CN114041422B - Honeysuckle tissue culture method based on low-mesh-number activated carbon culture medium - Google Patents
Honeysuckle tissue culture method based on low-mesh-number activated carbon culture medium Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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Abstract
The invention discloses a honeysuckle tissue culture method based on a low-mesh active carbon culture medium. The invention takes the leaves and stems of honeysuckle plants as explants for tissue culture, and the low-mesh granular active carbon with 10-40 meshes is added into a culture medium for inducing callus generation and proliferation to replace the common high-mesh active carbon (more than or equal to 100 meshes), so that the browning of the callus can be obviously reduced, the rooting and germination capacities of differentiated seedlings can be further improved, and the technical support is provided for the establishment of an optimized honeysuckle genetic transformation system. The method provided by the invention plays an important role in promoting the improvement of the honeysuckle tissue culture technology and the establishment of a genetic transformation system.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine tissue culture, and provides a function of adding low-mesh active carbon into a culture medium in optimizing a honeysuckle genetic transformation system and application thereof.
Background
Honeysuckle is a dried flower bud and a flower which is originally bloomed of Lonicera japonica Thunb of Caprifoliaceae, is a rare Chinese medicinal material in China, is widely used in traditional Chinese medicine clinical formulas and Chinese patent medicines, has cold and sweet taste, is known as a good medicine for clearing heat and removing toxicity, dispelling wind and dissipating heat since ancient times, and is also one of the main medicines of various recommended formulas in the period of pestilence. The honeysuckle contains abundant flavonoid and chlorogenic acid secondary metabolites, and is a main active ingredient type in the honeysuckle.
Due to the extremely high medicinal value and market value of honeysuckle, at present, honeysuckle is planted in many provinces in China, and more important planting areas are mainly concentrated in Shandong, shaanxi, henan, hebei, hubei, jiangxi, guangdong and other places. The main cultivation mode of honeysuckle is still field planting, but in recent years, the phenomena of germplasm mixing and degeneration are getting more and more serious in the honeysuckle planting process. Therefore, the honeysuckle industry is in urgent need of breeding new excellent varieties and improving the quality of medicinal materials to meet the requirements of the current honeysuckle industry.
The honeysuckle genetic transformation system established by using the tissue culture technology is an effective means for rapidly improving honeysuckle varieties and improving the quality of medicinal materials, and in recent years, certain progress is made on the research of the honeysuckle tissue culture technology. However, mature tissue culture techniques for honeysuckle are still lacking. The main reason is that when the honeysuckle explant is used for tissue culture, the callus generated by induction is easy to brown, so that the callus is difficult to further induce to bud and root. Similarly, a callus-based genetic transformation system for honeysuckle is lacking.
The activated carbon is an inert substance with a porous structure formed by heating an organic raw material under the condition of isolating air and then reacting with gas. Activated carbon is generally classified into two types, i.e., powder and granule, according to its shape. The granular activated carbon includes cylindrical, spherical, hollow cylindrical and hollow spherical and irregular shaped crushed carbon. The size of the activated carbon can be expressed by the mesh number, and the mesh number refers to the density of activated carbon particles which can pass through a screen. The mesh number of the activated carbon is generally from 4 meshes to 400 meshes, and the commonly used mesh number is from 100 meshes to 200 meshes.
Currently, it is known that adding activated carbon in the process of plant tissue culture can provide a dark environment for plant rooting, and adsorb plant growth regulators and other substances beneficial to rooting, thereby achieving the effects of promoting rooting and root system growth; in addition, the activated carbon is also used as an anti-browning agent in plant tissue culture. The current research on the application of the activated carbon in the tissue culture mainly focuses on the influence of the variety and the addition concentration of the activated carbon on the tissue culture, and no literature report on the influence of the mesh number of the activated carbon on the tissue culture is found.
The earlier-stage research of the invention finds that when honeysuckle stem segments are used as explants to develop the tissue culture research of honeysuckle, active carbon with different meshes is added, so that the browning of callus can be prevented. Based on the above, the invention provides a mesh range of active carbon for remarkably reducing browning of honeysuckle explants in the process of inducing callus, thereby providing a honeysuckle tissue culture technology with low browning rate and high rooting rate, and providing an important technical means for optimizing the construction of a genetic transformation system of honeysuckle, rapidly improving honeysuckle varieties and improving medicinal material quality.
Disclosure of Invention
The invention aims to solve the problems of high browning rate and low rooting rate of honeysuckle explants in the callus induction process, and provides a honeysuckle tissue culture method based on a low-mesh active carbon culture medium. According to the invention, by adding the low-mesh active carbon into the culture medium, a good technical support is laid for improving the honeysuckle tissue culture technology and optimizing the construction of a genetic transformation system of honeysuckle.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a honeysuckle tissue culture method based on a low-mesh-number activated carbon culture medium comprises the following steps:
firstly, placing a disinfected honeysuckle explant in a callus culture medium for culture, inducing to generate callus, and then transferring the callus to a proliferation culture medium for proliferation culture;
then, inoculating the callus subjected to proliferation culture into a differentiation culture medium for culture, after inducing differentiation and sprouting, separating the sprouts differentiated from the callus into single plants, inoculating the single plants into a rooting culture medium for culture, inducing roots, forming tissue culture seedlings capable of independently growing, and then using the tissue culture seedlings for domestication and transplantation;
wherein the callus culture medium and the proliferation culture medium are both added with low-mesh active carbon with the mesh number of 10-40, and the differentiation culture medium and the rooting culture medium are both added with high-mesh active carbon with the mesh number of 100-200.
Preferably, the honeysuckle explant is a leaf or a stem of a honeysuckle plant, and is preferably a tender stem which is healthy and has a leaf bud on the honeysuckle plant.
Preferably, the honeysuckle explant is disinfected by alternately soaking in a 75% ethanol solution and a 15% sodium hypochlorite solution.
Preferably, the formula of the callus culture medium is as follows: MS + KT 1.0mg/L +6-BA 1.5mg/L + NAA 0.1mg/L +0.3wt.% low-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L.
Preferably, the formula of the proliferation medium is as follows: MS + KT 0.1mg/L +6-BA 1.5mg/L + NAA 0.4mg/L +0.3wt.% low-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L.
Preferably, the formula of the differentiation medium is as follows: MS +6-BA 2.5mg/L + NAA 0.1mg/L +0.3wt.% high-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L.
Preferably, the rooting medium comprises the following formula: 1/2MS + NAA 1.5mg/L +0.3wt.% high-mesh active carbon + sucrose 20g/L + agar 9.3g/L.
Preferably, the method for culturing the disinfected honeysuckle explants in the callus culture medium comprises the following steps:
cutting the sterilized explant, inoculating the cut explant to a callus culture medium which is sterilized in advance, putting the cut explant into a culture room at the room temperature of 24 ℃ and keeping illumination for 12h and dark culture for 12h for alternate culture, wherein if the explant is a leaf, the cut explant is cut into a regular shape of 0.5cm multiplied by 0.5cm and is inoculated in a mode that the front side of the explant faces upwards, and if the explant is a stem, the cut explant is cut into stem sections of 0.5-1.0 cm; transferring the cultured callus to a proliferation culture medium after the cultured callus grows to 0.5cm multiplied by 0.5 cm.
Preferably, the method for separating the buds differentiated from the callus into single plants and inoculating the single plants into a rooting medium for culturing comprises the following steps:
the buds differentiated from the callus are respectively separated into single plants according to the types of the buds for culturing, wherein the terminal buds are vertically inoculated into a rooting culture medium for induced rooting culture to form young plants capable of living autonomously, and the buds generated by the differentiation of the axillary buds are cut from a mother plant and transferred to the rooting culture medium for culturing into seedlings.
Preferably, the method for domesticating and transplanting comprises the following steps: transferring the individual plants from the culture bottle to acclimatize when the roots of the tissue culture seedlings grow to 2-3cm and the plants on the upper parts of the roots grow to 3-5cm, and transplanting the plants into a pot for planting after adapting for 2-3 days.
Compared with the prior art, the beneficial effects possibly brought by the implementation of the invention are as follows:
compared with the traditional plant tissue culture medium added with 100-200 meshes of granular activated carbon, the scheme of the invention uses the granular activated carbon with low mesh number (10-40 meshes) to replace the callus induction and proliferation stages in the honeysuckle tissue culture process, and test results show that the method can effectively reduce the browning phenomenon of the callus and tissue culture seedlings in the honeysuckle tissue culture process, and prove that the addition of the low mesh number activated carbon in the culture medium is beneficial to the induction of callus and the prevention of tissue browning.
Compared with the method for disinfecting the explant by using mercury bichloride and sodium hypochlorite solution in the prior art, the invention adopts the method of sequentially soaking and washing by using sodium hypochlorite and 75% ethanol on the basis of multiple test verifications. Can more fully realize the disinfection of the honeysuckle explants, thereby avoiding the external pathogens from polluting the explants and the subsequent tissue culture links.
The method effectively overcomes the defects that callus is easy to brown and explants are incompletely disinfected in the honeysuckle tissue culture process, successfully constructs the tissue culture parameters of honeysuckle, and provides an important technical means for quickly improving honeysuckle varieties and improving the quality of medicinal materials.
Detailed Description
The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The technical characteristics in the embodiments of the present invention can be combined correspondingly without mutual conflict.
The invention provides application of low-mesh active carbon in optimizing a honeysuckle genetic transformation system, and the mesh number of the active carbon added in the stage of inducing callus is changed, so that the differentiation efficiency of honeysuckle explants is obviously improved.
The invention provides a honeysuckle tissue culture method based on a low-mesh active carbon culture medium, which comprises the following basic steps:
firstly, placing a disinfected honeysuckle explant in a callus culture medium for culture, inducing to generate callus, and then transferring the callus to a proliferation culture medium for proliferation culture;
then, inoculating the callus subjected to proliferation culture into a differentiation culture medium for culture, after inducing differentiation and sprouting, separating the sprouts differentiated from the callus into single plants, inoculating the single plants into a rooting culture medium for culture, inducing roots, forming tissue culture seedlings capable of independently growing, and then using the tissue culture seedlings for domestication and transplantation;
wherein the callus culture medium and the proliferation culture medium are both added with low-mesh active carbon with the mesh number of 10-40, and the differentiation culture medium and the rooting culture medium are both added with high-mesh active carbon with the mesh number of 100-200.
In the culture method, the specific formulas of the four culture media can be adjusted according to actual needs, and the tissue culture methods and operation details at different stages can also be optimized according to actual needs. The following provides a specific implementation manner of the honeysuckle tissue culture method, which comprises the following steps:
1) Preparing a culture medium: preparing an induced callus culture medium;
2) Explant selection and sterilization: selecting healthy and strong leaves or tender stems without diseases and insect pests as explants, cutting off mature parts, removing terminal buds and young leaves, and washing dust garbage attached to the surfaces of the explants in a super clean workbench by using sterile water; and then sequentially soaking for 1-5min by using 15% sodium hypochlorite, washing with sterile water for 1-3 times, soaking for 30s by using 75% ethanol, washing with sterile water for 1-3 times, finally soaking for 3s by using 15% sodium hypochlorite, repeating the steps for three times, washing with sterile water for 5-6 times, and placing on sterile filter paper for naturally ventilating and airing.
3) Inducing callus: inoculating the sterilized explant (cut into regular pattern of 0.5cm × 0.5cm if leaf, with right side up, or cut into 0.5-1.0cm segments if stem) into the sterilized callus culture medium, and culturing in culture room (keeping illumination for 12h, dark culture for 12h, and room temperature 24 deg.C). After the cultured callus grows to 0.5cm multiplied by 0.5cm, transferring the callus to a proliferation culture medium for proliferation culture.
4) Differentiation culture: inoculating the proliferated callus into a differentiation culture medium for culture, and inducing differentiation and sprouting; after the buds separated from the callus are separated into individual plants, the individual plants are inoculated into a rooting culture medium for culture, and roots are induced to form tissue culture seedlings capable of independently growing. Wherein, the terminal bud is vertically inoculated into a rooting culture medium for induced rooting culture to form a young plant which can independently live; the buds generated by the differentiation of the axillary buds are cut off from the stock plant and transferred to a rooting culture medium to be cultured into seedlings.
5) Domestication and transplantation: transferring the individual plants from the culture bottle to harden the seedlings when the roots grow to about 2-3cm and the plants on the upper parts of the roots grow to about 3-5 cm; after acclimation for 2-3 days, transplanting the seeds into an acclimated pot for planting.
The culture medium involved in the above-described embodiments may be formulated as follows:
i callus induction culture medium: MS + KT 1.0mg/L +6-BA 1.5mg/L + NAA 0.1mg/L +0.3wt.% active carbon (mesh number: 10-40 mesh) + sucrose 20.0g/L + agar 9.3g/L;
II proliferation medium: MS + KT 0.1mg/L +6-BA 1.5mg/L + NAA 0.4mg/L +0.3wt.% active carbon (mesh number: 10-40 mesh) +20.0g/L sucrose +9.3g/L agar;
III differentiation medium: MS +6-BA 2.5mg/L + NAA 0.1mg/L +0.3wt.% active carbon (mesh number: 100-200 mesh) +20.0g/L sucrose +9.3g/L agar;
IV rooting culture medium: 1/2MS + NAA 1.5mg/L +0.3wt.% active carbon (mesh number: 100-200 mesh) +20g/L sucrose +9.3g/L agar.
The culture method provided by the invention can effectively reduce the browning phenomenon of the callus in the tissue culture process of the honeysuckle.
In the present invention, the low mesh number of activated carbon added to the medium is mainly required to be controlled in the mesh number range, but the type of activated carbon may be adjusted as required. The activated carbon is preferably granular activated carbon, and can be one or more of coal granular activated carbon, wood granular activated carbon, shell granular activated carbon and the like in any proportion.
The above method is applied to specific examples to show specific technical effects in combination with data.
Examples
Preparing culture media with different functions, wherein the specific method comprises the following steps:
1. preparing induction callus culture medium
According to the formula: MS + KT 1.0mg/L +6-BA 1.5mg/L + NAA 0.1mg/L +0.3% active carbon +20.0g/L sucrose +9.3g/L agar, preparing an induction callus culture medium: weighing 4.74g MS and 20g sucrose, stirring in water to melt, adding 1.0mg KT, 1.5mg 6-BA and 0.1mg NAA, adjusting pH to 5.8, diluting the solution to 1L, pouring into a sterilization bottle containing 3g activated carbon (wood particle activated carbon, mesh number: 35 mesh) and 9.3g agar, and sterilizing at 121 ℃ for 20 minutes. And after the sterilization is finished, 25ml of the culture medium is filled into a culture dish in a super clean bench to obtain the induced callus culture medium.
2. Preparing proliferation culture medium
According to the formula: MS + KT 0.1mg/L +6-BA 1.5mg/L + NAA 0.4mg/L +0.3% active carbon +20.0g/L sucrose +9.3g/L agar, preparing a proliferation culture medium: weighing 4.74g MS and 20g sucrose, stirring in water to melt, adding 0.1mg KT, 1.5mg 6-BA and 0.4mg NAA, adjusting pH to 5.8, diluting the solution to 1L, pouring into a sterilization bottle containing 3g activated carbon (wood particle activated carbon, mesh number: 35 mesh) and 9.3g agar, and sterilizing at 121 ℃ for 20 minutes. And after the sterilization is finished, 25ml of the culture medium is filled into a culture dish in a super clean bench to obtain the proliferation culture medium.
3. Preparing differentiation culture medium
According to the formula: MS +6-BA 2.5mg/L + NAA 0.1mg/L +0.3% active carbon +20.0g/L sucrose +9.3g/L agar, preparing a differentiation culture medium: weighing 4.74g MS and 20g sucrose, stirring in water to melt, adding 2.5mg 6-BA and 0.1mg NAA, adjusting pH to 5.8, diluting to constant volume to 1L, pouring into a sterilization bottle containing 3g activated carbon (wood particle activated carbon, mesh number: 200 mesh) and 9.3g agar, and sterilizing at 121 deg.C for 20 min. And (5) after the sterilization is finished, filling 50ml of the culture solution into a culture bottle in a super clean bench to obtain the differentiation culture medium.
4. Preparing rooting culture medium
According to the formula: 1/2MS calcium carbonate NAA 1.5mg/L +0.3% active carbon +20g/L sucrose +9.3g/L agar, preparing a rooting culture medium: 2.37g of MS and 20g of sucrose were weighed out and melted in water under stirring, 1.5mg of NAA was added, the pH was adjusted to 5.8, the solution was made constant to 1L and poured into a sterilized flask containing 3g of activated carbon (wood-particle activated carbon, mesh number: 200 mesh) and 9.3g of agar, and sterilized at 121 ℃ for 20 minutes. And (5) after the sterilization is finished, filling 50ml of the culture solution into a culture bottle in a super clean bench to obtain the rooting culture medium.
Secondly, sterilizing the honeysuckle explants, wherein the specific method comprises the following steps:
the method comprises the steps of using plants growing indoors as selection objects, selecting strong and healthy tender stems without diseases and insect pests and with leaf buds as explants, separating leaves and stem segments, and washing dust garbage on the surfaces of the explants by running water. Further washing the dust waste with sterile water in a super-clean workbench, then sequentially soaking for 3min with 15% sodium hypochlorite, washing with sterile water for 3 times, soaking for 30s with 75% ethanol, washing with sterile water for 3 times, finally soaking for 3s with 15% sodium hypochlorite, washing with sterile water for 5 times after repeating for three times, and placing on sterile filter paper for natural ventilation and drying.
And (III) culturing the sterilized explants in the culture medium obtained in the step (I) for induced differentiation, wherein the specific method comprises the following steps:
cutting the disinfected leaves into regular patterns of 0.5cm multiplied by 0.5cm, inoculating the patterns into a pre-disinfected induced callus culture medium with the front side facing upwards, cutting stem sections without axillary buds into small sections of about 0.5cm, horizontally inoculating the small sections into the induced callus culture medium, and performing induced callus culture; and inoculating the stem segment with axillary buds into a differentiation culture medium for induced differentiation and bud culture. The culture process is carried out in a culture room (keeping illumination for 12h, dark culture for 12h alternately, room temperature 24 ℃). After the cultured callus grows to 0.5cm multiplied by 0.5cm, transferring the callus into a proliferation culture medium for proliferation culture.
Inoculating the proliferated callus into a differentiation culture medium for culture, and inducing differentiation and sprouting; after the buds separated from the callus are separated into individual plants, the individual plants are inoculated into a rooting culture medium for culture, and roots are induced to form tissue culture seedlings capable of independently growing. Wherein, the terminal bud is vertically inoculated into a rooting culture medium for induced rooting culture to form a young plant which can independently live; the buds generated by the axillary buds after differentiation are cut off from the stock plant and then transferred to a rooting culture medium to be cultured into seedlings.
For convenience of description, the tissue culture method of honeysuckle in the above steps (one) - (three) is referred to as low-mesh array. In addition, in order to show the influence of the mesh number of the activated carbon added in the callus induction and proliferation stages on the induction and differentiation of the honeysuckle explants, the example is also provided with a high mesh number group. Wherein, the difference between the high-mesh array and the low-mesh array is only that the low-mesh active carbon with the mesh number of 35 in the callus culture medium and the proliferation culture medium is replaced by the high-mesh active carbon with the same amount of 200 meshes, namely, the high-mesh active carbon is added in all four culture media, and the other methods are consistent with the low-mesh array. Through experiments, the influence of the high-mesh group and low-mesh group activated carbon on the induced differentiation of honeysuckle explants is shown in table 1.
TABLE 1 influence of active carbon in high mesh and low mesh groups on induced differentiation of honeysuckle explants
As can be seen from Table 1, different induction efficiencies of honeysuckle explants can be obtained by adding conventional high-mesh and low-mesh activated carbon to different culture media. In the callus induction stage and the proliferation stage, compared with the culture medium added with high-mesh active carbon, the culture medium added with low-mesh active carbon is more beneficial to inducing callus, can prevent the callus from browning, and improves the survival rate of the final tissue culture seedling. Meanwhile, comparison shows that the honeysuckle flower explant induced callus amount in the culture medium containing the low-mesh active carbon is more than that in the culture medium containing the high-mesh active carbon, and finally higher survival rate is shown.
And (IV) domesticating and transplanting the honeysuckle aseptic seedlings, wherein the specific method comprises the following steps:
when the root of the sterile tissue culture seedling of honeysuckle grows to about 3cm and the plant on the upper part of the root grows to about 3cm, unscrewing a culture bottle cap, hardening the seedling for 2 days, opening the culture bottle cap, hardening the seedling for 2 days, transferring to a subsequent growth environment, hardening the seedling for 3 days, then gently taking out individual plants from the culture bottle, washing off the culture medium on the root, transplanting the plants into sterilized turfy soil, building a small arched shed after being watered thoroughly, covering a plastic film for 5 days, and then growing in a natural environment.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (6)
1. A honeysuckle tissue culture method based on a low mesh number activated carbon culture medium is characterized by comprising the following steps:
firstly, placing a disinfected honeysuckle explant in a callus culture medium for culture, inducing to generate callus, and then transferring the callus to a proliferation culture medium for proliferation culture; the honeysuckle explant is a leaf or stem of a honeysuckle plant;
then, inoculating the callus subjected to proliferation culture into a differentiation culture medium for culture, after inducing differentiation and sprouting, separating the sprouts differentiated from the callus into single plants, inoculating the single plants into a rooting culture medium for culture, inducing roots, forming tissue culture seedlings capable of independently growing, and then using the tissue culture seedlings for domestication and transplantation;
wherein the callus culture medium and the proliferation culture medium are both added with low-mesh active carbon with the mesh number of 10-40, and the differentiation culture medium and the rooting culture medium are both added with high-mesh active carbon with the mesh number of 100-200;
the callus culture medium comprises the following components in percentage by weight: MS + KT 1.0mg/L +6-BA 1.5mg/L + NAA 0.1mg/L +0.3wt.% low-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L;
the formula of the proliferation culture medium is as follows: MS + KT 0.1mg/L +6-BA 1.5mg/L + NAA 0.4mg/L +0.3wt.% low-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L;
the formula of the differentiation medium is as follows: MS +6-BA 2.5mg/L + NAA 0.1mg/L +0.3wt.% high-mesh active carbon + sucrose 20.0g/L + agar 9.3g/L;
the formula of the rooting culture medium is as follows: 1/2MS + NAA 1.5mg/L +0.3wt.% high-mesh active carbon + sucrose 20g/L + agar 9.3g/L.
2. The method for tissue culture of honeysuckle based on low mesh activated carbon medium according to claim 1, wherein the honeysuckle explant is a tender stem with healthy leaf buds of honeysuckle plant.
3. The method for tissue culture of honeysuckle based on the low mesh activated carbon culture medium as claimed in claim 1, wherein the disinfection of the honeysuckle explant is performed by alternate soaking disinfection of 75% ethanol solution and 15% sodium hypochlorite solution.
4. The honeysuckle tissue culture method based on the low mesh activated carbon culture medium as claimed in claim 1, wherein the method for placing the disinfected honeysuckle explants in the callus culture medium is as follows:
cutting the sterilized explant, inoculating the cut explant into a callus culture medium which is sterilized in advance, putting the callus culture medium into a culture room at the room temperature of 24 ℃, and alternately culturing for 12h under illumination and 12h in dark, wherein if the explant is a leaf, the explant is cut into a regular shape of 0.5cm multiplied by 0.5cm and is inoculated in a manner that the front side of the explant faces upwards, and if the explant is a stem, the explant is cut into stem sections of 0.5 to 1.0 cm; transferring the cultured callus to a proliferation culture medium after the cultured callus grows to 0.5cm multiplied by 0.5 cm.
5. The method for culturing honeysuckle tissue based on the low mesh activated carbon culture medium as claimed in claim 1, wherein the method for separating the buds differentiated from the callus into single plants and inoculating the single plants into a rooting culture medium for culturing is as follows:
the buds differentiated from the callus are respectively separated into single plants according to the types of the buds for culturing, wherein the terminal buds are vertically inoculated into a rooting culture medium for induced rooting culture to form young plants capable of living autonomously, and the buds generated by the differentiation of the axillary buds are cut from a mother plant and then transferred to the rooting culture medium for culturing into seedlings.
6. The honeysuckle tissue culture method based on the low mesh activated carbon culture medium according to claim 1, wherein the domestication and transplantation method comprises the following steps: transferring the individual plants from the culture bottle to acclimatize when the root of the tissue culture seedling grows to 2-3cm and the plant on the upper part of the root grows to 3-5cm, and transplanting the individual plants into a pot for planting after adapting for 2-3 days.
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