CN112154918A - Common culture medium suitable for woody plant tissue culture - Google Patents
Common culture medium suitable for woody plant tissue culture Download PDFInfo
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- CN112154918A CN112154918A CN202011181492.0A CN202011181492A CN112154918A CN 112154918 A CN112154918 A CN 112154918A CN 202011181492 A CN202011181492 A CN 202011181492A CN 112154918 A CN112154918 A CN 112154918A
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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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Abstract
The invention discloses a common culture medium suitable for woody plant tissue culture, which relates to the technical field of plant tissue culture and comprises the following components in percentage by weight: macroelements: NH (NH)4NO3、KNO3、Ca(NO3)2·4H2O、MgSO4·7H2O and KH2PO4(ii) a Iron salt: FeSO4·7H2O and Na2-EDTA; trace elements: MnSO4·4H2O、ZnSO4·7H2O、CuSO4·5H2O、Na2MoO4·2H2O, KI and H3BO3(ii) a Organic matter: VB5、VB6、VB1Inositol, glycine, L-cysteine and VB2VH, calcium pantothenate, VC and sucrose, and has comprehensive nutrition components, salt concentration and NO3+And NH4 +The proportion is moderate, and the culture medium is suitable for being used as a basic culture medium in a plurality of plant tissue culture researches and is suitable for the growth of most plants.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a common culture medium suitable for woody plant tissue culture.
Background
The tissue culture of plants is based on the theory that plant cells have totipotency, and starts in the 30's of the 20 th century, and is subject to over 80 years of basic research and technical perfection. Tissue culture research has been successfully carried out on thousands of plants such as crops, ornamental plants, horticultural crops, economic trees and the like. Commercial test-tube plantlet production systems are available for potatoes, strawberries, bananas, sugarcane, eucalyptus, poplar and some flowers, good economic benefits and social benefits are generated, and the orchid industry and the banana industry are formed. The plant tissue culture can rapidly improve the propagation speed of perennial or plant with small seed propagation coefficient and great difficulty. In addition, the tissue culture detoxification technology improves the yield of plants, reduces the use of pesticide and chemical fertilizer, and has wide application in detoxified potatoes and detoxified strawberries. The tissue culture has great technical advantages for a plurality of fruit trees, nursery stocks, forest trees and traditional Chinese medicinal materials, and can rapidly carry out factory and batch production on the dominant single plants, reduce the propagation period and reduce the time cost. The plant tissue culture has good application in the research of plant breeding, forest germplasm resource preservation and exchange, genetics, molecular biology, genetic engineering, biological engineering and the like.
The applicant develops tissue culture technical research of excellent single eucalyptus plants from 2004, and in the research process, an MS culture medium with high salt concentration is used, so that adverse effects are easily caused on plants with slow growth speed and low requirements on inorganic salt concentration; using White medium with low salt concentration, WPM medium with high nitrate nitrogen content, high calcium content, high potassium content and no iodine or molybdenum, and low NH4 +The B5 culture medium with the content is not ideal, and the situations of browning, death, weak seedlings, abnormal leaf color or slow growth and the like often occur.
Through continuous adjustment of the basic culture medium, a culture medium named as B1 is finally found, and the culture medium is very suitable for growth of eucalyptus, strong in seedlings and normal in growth vigor. In the subsequent tissue culture researches of various plants such as bean curd vegetable, robinia pseudoacacia, photinia fraseri, Caryopteris incana, mallow floral tape, mallow red seed, mangnolia officinalis, Changshan, camptotheca acuminata, broussonetia papyrifera, walnut, radix puerariae thomsonii, kiwi fruit and the like, B1 is used as a basic culture medium, and the good effect is achieved.
Disclosure of Invention
The invention provides a common culture medium (namely B1 culture medium) suitable for woody plant tissue culture, which has comprehensive nutrient components, salt concentration and NO3+And NH4 +The proportion is moderate, and the culture medium is suitable for being used as a basic culture medium in a plurality of plant tissue culture researches and is suitable for the growth of most plants.
The technical scheme adopted by the invention for solving the problems is as follows:
a common culture medium suitable for woody plant tissue culture comprises the following components and contents:
macroelements: NH (NH)4NO3 240~480 mg/L、KNO3 1200~2400 mg/L、Ca(NO3)2·4H2O 400~800 mg/L、MgSO4·7H2O240-480 mg/L and KH2PO4 183~366mg/L；
Iron salt: FeSO4·7H219.46 to 36.14 mg/L of O and Na2-EDTA 26.11~48.49 mg/L ；
Trace elements: MnSO4·4H2O 18 ~27 mg/L、 ZnSO4·7H2O 8.64~12.96 mg/L、CuSO4·5H2O 0.02~0.03 mg/L、Na2MoO4·2H20.2-0.3 mg/L, KI 0.664.664-0.996 mg/L of O and H3BO3 4.64~6.96 mg/L；
Organic matter: VB5 2.5mg/L、VB6 1.2 mg/L、VB14.795 mg/L, inositol 100mg/L, glycine 3.997 mg/L, L-cysteine 7.5 mg/L, VB210mg/L, VH 0.3.3 mg/L, calcium pantothenate 2.395 mg/L and VC 2 mg/L;
and 10-50 g/L of sucrose.
Further, the basic culture medium comprises the following components in percentage by weight:
macroelements: NH (NH)4NO3 360 mg/L、KNO3 1800 mg/L、Ca(NO3)2·4H2O 600mg/L、MgSO4·7H2O360 mg/L and KH2PO4 275mg/L；
Iron salt: FeSO4·7H2O27.8 mg/L and Na2-EDTA 37.3 mg/L ；
Trace elements: MnSO4·4H2O 22.5mg/L、ZnSO4·7H2O 10.8mg/L、CuSO4·5H2O 0.025mg/L、Na2MoO4·2H2O0.25 mg/L, KI 0.83.83 mg/L and H3BO3 5.8 mg/L；
Organic matter: VB5 2.5mg/L、VB6 1.2 mg/L、VB14.795 mg/L, inositol 100mg/L, glycine 3.997 mg/L, L-cysteine 7.5 mg/L, VB210mg/L, VH 0.3.3 mg/L, calcium pantothenate 2.395 mg/L and VC 2 mg/L;
and sucrose 30 g/L.
Further, the basic culture medium also comprises agar for fixing, and the specific dosage is determined according to the coagulation capacity of the selected agar.
Further, the trace elements also comprise Alcl3Said Alcl being3The content of (A) is 0.024-0.036 mg/L, the specific content can be varied up and down according to the requirement of the selected plant species on chlorine, preferably, Alcl3The content of (B) is 0.03 mg/L.
Further, the basic culture medium also comprises hormones, and the hormones and the content are as follows: 6-BA 0.4 mg/L and NAA 0.2 mg/L, the content is only taken as a preference, and the content can be specifically selected according to the requirement of the selected plant species on hormones.
Further, the pH value of the basic culture medium is in the range of 5.4-6.0, which is only a preferred range and can be changed according to the requirement of the selected plant species on the pH value of the environment.
Further, the culture temperature for the plant tissue culture using the basic medium is 20 to 25 ℃, which is only a preferred range and can be changed according to the requirement of the selected plant species for the environmental temperature.
Further, the light condition for culturing the plant tissue using the basic culture medium is dark culture for 7-10 days, and then the light intensity is2000 lx-3000 lx, and 14 h/d illumination time. The reasons why dark culture is required first are: the basic culture medium is added with riboflavin VB２Riboflavin VB２The protein is involved in the metabolism of carbohydrate, protein, nucleic acid and fat, can improve the utilization rate of protein by the body, promote growth and development, can be involved in the growth and metabolism of cells, and is an essential nutrient for the metabolism and repair of tissues of the body. However, the disadvantage is that the culture medium is destroyed by light, so that the culture effect is only obvious when the culture medium is cultured under weak light.
The basic culture medium provided by the invention has the technical characteristics that:
(1) the minimal medium provided by the invention has comprehensive nutrient components and moderate salt concentration, is suitable for being used as a minimal medium in a plurality of plant tissue culture researches, is suitable for the growth of most plants, and has the characteristics of normal growth, healthy tissue culture seedlings, strong environmental adaptability, high transplanting survival rate and the like. Compared with the MS and White which are common culture media, the salt concentration is moderate, the conditions of salt poisoning caused by too high salt concentration or malnutrition caused by low salt concentration are avoided, and the incidence rate of seedling death or weak growth is reduced.
(2) NO in the minimal medium provided by the invention3+And NH4 +Moderate proportion and prevention of NH4 +The phenomenon of vitrification, edema, blackening of the top of the seedling, blackening of the cut and root system, leaf drop, yellowing of leaves or falling off into a smooth stick after browning and the like can be caused by over-high concentration.
(3) The thiamine hydrochloride VB in the organic matter of the minimal medium provided by the invention1The content of the L-cysteine and the riboflavin VB are increased when the content of the L-cysteine and the glycine are higher than that of other minimal culture media2Biotin VH and calcium pantothenate (VB)5Calcium salt of (ii). Wherein, thiamine hydrochloride VB1The function in vivo is that the coenzyme of carboxylase and glycolaldehyde transferase takes part in carbohydrate metabolism, is a key material basis in material metabolism and energy metabolism, participates in vivo oxidative decarboxylation, and is necessary for branched chain amino acid metabolism; glycine promotes the growth of isolated roots; l-cysteine for preventing browning and riboflavin VB2The biotin VH and the calcium pantothenate are beneficial to promoting the growth of test-tube plantlets; riboflavin VB2Is a component of the prosthetic group of the internal yellow enzymes (the yellow enzymes play a role of hydrogen delivery in biological oxidation-reduction), and influences the biological oxidation of the organism when lacking, so that the metabolism is disturbed; biotin VH is an essential substance for the synthesis of vitamin C, is an indispensable substance for the normal metabolism of fats and proteins, is an important factor for the fixation of carbon dioxide by living organisms, is a coenzyme for various carboxylases, and plays a role in CO in carboxylase reaction2The function of a carrier; calcium pantothenate is a coenzyme A component, and is involved in the metabolism of proteins, fats, and sugars in the body.
(4) The basic culture medium provided by the invention is also added with Alcl which is a nutrient component not contained in other common culture media3The chlorine demand of general plants is very small, while the chlorine content of halophytes is relatively high, about 70-100 mg/L, but Cl is generated in photosynthesis-Involved in photolysis of water, the division of leaf and root cells also requires Cl-In the presence of Cl-And also with K+Together with the regulation of osmotic potential, e.g. with K+The fertilizer and malic acid regulate the open and close of air holes, so that the plant lacks chlorine, which causes leaf wilting, green loss and necrosis, and finally turns brown, and simultaneously, the root system growth is blocked and becomes coarse, and the root tip turns into rod shape. Only a few pairs of Cl-Sensitive plants (such as blueberry tissue culture) do not require addition of Cl during culture-In addition, Cl was added to the minimal medium for tissue culture of other plants-Can promote the culture effect.
The invention has the beneficial effects that: the basic culture medium provided by the invention has comprehensive nutrient components, salt concentration and NO3+And NH4 +Moderate proportion, thiamine hydrochloride VB in organic matter1The content of the L-cysteine and the riboflavin VB are increased when the content of the L-cysteine and the glycine are higher than that of other minimal culture media2Biotin VH and calcium pantothenate (VB)5Calcium salt) is more suitable for the growth requirements of plants, is suitable for being used as a basic culture medium in the research of tissue culture of a plurality of plants, and is suitable for the growth of most plants.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
A common culture medium suitable for woody plant tissue culture comprises the following components in percentage by weight:
macroelements: NH (NH)4NO3 360 mg/L、KNO3 1800 mg/L、Ca(NO3)2·4H2O 600mg/L、MgSO4·7H2O360 mg/L and KH2PO4 275mg/L；
Iron salt: FeSO4·7H2O27.8 mg/L and Na2-EDTA 37.3 mg/L ；
Trace elements: MnSO4·4H2O 22.5mg/L、ZnSO4·7H2O 10.8mg/L、CuSO4·5H2O 0.025mg/L、Na2MoO4·2H2O 0.25 mg/L、KI 0.83 mg/L、H3BO35.8 mg/L and Alcl3 0.03 mg/L；
Organic matter: VB5 2.5mg/L、VB6 1.2 mg/L、VB14.795 mg/L, inositol 100mg/L, glycine 3.997 mg/L, L-cysteine 7.5 mg/L, VB210mg/L, VH 0.3.3 mg/L, calcium pantothenate 2.395 mg/L and VC 2 mg/L;
hormones: 0.4 mg/L of 6-BA and 0.2 mg/L of NAA;
and sucrose 30 g/L.
The preparation method of the basic culture medium comprises the following steps: preparing concentrated mother liquor from macroelements, microelements, ferric salt, organic matters and hormone respectively, measuring the concentrated mother liquor of the macroelements, the microelements, the ferric salt and the organic matters according to a proportion, and adding a second concentrated mother liquor after fully mixing every two concentrated mother liquors; designing different hormone formulas according to different culture purposes, and adding hormone according to the formulas; adding 30g/L of sucrose and 3.6-4.2g/L of agar, melting, and fixing the volume; then adjusting the pH value to 5.4-6.0 by using 0.1% of NaOH and 0.1% of HCl; cooling to 50 deg.C, packaging, placing into autoclave, sterilizing at 1.1 atmospheric pressure for 20min, and placing into inoculation chamber.
Example 2
The procedure for tissue culture of plants using the medium described in example 1 was as follows:
(1) explant selection and disinfection
Collecting young stem segments, scapes, leaves, tubers and the like of plants to be cultured as explants, washing the explants cleanly by flowing tap water, soaking the explants in 75% alcohol for 0 to 30 seconds, taking the explants out, washing the explants for 2 to 3 times by using sterile water, then disinfecting the explants for 3 to 10 minutes by using 0.1% mercuric chloride, washing the explants for 6 to 8 times by using the sterile water, sucking up the surface moisture by using sterile filter paper, properly trimming the explants by using surgical scissors, and then inoculating the disinfected explants into the culture medium prepared in the example 1 by using tweezers;
(2) culturing
Culturing in the dark for 7-10 days at 20-25 deg.C, and culturing under the conditions of illumination intensity of 2000 lx-3000 lx and illumination time of 14 h/d. Observing every 3 days, recording the growth condition, and removing pollution and dead culture bottles at any time.
The explant is cultured to generate multiple buds after axillary buds germinate and grow high, the buds are cut off and are continuously transferred into a new culture medium for culture, and the purpose of proliferation is achieved. When the explants are cultured in a proliferation way until enough explants are available, rooting induction is needed to ensure that the explants grow into a complete plant.
Example 3
Compared with the conventional culture medium, the basic culture medium provided by the invention has obvious effect, and the specific expression examples are as follows:
(1) in the culture process of the test seedling, if the used culture medium is an MS culture medium, brown water drops appear at the growth point at the top of the plant, and the conditions of blackening and death of the top end of the root system of the rooting seedling, stopping growth and the like appear at the same time.
(2) In the culture process of the test seedling, if the used culture medium is an MS culture medium or other culture media, the phenomena of plant vitrification and edema are very serious, the condition is gradually improved after the culture medium is changed into the basic culture medium provided by the invention, and finally the edema and the vitrification completely disappear, the test seedling grows vigorously and is more prominent in the tissue culture research of plants such as Ailanthus altissima, premna microphylla, Caryopteris incana, Magnolia officinalis, Broussonetia papyrifera, Actinidia chinensis and the like.
(3) In the process of culturing the test seedlings, if the used culture medium is an MS culture medium or other culture media, the conditions that the plants fall off into a light stick after leaves are yellowed and the like often occur. The condition is improved after the culture medium is changed into the basic culture medium provided by the invention, and new leaves grow from the newly grown terminal buds, and the appearance is obvious in tissue culture tests of walnuts and olea europaea.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A common culture medium suitable for woody plant tissue culture is characterized in that: the basic culture medium comprises the following components in percentage by weight:
macroelements：NH4NO3 360 mg/L、KNO3 1800 mg/L、Ca(NO3)2·4H2O 600mg/L、MgSO4·7H2O360 mg/L and KH2PO4 275mg/L；
Iron salt: FeSO4·7H2O27.8 mg/L and Na2-EDTA 37.3 mg/L ；
Trace elements: MnSO4·4H2O 22.5mg/L、ZnSO4·7H2O 10.8mg/L、CuSO4·5H2O 0.025mg/L、Na2MoO4·2H2O0.25 mg/L, KI 0.83.83 mg/L and H3BO3 5.8 mg/L；
Organic matter: VB5 2.5mg/L、VB6 1.2 mg/L、VB14.795 mg/L, inositol 100mg/L, glycine 3.997 mg/L, L-cysteine 7.5 mg/L, VB210mg/L, VH 0.3.3 mg/L, calcium pantothenate 2.395 mg/L and VC 2 mg/L;
and sucrose 30 g/L.
2. A common culture medium suitable for tissue culture of woody plants according to claim 1, wherein: the basic culture medium also comprises agar for fixing.
3. A common culture medium suitable for tissue culture of woody plants according to claim 1, wherein: the trace elements also comprise Alcl3Said Alcl being3The content of (b) is 0.024-0.036 mg/L.
4. A common culture medium suitable for tissue culture of woody plants according to claim 1, wherein: the basic culture medium also comprises hormones, wherein the hormones comprise the following components in percentage by weight: 0.4 mg/L of 6-BA and 0.2 mg/L of NAA.
5. A common culture medium suitable for tissue culture of woody plants according to claim 1, wherein: the pH value range of the basic culture medium is 5.4-6.0.
6. A common culture medium suitable for tissue culture of woody plants according to claim 1, wherein: the culture temperature when the basic culture medium is used for plant tissue culture is 20-25 ℃.
7. A general culture medium suitable for tissue culture of woody plants according to claim 6, wherein: the illumination condition when the basic culture medium is used for plant tissue culture is dark culture for 7-10 days, and then culture is carried out under the conditions that the illumination intensity is 2000 lx-3000 lx and the illumination time is 14 h/d.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114731950A (en) * | 2022-03-11 | 2022-07-12 | 山东省林草种质资源中心(山东省药乡林场) | Culture medium for tissue culture of pterocarpus santalinus and tissue culture method thereof |
| CN114868652A (en) * | 2022-06-07 | 2022-08-09 | 山东省果树研究所 | Culture medium for obtaining clump seedlings from jujube tree stock tissue culture seedlings and method for jujube tree stock tissue culture |
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| CN106258972A (en) * | 2016-08-17 | 2017-01-04 | 宜宾中绿林业科技开发有限公司 | A kind of method of Cinnamomum langepaniculatum tissue culture |
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Patent Citations (3)
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| CN102763596A (en) * | 2012-08-08 | 2012-11-07 | 广东省林业科学研究院 | High-efficiency quick dendrobium candidum tissue culture and propagation method |
| CN106258972A (en) * | 2016-08-17 | 2017-01-04 | 宜宾中绿林业科技开发有限公司 | A kind of method of Cinnamomum langepaniculatum tissue culture |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114731950A (en) * | 2022-03-11 | 2022-07-12 | 山东省林草种质资源中心(山东省药乡林场) | Culture medium for tissue culture of pterocarpus santalinus and tissue culture method thereof |
| CN114731950B (en) * | 2022-03-11 | 2023-01-24 | 山东省林草种质资源中心(山东省药乡林场) | Culture medium for tissue culture of pterocarpus santalinus and tissue culture method thereof |
| CN114868652A (en) * | 2022-06-07 | 2022-08-09 | 山东省果树研究所 | Culture medium for obtaining clump seedlings from jujube tree stock tissue culture seedlings and method for jujube tree stock tissue culture |
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