CN113678737B - Open tissue culture method for polygonatum cyrtonema - Google Patents
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to an open tissue culture method of polygonatum cyrtonema, which utilizes the bacteriostasis of YJ104 bacteriostat, and adds YJ104 bacteriostat into polygonatum cyrtonema tissue culture medium to inhibit the growth and propagation of fungus bacteria in the polygonatum cyrtonema tissue culture process, thereby realizing the proliferation and rooting culture of polygonatum cyrtonema under the condition of bacteria. The method has the advantages of simple operation, time and labor saving, and greatly reduced cost, and lays an important foundation for the development of the polygonatum cyrtonema's industrialized seedling technology.
Description
Technical Field
The invention relates to an open tissue culture method of polygonatum cyrtonema.
Background
Polygonatum cyrtonema (Polygonatum cyrtonema) is a perennial herb of Polygonatum (Polygonatum) in Liliaceae (Liliaceae), and dried rhizome of the herb is used as the medicine, and is also called as rhizoma Polygonati according to the shape. Polygonatum cyrtonema is a traditional medicine-food dual-purpose tonifying medicinal material, and along with the continuous expansion of market demands, wild resources are reduced rapidly due to the artificial excessive mining and digging, so that the growing market demands cannot be met. Although artificial planting is one of effective ways for solving the problem of resource shortage of polygonatum cyrtonema at present, the phenomena of quality reduction and aggravation of plant diseases and insect pests can be caused by long-term artificial cultivation; in addition, the dormancy characteristic of polygonatum cyrtonema seeds also causes certain difficulty for artificial cultivation. Therefore, constructing a polygonatum cyrtonema tissue culture rapid propagation system and improving the propagation coefficient are effective ways for solving the problems of seed source shortage and artificial cultivation. Although polygonatum cyrtonema tissue culture can provide huge seedlings in a short period, the problem of high production cost is increasingly highlighted after tissue culture production is put into commercial scale production, and a plurality of reports are reported on polygonatum cyrtonema tissue culture propagation research, but no better solution is provided for solving the problem of cost increase caused by frequent transfer and sterilization.
The open tissue culture is to separate the plant tissue culture from the strict aseptic operation environment, and operate in the natural and open aseptic environment, thus simplifying the tissue culture link fundamentally and reducing the tissue culture cost. Through the application of the inhibin, autoclaving and superclean bench inoculation are not needed, and the pollution rate can be controlled within an acceptable range of less than 10% under the condition of not influencing the tissue culture effect. The tissue culture cost can be greatly reduced through open tissue culture, so that large-scale commodity production with high benefit is realized. Compared with the traditional tissue culture, the open tissue culture does not need aseptic inoculation and aseptic environment culture, only needs to add a safe and efficient antibacterial agent into a culture medium, can be inoculated and cultured under the aseptic environment condition, and saves an autoclave and an ultra-clean workbench with larger investment proportion. At present, the open tissue culture has been successful on dozens of plants, but the polygonatum cyrtonema open tissue culture has not been reported at present.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a continuous rotation-rotation reciprocating rotation mechanism for a vibrating aircraft.
In order to achieve the purpose, the invention adopts the following technical scheme: an open tissue culture method of polygonatum cyrtonema includes the following steps:
(1) sterilization of culture bags
The culture bag is a special self-supporting self-sealing bag with a breathable film, is cleaned by tap water and then is soaked in 0.1% sodium hypochlorite, and is completely immersed by being pressed by a weight for 2-3 hours; after soaking, fishing out the culture bag, airing and draining;
(2) preparation of culture Medium
Preparing a basic culture medium according to a preparation method of a conventional culture medium, wherein the basic culture medium comprises MS, 0.3% of cane sugar, 7.2% of agar, 1.0-3.0 mg/L of 6-BA and 0.2-0.5 mg/L of NAA, adding different types of hormones and concentrations according to the requirements of different growth periods of polygonatum cyrtonema tissue culture, adding water to fix the volume, covering a cover to boil for 2-3 min, adjusting the pH to 5.8-6.0, and adding 30-60 mg/L of YJ104 bacteriostatic agent;
(3) inoculation operation
Sterilizing an inoculation chamber by irradiating with an ultraviolet lamp for 20-30 min, ventilating for 30min +/-5 min, cleaning an inoculated table, wiping with 75% alcohol for sterilization, and sterilizing inoculated instruments by using an infrared ray inoculation sterilizer;
(4) induced culture
Putting strong polygonatum cyrtonema tubers without diseases and insect pests in an indoor sand heap, thoroughly watering the strong polygonatum cyrtonema tubers with 1000 times of 50% carbendazim solution, covering with a mulching film, generating tender stems in 30-40 days, taking the tender stems of polygonatum cyrtonema as an induction material, removing surface soil, drying in the air, cutting into tubers with the size of 2cm multiplied by 4cm, soaking and disinfecting for 10-15 min by 0.1% dodecyl dimethyl benzyl ammonium bromide, cleaning for 5-6 times by sterile water, and cutting into stem tips with the size of 1cm multiplied by 2 cm; shaking and disinfecting the mixture for 6 to 8min by using 2 percent sodium hypochlorite, washing the mixture for 5 to 6 times by using sterile water, cutting the mixture into stem tips with the size of 0.5cm multiplied by 0.5cm, and inoculating the stem tips into a culture medium of MS, 0.3 percent of cane sugar, 7.2 percent of agar, 1.0 to 3.0mg/L of 6-BA, 0.2 to 0.5mg/L of NAA and 30 to 60mg/L of bacteriostatic agent; the temperature of the culture room is 24 +/-1 ℃, the illumination is carried out for 18h every day, the illumination intensity in the first 7 days is 500-1500 lx, the illumination intensity in the later days is 1000-1500lx, and the air humidity is 60-70 percent;
(5) proliferation culture
After induction culture and growth of cluster buds, cutting the cluster buds according to 2-3 strains, inoculating the cut cluster buds into a multiplication culture medium, wherein the multiplication culture medium is MS + 1.0-3.0 mg/L6-BA + 0.2-0.5 mg/L NAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times in subsequent open tissue culture can be properly adjusted to MS +1.0mg/L6-BA +0.1mg/L NAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times are carried out once in 20 days; the temperature of the culture room is 24 +/-1 ℃, dark culture is carried out for the first 7 days, then the illumination is carried out for 18h every day, the illumination intensity is 1000-1500lx, and the air humidity is 60-70 percent;
(6) rooting culture
When the height of the seedlings is not less than 3cm, cutting the seedlings into individual plants, transferring the individual plants into a rooting culture medium of 1/2MS + 0.2-0.5 mg/L NAA + antibacterial agent, and culturing for 20-30 days to obtain transplantable seedlings; the temperature of the culture room is 24 +/-1 ℃, the illumination is 12h every day, the illumination intensity is 2000-;
after the adventitious buds of the polygonatum cyrtonema are induced in the previous step, proliferation and rooting culture are directly carried out under the condition of opening bacteria.
Further, the polygonatum cyrtonema proliferation culture medium is as follows: MS + 1.0-3.0 mg/L6-BA + 0.2-0.5 mg/L NAA, and can be properly adjusted to MS +1.0mg/L6-BA +0.1mg/L NAA when the number of passages is large in subsequent open tissue culture.
Further, the YJ104 bacteriostatic agent is prepared by the following formula: solution A: 150ml of plant extract and 50ml of penicillin; mixing rhizoma Atractylodis tuber, rhizoma Zingiberis recens rhizome, folium Aloe, folium Artemisiae Argyi, cortex Cinnamomi Camphorae and flos Lonicerae, adding ethanol, stirring, and pulverizing to obtain mixed solution; carrying out ultrasonic treatment on the obtained mixed solution, filtering to remove solid impurities, and then evaporating and concentrating to remove ethanol to obtain a plant extracting solution; and B, liquid B: 50ml of surfactant and 150ml of deionized water; when in use, the liquid A and the liquid B are mixed and shaken uniformly to obtain the YJ104 bacteriostatic agent.
The invention has the beneficial effects that: the YJ104 bacteriostatic agent is added into the polygonatum cyrtonema tissue culture medium, so that the polygonatum cyrtonema can proliferate and root under the condition of bacteria. The tissue culture method has the advantages that strict aseptic conditions are avoided, large instruments such as a pressure cooker and an ultra-clean workbench are not needed, the tissue culture link is fundamentally simplified, and the tissue culture cost is greatly reduced.
Drawings
FIG. 1 example 1 tissue culture of Polygonatum cyrtonema tissue culture seedling
FIG. 2 Polygonatum cyrtonema explant selected in comparative example 1
FIG. 3 is a process for establishing a sterile line by using polygonatum cyrtonema seed embryo as an explant in comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to fig. 1 to 3 in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Embodiment 1 referring to fig. 1 to 3, an open tissue culture method of polygonatum cyrtonema includes the following steps:
1. and (3) sterilizing the culture bag: the culture bag is a special self-supporting self-sealing bag (with a breathable film), is cleaned by tap water and then is soaked in 0.1% sodium hypochlorite, and is completely immersed by being pressed by a weight for 2-3 hours. And (5) fishing out the culture bag after soaking, airing, and draining.
2. Preparation of a culture medium: preparing a basic culture medium according to a preparation method of a conventional culture medium, wherein the basic culture medium comprises MS, 0.3% of cane sugar, 7.2% of agar, 1.0-3.0 mg/L of 6-BA and 0.2-0.5 mg/L of NAA, adding different types of hormones and concentrations according to the requirements of different growth periods of polygonatum cyrtonema tissue culture, adding water to fix the volume, covering a cover to boil for 2-3 min, adjusting the pH to 5.8-6.0, and adding 30-60 mg/L of YJ104 bacteriostatic agent.
3. And (3) inoculation operation: sterilizing the inoculation chamber by ultraviolet lamp irradiation for 20-30 min, ventilating for about 30min, cleaning the inoculated table, wiping with 75% alcohol for sterilization, and sterilizing the inoculated apparatus by an infrared ray inoculation sterilizer.
4. And (3) induction culture: putting strong polygonatum cyrtonema tubers without diseases and insect pests in an indoor sand heap, thoroughly watering the strong polygonatum cyrtonema tubers with 1000 times of 50% carbendazim solution, covering with a mulching film, generating tender stems in 30-40 days, taking the tender stems of polygonatum cyrtonema as an induction material, removing surface soil, drying in the air, cutting into tubers with the size of 2cm multiplied by 4cm, soaking and disinfecting for 10-15 min by 0.1% dodecyl dimethyl benzyl ammonium bromide (benzalkonium bromide), cleaning for 5-6 times by sterile water, and cutting into stem tips with the size of 1cm multiplied by 2 cm; shaking and disinfecting the mixture for 6-8 min by using 2% sodium hypochlorite, cleaning the mixture for 5-6 times by using sterile water, cutting the mixture into stem tips with the size of 0.5cm multiplied by 0.5cm, and inoculating the stem tips into a culture medium of MS, 0.3% of cane sugar, 7.2% of agar, 1.0-3.0 mg/L of 6-BA, 0.2-0.5 mg/L of NAA and 30 mg/L of an antibacterial agent. The temperature of the culture room is 24 +/-1 ℃, the daily illumination is 18h, the illumination intensity in the first 7 days is 500-1500 lx, the illumination intensity in the later days is 1000-1500lx, and the air humidity is 60-70%.
5. And (3) proliferation culture: after induction culture and growth of cluster buds, cutting the cluster buds according to 2-3 strains, inoculating the cut cluster buds into a multiplication culture medium, wherein the multiplication culture medium is MS + 1.0-3.0 mg/L6-BA + 0.2-0.5 mg/L NAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times in subsequent open tissue culture can be properly adjusted to MS +1.0mg/L6-BA +0.1mg/L NAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times are transferred once in 20 days. The temperature of the culture room is 24 +/-1 ℃, dark culture is carried out for the first 7 days, then illumination is carried out for 18h every day, the illumination intensity is 1000-.
6. Rooting culture: when the height of the seedling is not less than 3cm, cutting the seedling into single plants, transferring the single plants into a rooting culture medium of 1/2MS + 0.2-0.5 mg/L NAA (activated carbon can be added or 1 g/L) + an antibacterial agent, and culturing for 20-30 days, thus obtaining the seedlings for transplantation. The temperature of the culture room is 24 +/-1 ℃, the daily illumination is 12 hours, the illumination intensity is 2000-.
7. Selecting an open polygonatum cyrtonema test-tube seedling with 5-6 roots and a seedling height of 4-6 cm, transplanting the test-tube seedling into an outdoor shade place, uncovering, domesticating and culturing for 5-7 d (the temperature is 10-15 ℃, the humidity is 60% -70%, direct sunlight is avoided), taking out, washing off a residual culture medium on the seedling with clear water, then soaking in 400 mg/L of carbendazim solution for 20-30 min, finally transplanting the test-tube seedling into a seedling culture tray (32 hole tray, single tray is 540 mm x 280 mm x 100 mm long x wide x high) containing a seedling culture substrate (vermiculite: river sand = 2: 1: 1), culturing the test-tube seedling in a greenhouse with the temperature of 21-25 ℃, the illumination intensity of 2000-3000 lx and the humidity of 60% -70% for 28-35 d, transplanting the test-tube seedling in an outdoor field when the plant length is 10 cm high, and selecting the transplanting time to be between 5: 00-6: 00 pm, and watering thoroughly. In this example 1, the seedlings grew very strongly, and the survival rate of transplantation was more than 90%.
The culture bag is made of PEG, is made of pure plant materials, and has no pollution to the environment. Low cost and suitability for large-scale use.
Example 2
A method for openly culturing polygonatum cyrtonema tissue culture seedlings comprises the following steps:
the operation of this embodiment is the same as that of embodiment 1 except for the following operation, which is not described again.
The YJ104 bacteriostatic agent is added into each culture medium at the concentration of 40 mg/L.
Example 3
A method for openly culturing polygonatum cyrtonema tissue culture seedlings comprises the following steps:
the operation of this embodiment is the same as that of embodiment 1 except for the following operation, which is not described again.
The YJ104 bacteriostatic agent is added into each culture medium at the concentration of 50 mg/L.
Example 4
A method for openly culturing polygonatum cyrtonema tissue culture seedlings comprises the following steps:
the operation of this embodiment is the same as that of embodiment 1 except for the following operation, which is not described again.
The YJ104 bacteriostatic agent is added into each culture medium at the concentration of 60 mg/L.
Comparative example 1
1. Explants (polygonatum cyrtonema seed embryos): mature polygonatum cyrtonema seeds are used as starting materials for tissue culture and rapid propagation, and explants are polygonatum cyrtonema seeds which break dormancy (preferably, radicle is exposed from hypocotyl).
2. Seed pretreatment: the polygonatum cyrtonema seeds are divided into a plurality of groups, 10 seeds are used in each group, and then the seeds are soaked in 200, 300, 500 and 800 mg/L GA3 solution respectively.
3. And (3) disinfection: binding Polygonatum cyrtonema seeds with a 20 cm2 nylon mesh bag by a stapler, washing the Polygonatum cyrtonema seed mesh bag for 3 times by washing powder, then cleaning for 3 times by sterile water, absorbing the surface moisture of the seeds, putting 75% alcohol into a superclean workbench for disinfection for 30 s, and washing for 2-3 times by sterile water; sterilizing with 0.1% mercuric chloride for 5min, washing with sterile water for 2-3 times, placing on a seed inoculation tray, sucking water from explant surface with filter paper, cutting off lower end of seed hypocotyl (contact end with mercuric chloride) with scalpel, and inoculating. Boiling the prepared culture medium, heating to dissolve, fixing the volume, adjusting the pH to 5.8-6.0, subpackaging, and sterilizing at high temperature and high pressure.
4. Inoculation: the starting culture medium for polygonatum cyrtonema seeds is as follows: MS +6-BA (0.2 mg/L) +2,4-D (0.2 mg/L) + GA3 (2.0 mg/L); and (3) inoculating 1-3 grains (preferably 1 grain, and can be directly discarded when the pollution exists) in each bottle, and then carrying out transfer (one bottle with multiple plants) after the pollution is avoided.
5. And (3) proliferation medium selection: MS +2.0mg/L6-BA +0.2mg/L NAA is used at the early stage of establishment of the polygonatum cyrtonema sterile line, and when the number of passages is large, the MS +1.0mg/L6-BA +0.1mg/L NAA can be properly adjusted.
6, selecting a rooting culture medium: the rooting medium used was 1/2MS +0.2mg/L NAA (1 g/L with charcoal).
Comparative example 2
1. Putting strong polygonatum cyrtonema tubers without diseases and insect pests in an indoor sand heap, watering the strong polygonatum cyrtonema tubers with 1000 times of 50% carbendazim solution, covering with a mulching film, generating tender stems in 30-40 days, taking the tender stems of polygonatum cyrtonema in the period as explants, cutting the tender stems of polygonatum cyrtonema into stem tips with the size of 0.5cm multiplied by 0.5cm, sequentially cleaning with washing powder and distilled water, preliminarily sterilizing, washing with running water, sucking water on the surfaces of the tender stems of polygonatum cyrtonema, soaking the explants in a bacteriostatic agent for 2-5 h, taking out and washing with sterile water to be clean. Starting ventilation ultraviolet for 20-30 min, putting the polygonatum cyrtonema explant into 75% alcohol to be disinfected for 30 s in a super-clean workbench, and washing with sterile water for 2-3 times; and then 0.1% mercuric chloride is put into the seed culture tray for disinfection for 5min, the seed culture tray is put on a seed inoculation tray after being washed for 2-3 times by sterile water, and the seed culture tray is inoculated into a bacteriostatic agent culture medium of MS, 0.3% sucrose, 7.2% agar, 1.0-3.0 mg/L6-BA and 0.2-0.5 mg/L NAA and 40 mg/LYJ 104. When the height of the seedlings is not less than 3cm, the seedlings are cut into single plants, the single plants are transferred into a rooting culture medium of 1/2MS + 0.2-0.5 mg/L NAA (activated carbon can be added at 1 g/L) +40 mg/LYJ104 bacteriostatic agent, the culture is carried out for 20-30 days, and then the seedlings can be transplanted, wherein the hardening seedling transplanting method is the same as that of the embodiment 1. The temperature of the culture room is 24 +/-1 ℃, the daily illumination is 12 hours, the illumination intensity is 2000-. Boiling the prepared culture medium, heating to dissolve, fixing the volume, adjusting the pH to 5.8-6.0, subpackaging, and sterilizing at high temperature and high pressure.
Comparative example 3
The comparative example is the same as comparative example 2 except for the following operations, which are not repeated.
The culture medium is prepared without adding 40 mg/LYJ104 bacteriostatic agent.
Comparative example 4
The comparative example is the same as comparative example 2 except for the following operations, which are not repeated.
The media prepared were the same as those of example 1, but were not autoclaved in an autoclave
TABLE 1 multiplication and rooting of tissue culture seedlings of Polygonatum cyrtonema in open culture
Adding bacteriostatic agents in different species into PDA culture medium respectively, setting control group at the same time, wherein the control group is PDA culture medium without combined bactericide, and repeating every level for 5 times. Culturing in natural condition in seedling factory workshop, periodically observing the appearance time and growth condition of colony on the surface of culture medium, and recording the observation data of 2 nd and 13 d. The result shows that the inhibition effect on colony formation under the open condition shows that the inhibition effect of the YJ104 bacteriostatic agent is optimal, so that the YJ104 bacteriostatic agent is selected as the polygonatum cyrtonema open tissue culture bacteriostatic agent.
TABLE 2 evaluation of bacteriostatic effect of plate
Table 3 comparison of test-tube plantlet of three Polygonatum cyrtonema tissue culture modes of example 1, comparative example 1 and comparative example 2
| Treatment method | Number of transplants | Survival rate (%) | Growth conditions |
| Example 1 | 100 | 93 | The plant recovery is fast, the leaf color is verdure, and the growth vigor is excellent |
| COMPARATIVE EXAMPLE 2 (CK) | 100 | 95 | The plant recovers quickly, leaves are light green, and the growth vigor is general |
| Comparative example 1 | 100 | 98 | Fast recovery of plant, light green leaf color and excellent growthGood wine |
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Claims (1)
1. An open tissue culture method of polygonatum cyrtonema is characterized by comprising the following steps:
(1) sterilization of culture bags
The culture bag is a special self-supporting self-sealing bag with a breathable film, is cleaned by tap water and then is soaked in 0.1% sodium hypochlorite, and is completely immersed by being pressed by a weight for 2-3 hours; after soaking, fishing out the culture bag, airing and draining;
(2) preparation of culture Medium
Preparing a basic culture medium according to a preparation method of a conventional culture medium, wherein the basic culture medium comprises MS, 0.3% of cane sugar, 7.2% of agar, 1.0-3.0 mg/L of 6-BA and 0.2-0.5 mg/L of NAA, adding different types of hormones and concentrations according to the requirements of different growth periods of polygonatum cyrtonema tissue culture, adding water to fix the volume, covering a cover to boil for 2-3 min, adjusting the pH to 5.8-6.0, and adding 30-60 mg/L of YJ104 bacteriostatic agent;
the polygonatum cyrtonema proliferation culture medium comprises: MS + 1.0-3.0 mg/L6-BA + 0.2-0.5 mg/L NAA, and the content can be properly adjusted to MS +1.0mg/L6-BA +0.1mg/L NAA when the number of passages is large in subsequent open tissue culture;
(3) inoculation operation
Sterilizing an inoculation chamber by irradiating with an ultraviolet lamp for 20-30 min, ventilating for 30min +/-5 min, cleaning an inoculated table, wiping with 75% alcohol for sterilization, and sterilizing inoculated instruments by using an infrared ray inoculation sterilizer;
(4) induced culture
Putting strong polygonatum cyrtonema tubers without diseases and insect pests in an indoor sand heap, watering the polygonatum cyrtonema tubers thoroughly by using 1000 times of 50% carbendazim solution, covering with a mulching film, generating tender stems in 30-40 days, taking the tender stems of polygonatum cyrtonema as an induction material, removing surface soil, drying in the air, cutting into tubers with the sizes of 2cm multiplied by 4cm, soaking and disinfecting for 10-15 min by using 0.1% dodecyl dimethyl benzyl ammonium bromide, washing for 5-6 times by using sterile water, and cutting into stem tips with the sizes of 1cm multiplied by 2 cm; shaking and disinfecting the mixture for 6 to 8min by using 2 percent sodium hypochlorite, washing the mixture for 5 to 6 times by using sterile water, cutting the mixture into stem tips with the size of 0.5cm multiplied by 0.5cm, and inoculating the stem tips into a culture medium of MS, 0.3 percent of cane sugar, 7.2 percent of agar, 1.0 to 3.0mg/L of 6-BA, 0.2 to 0.5mg/L of NAA and 30 to 60mg/L of bacteriostatic agent; the temperature of the culture room is 24 +/-1 ℃, the illumination is carried out for 18h every day, the illumination intensity in the first 7 days is 500-1500 lx, the illumination intensity in the later days is 1000-1500lx, and the air humidity is 60-70 percent;
(5) proliferation culture
After induction culture and growth of cluster buds, cutting the cluster buds according to 2-3 strains, inoculating the cut cluster buds into a multiplication culture medium, wherein the multiplication culture medium is MS + 1.0-3.0 mg/L6-BA + 0.2-0.5 mg/LNAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times in subsequent open tissue culture can be properly adjusted to MS +1.0mg/L6-BA +0.1mg/L NAA + 30-60 mg/LYJ104 bacteriostatic agent, and the multiple passage times are transferred once in 20 days; the temperature of the culture room is 24 +/-1 ℃, dark culture is carried out for the first 7 days, then illumination is carried out for 18h every day, the illumination intensity is 1000-;
(6) rooting culture
When the height of the seedlings is not less than 3cm, cutting the seedlings into single plants, transferring the single plants into a rooting culture medium of 1/2MS + 0.2-0.5 mg/L NAA + antibacterial agent, and culturing for 20-30 days to obtain transplantable seedlings; the temperature of the culture room is 24 +/-1 ℃, the illumination is 12h every day, the illumination intensity is 2000-;
the YJ104 bacteriostatic agent comprises the following components in percentage by weight: solution A: 150ml of plant extract and 50ml of penicillin; mixing rhizoma Atractylodis tuber, rhizoma Zingiberis recens rhizome, folium Aloe, folium Artemisiae Argyi, cortex Cinnamomi Camphorae and flos Lonicerae, adding ethanol, stirring, and pulverizing to obtain mixed solution; carrying out ultrasonic treatment on the obtained mixed solution, filtering to remove solid impurities, and then evaporating and concentrating to remove ethanol to obtain a plant extracting solution; and B, liquid B: 50ml of surfactant and 150ml of deionized water; when in use, the liquid A and the liquid B are mixed and shaken uniformly to obtain the YJ104 bacteriostatic agent;
after the adventitious buds of the polygonatum cyrtonema are induced in the previous step, proliferation and rooting culture are directly carried out under the condition of opening bacteria.
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| CN106359107A (en) * | 2016-10-28 | 2017-02-01 | 广西大学 | Tissue culture method of wild lilium |
| CN106332784A (en) * | 2016-11-10 | 2017-01-18 | 福建农林大学 | Open gerbera jamesonii tissue culture method |
| CN110771511A (en) * | 2019-11-26 | 2020-02-11 | 广州市农业科学研究院 | Method for open culture of banana tissue culture seedlings |
| CN113412788A (en) * | 2021-07-28 | 2021-09-21 | 广西壮族自治区农业科学院 | Method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems |
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