CN112493133B - Plant open tissue culture method and special culture medium - Google Patents
Plant open tissue culture method and special 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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to the technical field of plant biology, and particularly relates to a method for carrying out culture flask open culture in open tissue culture of plants and a special culture medium. When the open tissue culture medium is prepared, the components of the culture medium are subpackaged into culture bottles, oxygen slow release particles are added, the culture bottles are horizontally placed, cooled and solidified, after the bud seedling explants are inoculated on the culture medium, a layer of non-volatile oily liquid is perfused on the surface of the culture medium, and the culture is carried out in an open mode. The invention enhances the photosynthesis of the bottle seedlings, obtains robust bottle seedlings, reduces the transpiration, improves the transplanting survival rate of the bottle seedlings, and can also reduce the usage amount of the antibacterial agent of the culture medium and inhibit the over-acidification of the culture medium. The invention has the advantages of low cost, simple preparation, convenient application and obvious effect.
Description
Technical Field
The present invention belongs to the field of plant biotechnology. Relates to a plant open tissue culture method and a special culture medium, in particular to a method for improving the gas microenvironment of a bottle seedling by bottle mouth open culture in plant open tissue culture.
Background
The plant tissue culture technology has wide market prospect in seedling production, virus removal, germplasm improvement and the like. In the open tissue culture (open tissue culture) of plants, which has attracted much attention in recent years, an antimicrobial agent is added to a culture medium, autoclaving is not required, inoculation is performed in an open environment (no need of a clean bench), and conventional culture is performed by covering. Open tissue culture has great significance in improving operation efficiency and reducing tissue culture cost, and related papers (Liu Fu Ping, research method of antibacterial agent application in plant antibacterial tissue culture, genomics and application biology, 2014, 33 (4): 910-doped 915, introduction) and patent applications are increasing, such as application numbers 200810047258.1, 200910060484.8, 200910088122.X, 201010257107.6, 201110195928.6, 201110410305.6, 201110134608.X, 201110311185.4, 201310238845.X, 201310204499.3, 201410097129.9, 201510151118.9, 201510933918.6, 201610696644.8, 201710207906.4, 201911177574.5, 202010698173.0 and the like.
The tissue culture seedling is cultured in sealed test tube or glass bottle due to overhigh humidity and CO content in the container2Deficiency causes that the cultured plantlet can not normally carry out photosynthesis, but has to depend on sugar in a culture medium for heterotrophic growth, which causes that the culture time is too long, the growth of the plantlet is slow, the growth vigor of the plantlet is weak, and the transplanting survival rate is low; the sugar in the culture medium can also promote the reproduction of microorganisms and increase the pollution rate of plants; environmental abnormalities in the container, such as increase of harmful gas, higher temperature and higher humidity, can cause less plant leaves, excessive growth, vitrification, difficult transplantation, or slow plant development, high plant death rate in the domestication stage and the like [ Mamingjian, Song overwintering, a tissue culture seedling sugar-free culture system based on environmental control, agricultural engineering report, 2009,25 (6): 192-]. Open tissue culture seedlings are also cultured in closed vessels, and the obtained bottle seedlings also have the defects.
"test tube seedling pore density under conventional culture condition is great, and the function is imperfect, hardly adapts to external environment at the transplantation domestication in-process, makes photosynthetic capacity reduce, appears excessive dehydration scheduling problem moreover easily, directly influences the transplantation survival rate of test tube seedling. … … the test-tube plantlet has maximum pore opening at the initial stage of transplantation, and the transpiration rate reaches the peak value, and the test-tube plantlet is in the state of fastest water loss. "(Sun Dong et al. influence of tissue culture microenvironment on physiological characteristics of the cultured seedlings of the hyacinths group after transplantation. northwest agronomy report, 2008, 17 (4): 244-.
In open tissue culture, culture medium microbial contamination comes from microorganisms mixed in a culture medium and microorganisms in a bottle are settled to the culture medium, an antibacterial agent for open tissue culture is dissolved in an agar culture medium and only partially resists bacteria of the culture medium, and does not aim at the microorganisms in the bottle, so that the antibacterial agent attached to the culture medium is required to be kept at a higher concentration, toxic and side effects on cultured plant materials are avoided, the contradiction is a technical difficulty (key) of open tissue culture, and the wide application of the open tissue culture is limited.
Disclosure of Invention
The invention aims to provide a plant open culture method and a special culture medium, and a method for improving the gas microenvironment of bottle seedlings by culturing in an open bottle mouth after explants are inoculated.
The technical scheme of the invention is as follows:
an open culture method for plant comprises heating the culture medium, agar, antibacterial agent and water to melt, adjusting pH, packaging into culture bottle, adding oxygen slow-release granules, covering the bottle opening with clean film or panel, and cooling for solidification. Inoculating bud seedling explant by conventional open tissue culture method, pouring a layer of non-volatile oily liquid with thickness of 0.2-0.5cm on the surface of culture medium, and culturing in open place without bottle cap.
The oxygen slow-release granular material is prepared by mixing a filling agent, an adhesive and an oxygen release agent, and performing cold sterilization treatment in advance, wherein the filling agent comprises the following components in parts by weight: adhesive: the weight ratio of the oxygen releasing agent is 1: 0.2: 0.2-0.8, adding ice water for granulation, and freezing and storing under aseptic condition, wherein the particle size is 0.3-0.8 cm. 10-25 granules of oxygen slow release granules are put in each 100ml of culture medium.
The filler is formed by mixing kaolin and perlite in equal volume, the adhesive is carboxymethyl cellulose or polyvinyl acetate, and the oxygen release agent is sodium percarbonate (Na)2CO3·3H2O2) Sodium perborate (NaBO)3·4H2O).
The volatile oily liquid is liquid paraffin or methyl silicone oil.
The invention also provides a special culture medium for open tissue culture of plants, which comprises conventional components, agar and an antibacterial agent, wherein the agar culture medium contains oxygen slow-release particles, and a layer of non-volatile oily liquid is poured on the surface of the agar culture medium.
The oxygen slow-release granular material is prepared by mixing a filler, an adhesive and an oxygen release agent, wherein the filler: adhesive: the weight ratio of the oxygen releasing agent is 1: 0.2: 0.2-0.8, adding ice water for granulation, and the particle size is 0.3-0.8 cm. 10-25 granules of oxygen slow release granules are put in each 100ml of culture medium.
The filler is formed by mixing kaolin and perlite in equal volume, the adhesive is carboxymethyl cellulose or polyvinyl acetate, and the oxygen release agent is sodium percarbonate (Na)2CO3·3H2O2) Sodium perborate (NaBO)3·4H2O).
The principle and the technical effect of the invention are as follows:
compared with the conventional capped culture, the open culture after the explant is inoculated by the culture medium has the technical problems that the moisture in a culture bottle is lower, the moisture of the agar in the culture medium and the water vapor pressure difference of the space in the bottle are higher, the evaporation of the moisture of the agar is accelerated, and the growth of the bottle seedlings is possibly influenced due to insufficient water supply of a root system.
The oil layer on the surface of the culture medium prevents agar water from evaporating to keep the water of the culture medium, and the oil layer prevents the space in the bottle from depositing the mixed bacteria on the surface of the culture medium to avoid pollution. But the oil layer also prevents the exchange of the agar culture medium and the outside gas and reduces the oxygen level of the culture, so that the oxygen slow-release particles are put into the culture medium, and the oxygen is dissolved and diffused in the agar culture medium to improve the root respiration.
The oxygen releasing agent of the oxygen sustained-release particles is sodium percarbonate (Na)2CO3·3H2O2) Sodium perborate (NaBO)3·4H2O) oxygen release equation is 2Na2CO3·3H2O2=4NaOH+2H2CO3+2H2O+3O2And 4 (NaBO)3·4H2O) = Na2B4O7+ 2NaOH+ 4H2O2+ 11H2O，2H2O2=2 H2O+O2. Produced O2The particles are discharged into an agar medium, and when O is accumulated inside the particles2When the pressure reaches a certain value, the expansion causes the particles to crack, and the O in the particles is also generated2Is discharged out.
Another problem caused by low air humidity in the bottle is that the transpiration of the bottle seedlings is large, so that the bottle seedlings need to absorb more water from the culture medium, and the reaction of the two oxygen release agents releasing oxygen also generates water to slowly supplement water for the agar culture medium.
As the culture time in the tissue culture continues, the carbon dioxide released by the respiration of the plant roots is increasingly accumulated in the culture medium, and CO is generated2+H2O = H2CO3 = H+ + HCO3 -1The culture medium is over-acidified, the acid-base balance of the culture medium is damaged, and the growth of the root system is influenced. The other product of the two oxygen releasing agents is NaOH, OH-1Can neutralize H+And the over acidification of the culture medium is relieved, so that the growth and absorption functions of the root system are not obviously influenced.
The invention has the beneficial effects that (1) the open culture improves the CO in the bottle2The supply amount is favorable for photosynthesis of the bottle seedlings to obtain robust bottle seedlings, the humidity in the culture bottle is reduced, the cuticle of the leaf of the bottle seedlings is developed, the function of opening and closing pores is enhanced, and the transpiration is favorably reduced, so that the survival rate of the bottle seedlings in transplantation can be improved. (2) Increase CO in the bottle2The level is favorable for photosynthesis, the carbon source sucrose supply of the culture medium is reduced, the pollution rate of the culture medium is reduced, and in addition, an oil layer is covered on the surface of the culture medium to block micro space in a bottleThe biological sedimentation to the culture medium shows the pollution caused, the reduction of the pollution rate of the culture medium can reduce the usage amount of the antibacterial agent of the culture medium, and the toxic and side effects of the antibacterial agent on the bottle seedlings are reduced or eliminated, thereby being beneficial to the wide application of the open tissue culture technology. (3) Compared with the conventional cover-added culture, the open culture bottle seedling has the advantages that the transpiration effect is greater, the root part of the transpiration effect absorbs more water, and the oxygen release agent can supplement water for the agar culture medium. (4) The oxygen release agent relieves the over acidification of the culture medium at the later stage of culture, and the growth and absorption functions of the root system are not obviously influenced. (5) The bottles are not required to be covered in the tissue culture operation process, so that the material investment of the bottle covers is reduced, and the operation efficiency is improved.
Detailed description of the invention
Example one
Method (A)
1. (1) preparation of a culture medium of a control group (conventional open tissue culture), 200ml of culture bottles (cylindrical, the diameter of the bottom of the culture bottles is 6 cm), 0.2 mg/L of MS + NAA + 3% of sucrose, 0.6% of agar, 0.3% of antibacterial agent potassium sorbate and 0.1% of benzalkonium bromide (the concentration of the antibacterial agent is the lowest concentration of the open tissue culture medium without microbial pollution), mixing with water, boiling until the antibacterial agent is completely dissolved, fixing the volume, adjusting the pH to 5.6, subpackaging the culture bottles when the culture medium is hot, subpackaging each bottle with about 25ml of the culture medium, covering, horizontally placing, cooling and solidifying.
(2) Preparing oxygen slow-release particles, wherein the filler is formed by mixing kaolin and perlite in equal volume, the adhesive is carboxymethyl cellulose, and the oxygen release agent is sodium percarbonate (Na)2CO3·3H2O2) The raw materials are subjected to ultraviolet treatment in advance, and the filler: adhesive: the weight ratio of the oxygen releasing agent is 1: 0.2: 0.4, mixing, adding ice water, granulating to obtain granules with a particle size of 0.4cm, and freezing and storing under aseptic conditions.
Treating a special culture medium for the group (the invention), wherein culture bottles are the same as a control, the culture medium MS + NAA is 0.2 mg/L + sucrose is 1.5%, agar is 0.6%, antibacterial agent potassium sorbate is 0.3%, boiling to completely melt, fixing the volume, adjusting the pH to 5.6, subpackaging the obtained product into the culture bottles when the product is hot, subpackaging each bottle with about 25ml of the culture medium, adding 5 oxygen slow-release particles into each culture bottle, covering the culture bottles with a clean panel without a bottle cap, flatly placing, cooling and solidifying.
2. 70% alcohol spraying dust settling of inoculation chamberIrradiating with ultraviolet lamp for 20 min, wiping the inoculated table and hands with 70% alcohol, inoculating flos Chrysanthemi (chrysanthemum flower) in culture mediumDendranthema morifolium) And (3) inoculating 12-14 explants to each bottle of bud seedlings (no obvious root growth, average fresh weight of 0.084 g/plant and average leaf number of 2.1/plant), covering a control group for conventional culture, pouring a layer of liquid paraffin on the surface of a culture medium in a treatment group, wherein the thickness of the liquid paraffin is about 0.3cm, and the liquid paraffin is not added to a culture bottle and is horizontally cultured in an open manner. The humidity in the culture room is 65-80%.
3. Culturing for 30 days, opening the cover of the control group culture bottle, exercising the control group culture bottle and the treatment group culture bottle in a greenhouse for 5 days, transplanting bottle seedlings into the soil, and performing conventional management in the greenhouse.
(II) results
1. The concentration of potassium sorbate in the treated medium was the same as the control, with less benzalkonium bromide. After 30 days of culture, no microbial colonies were found on the surface and in the medium of the control and treated groups.
2. The growth indexes of the bottle seedlings are shown in the table.
TABLE. growth and transplantation of bottle seedlings of Chrysanthemum
The net photosynthetic rate of the bottle seedlings is obviously increased compared with that of the control group. The average fresh weight of the stems and leaves of the treated group is heavier, the difference with the control is extremely obvious, and the average leaf number of the treated group is more than that of the control, and the difference is obvious. The survival rate of the bottle seedlings is 100 percent for both groups.
The average fresh weight of the root system of the treatment group is heavier, the difference with the control is obvious, and the average number of the root of the treatment group is obviously improved compared with the control. The transpiration rate treated group was significantly lower than the control. After 1 month after the transplantation, the survival rate of the treated group seedlings is obviously improved compared with that of the control group seedlings
3. 1 month after the transplanting, the survival rate of the processed components is higher, and the difference reaches a very significant level.
(III) conclusion
The surface of the culture medium is covered with an oil layer to block the space in the bottle from microbial contamination, and the sucrose content of the culture medium of the treatment group is reduced by half to reduce the propagation nutrition of the microbes, so the pollution rate of the culture medium can be reduced, and the invention reduces the usage amount of the antibacterial agent of the culture medium, and the two groups of culture media have no pollution after culture.
The average fresh weight of the stem leaves is heavier, the average number of the leaves is more, and the method is related to the bottle seedling open culture of the treatment group, the enhancement of photosynthesis and the promotion of growth. The average fresh weight of the root system is heavier, the number of the root system is more, the invention can reduce the usage amount of the antibacterial agent of the culture medium and reduce the toxic and side effect to the root system.
1 month after the chrysanthemum is taken out of the bottle and transplanted, the treatment composition survival rate is higher, and the method is related to that the bottle seedlings grow in a lower humidity environment, the transpiration capability is reduced, the transpiration water loss of the transplanted plants can be reduced, and is also related to that the roots of the robust seedlings obtained by the method are developed.
Example two
Method (A)
1. (1) open tissue culture medium (control group) preparation, 650ml blue vase (bottom diameter 8cm, bottleneck diameter about 4 cm), culture medium 1/2MS +6-BA3mL/L + sucrose 3% + activated carbon 0.1% + agar 0.6%. 0.3 percent of antibacterial agent sodium hypochlorite and 0.2 percent of benzalkonium bromide (the concentration of the antibacterial agent is the lowest concentration of the open tissue culture medium without microbial pollution), mixing with water, heating until the antibacterial agent is completely dissolved, fixing the volume, adjusting the pH to 5.6, subpackaging into culture bottles while hot, subpackaging each bottle with about 100ml of culture medium, covering, horizontally placing, cooling and solidifying.
(2) The oxygen slow-release particles are prepared by mixing kaolin and perlite in equal volume as filler, polyvinyl acetate as adhesive, and sodium perborate (NaBO) as oxygen release agent3·4H2O), pre-cooling and sterilizing all raw materials, and filling agent: adhesive: the weight ratio of the oxygen releasing agent is 1: 0.2: 0.6 mixing, adding ice water for granulation, and freezing and storing under aseptic condition, wherein the particle size is 0.6 cm.
The special culture medium for the treatment group (open tissue culture) is prepared by using a culture bottle and a control, wherein the culture bottle is used for preparing 1/2MS +6-BA3mL/L +1.5% of cane sugar +0.1% of active carbon + agar 0.6%, an antibacterial agent sodium hypochlorite 0.15% + benzalkonium bromide 0.1%, boiling until the mixture is completely melted, fixing the volume, adjusting the pH to 5.6, subpackaging the mixture into the culture bottle when the mixture is hot, subpackaging each bottle with about 100ml of the culture medium, adding 15 oxygen slow-release particles into the culture bottle, not adding a bottle cap, covering the culture bottle with a clean film, and flatly placing, cooling and solidifying the culture bottle.
2. Spraying 70% alcohol in the inoculation chamber for dust reduction, irradiating with ultraviolet lamp for 20 min, wiping the table top and hands with 70% alcohol, and opening the bottle cap for inoculation. Inoculating phalaenopsis into culture medium (Phalaenopsisssp.) sprouts (average fresh weight 0.215 g/strain, leaf number 2-3, root 1-2, all shorter than 0.5 cm), 16-18 strains were inoculated per bottle, control group was cultured by conventional culture with cap, treatment group was perfused with a layer of methyl silicone oil on the surface of the medium, the thickness was about 0.4cm, the culture bottle was not capped, and culture was carried out with open place. The humidity in the culture room is 65-80%.
3. Culturing for 90 days, opening the cover of the control group culture bottle, hardening off the seedlings of the control group culture bottle and the treatment group culture bottle in a greenhouse for 7 days, transplanting the seedlings in the bottle into a soilless substrate, and performing conventional management in the greenhouse.
(II) results
1. The concentration of the antibacterial agent in the culture medium of the treated group is reduced by half compared with that of the control group. The culture medium pollution rates of the control group and the treatment group are respectively 3.05 +/-0.30 percent and 3.58 +/-0.23 percent after being cultured in the bottle for 90 days, and the difference is not significant.
2. The growth indexes of the bottle seedlings are shown in the table.
Conditions of growth and transplantation of bottle seedlings of butterfly orchid
The net photosynthetic rate of the bottle seedlings is obviously increased compared with that of the control group. The average fresh weight of the stems and leaves of the treated group is heavier, the difference with the control is extremely obvious, and the average leaf number of the treated group is more than that of the control, and the difference is obvious. The survival rate of the bottle seedlings is remarkably improved compared with the control group.
The average fresh weight of the root system of the treated group is heavier, the difference with the control is obvious, and the difference of the average root number and the two groups is not obvious. The transpiration rate treated group was significantly lower than the control. After 1 month after the transplantation, the survival rate of the seedlings in the treatment group is remarkably improved compared with that in the control group
(III) conclusion
The surface of the culture medium is covered with an oil layer to block the space in the bottle from microbial contamination, and the culture medium sucrose amount of the treated group is reduced by half to reduce the propagation nutrition of the microbes, so the pollution rate of the culture medium can be reduced, so the invention reduces the usage amount of the antibacterial agent of the culture medium, and the difference of the pollution rates of the two groups of culture media after 90 days of culture is not obvious.
The invention is open culture, the average fresh weight of the stem leaves is heavier, the average number of the leaves is more, and the invention is related to strengthening photosynthesis and promoting growth. The fresh weight of the root system is heavier, and the number of the root system is not different from that of the root system of the conventional open tissue culture, which shows that the invention reduces the usage amount of the culture medium antibacterial agent and lightens the toxic and side effect on the root system. The culture bottle is open, so that air circulation is facilitated, stems and roots of the bottle seedlings are developed, and the survival rate of the bottle seedlings is improved.
The survival rate of the invention is higher after 1 month after the phalaenopsis is taken out of the bottle and transplanted, which is related to the robust plantlet obtained by the method of the invention and developed root system, and is also related to the fact that the bottle plantlet grows in a lower humidity environment, the transpiration capability is reduced, and the transpiration water loss of the transplanted plant can be reduced.
Claims (2)
1. An open tissue culture method for plants is characterized in that: (1) when preparing the open tissue culture medium, subpackaging each component of the culture medium into culture bottles, adding the oxygen slow release particles, flatly placing, cooling and solidifying; (2) after the bud seedling explant is inoculated on the culture medium, a layer of non-volatile oily liquid is poured on the surface of the culture medium, and the bud seedling explant is subjected to open culture;
the raw materials for preparing the oxygen slow-release particles comprise a filling agent, an adhesive and an oxygen release agent, and the filling agent is subjected to cold sterilization treatment in advance: adhesive: the weight ratio of the oxygen releasing agent is 1: 0.2: 0.2-0.8, adding ice water, mixing and granulating, wherein the grain diameter is 0.3-0.8cm, and the adding amount of the oxygen slow release granules is 10-25 granules per 100ml of culture medium; the filler is formed by mixing kaolin and perlite in equal volume, the adhesive is carboxymethyl cellulose or polyvinyl acetate, and the oxygen release agent is at least one of sodium percarbonate and sodium perborate;
the non-volatile oily liquid is liquid paraffin or methyl silicone oil, and the thickness of the non-volatile oily liquid in the culture medium is 0.2-0.5 cm.
2. A special culture medium for plant open tissue culture is characterized in that: oxygen-containing slow-release particles are arranged in a solid culture medium for open tissue culture, 10-25 oxygen-containing slow-release particles are arranged in each 100ml of the culture medium, a layer of non-volatile oily liquid is paved on the surface of the culture medium, the thickness is 0.2-0.5cm, and a bottle cap is not needed;
the weight ratio of the prepared raw materials of the oxygen sustained-release particles is as follows: adhesive: oxygen release agent 1: 0.2: 0.2-0.8, and the grain diameter is 0.3-0.8 cm; the filler is formed by mixing kaolin and perlite in equal volume, the adhesive is carboxymethyl cellulose or polyvinyl acetate, and the oxygen release agent is at least one of sodium percarbonate and sodium perborate;
the non-volatile oily liquid is liquid paraffin or methyl silicone oil.
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| CN87104410A (en) * | 1987-06-29 | 1988-02-03 | 北京林业大学 | Plant organism exposing breeding method and equipment |
| CN106676055A (en) * | 2017-03-10 | 2017-05-17 | 福建省亚热带植物研究所 | Method for reducing culture medium surface contamination of plant tissue culture |
| CN107586718A (en) * | 2017-10-20 | 2018-01-16 | 刘泽洋 | A kind of oxygen slow release particle preparation method applied to microbial fermentation culture |
| CN109329048A (en) * | 2018-09-25 | 2019-02-15 | 谭兴江 | A kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104410A (en) * | 1987-06-29 | 1988-02-03 | 北京林业大学 | Plant organism exposing breeding method and equipment |
| CN106676055A (en) * | 2017-03-10 | 2017-05-17 | 福建省亚热带植物研究所 | Method for reducing culture medium surface contamination of plant tissue culture |
| CN107586718A (en) * | 2017-10-20 | 2018-01-16 | 刘泽洋 | A kind of oxygen slow release particle preparation method applied to microbial fermentation culture |
| CN109329048A (en) * | 2018-09-25 | 2019-02-15 | 谭兴江 | A kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly |
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