CN115589945A - Method for openly culturing ginger test-tube plantlets - Google Patents
Method for openly culturing ginger test-tube plantlets Download PDFInfo
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Abstract
The invention belongs to the technical field of horticultural plant biology, and discloses an open type ginger test-tube seedling culture method which comprises the steps of ginger root stem germination acceleration, compound bacteriostatic agent preparation, culture medium preparation, inoculation tool and inoculation chamber disinfection, cluster bud induction, multiplication, strong seedling culture and test-tube seedling domestication and transplantation. According to the invention, the compound bacteriostatic agent is added into the culture medium to establish the ginger open tissue culture system, so that the occurrence of pollution of the test-tube plantlet can be effectively inhibited, the culture medium does not need high-temperature and high-pressure sterilization, the inoculation operation does not need a superclean workbench, and the inoculation operation only needs to be carried out in a clean room, so that the ginger tissue culture link is fundamentally simplified, and the production cost of the test-tube plantlet is reduced. Compared with the traditional tissue culture, the method has the advantages of less production investment, strong practicability and cost reduction of more than 30 percent, and is suitable for the commercial production of the ginger test-tube plantlet.
Description
Technical Field
The invention belongs to the technical field of horticultural plant biology, and particularly relates to an open method for culturing ginger test-tube plantlets.
Background
Ginger (Zingiber officinale Roscoe) is a perennial root herbaceous plant in Zingiberaceae, has the characteristics of homology of medicine and food, has unit area economic benefit in the front of crops, and has become a high-efficiency supporting industry for increasing income of farmers, improving efficiency of agriculture and promoting the pleasure of national villages. In the production of the ginger, mature rhizome is mainly used for asexual propagation, the propagation coefficient is low, and continuous years of planting causes soil-borne diseases such as ginger blast and the like to be aggravated year by year, so that the growth vigor is reduced, the seed nature is degraded, the yield and the quality are reduced, and the method becomes an important factor for restricting the production of the ginger.
In order to prevent diseases and improve the propagation coefficient, at present, shoot apical meristem is generally adopted to culture and remove ginger germ virus in vitro, and then a seedling and ginger seed rapid propagation system is established. The ginger seedlings obtained by detoxification, bacterium removal and tissue culture can obviously improve the quality of the ginger, greatly reduce virus and germ infection and reduce the cost investment in planting. The prior art shows that the yield of the ginger virus-free seedling planting land is 10 times of that of the traditional cultivation, and the production seed ratio coefficient of the test-tube seedlings to the ginger is 1:300, the average yield of a single plant reaches 800g, the yield per mu reaches 5260kg, and the yield is improved by more than 30-45 percent compared with the traditional planting of ginger. However, the ginger in-vitro rapid propagation system requires strict sterile environment and sterile operation, the operation steps are complicated, and pollution is often caused by negligence of one small link. In addition, the aseptic operation of traditional group banks up needs to be carried out in superclean bench, and the culture medium of configuration needs to consume the electric energy through high temperature autoclaving, and is also higher to the requirement of culture vessel. These factors result in higher production cost of traditional tissue culture seedlings of ginger, which limits the commercial development of ginger.
In order to reduce the production cost of tissue culture seedlings and simplify the working procedures, researchers have been striving to explore a new technology capable of producing test-tube seedlings efficiently at low cost and on a large scale. Open tissue culture of plants refers to that under the action of a bacteriostatic agent, the in vitro culture of plants is separated from a strict sterile operating environment, a culture medium does not need high-temperature high-pressure sterilization, inoculation can be carried out in a super clean bench, a common container is used for replacing a traditional tissue culture bottle, and the in vitro culture of plants is carried out in a natural and open sterile environment. Compared with the traditional tissue culture technology, the open tissue culture can simplify the operation procedure, reduce the tissue culture cost, control the pollution rate in an acceptable range and does not influence the growth and development of explants. In addition, high-temperature and high-pressure sterilization is not needed in the open tissue culture, the loss of plant growth regulators and nutrient elements in the culture medium is less, and the light transmittance of the culture container is better than that of the traditional tissue culture container. Many researches show that the regenerated plants of open tissue culture grow strongly, the differentiation rate is relatively high, and the survival rate of transplanted plants is also high. Therefore, plant open tissue culture has been favored by researchers since its birth, and has attracted much attention to plants such as sugarcane, acacia, potato, peony, and the like.
The screening of effective bacteriostats (combinations) and their optimal concentrations is the key to the successful establishment of an open tissue culture system. The ideal bacteriostatic agent formula can effectively inhibit the microbial contamination of the culture medium, and has no obvious negative effect on the growth and development of the culture in the bottle. Many researches have carried out a large number of tests of singly or compositely adding common antibiotics, antiseptics or plant active substances into a culture medium, and the results show that the antibiotics, the antiseptics or the plant active substances have certain effects on the prevention and treatment of the tissue culture pollution of plants. However, it is difficult to find a suitable combination of bacteriostats for different species, and often the bacteriostats in the culture medium have an inhibitory effect on the growth of plant tissue or have poor antibacterial effect. This is probably because there is no bacteriostatic agent which is effective against all bacteria or fungi at present, and some antibiotics and antiseptics have bactericidal and antibacterial components which are harmful to plant tissues directly under effective concentration, and some have salt harm. Aiming at the problems in the screening of the bacteriostatic agent (combination), a successful open tissue culture system can be established only by selecting substances which are compatible and friendly with plant tissues, have long drug effect period (at least one growth period), do not generate salt damage and have bactericidal and antibacterial activities.
The ginger has the functions of diminishing inflammation and resisting bacteria, and the ginger ether extract has obvious inhibiting effect on various microorganisms such as staphylococcus aureus, bacillus subtilis, aspergillus niger, penicillium, escherichia coli and saccharomyces cerevisiae. Studies find that the ethanol extract of ginger with the concentration of 0.25-4.0% has the inhibiting effect on bacteria, saccharomycetes and mould at the pH value of 6-8. Yipelong is a long-acting, broad-spectrum, high-activity, novel anti-pollution bactericide special for tissue culture. After Yipelong is contacted with microbes, the cells can be killed, so that the growth of the cells can be quickly killed or inhibited. The sodium hypochlorite is a broad-spectrum bactericide with rapid action, low toxicity and low price, and has strong killing capability on viruses, bacteria, fungi and spores. The effective ginger extract, yipelong and sodium hypochlorite are screened to compound the bacteriostatic agent and applied to the production of the ginger detoxified seedlings, so that the production cost can be effectively reduced. However, at present, an open ginger tissue culture system is not established, and a proper bacteriostatic agent formula is not screened.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) The conventional ginger test-tube plantlet culture method needs operations such as explant disinfection, culture medium sterilization, sterile inoculation, sterile culture and the like, and has the disadvantages of high cost, more complicated operation steps, high environmental requirement and high possibility of pollution. These factors limit the commercialization and industrialization of ginger tissue culture seedlings;
(2) The prior bacteriostatic agent formula has poor bacteriostatic effect when applied to open tissue culture of ginger, can inhibit and damage the growth and development of tissues or organs of ginger, and cannot show the superiority of the open tissue culture technology.
Disclosure of Invention
Aiming at the problems of complicated operation steps, high cost and the like of the conventional ginger tissue culture technology, the invention provides an open type method for culturing ginger test-tube plantlets.
The invention is realized in such a way that the method for openly culturing the ginger test-tube plantlet comprises the following steps:
after the surfaces of the roots and the stems of the clean ginger are disinfected, the roots and the stems of the clean ginger are cleaned by clear water and then placed in wet sand for accelerating germination;
preparing a ginger extracting solution and a Yipeilong mother solution, and preparing a compound bacteriostatic agent;
cleaning a tissue culture container, airing, sterilizing by using an ultraviolet lamp, and preparing a cluster bud induction culture medium and a propagation and seedling strengthening culture medium containing a compound bacteriostatic agent;
after dust fall and sterilization, the inoculation chamber is irradiated by an ultraviolet lamp, and an inoculation tool is sterilized by ethanol and then is soaked in the compound bacteriostatic agent aqueous solution;
disinfecting young buds of ginger rootstock by mercuric chloride solution, peeling off outer leaf sheaths by using a scalpel, and inoculating cut bud tips into a cluster bud induction culture medium for culture;
trimming the induced cluster buds into micro-blocks, and transferring the micro-blocks into a proliferation and strong seedling culture medium for culture;
cleaning the regenerated aseptic seedlings with a root culture medium, transplanting the aseptic seedlings into a plug tray filled with a seedling culture substrate, watering the aseptic seedlings thoroughly, putting the aseptic seedlings into an intelligent glass greenhouse for hardening the seedlings, and planting the aseptic seedlings in the field when new leaves grow out;
further, when accelerating germination of ginger rhizome, soaking the cleaned ginger rhizome in 1000 times diluted carbendazim for 30min, cleaning with clear water, placing in sterilized sand, adding a proper amount of tap water to keep humidity, and accelerating germination for 20-30d under the dark condition of 25 +/-2 ℃;
further, when preparing the crude ginger extract, ginger blocks are cleaned and peeled, cut into ginger slices with the thickness of 5mm, placed in a constant-temperature drying oven for drying for 15 hours at the temperature of 60 ℃, crushed by a crusher, sieved by a 60-mesh sieve, and weighed into ginger powder according to the material-liquid ratio of 1:10 Adding 50ml 70% ethanol solution (g/mL), soaking for 0.5h, refluxing with Soxhlet extractor until the liquid in the extractor is colorless, extracting at 60 deg.C, and filtering with 0.22 μm pore size filter membrane to obtain yellow oily crude extractive solution. Extracting multiple parts in parallel, mixing the crude extractive solutions, and rotary evaporating with rotary evaporator (at 45 deg.C) until ethanol is completely volatilized. Preparing 1g (ginger powder mass)/mL of ginger extract solution from the paste by using 50% ethanol as a solvent, and storing in a refrigerator at 4 ℃;
further, when preparing Yipelong mother liquor, yipelong B bottle white powder is dissolved in Yipelong A bottle buffer solution, and 10mL of distilled water is added to dilute to 4.35 × 10 4 mg/L mother liquor;
further, when the compound bacteriostatic agent is prepared, taking 15.5mL of the ginger extract solution, 1.0mL of the Yipelong mother liquor and 0.05mL of a 10% sodium hypochlorite solution, mixing, and then using distilled water to fix the volume to 100mL, thus obtaining a compound bacteriostatic agent aqueous solution;
further, the culture medium for inducing, proliferating and culturing the cluster buds and the strong seedlings is heated to boiling after being added with all the components, 1mL/L of compound bacteriostatic agent is added into the culture medium when the temperature of the culture medium is reduced to 50 ℃, and the culture medium is immediately subpackaged into a culture container which is cleaned in advance, dried in the air and sterilized by an ultraviolet lamp for 30min for standby after being uniformly mixed, without high-temperature high-pressure sterilization;
further, spraying 75% ethanol on an inoculation chamber for dust reduction, irradiating for 30min by using an ultraviolet lamp, disinfecting an inoculation tool by using 75% ethanol, soaking in 1.0% (v/v) of a compound bacteriostatic agent aqueous solution for at least more than 1h, and soaking in the bacteriostatic agent aqueous solution all the time during inoculation;
further, when the cluster buds are induced, the newly germinated 1.5-2 cm tender shoots obtained by accelerating germination of ginger rootstocks are taken, washed by tap water for 15min, soaked by 70% ethanol for 15s, then soaked by 0.1% (m/v) mercuric chloride solution for 15min, washed by sterile water for 5 times after disinfection treatment, and then the water on the surfaces of the tender shoots is sucked by sterile filter paper. Stripping off outer leaf sheath with scalpel, cutting 0.3-0.5cm bud tip, and inoculating into MS +0.5mg/LNAA +2.0 mg/L6-BA +1mL/L complex bacteriostatic agent +30g/L sucrose +7.0g/L agar (pH 5.8) cluster bud induction culture medium;
further, after inducing the cluster buds for 2 months, cutting off the part of the base part of the induced cluster buds more than 0.5cm to form micro blocks, and transferring the micro blocks to a multiplication and strong seedling culture medium of MS +1 mg/L6-BA +0.2mg/L IBA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar (pH 5.8);
further, the inoculation materials in the induction, proliferation and strong seedling culture of the cluster buds are all cultured in a culture room with the illumination intensity of about 2000lx, the photoperiod of 14h/10h and the temperature of 25 +/-2 ℃;
further, after 2 months of proliferation and strong seedling culture, opening the bottle cap of a can bottle after the height of the seedling is 5-6cm, injecting a small amount of tap water to enable the aseptic seedling to slowly adapt to the aseptic environment, cleaning the culture medium at the root of the aseptic seedling with the tap water after 3 days, transplanting the seedling into a 32-hole plug tray filled with a seedling culture medium, planting 1 plant in each hole, wherein the volume ratio of the medium is 3:1, pindstrup peat soil and perlite. After being watered thoroughly, the seedlings are placed in an intelligent glass greenhouse for hardening, and the domestication conditions are that the temperature is 25 ℃, the relative humidity is 80 percent, and the light intensity is about 3000 lx.
In combination with the technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:
according to the invention, the screened compound bacteriostatic agent is used for open tissue culture seedling raising of the ginger, so that the pollution of test-tube seedlings can be effectively avoided. In the process of inducing and proliferating the cluster buds, compared with the traditional tissue culture, the pollution rate, the cluster bud induction rate and the proliferation rate of the open tissue culture have no obvious difference. From the growth condition of the test-tube plantlet, because the open tissue culture medium is not subjected to high-temperature high-pressure sterilization, the loss of nutrient elements and hormones is less, and the growth of the test-tube plantlet is more facilitated.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:
the ginger open tissue culture technical system established by the invention can effectively reduce the production cost of the tissue culture seedlings by more than 30 percent, and has wide application prospect.
Third, as an inventive supplementary proof of the claims of the present invention, there are also presented several important aspects:
(1) The expected income and commercial value after the technical scheme of the invention is converted are as follows: the compound bacteriostatic agent screened by the invention has the characteristics of high efficiency and low toxicity in ginger open tissue culture, and has wide commercial application prospect; the established open tissue culture system can reduce the production cost of the ginger test-tube plantlet and promote the commercial production of the ginger test-tube plantlet.
(2) The technical scheme of the invention fills the technical blank in the industry at home and abroad: the ginger extract has the functions of diminishing inflammation and resisting bacteria, and has obvious inhibiting effect on various microbes, such as staphylococcus aureus, bacillus subtilis, aspergillus niger, penicillium, escherichia coli and saccharomyces cerevisiae. The invention applies the ginger extract to open tissue culture for the first time, and obtains better effect.
(3) The technical scheme of the invention solves the technical problems which are always desired to be solved but are not successfully achieved:
the traditional ginger in-vitro rapid propagation system requires strict sterile environment and sterile operation, the operation steps are complex, and pollution is often caused by negligence of one small link. In addition, the aseptic operation of traditional group banks up needs to be carried out in superclean bench, and the culture medium of configuration needs to consume the electric energy through high temperature autoclaving, and is also higher to the requirement of culture vessel. These factors result in high production cost of traditional tissue culture seedlings of ginger, and limit the commercial development of tissue culture seedlings of ginger. According to the invention, the compound bacteriostatic agent is added into the culture medium to establish the ginger open tissue culture system, so that the occurrence of pollution can be effectively inhibited, the growth of plants is not influenced, and the production cost of test-tube plantlets is reduced.
Drawings
FIG. 1 is a flow chart of an open tissue culture method for ginger provided by an embodiment of the present invention;
FIG. 2 is the tender shoot of the peltate yam provided by the embodiment of the invention after being stored in dark and sand for 20 days at 25 +/-2 ℃;
FIG. 3 is a schematic representation of a shoot tip as inoculated onto a multiple shoot induction medium according to an embodiment of the present invention;
FIG. 4 shows multiple shoots formed after 2 months of culturing on a multiple shoot induction medium according to an embodiment of the present invention
FIG. 5 shows a plant regenerated by culturing for 2 months on a proliferation and strong seedling medium according to an embodiment of the present invention;
FIG. 6 shows test-tube plantlets after 1 month acclimation and culture according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Illustrative embodiments are explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
As shown in fig. 1, the method for open-culture of ginger test-tube plantlets provided by the embodiment of the invention comprises the following steps:
s101: and (4) accelerating germination of ginger rhizome. Soaking cleaned rhizoma Zingiberis recens rhizome in 1000 times diluted carbendazim for 30min, cleaning with clear water, placing in sterilized sand, adding appropriate amount of tap water to maintain humidity, and performing germination at 25 + -2 deg.C in dark for 20-30d;
s102: and (4) preparing a compound bacteriostatic agent.
(1) Preparing a ginger crude extract: cleaning ginger blocks, peeling, cutting into ginger slices with the thickness of 5mm, placing the ginger slices in a constant-temperature drying oven at the temperature of 60 ℃ for drying for 15 hours, crushing the ginger slices by using a crusher, sieving the ginger slices by using a 60-mesh sieve, weighing ginger powder, and mixing the ginger powder and the ginger powder according to a material-liquid ratio of 1:10 Adding 50ml 70% ethanol solution (g/mL), soaking for 0.5h, refluxing with Soxhlet extractor until the liquid in the extractor is colorless, extracting at 60 deg.C, and filtering with 0.22 μm pore size filter membrane to obtain yellow oily crude extractive solution. Extracting multiple parts in parallel, mixing the crude extractive solutions, and rotary evaporating with rotary evaporator (at 45 deg.C) until ethanol is completely volatilized. Preparing 1g (ginger powder mass)/mL of ginger extract solution from the paste by using 50% ethanol as a solvent, and storing in a refrigerator at 4 ℃;
(2) Preparing Yipeheng mother liquor: dissolving white powder in Yipelong B bottle in Yipelong A bottle buffer solution, adding 10mL of distilled water, and diluting to 4.35 × 10 4 mg/L mother liquor;
(3) Preparing a compound bacteriostatic agent: taking 15.5mL of the ginger extract solution, 1.0mL of the Yipelong mother liquor and 0.05mL of a 10% sodium hypochlorite solution, mixing, and then adding distilled water to a constant volume of 100mL to obtain a compound bacteriostatic agent aqueous solution;
s103: culture medium configuration and culture conditions. Adding the components into a culture medium for inducing, proliferating and culturing strong seedlings of the cluster buds, heating to boil, adding 1mL/L of compound bacteriostatic agent into the culture medium when the temperature of the culture medium is reduced to 50 ℃, mixing uniformly, and immediately subpackaging into a can bottle or a triangular bottle for later use; after inoculation, the seeds are all cultured in a culture room with the illumination intensity of about 2000lx, the photoperiod of 14h/10h and the temperature of 25 +/-2 ℃.
S104: disinfecting utensils, appliances and inoculation rooms. Cleaning tissue culture bottle, air drying, sterilizing with ultraviolet lamp for 30min, spraying with 75% ethanol to remove dust, sterilizing, irradiating with ultraviolet lamp for 30min, sterilizing with 75% ethanol, and soaking in 1.0% (v/v) water solution of bacteriostatic agent for at least 1 hr;
s105: and (4) inducing cluster buds. Taking a tender bud with the length of 1.5-2 cm newly germinated by accelerating germination of ginger rhizome, washing for 15min by tap water, soaking for 15s by 75% ethanol, soaking for 15min by 0.1% (m/v) mercuric chloride solution, washing for 5 times by sterile water after disinfection treatment, and then sucking water on the surface of the tender bud by sterile filter paper. Stripping off outer leaf sheath with scalpel, cutting 0.3-0.5cm bud tip, and inoculating into MS +0.5mg/LNAA +2.0 mg/L6-BA +1mL/L complex bacteriostatic agent +30g/L sucrose +7.0g/L agar cluster bud induction culture medium (pH 5.8);
s106: proliferation and strong seedling culture. After the cluster buds are induced for 2 months, cutting off the part of more than 0.5cm of the base part of the induced cluster buds to form a micro lump, and transferring the micro lump to an MS +1 mg/L6-BA +0.2mg/L IBA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar (pH 5.8) proliferation and seedling strengthening culture medium;
s107: transplanting and domesticating the test-tube plantlets. After the propagation and strong seedling culture is carried out for 2 months, after the height of the seedling is 5-6cm, the bottle cap of a can bottle is opened, a small amount of tap water is injected to ensure that the aseptic seedling slowly adapts to a sterile environment, the tap water is used for cleaning the culture medium of the root part of the aseptic seedling after 3 days, the small seedling is transplanted into a 32-hole plug tray filled with a seedling culture medium, 1 plant is planted in each hole, and the volume ratio of the medium is 3:1, pindstrup peat soil and perlite. After being watered thoroughly, the seedlings are placed in an intelligent glass greenhouse for hardening, and the domestication conditions are that the temperature is 25 ℃, the relative humidity is 80 percent, and the light intensity is about 3000 lx.
2. Application examples. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
Application example 1 establishment of open tissue culture System of Kaempferia Craib (FIGS. 2-6)
(1) Placing cleaned and aged rhizome of Alpinia zerumbet Hance in sterilized sand, adding appropriate amount of tap water to maintain sand humidity, and culturing in artificial climate box at 25 + -2 deg.C in dark;
(2) Preparing a compound bacteriostatic agent: collecting 15.5mL of 1g (weight of rhizoma Zingiberis recens powder)/mL of rhizoma Zingiberis recens extract solution, and collecting 1.0mL of 4.35 × 10 4 And mixing the Yipeilong mother liquor in mg/L and sodium hypochlorite solution with the concentration of 0.05mL and 10%, and then fixing the volume to 100mL by using distilled water to obtain the compound bacteriostatic agent aqueous solution.
The preparation method of the ginger crude extract comprises the following steps: weighing 1.0Kg of ginger blocks of phoenix-fern, cleaning, peeling, cutting into ginger slices with the thickness of 5mm, placing the ginger slices in a constant-temperature drying oven, drying for 15 hours at the temperature of 60 ℃, crushing by using a crusher, sieving by using a 60-mesh sieve, weighing ginger powder, and mixing according to a material-liquid ratio of 1:10 Adding 50ml 70% ethanol solution (g/mL), soaking for 0.5h, refluxing with Soxhlet extractor until the liquid in the extractor is colorless, extracting at 60 deg.C, and filtering with 0.22um pore size filter membrane to obtain yellow oily crude extract. Extracting multiple parts in parallel, combining the crude extract, and performing rotary evaporation (at 45 ℃) by using a rotary evaporator until ethanol is completely volatilized. Preparing 1g (ginger powder mass)/mL of ginger extract solution from the paste by using 50% ethanol as a solvent, and storing in a refrigerator at 4 ℃;
the preparation method of the Yipeheng mother liquor comprises the following steps: dissolving Yipelong B bottle white powder in Yipelong A bottle buffer solution, adding 10mL distilled water to dilute to 4.35 × 10 4 mg/L mother liquor;
(3) Preparing a culture medium for inducing, proliferating and culturing strong seedlings of the cluster buds. The cluster bud induction culture medium is MS +0.5mg/L NAA +2.0 mg/L6-BA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar, and the pH value is 5.8; the formula of the culture medium for proliferation and strong seedling culture comprises MS, 1 mg/L6-BA, 0.2mg/LIBA, 1mL/L compound bacteriostatic agent, 30g/L sucrose and 7.0g/L agar proliferation medium (pH is 5.8). The culture medium for inducing, proliferating and culturing the cluster buds and the strong seedlings is heated to boiling after being added with the components, 1mL/L of compound bacteriostatic agent is added into the culture medium when the temperature of the culture medium is reduced to 50 ℃, and the mixture is uniformly mixed and immediately subpackaged into a can bottle or a triangular flask for later use. Wherein the canned bottle or triangular bottle is cleaned, air dried, sterilized with ultraviolet lamp for 30min, and packaged with culture medium without high temperature and high pressure sterilization;
(4) Spraying 75% ethanol to the inoculation room before inoculation, performing dust settling sterilization, irradiating with ultraviolet lamp for 30min, sterilizing the inoculation tool with 75% ethanol, and soaking in 1.0% (v/v) water solution of formulated bacteriostatic agent for at least 1 hr;
(5) Taking 1.5-2 cm tender shoots newly germinated from the peltate yam rhizome, washing the tender shoots with tap water for 15min, soaking the tender shoots with 75% ethanol for 15s, soaking the tender shoots with 0.1% (m/v) mercuric chloride solution for 15min, washing the tender shoots with sterile water for 5 times after disinfection treatment, and then sucking the water on the surfaces of the tender shoots with sterile filter paper. Peeling off outer leaf sheaths by using a scalpel, cutting bud tips with the length of 0.5cm, inoculating the bud tips into a cluster bud induction culture medium, and culturing the inoculated bud tips in a culture chamber with the illumination intensity of about 2000lx, the photoperiod of 14h/10h and the temperature of 25 +/-2 ℃;
(6) After the cluster buds are induced for 2 months, cutting off the part of the induced cluster bud base part of more than 0.5cm, cutting off the induced root system by using surgical scissors to form a miniature lump, and transferring the miniature lump to MS +1 mg/L6-BA +0.2mg/LIBA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar proliferation culture medium (pH 5.8);
(7) After 2 months of proliferation and strong seedling culture, opening the bottle cap of a can bottle after the height of the seedling is 5-6cm, injecting a few drops of tap water to ensure that the aseptic seedling slowly adapts to a sterile environment, cleaning a culture medium adhered to the root of the aseptic seedling by using the tap water after 3 days, then transplanting the seedling into a 32-hole plug tray filled with a seedling culture medium, planting 1 seedling in each hole, wherein the volume ratio of the matrix is 3:1 danish Pindstrup peat soil and perlite. Watering thoroughly, and hardening in intelligent glass greenhouse at 25 deg.C, 80% relative humidity and 3000lx light intensity
The method for establishing the open tissue culture system of the alpinia speciosa provided by the embodiment of the invention comprises the following steps: 1mL/L of screened compound bacteriostatic agent is added into a culture medium, 1.5-2.0cm long tender buds germinated are taken as explants, and an open tissue culture system of the peltate yam rhizome is established through the processes of explant disinfection, cluster bud induction, proliferation, strong seedling culture, domestication and transplantation and the like.
3. Evidence of the relevant effects of the examples. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.
1. Experimental protocol
1.1 materials, reagents and instruments
1.1.1 materials
Uses ginger germplasm resources preserved in cell engineering laboratories of Xiangxin crop research institute of Yangtze university as materials.
1.1.2 Primary reagents
Yipelong, 6-benzyladenine (6-BA), indoleacetic acid (IBA) and the like.
1.1.3 Main instrumentation
A Soxhlet extractor, a rotary evaporator, an electronic balance, a liquid-transfering gun, a pH meter, a pure water meter and the like.
1.2 establishment of open tissue culture System
1.2.1 screening of ginger extract and Yipeilong Compound bacteriostatic agent formulation
1) Extraction of ginger active substance
Selecting 1kg of ginger of Phoenix cephalanoplos, cleaning, cutting into 5mm thick ginger slices, placing in a constant temperature drying oven, drying at 60 deg.C for 15h until the water content is about 15%, pulverizing with a pulverizer, sieving with 60 mesh sieve, and packaging into plastic bottles. Sealing and placing in a cool and dry place for later use. Weighing 3g of ginger powder, and mixing the raw materials according to a material-liquid ratio of 1:10 (w/v) soaking in 70% ethanol solution for 30min, and extracting under reflux in Soxhlet extractor until the liquid in the extractor is colorless, at 60 deg.C. Filtering with 0.22 μm filter membrane to obtain yellow oily crude extractive solution, rotary evaporating with rotary evaporator (at 45 deg.C) until ethanol is completely volatilized, making the obtained paste into stock solution with mass concentration of 1.0g/mL with 50% ethanol as solvent, and storing in 4 deg.C refrigerator.
2) Natural colony test
The test adopts three-factor four-horizontal orthogonal test treatment for natural bacteria falling test.
The factor one is as follows: ginger extract concentration
Level 1:0mg/mL
Level 2:7.65mg/mL
Level 3:15.5mg/mL
Level 4:25.0mg/mL
Factor two: concentration of Yipeheng
Level 1:0.0mg/mL
Level 2:0.155mg/mL
Level 3:0.25mg/mL
Level 4:0.50mg/mL
Factor three: sodium hypochlorite concentration
Level 1:0.0mg/mL
Level 2:0.155mg/mL
Level 3:0.25mg/mL
Level 4:0.50mg/mL
Selecting a clean room as an operation room, closing doors and windows, spraying with 75% ethanol, removing dust, sterilizing, and irradiating with ultraviolet lamp for 30min. The culture medium is a common ginger proliferation culture medium: MS +1 mg/L6-BA +0.2mg/L IBA +30g/L sucrose +7.0g/L agar, pH =5.8. Cleaning a culture dish (60 mm multiplied by 15 mm), airing, sterilizing by an ultraviolet lamp for 30min, adding all components of an MS culture medium, heating to boil according to a conventional method, adding ginger extracts and Yipelong compound bacteriostatic agents with different concentrations when the temperature of the culture medium is reduced to about 50 ℃ (before agar is not solidified), fully stirring the culture medium, respectively adding 5mL of multiplication culture medium added with the compound bacteriostatic agents with different formulas into the culture dish, exposing in the air for l h without autoclaving, sealing, and then placing in a culture room for culturing. The experiment was repeated 3 times for a total of 16 treatments, 64 with 4 dishes per treatment.
3) Influence of compound bacteriostatic agent on isolated culture of different explants of zingiber officinale roscoe
By adopting the screened optimal compound bacteriostatic agent formula, the influence of the compound bacteriostatic agent on the in vitro culture of different explants of the ginger of the phoenix-head ginger is discussed by taking the ginger bud of the ginger of the phoenix-head ginger, the sterile test-tube plantlet, the test-tube plantlet polluted by bacteria and the test-tube plantlet polluted by fungi as materials. The method for inducing, proliferating and strengthening the seedling culture medium is the same as the above, and the inoculation method is the same as the above with the culture medium without adding the bacteriostatic agent as a control. The inoculation tool is always soaked in 75% ethanol, and is burned with alcohol burner when in use. Selecting a clean room as an inoculation room, closing doors and windows, spraying 75% ethanol for dust suppression and sterilization, irradiating 30min by using an ultraviolet lamp, wiping off an inoculation table top and hands by using 75% ethanol, inoculating a treatment group on a conventional experiment table top, and inoculating in a super clean table in contrast. 4 explants are respectively inoculated to each can bottle, and the can bottles are transferred to an illumination culture room for culture after inoculation, and the culture conditions are the same as the above. 4 flasks were inoculated per treatment and repeated 3 times. 4) Transplanting and domesticating of explant-contaminated, growing and regenerated plants
And (5) observing the pollution and growth condition of the inoculated material after the explant is inoculated for 15 days. Opening the bottle cap of the can bottle after the height of the regenerated plant seedling is 5-6cm, injecting a few drops of tap water to enable the aseptic seedling to slowly adapt to the sterile environment, cleaning the culture medium adhered to the root of the aseptic seedling by using the tap water after 3d, then transplanting the seedling into a 32-hole plug tray filled with a seedling culture substrate, planting 1 plant in each hole, wherein the volume ratio of the substrate is 3:1 danish Pindstrup peat soil and perlite. After being watered thoroughly, the seedlings are placed in an intelligent glass greenhouse for hardening, and the domestication conditions are that the temperature is 25 ℃, the relative humidity is 80 percent, and the light intensity is about 3000 lx. Transplanting 16 plantlets for each treatment, counting the transplanting survival rate after 1 month of transplanting, and repeating the test for 3 times.
2. Test results
2.1 Effect of different formulations of compounded bacteriostat on contamination of culture Medium under Natural colony conditions
The analysis result of the mean value of the concentration of the crude ginger extract (table 1) shows that K2 is more than K4 and more than K3 is more than K1, so that the optimal level of A2 as the factor A is judged, namely the optimal concentration of 1.25mg/mL of the crude ginger extract. The same principle confirms that B4 (4.35 mg/L) is the optimal concentration of Yipesulfuron, and C3 (0.005 mg/L) or C4 (0.01 mg/L) is the optimal concentration of sodium hypochlorite. However, considering the damage effect of sodium hypochlorite in the culture medium on explants, the optimum concentration combination of the compound bacteriostatic agent obtained by analyzing the orthogonal experimental data is as follows: 1.25mg/mL ginger extract +4.35mg/L Yipeong +0.005mg/L sodium hypochlorite. The larger the difference value is, the more important the level change of the factors has on the test indexes, and the factors influencing the effect of the compound bacteriostatic agent are ranked as Yipeong > sodium hypochlorite > ginger crude extract (table 1).
TABLE 1 Effect of different formulations of compounded bacteriostats on Medium contamination under Natural colony conditions
Note: factor A is crude extract of rhizoma Zingiberis recens (mg/mL), factor B is Yipeilong (mg/L), and factor C is crude extract of rhizoma Zingiberis recens (mg/mL)
2.2 Effect of Compound bacteriostatic agent on in vitro regeneration of shoot of Litsea pungens
As shown in FIG. 2-FIG. 6, tender shoots of the Pimenta dioica (FIG. 2) with a length of 0.5-1.0cm are germinated after storing in dark and sand at 25 + -2 deg.C for 20 days. Tender bud explants were inoculated into clumpy bud medium supplemented with the best complex bacteriostatic (1.25 mg/mL ginger extract +4.35mg/L oxyphenuron +0.005mg/L sodium hypochlorite) (fig. 3). After 15 days of culture, the statistical result of the pollution rate shows that the pollution rate of the primary open tissue culture of the peltate yam is 25.9 percent on average, and the pollution rate is within an acceptable range in the primary culture. After the uncontaminated buds are cultured for 15 days, the base parts of the buds are expanded, and the bud tips gradually turn green. After 60 days of culture, clumpy shoots formed with a height of 5-6cm (FIG. 4). Cutting cluster buds, then performing strong seedling and propagation culture, and growing up the can bottle after 2 months, wherein the leaves are dark green and the root system is developed (figure 5). After acclimatization and culture in a glass greenhouse for 1 month, most plants grow 2-3 new leaves (figure 6), and the transplanting survival rate reaches 92.6 percent.
2.3 Effect of compounded bacteriostat on non-polluted and polluted test-tube plantlet subculture of Spanish-head ginger
As shown in Table 2, the contamination rate of the explant of the sterile test-tube plantlet is the lowest (5.56%) under the treatment condition of the screened compound bacteriostatic agent for different explants. The culture medium is not sterilized at high temperature and high pressure to cause nutrition loss, and the plant height of the uncontaminated test-tube plantlet is increased by 7.21 percent compared with that of a control plant. When the test-tube plantlet polluted by bacteria or fungi is inoculated, the pollution rate reaches 100 percent compared with the control (the test-tube plantlet polluted by bacteria or fungi), bacterial plaque in a culture medium is smaller due to the existence of the bacteriostatic agent, and the plant can still grow better.
TABLE 2 influence of the compounded bacteriostatic agent on the contamination rate and growth of the explants from different sources of the ginger of Eichhornia crassipes
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The method for openly culturing the ginger test-tube plantlets is characterized by comprising the following steps:
after the surfaces of the roots and the stems of the clean ginger are disinfected, the roots and the stems of the clean ginger are cleaned by clear water and then placed in wet sand for accelerating germination;
preparing a ginger extracting solution and a Yipeilong mother solution, and preparing a compound bacteriostatic agent;
cleaning and airing a tissue culture bottle, sterilizing the tissue culture bottle by using an ultraviolet lamp, and preparing a cluster bud induction culture medium and a propagation and seedling strengthening culture medium containing a compound bacteriostatic agent;
sterilizing the inoculation chamber by dust fall, irradiating by using an ultraviolet lamp, sterilizing the inoculation tool by using ethanol, and soaking in a compound bacteriostatic agent aqueous solution;
disinfecting young buds of ginger rootstock by mercuric chloride solution, peeling off outer leaf sheaths by using a scalpel, and inoculating cut bud tips into a cluster bud induction culture medium for culture;
trimming the induced cluster buds into micro-blocks, and transferring the micro-blocks into a proliferation and strong seedling culture medium for culture;
cleaning the regenerated aseptic seedlings with a root culture medium, transplanting the seedlings into a plug tray filled with a seedling culture medium, watering the seedlings thoroughly, putting the seedlings into an intelligent glass greenhouse for hardening, and planting the seedlings in the field when new leaves grow out.
2. The method for open-culture of ginger test-tube plantlets according to claim 1, wherein the ginger rhizome germination accelerating method comprises: soaking cleaned rhizoma Zingiberis recens rhizome in 1000 times diluted carbendazim for 30min, cleaning with clear water, placing in sterilized sand, adding appropriate amount of tap water to maintain humidity, and performing germination acceleration at 25 + -2 deg.C in dark for 20-30d.
3. The method for open culture of ginger test-tube plantlets according to claim 1, wherein the preparation method of the compound bacteriostatic agent comprises the following steps:
step one, cleaning ginger blocks, peeling, cutting into ginger slices with the thickness of 5mm, placing the ginger slices in a constant-temperature drying oven for drying for 12 hours at the temperature of 60 ℃, crushing the ginger slices by using a crusher, sieving the ginger slices by using a 60-mesh sieve, weighing ginger powder, and mixing the ginger powder and the ginger powder according to a material-liquid ratio of 1:10 Adding 50ml 70% ethanol solution (g/mL), soaking for 0.5h, refluxing with Soxhlet extractor until the liquid in the extractor is colorless, extracting at 60 deg.C, and filtering with 0.22um pore size filter membrane to obtain yellow oily crude extractive solution; extracting multiple parts in parallel, mixing the crude extractive solutions, and rotary evaporating with rotary evaporator (at 45 deg.C) until ethanol is completely volatilized; preparing 1g (ginger powder mass)/mL of ginger extract solution from the paste by using 50% ethanol as a solvent, and storing in a refrigerator at 4 ℃;
step two, dissolving Yipelong B bottle white powder in Yipelong A bottle buffer solution, adding 10mL of distilled water to dilute to the concentration of 4.35 multiplied by 10 4 mg/L mother liquor;
and step three, mixing 12.5mL of the ginger extract solution, 1.0mL of the Yipelong mother liquor and 0.05mL of 10% sodium hypochlorite solution, and then adding distilled water to a constant volume of 100mL to obtain the compound bacteriostatic agent aqueous solution.
4. The method for open-culture of ginger test-tube plantlets according to claim 1, wherein the medium configuration method comprises: the culture medium for the induction, proliferation and strong seedling culture of the cluster buds is heated to boiling after being added with each component, 1mL/L of compound bacteriostatic agent is added into the culture medium when the temperature of the culture medium is reduced to 50 ℃, and the culture medium is immediately subpackaged into a can bottle or a triangular bottle which is cleaned in advance, dried in the air and sterilized by an ultraviolet lamp for 30min after being uniformly mixed without high-temperature and high-pressure sterilization.
5. The method for open-type cultivation of ginger test-tube plantlets according to claim 1, wherein the method for disinfecting the inoculation chamber and the inoculation tool comprises the following steps: spraying 75% ethanol on the inoculation chamber, performing dust settling sterilization, irradiating with an ultraviolet lamp for 30min, sterilizing the inoculation tool with 75% ethanol, soaking in 1.0% (v/v) of the compound bacteriostatic agent aqueous solution for at least more than 1h, and soaking in the bacteriostatic agent aqueous solution all the time during inoculation.
6. The method for open-culture of ginger test-tube plantlets according to claim 1, wherein the ginger clustered shoot induction method comprises: taking 1.5-2 cm tender shoots newly germinated by sprouting of ginger rhizome, washing with tap water for 15min, soaking with 70% ethanol for 15s, soaking with 0.1% (m/v) mercuric chloride solution for 12min, washing with sterile water for 5 times after disinfection treatment, and then sucking water on the surfaces of the tender shoots with sterile filter paper; peeling off outer leaf sheath with a scalpel, cutting 0.3-0.5cm bud tip, inoculating into MS +0.5mg/L NAA +2.0 mg/L6-BA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar (pH 5.8) cluster bud induction culture medium, and culturing in a culture room with illumination intensity of about 2000lx, photoperiod of 14h/10h and temperature of 25 ℃ +/-2 ℃.
7. The method for open-culture of ginger test-tube plantlets according to claim 1, wherein the ginger propagation and strong seedling culture method comprises: after the cluster buds are induced for 2 months, cutting off the part of more than 0.5cm of the base part of the induced cluster buds to form a micro lump, and transferring the micro lump to a proliferation and strong seedling culture medium of MS +1 mg/L6-BA +0.2mg/L IBA +1mL/L compound bacteriostatic agent +30g/L sucrose +7.0g/L agar (pH 5.8); subculturing for 1 time every 2 months in a culture room with illumination intensity of about 2000lx, photoperiod of 14h/10h and temperature of 25 + -2 deg.C, and performing acclimatization and transplantation on the plantlets after 2 times of subculture.
8. The method for open culture of ginger tube plantlets according to claim 1, wherein the domestication and transplantation method of ginger tube plantlets comprises: after the propagation and strong seedling culture is carried out for 2 months, after the height of the seedling is 5-6cm, the bottle cap of a can bottle is opened, a small amount of tap water is injected to ensure that the aseptic seedling slowly adapts to a sterile environment, the tap water is used for cleaning the culture medium of the root part of the aseptic seedling after 3 days, the small seedling is transplanted into a 32-hole plug tray filled with a seedling culture medium, 1 plant is planted in each hole, and the volume ratio of the medium is 3:1 danish Pindstrup peat soil and perlite; watering thoroughly, and then putting in an intelligent glass greenhouse for acclimatization under the conditions of 25 +/-2 ℃, 80% of relative humidity and 3000lx of light intensity.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101836586A (en) * | 2010-01-15 | 2010-09-22 | 重庆市澳桉花木种植有限公司 | Zhugen ginger detoxification tissue culture seedling industrialized breeding method |
| CN102405842A (en) * | 2011-10-14 | 2012-04-11 | 福建农林大学 | Open type method for cultivating toxin-free seedlings of sugarcanes |
| CN102577976A (en) * | 2012-03-17 | 2012-07-18 | 福建农林大学 | Simple tissue culture method for broussonetia papyrifera |
| CN103348917A (en) * | 2013-07-18 | 2013-10-16 | 湖北蔬谷农业科技有限公司 | Rapid propagation method of ginger virus-free seedlings by one step |
| CN103651124A (en) * | 2013-11-22 | 2014-03-26 | 张明 | Inducing method for plant regeneration of zingiber officinale |
| CN105123750A (en) * | 2015-10-15 | 2015-12-09 | 贵州省生物技术研究所 | Mold bacteriostatic agent for open culture of potato tissue culture seedlings and using method thereof |
| CN112704011A (en) * | 2021-01-08 | 2021-04-27 | 重庆市幅沅农业生物技术研究院有限公司 | Ginger tissue culture method |
-
2022
- 2022-10-10 CN CN202211237430.6A patent/CN115589945A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101836586A (en) * | 2010-01-15 | 2010-09-22 | 重庆市澳桉花木种植有限公司 | Zhugen ginger detoxification tissue culture seedling industrialized breeding method |
| CN102405842A (en) * | 2011-10-14 | 2012-04-11 | 福建农林大学 | Open type method for cultivating toxin-free seedlings of sugarcanes |
| CN102577976A (en) * | 2012-03-17 | 2012-07-18 | 福建农林大学 | Simple tissue culture method for broussonetia papyrifera |
| CN103348917A (en) * | 2013-07-18 | 2013-10-16 | 湖北蔬谷农业科技有限公司 | Rapid propagation method of ginger virus-free seedlings by one step |
| CN103651124A (en) * | 2013-11-22 | 2014-03-26 | 张明 | Inducing method for plant regeneration of zingiber officinale |
| CN105123750A (en) * | 2015-10-15 | 2015-12-09 | 贵州省生物技术研究所 | Mold bacteriostatic agent for open culture of potato tissue culture seedlings and using method thereof |
| CN112704011A (en) * | 2021-01-08 | 2021-04-27 | 重庆市幅沅农业生物技术研究院有限公司 | Ginger tissue culture method |
Non-Patent Citations (1)
| Title |
|---|
| 李白等: ""四种蔬菜食用器官提取物对植物组培污染细菌的抑制作用"", 《浙江农业学报》, vol. 29, no. 11, pages 1854 - 1861 * |
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