulfate and the supported modified rice husk biochar is 70-80 g: 80-90 g: 400-500 mL: 1-2 g: 3-4 L: 1-2 g: 50-60 g.

[0012] Furthermore, the supported modified rice husk biochar is prepared by the following steps:

[0013] Add copper sulfate pentahydrate and deionized water into a reaction kettle, stir for 20 - 30 min under the conditions of 50 - 60 °C and 400 - 500 r / min, then add modified rice husk biochar, continue to stir for 25 - 40 min, add an ammonia water solution with a mass fraction of 20 - 30%, adjust the pH value to 10.5 - 11, then add a hydrogen peroxide solution with a mass fraction of 25 - 30%, continue to stir for 1 - 2 h, age for 24 - 26 h, filter, wash the filter cake with deionized water and absolute ethanol until the last washing liquid is neutral, and dry it in vacuum at 60 - 80 °C for 1 - 2 h to obtain the supported modified rice husk biochar.

[0014] Further, the dosage ratio of copper sulfate pentahydrate, deionized water, modified rice husk biochar, ammonia water solution and hydrogen peroxide solution is 20 - 30 g : 800 - 900 mL : 35 - 45 g : 10 - 20 mL : 50 - 60 mL.

[0015] Further, the modified rice husk biochar is prepared by the following steps:

[0016] Place rice husks in a muffle furnace, calcine at 500 - 600 °C for 1 - 2 h under a nitrogen atmosphere, crush, and pass through a 200 - 300 - mesh sieve to obtain rice husk biochar; add the rice husk biochar, glucose, acrylic acid and deionized water into a reaction kettle, ultrasonically disperse for 30 - 40 min, stir at 170 - 180 °C and 400 - 500 r / min for 6 - 7 h, then add chitosan and an acetic acid solution with a mass fraction of 5 - 6% and continue to stir for 2 - 3 h, filter, wash the filter cake with deionized water and absolute ethanol 2 - 3 times respectively, and dry it in vacuum at 60 - 80 °C for 1 - 2 h to obtain the modified rice husk biochar.

[0017] Further, the dosage ratio of rice husk biochar, glucose, acrylic acid, deionized water, chitosan and acetic acid solution is 50 - 60 g : 70 - 80 g : 100 - 120 mL : 600 - 800 mL : 50 - 60 g : 12 - 15 mL.

[0018] Further, the polythioether - acrylate is prepared by the following steps:

[0019] Add ethylene glycol diacrylate and 1,2 - benzenedithiol into a reaction kettle, stir for 20 - 30 min under the conditions of 20 - 25 °C and 400 - 500 r / min, then add dichloromethane, 4,4'-diaminodiphenylmethane, ammonium persulfate and triethylamine, heat to 50 - 60 °C, continue to stir and react for 24 - 26 h, rotary evaporate to remove dichloromethane, transfer to 4 - 6 times the volume of ether for precipitation, centrifuge at 2000 - 2500 r / min, discard the supernatant, wash the product with ether and deionized water 2 - 3 times, and dry it in vacuum at 60 - 80 °C for 1 - 2 h to obtain the polythioether - acrylate.

[0020] Further, the dosage ratio of ethylene glycol diacrylate, 1,2-benzenedithiol, dichloromethane, 4,4'-diaminodiphenylmethane, ammonium persulfate and triethylamine is 80-90 g: 50-60 mL: 200-300 mL: 30-40 g: 1-2 g: 2-3 mL.

[0021] Advantages of the present invention:

[0022] 1. The culture medium composition for garlic gene editing and breeding prepared by the present invention generates cupric peroxide on the surface of modified rice husk biochar. In the culture medium, cupric peroxide slowly reacts with water to generate copper hydroxide; the hydrogen atom of thiol has high nucleophilicity and will attack the double bond of acrylate to form a thioether bond, obtaining polythioether-acrylate. Polythioether-acrylate and 2-hydroxyethyl acrylate copolymerize under the action of an initiator to obtain a hydrogel film containing thioether bonds covering the surface of the supported modified rice husk biochar, obtaining a porous rice husk biochar adsorbent, which is added to the culture medium, enabling the culture medium to have a good growth environment, promoting the growth of garlic, reducing harmful substances such as phenols released during the growth of garlic, and avoiding the growth of bacteria.

[0023] 2. The supported modified rice husk biochar of the present invention has a chitosan film covering the surface of the rice husk biochar, and contains a large number of groups on the surface, which can adsorb copper ions. Under the action of copper ions in an alkaline condition and hydrogen peroxide, cupric peroxide is generated and adheres to the surface of the modified rice husk biochar. In the culture medium, cupric peroxide slowly reacts with water to generate copper hydroxide, and copper hydroxide has bactericidal properties and can effectively prevent common diseases of garlic. By being supported on the surface of the modified rice husk biochar, it can avoid the problem of local concentration being too high caused by directly adding copper hydroxide traditionally, which may lead to plant poisoning.

[0024] 3. The porous rice husk biochar adsorbent of the present invention copolymerizes with 2-hydroxyethyl acrylate using polythioether-acrylate containing thioether bonds as a monomer to obtain a hydrogel film containing thioether bonds covering the surface of the supported modified rice husk biochar. In a high reactive oxygen species (ROS) environment, the thioether bond is broken, and then the hydrogel film ruptures. The ruptured hydrogel film will expose the internal supported modified rice husk biochar, thereby playing an antibacterial role and an adsorption role for metabolites; by adding the porous rice husk biochar adsorbent to the culture medium, it can effectively prevent garlic browning, effectively adsorb harmful substances such as phenols released during the garlic breeding process, and reduce the occurrence probability of browning seedlings during the garlic breeding process. In this way, it can avoid the adsorption of beneficial substances such as vitamin B6, folic acid, and niacin caused by directly adding activated carbon traditionally. Specific embodiments

[0025] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] Embodiment 1: A preparation method of a culture medium composition for garlic gene editing and breeding is prepared by the following steps:

[0027] S1: Place 100 g of rice husks in a muffle furnace, heat to 500 °C under a nitrogen atmosphere, calcine for 1 h, crush, and pass through a 200-mesh sieve to obtain rice husk biochar; add 50 g of rice husk biochar, 70 g of glucose, 100 mL of acrylic acid, and 600 mL of deionized water to a reaction kettle, ultrasonically disperse for 30 min, stir at 170 °C and 400 r / min for 6 h, then add 50 g of chitosan and 12 mL of a 5% acetic acid solution by mass fraction and continue to stir for 2 h, filter, wash the filter cake twice with deionized water and absolute ethanol respectively, and vacuum dry at 60 °C for 1 h to obtain modified rice husk biochar.

[0028] Glucose is carbonized at high temperature to form an amorphous carbon layer, which tightly wraps the rice husk biochar. The carbon-carbon double bond (C=C) of acrylic acid undergoes an addition reaction with the free radicals generated by the pyrolysis of glucose under high-temperature hydrothermal conditions to fix the carboxyl group on the surface of the rice husk biochar.

[0029] S2: Add 20 g of copper sulfate pentahydrate and 800 mL of deionized water to a reaction kettle, stir at 50 °C and 400 r / min for 20 min, then add 35 g of modified rice husk biochar, continue to stir for 25 min, add 10 mL of a 20% ammonia water solution by mass fraction to adjust the pH value to 10.5, then add 50 mL of a 25-30% hydrogen peroxide solution by mass fraction, continue to stir for 1 h, age for 24 h, filter, wash the filter cake with deionized water and absolute ethanol until the last washing liquid is neutral, and vacuum dry at 60 °C for 1 h to obtain supported modified rice husk biochar.

[0030] Rice husk biochar contains abundant amorphous silicon. This is because the silicon content of rice husks itself is relatively high, and silicon is retained during the pyrolysis process. Silicon crosslinks with the cell wall components, making the cell wall thicker and harder. The enhanced cell wall can improve the resistance of garlic to pathogenic bacteria.

[0031] The surface of the modified rice husk biochar is covered with a layer of chitosan film and contains a large number of groups. The negatively charged carboxyl groups can adsorb positively charged copper ions. Under the action of copper ions and hydrogen peroxide under alkaline conditions, cupric peroxide is generated and adheres to the surface of the modified rice husk biochar. In the culture medium, cupric peroxide slowly reacts with water to form copper hydroxide.

[0032] S3: Add 80 g of ethylene glycol diacrylate and 50 mL of 1,2-benzenedithiol into the reaction kettle, stir for 20 min under the conditions of 20 °C and 400 r / min, then add 200 mL of dichloromethane, 30 g of 4,4'-diaminodiphenylmethane, 1 g of ammonium persulfate and 2 mL of triethylamine, heat to 50 °C, continue to stir and react for 24 h, rotary evaporate to remove dichloromethane, transfer to 4 times the volume of diethyl ether for precipitation, centrifuge at 2000 r / min, discard the supernatant, wash the product twice with diethyl ether and deionized water, and vacuum dry at 60 °C for 1 h to obtain polythioether-acrylate.

[0033] The hydrogen atom of thiol (-SH) has high nucleophilicity and will attack the double bond (C=C) of acrylate. By controlling the dosage, Michael addition reaction occurs between part of the double bonds in 1,2-benzenedithiol and ethylene glycol diacrylate to form a hydrogel film containing thioether bonds covering the surface of the supported modified rice husk biochar. In a high reactive oxygen species (ROS) environment, this process causes the cleavage of thioether bonds, which in turn leads to the rupture of the hydrogel film, and the ruptured hydrogel film will expose the internal supported modified rice husk biochar.

[0034] S4: Add 70 g of polythioether-acrylate, 80 g of 2-hydroxyethyl acrylate and 400 mL of dimethyl sulfoxide into the reaction kettle, stir for 1 h under the conditions of 40 °C and 400 r / min, then add 1 g of sodium dodecyl sulfate and 3 L of deionized water, heat to 40 °C, continue to stir for 20 min, introduce ammonia gas at a flow rate of 10 min / L for 30 min, heat to 70 °C, add 1 g of ammonium persulfate, and continue to stir and react for 6 h under a nitrogen atmosphere. Then add 50 g of the supported modified rice husk biochar into the reaction kettle, continue to stir for 3 h, filter, wash the filter cake twice with deionized water and absolute ethanol respectively, and vacuum dry at 60 °C for 1 h to obtain a ROS-responsive porous rice husk biochar adsorbent containing thioether bonds.

[0035] Polythioether-acrylate and 2-hydroxyethyl acrylate copolymerize under the action of an initiator to obtain a hydrogel film containing thioether bonds covering the surface of the supported modified rice husk biochar.

[0036] S5: Stir 20 g of sucrose, 2 g of plant gel, 1 mg of 2,4-dichlorophenoxyacetic acid, 1 mg of indoleacetic acid, 0.25 g of ticarcillin sodium clavulanate, 0.1 g of kanamycin sulfate, and 5 g of porous rice husk biochar adsorbent evenly and dissolve them in MS medium to obtain a culture medium composition for garlic gene editing breeding.

[0037] Example 2: A preparation method of a culture medium composition for garlic gene editing breeding, which is prepared by the following steps:

[0038] S1: Place 125 g of rice husks in a muffle furnace, heat them to 550 °C in a nitrogen atmosphere, calcine for 1.5 h, crush them, and pass through a 250-mesh sieve to obtain rice husk biochar; add 55 g of rice husk biochar, 75 g of glucose, 110 mL of acrylic acid, and 700 mL of deionized water to a reaction kettle, ultrasonically disperse for 35 min, stir at 175 °C and 450 r / min for 6.5 h, then add 55 g of chitosan and 13.5 mL of acetic acid solution with a mass fraction of 5.5% and continue to stir for 2.5 h, filter, wash the filter cake twice with deionized water and anhydrous ethanol respectively, and vacuum dry at 70 °C for 1.5 h to obtain modified rice husk biochar.

[0039] S2: Add 25 g of copper sulfate pentahydrate and 850 mL of deionized water to a reaction kettle, stir at 55 °C and 450 r / min for 25 min, then add 40 g of modified rice husk biochar, continue to stir for 32.5 min, add 15 mL of ammonia water with a mass fraction of 25%, adjust the pH value to 10.75, then add 55 mL of hydrogen peroxide solution with a mass fraction of 25-30%, continue to stir for 1.5 h, age for 25 h, filter, wash the filter cake with deionized water and anhydrous ethanol until the last washing liquid is neutral, and vacuum dry at 70 °C for 1.5 h to obtain supported modified rice husk biochar.

[0040] S3: Add 85 g of ethylene glycol diacrylate and 55 mL of 1,2-benzenedithiol to a reaction kettle, stir at 22.5 °C and 450 r / min for 25 min, then add 250 mL of dichloromethane, 35 g of 4,4'-diaminodiphenylmethane, 1.5 g of ammonium persulfate, and 2.5 mL of triethylamine, heat to 55 °C, continue to stir and react for 25 h, rotary evaporate to remove dichloromethane, transfer to 5 times the volume of ether for precipitation, centrifuge at 2250 r / min, discard the supernatant, wash the product twice with ether and deionized water, and vacuum dry at 70 °C for 1.5 h to obtain polythioether-acrylate.

[0041] S4: Add 75 g of polysulfide-acrylate, 85 g of 2-hydroxyethyl acrylate, and 450 mL of dimethyl sulfoxide into a reaction kettle, stir for 1.5 h under the conditions of 47.5 °C and 450 r / min, then add 1.5 g of sodium dodecyl sulfate and 3.5 L of deionized water, heat to 45 °C, continue to stir for 25 min, introduce ammonia gas at a flow rate of 11 min / L for 35 min, heat to 75 °C, add 1.5 g of ammonium persulfate, and continue to stir and react for 6.5 h under a nitrogen atmosphere. Then add 55 g of supported modified rice husk biochar into the reaction kettle, continue to stir for 3.5 h, filter, wash the filter cake twice with deionized water and anhydrous ethanol respectively, and dry it in vacuum at 70 °C for 1.5 h to obtain a ROS-responsive porous rice husk biochar adsorbent containing thioether bonds.

[0042] S5: Stir and dissolve 25 g of sucrose, 2.5 g of plant gel, 1.2 mg of 2,4-dichlorophenoxyacetic acid, 1.2 mg of indoleacetic acid, 0.28 g of ticarcillin sodium clavulanate, 0.15 g of kanamycin sulfate, and 5.6 g of porous rice husk biochar adsorbent evenly in MS medium to obtain a culture medium composition for garlic gene editing and breeding.

[0043] Example 3: A preparation method of a culture medium composition for garlic gene editing and breeding is prepared through the following steps:

[0044] S1: Place 150 g of rice husks in a muffle furnace, heat to 600 °C and calcine for 2 h under a nitrogen atmosphere, crush, and pass through a 300-mesh sieve to obtain rice husk biochar; add 60 g of rice husk biochar, 80 g of glucose, 120 mL of acrylic acid, and 800 mL of deionized water into a reaction kettle, ultrasonically disperse for 40 min, stir at 180 °C and 500 r / min for 7 h, then add 60 g of chitosan and 15 mL of acetic acid solution with a mass fraction of 6% and continue to stir for 3 h, filter, wash the filter cake three times with deionized water and anhydrous ethanol respectively, and dry it in vacuum at 80 °C for 2 h to obtain modified rice husk biochar.

[0045] S2: Add 30 g of copper sulfate pentahydrate and 900 mL of deionized water into a reaction kettle, stir for 30 min at 60 °C and 500 r / min, then add 45 g of modified rice husk biochar, continue to stir for 40 min, add 20 mL of ammonia water solution with a mass fraction of 30% to adjust the pH value to 11, then add 60 mL of hydrogen peroxide solution with a mass fraction of 25 - 30%, continue to stir for 2 h, age for 26 h, filter, wash the filter cake with deionized water and anhydrous ethanol until the last washing liquid is neutral, and dry it in vacuum at 80 °C for 2 h to obtain supported modified rice husk biochar.

[0046] S3: Add 90 g of ethylene glycol diacrylate and 60 mL of 1,2-benzenedithiol into a reaction kettle, stir for 30 min under the conditions of 25 °C and 500 r / min, then add 300 mL of dichloromethane, 40 g of 4,4'-diaminodiphenylmethane, 2 g of ammonium persulfate and 3 mL of triethylamine, heat to 60 °C, continue to stir and react for 26 h, rotary evaporate to remove dichloromethane, transfer to 6 times the volume of diethyl ether for precipitation, centrifuge at 2500 r / min, discard the supernatant, wash the product 3 times with diethyl ether and deionized water, and dry in vacuum at 80 °C for 2 h to obtain polythioether-acrylate.

[0047] S4: Add 80 g of polythioether-acrylate, 90 g of 2-hydroxyethyl acrylate and 500 mL of dimethyl sulfoxide into a reaction kettle, stir for 2 h under the conditions of 55 °C and 500 r / min, then add 2 g of sodium dodecyl sulfate and 4 L of deionized water, heat to 50 °C, continue to stir for 30 min, introduce ammonia gas at a flow rate of 12 min / L for 40 min, heat to 80 °C, add 2 g of ammonium persulfate, under a nitrogen atmosphere, continue to stir and react for 7 h, then add 60 g of supported modified rice husk biochar into the reaction kettle, continue to stir for 4 h, filter, wash the filter cake 3 times with deionized water and absolute ethanol respectively, and dry in vacuum at 80 °C for 2 h to obtain a ROS-responsive porous rice husk biochar adsorbent containing thioether bonds.

[0048] S5: Stir and dissolve 30 g of sucrose, 3 g of phytagel, 2 mg of 2,4-dichlorophenoxyacetic acid, 2 mg of indoleacetic acid, 0.3 g of ticarcillin sodium clavulanate, 0.2 g of kanamycin sulfate and 6 g of porous rice husk biochar adsorbent evenly in MS medium to obtain a culture medium composition for garlic gene editing and breeding.

[0049] Comparative Example 1: On the basis of Example 3, replace the modified rice husk biochar in step S2 with commercially available activated carbon of the same mass, and keep the other steps unchanged to prepare a culture medium composition for garlic gene editing and breeding.

[0050] Comparative Example 2: On the basis of Example 3, replace the supported modified rice husk biochar in step S4 with the modified rice husk biochar in step S1 of the same mass, and keep the other steps unchanged to prepare a culture medium composition for garlic gene editing and breeding.

[0051] Comparative Example 3: On the basis of Example 3, replace the polythioether-acrylate in step S4 with commercially available N,N'-methylenebis(propylamide) of the same mass, and keep the other steps unchanged to prepare a culture medium composition for garlic gene editing and breeding.

[0052] In the examples and comparative examples:

[0053] The MS medium was purchased from Jinclon (Beijing) Biotechnology Co., Ltd.

[0054] Ticarcillin sodium and clavulanate potassium (Timentin), with the composition of ticarcillin and clavulanic acid in a mass ratio of 15:1, was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0055] 2-Hydroxyethyl acrylate was purchased from Sigma-Aldrich.

[0056] Kanamycin sulfate was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0057] Performance tests were conducted on the culture medium compositions for garlic gene editing breeding prepared in Examples 1 - 3 and Comparative Examples 1 - 3. The results are shown in Table 1:

[0058] The callus was infected with the GV3101 strain. After co-cultivation, resistant callus was obtained through screening with Timentin. Finally, transgenic seedlings were differentiated. The garlic bulbs that had passed the dormancy period and had intact surfaces were peeled, rinsed 3 times with sterile water, soaked in 70% ethanol for 3 min, then soaked in 50% sodium hypochlorite solution for disinfection for 30 min, and finally rinsed 3 times with sterile water. They were placed on the MS solid medium and cultured at 24°C with 16 h of light and 8 h of darkness to root. When the garlic roots grew to 5 cm, the roots were cut into 1-cm lengths and placed in the callus induction MS1 medium (4.4 g / L MS medium + 30 g / L sucrose + 1 mg / L 2,4-dichlorophenoxyacetic acid + 0.1 mg / L indoleacetic acid, pH 5.8) for culture. After 1 month, callus was obtained. The callus was transferred to the culture medium composition for garlic gene editing breeding and cultured at 24°C with light (12 h of light and 12 h of darkness). The medium was changed once every 3 weeks until buds were differentiated.

[0059] Table 1 Results of garlic growth

[0060] Project Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Number of inoculations (pcs) 50 50 50 50 50 50 Induction rate (%) 70 72 74 36 42 48 Number of somatic embryos produced (pcs) 35 36 37 18 21 24 Number of buds produced (pcs) 45 46 47 32 38 35 Bud formation rate (%) 90 92 94 64 76 70

[0061] As can be seen from Table 1, for the garlic bred with the culture medium compositions for garlic gene editing breeding prepared in Examples 1 - 3, the induction rate, the number of somatic embryos, the number of buds, and the budding rate are all significantly higher than those of the comparative examples. This shows that the culture medium composition for garlic gene editing breeding prepared in the present invention has a good growth environment, can promote the growth of garlic, can reduce the phenolic harmful substances released during the growth of garlic, and can avoid the growth of bacteria.

[0062] On the basis of Example 3 in Comparative Example 1, the modified rice husk biochar in Step S2 was replaced with commercially available activated carbon of the same mass. A layer of chitosan film was covered on the surface of the rice husk biochar. The surface contains a large number of groups, which can adsorb copper ions and enable copper ions to be more stably generated on the surface of the modified rice husk biochar under the action of hydrogen peroxide under alkaline conditions. Moreover, the rice husk biochar contains abundant amorphous silicon. This is because the silicon content of the rice husk itself is relatively high, and silicon is retained during the pyrolysis process. Silicon crosslinks with the cell wall components, making the cell wall thicker and harder. The enhanced cell wall can improve the resistance of garlic to pathogenic bacteria.

[0063] In Comparative Example 2, the supported modified rice husk biochar was replaced with modified rice husk biochar of the same mass. Cupric peroxide was generated on the surface of the supported modified rice husk biochar. In the culture medium, cupric peroxide slowly reacts with water to form copper hydroxide. Copper hydroxide has bactericidal properties and can effectively prevent common diseases of garlic. By being supported on the surface of the modified rice husk biochar, it can avoid the excessive local concentration caused by traditional direct addition, which may lead to plant poisoning.

[0064] In Comparative Example 3, the polythioether-acrylate was replaced with N,N'-methylenebispropylamide of the same mass. The hydrogen atom of the mercaptan (-SH) has high nucleophilicity and will attack the double bond (C=C) of the acrylate, undergoing a Michael addition reaction to form a thioether bond. In a highly reactive oxygen species (ROS) environment, this process causes the thioether bond to break. By adding a porous rice husk biochar adsorbent to the culture medium, it can effectively prevent the browning of explants, effectively adsorb the phenols released during the breeding process of sweet garlic, and reduce the occurrence probability of browning seedlings during the garlic breeding process. In this way, it can avoid the adsorption of beneficial substances such as vitamin B6, folic acid, and niacin caused by traditional direct addition of activated carbon.

[0065] It should be noted that in this article, terms such as "including", "comprising", or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device including a series of elements not only includes those elements but also includes other elements not explicitly listed, or elements inherent to such a process, method, article, or device.

[0066] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

Claims

1. A preparation method of a culture medium composition for garlic gene editing breeding, characterized in that, It is prepared by the following steps: Generate a ROS-responsive porous rice husk biochar adsorbent containing thioether bonds on the surface of the supported modified rice husk biochar; stir and dissolve sucrose, phytagel, 2,4-dichlorophenoxyacetic acid, indoleacetic acid, ticarcillin sodium clavulanate, kanamycin sulfate and the porous rice husk biochar adsorbent evenly in MS medium to obtain a culture medium composition for garlic gene editing and breeding.

2. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 1, characterized in that, The dosage ratio of the sucrose, phytagel, 2,4-dichlorophenoxyacetic acid, indoleacetic acid, Timentin, kanamycin sulfate and the porous rice husk biochar adsorbent is 20 - 30 g: 2 - 3 g: 1 - 2 mg: 1 - 2 mg: 0.2 - 0.3 g: 0.1 - 0.2 g: 5 - 6 g.

3. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 1, characterized in that, The porous rice husk biochar adsorbent is specifically prepared by the following steps: Add polythioether-acrylate, 2-hydroxyethyl acrylate and dimethyl sulfoxide into the reaction kettle, stir at 40 - 55 °C and 400 - 500 r / min for 1 - 2 h, then add sodium dodecyl sulfate and deionized water, heat to 40 - 50 °C, continue to stir for 20 - 30 min, introduce ammonia gas at a flow rate of 10 - 12 min / L for 30 - 40 min, heat to 70 - 80 °C, add ammonium persulfate, and continue to stir and react for 6 - 7 h under a nitrogen atmosphere. Then add the supported modified rice husk biochar into the reaction kettle, continue to stir for 3 - 4 h, filter, wash, and vacuum dry to obtain the porous rice husk biochar adsorbent.

4. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 3, characterized in that, The dosage ratio of the polythioether-acrylate, 2-hydroxyethyl acrylate, dimethyl sulfoxide, sodium dodecyl sulfate, deionized water, ammonium persulfate and the supported modified rice husk biochar is 70 - 80 g: 80 - 90 g: 400 - 500 mL: 1 - 2 g: 3 - 4 L: 1 - 2 g: 50 - 60 g.

5. The preparation method of a culture medium composition for garlic gene editing and breeding according to claim 3, characterized in that, The supported modified rice husk biochar is prepared by the following steps: Add copper sulfate pentahydrate and deionized water into the reaction kettle, stir at 50 - 60 °C and 400 - 500 r / min for 20 - 30 min, then add the modified rice husk biochar, continue to stir for 25 - 40 min, add 20 - 30 wt% ammonia water solution to adjust the pH value to 10.5 - 11, then add 25 - 30 wt% hydrogen peroxide solution, continue to stir for 1 - 2 h, age for 24 - 26 h, filter, wash, and vacuum dry to obtain the supported modified rice husk biochar.

6. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 5, characterized in that, The dosage ratio of the copper sulfate pentahydrate, deionized water, modified rice husk biochar, ammonia water solution and hydrogen peroxide solution is 20 - 30 g: 800 - 900 mL: 35 - 45 g: 10 - 20 mL: 50 - 60 mL.

7. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 5, characterized in that, The modified rice husk biochar is prepared by the following steps: The rice husk is placed in a muffle furnace and calcined at 500 - 600 °C for 1 - 2 h under a nitrogen atmosphere, then crushed and sieved through a 200 - 300 mesh sieve to obtain rice husk biochar; the rice husk biochar, glucose, acrylic acid and deionized water are added to a reaction kettle, ultrasonically dispersed for 30 - 40 min, stirred at 170 - 180 °C and 400 - 500 r / min for 6 - 7 h, then chitosan and a 5 - 6 wt% acetic acid solution are added and stirring is continued for 2 - 3 h, followed by filtration, washing and vacuum drying to obtain modified rice husk biochar.

8. The preparation method of a culture medium composition for garlic gene editing and breeding according to claim 7, characterized in that, The dosage ratio of the rice husk biochar, glucose, acrylic acid, deionized water, chitosan and acetic acid solution is 50 - 60 g : 70 - 80 g : 100 - 120 mL : 600 - 800 mL : 50 - 60 g : 12 - 15 mL.

9. The preparation method of a culture medium composition for garlic gene editing breeding according to claim 3, characterized in that, The polythioether - acrylate is prepared by the following steps: Ethylene glycol diacrylate and 1,2 - benzenedithiol are added to a reaction kettle, stirred at 20 - 25 °C and 400 - 500 r / min for 20 - 30 min, then dichloromethane, 4,4'-diaminodiphenylmethane, ammonium persulfate and triethylamine are added, heated to 50 - 60 °C and stirring reaction is continued for 24 - 26 h, the dichloromethane is removed by rotary evaporation, transferred to 4 - 6 times the volume of ether for precipitation, centrifuged at 2000 - 2500 r / min, the supernatant is discarded, the product is washed 2 - 3 times with ether and deionized water, and vacuum dried to obtain polythioether - acrylate; The dosage ratio of ethylene glycol diacrylate, 1,2 - benzenedithiol, dichloromethane, 4,4'-diaminodiphenylmethane, ammonium persulfate and triethylamine is 80 - 90 g : 50 - 60 mL : 200 - 300 mL : 30 - 40 g : 1 - 2 g : 2 - 3 mL.

10. A culture medium composition for garlic gene editing breeding, characterized in that, Prepared by the preparation method according to any one of claims 1 - 9.