CN112655563B - Method for in vitro preservation of tetrastigma hemsleyanum test-tube plantlet - Google Patents
Method for in vitro preservation of tetrastigma hemsleyanum test-tube plantlet Download PDFInfo
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Abstract
The invention discloses a method for in vitro preservation of a tetrastigma hemsleyanum Diels et Gilg test-tube plantlet, which belongs to the technical field of plant cultivation and comprises the following steps: selecting a radix tetrastigme stem tip as an explant, and performing secondary culture after pre-culture; embedding the stem tip of the second culture with sodium alginate; taking out the embedding balls containing the stem tips, placing the embedding balls on sterile filter paper on a sterile culture dish, drying the embedding balls in sterile air flow for 1 to 2 hours, and then filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 10 to 20 embedding balls; and putting the cryovial filled with the embedded balls into a cryovial rack or a sand bag, and then putting the cryovial into liquid nitrogen for ultralow-temperature preservation. The invention does not form callus, avoids generating genetic variation, has good genetic stability, good recovery and growth of regenerated seedlings, and the regeneration rate of plants can reach more than 99 percent.
Description
Technical Field
The invention relates to the technical field of plant cultivation, in particular to a method for in vitro preservation of a tetrastigma hemsleyanum Diels et Gilg test-tube plantlet.
Background
Radix tetrastigme (Tetrastigma hemsleyanum Diels et Gilg) is called as radix seu caulis Tetrastigmatis trifolii and herba Anoectochili Roxburghii, belongs to mice Li Mu, and is called as grapevine. The clover is vine, small branches are fine, longitudinal ridges are arranged, and no hair or quilt is fluffy. The tendrils are not branched and are separated by 2 internodes and leaf pairs. The leaves are 3 small leaves, the small She Pi is needle-shaped, long ellipse is needle-shaped or egg is needle-shaped, the length is 3-lOcm, the width is 1.5-3 cm, the top end is gradually sharp, the tip is thin and sharp, the base part is wedge-shaped or circular, the base part of the lateral small leaves is asymmetric and is approximately circular, each side of the edge is provided with 4-6 sawteeth, the sawteeth are thin or thick sometimes, the upper part is green, the lower part is light green, and both sides have no hair; 5-6 pairs of lateral veins are not obvious on both sides of the network vein and have no hair; the petiole is 2-7.5 cm long, the central petiole is 0.5-1.8 cm long, the lateral petiole is shorter and 0.3-0.5 cm long, and has no hair or is axillary by the frigid hair, the lateral petiole is 1-5 cm long, is shorter than, nearly equal to or longer than the petiole, the lower part has a node, the node has a bud, or the false top grows without a node and a bud at the base, and the secondary branch is usually 4, and is collected to form an umbrella shape. The flowering period is 4-6 months, and the fruit period is 8-11 months. The plant is a unique medicinal plant in China, is widely distributed in the southern provinces of the Yangtze river, mainly comprises Zhejiang, jiangxi, fujian, hunan, hubei, guangdong, guangxi, guizhou, chongqing, sichuan, tibet, yunnan, hainan, taiwan and other areas, is usually grown in rocky seams in the shrubby under mountain slope forests or in the mountain valley creek forests, and has the growth altitude of 300-1300 m.
The whole herb of radix tetrastigme can be used as medicine, and the medicinal effect of underground root tuber and fruit is best. It has mild nature and slightly bitter taste, has effects of clearing heat and detoxicating, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and relieving pain, and can be used for treating venomous snake bite, tonsillitis, lymphoid tuberculosis, traumatic injury, infantile febrile convulsion, etc. At present, the traditional Chinese medicine composition is also clinically applied to resisting cancers and AIDS viruses and treating diseases such as hematopathy, cardiovascular and cerebrovascular diseases, hepatitis, viral meningitis and the like.
At present, the hemsley rockvine root is mainly dependent on wild excavation, in recent years, due to excessive excavation and habitat damage, the wild resources of the hemsley rockvine root are endangered to be extinct, artificial cultivation is not slow, but seedling supply is insufficient in the artificial cultivation, and the rapid propagation of seedlings by a tissue culture method has great market value.
In vitro subculture technology is increasingly becoming a common means for preserving test-tube plantlets. However, the method of subculture is used for tube seedling preservation, and a common problem is that subculture is frequent. Each subculture may cause cross contamination, and when the storage amount is large, the cost of manpower and material resources consumed for subculture is high. Moreover, some preserved plant tissue cultures can also cause genetic variation through multiple subcultures. The research on the radix tetrastigme at home and abroad mainly focuses on the aspects of clinical application, chemical components and pharmacological action, a few reports are made on tissue culture and rapid propagation technologies at present, but the in vitro preservation technology related to the preservation of test-tube plantlets and the genetic evaluation thereof are not reported. Moreover, the radix tetrastigme germplasm resource has high field planting difficulty, large storage workload and high cost, and is easily influenced by environmental climate and plant diseases and insect pests, and the tissue culture normal subculture is frequently transferred.
Disclosure of Invention
The invention aims to provide an in vitro preservation method of a radix tetrastigme test-tube plantlet, which is an in vitro preservation method of the radix tetrastigme test-tube plantlet, wherein the plant grows normally after being preserved for 15 months, and the progeny of the preserved material has no genetic variation.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for in vitro preservation of a radix tetrastigme test-tube plantlet, which comprises the following steps:
(1) Selecting radix tetrastigme stem tips as explants, soaking in 75% alcohol by mass concentration, washing with sterile water, dropwise adding 0.1% mercuric chloride by mass concentration, washing with sterile water, cutting into 0.5-1.0cm long, inoculating in a first culture medium, and culturing;
(2) Cutting in vitro stem tips with the diameter of 1-3mm from the strong aseptic seedlings under aseptic conditions to obtain materials, placing the in vitro stem tips into a second culture medium, and culturing;
(3) Placing the stem tip cultured in the second culture medium in a calcium-free MS culture solution of 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution containing one stem tip by using a sterile dropper or a pipette into a culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose, and keeping the culture solution at 25 ℃ for 2-30min to form an embedding ball;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying in sterile air flow for 1-2h;
(5) After drying by sterile air flow, filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 10-20 embedding balls;
(6) And putting the cryovial filled with the embedded balls into a cryovial rack or a sand bag, and then putting the cryovial into liquid nitrogen for ultralow-temperature preservation.
Preferably, the first medium comprises 500-650mg/L NH 4 NO 3 、600-700mg/L KNO 3 、100-150mg/L MgSO 4 ·7H 2 O、60-100mg/L KH 2 PO 4 0.1mg/L BA, 20-25g/L sucrose and 5-10g/L agar.
Preferably, the first culture medium is cultured at 20 + -2 deg.C under illumination intensity of 1500-2000lx for 16h/d, and subcultured for 1 time every 280-300 d.
Preferably, the second culture medium is MS solid culture medium containing 0.3-0.5mg/L sucrose.
Preferably, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 16h/d.
Preferably, each embedding sphere in step (3) is 4-5mm in size and contains a stem tip. The liquid drop containing a stem tip is dripped into the culture solution, the liquid drop reacts with calcium ions in the culture solution to generate a solidified embedding sphere, the culture solution with higher osmotic pressure further reduces the water content of the embedding sphere and the stem tip therein, and further prevents water in cells from generating ice crystals during ultralow temperature freezing.
Preferably, the ultra-low temperature preservation in step (6) is preservation at-196 ℃.
The invention discloses the following technical effects:
1. the method provided by the invention is used for in vitro preservation of radix tetrastigme, the survival rate of the test-tube plantlet is still 100% after the test-tube plantlet is preserved for 15 months, the plant growth is normal, and compared with the test-tube plantlet on a conventional normal culture medium which can only survive for 3 months, the subculture time is greatly prolonged, so that the subculture interval is changed from 1-2 month subculture at ordinary times to 1 subculture at about 10 months. Therefore, the method can prevent the seed nature degeneration and virus infection caused by multiple passages in the common passage preservation method, and ensure the excellent property and purity of the seed quality.
2. The invention provides a safe, stable, reliable, simple and effective method for preserving the tetrastigma hemsleyanum Diels et Gilg test-tube plantlet for a long time, after the in vitro stem tip of the tetrastigma hemsleyanum Diels et Gilg test-tube plantlet is preserved at ultralow temperature, the in vitro stem tip is processed by thawing treatment and regeneration recovery culture to directly form the plantlet, callus is not formed, genetic variation is avoided, the genetic stability is good, the regeneration plantlet is good in recovery and growth, and the plant regeneration rate can reach more than 99%.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
A method for in vitro preservation of radix tetrastigme test-tube plantlets comprises the following steps:
(1) Selecting a radix tetrastigme stem tip as an explant, soaking the radix tetrastigme stem tip in 75% alcohol by mass concentration, washing with sterile water for 2 times, dropwise adding 0.1% mercuric chloride by mass concentration, washing with sterile water for 8 times, cutting into 0.5cm long, inoculating the cut stems into a first culture medium, and culturing; the first culture medium comprises 500mg/L NH 4 NO 3 、700mg/L KNO 3 、150mg/L MgSO 4 ·7H 2 O、70mg/L KH 2 PO 4 0.1mg/L BA, 22g/L sucrose and 8g/L agar; at 20 +/-2 ℃, the illumination intensity is 1500lx, the illumination time is 16h/d, and 1 subculture is carried out every 280 d;
(2) Cutting an in-vitro stem tip with the diameter of 1mm from a strong aseptic seedling under aseptic conditions to be used as a material, putting the in-vitro stem tip into a second culture medium, and culturing, wherein the second culture medium is an MS solid culture medium containing 0.3mg/L sucrose, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 1800lx, and the illumination time is 16h/d;
(3) Placing the stem tip cultured in the second culture medium in a calcium-free MS culture solution containing 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution containing one stem tip into a culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose by using a sterile dropper or a pipettor, and keeping the culture solution at 25 ℃ for 15min to embed the stem tip into embedding balls, wherein each embedding ball is 5mm in size and contains one stem tip;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying for 2h in sterile air flow;
(5) After drying by sterile air flow, filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 15 embedding balls;
(6) Placing the cryovial filled with the embedded spheres on a cryovial rack, and then adding liquid nitrogen for preservation at-196 ℃.
The survival rate of the test-tube plantlet is still 100% after the test-tube plantlet is stored for 15 months, the plant grows normally, and the plant regeneration rate can reach 99%.
Example 2
A method for in vitro preservation of radix tetrastigme test-tube plantlets comprises the following steps:
(1) Selecting a radix tetrastigme stem tip as an explant, soaking the radix tetrastigme stem tip in 75% alcohol by mass concentration, washing with sterile water for 2 times, dripping mercuric chloride with the mass concentration of 0.1%, washing with sterile water for 7 times, dripping mercuric chloride with the mass concentration of 0.1%, washing with sterile water for 8 times, cutting into 0.8cm long, inoculating the cut stems into a first culture medium, and culturing; the first culture medium comprises 650mg/L NH 4 NO 3 、600mg/L KNO 3 、120mg/L MgSO 4 ·7H 2 O、100mg/L KH 2 PO 4 0.1mg/L BA, 20g/L sucrose and 10g/L agar; at 20 +/-2 ℃, the illumination intensity is 1800lx, the illumination time is 16h/d, and the subculture is carried out for 1 time every 280 d;
(2) Cutting an in-vitro stem tip with the diameter of 1mm from a strong aseptic seedling under aseptic conditions to be used as a material, putting the in-vitro stem tip into a second culture medium for culture, wherein the second culture medium is an MS solid culture medium containing 0.3mg/L sucrose, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 2000lx, and the illumination time is 16h/d;
(3) Placing the stem tip cultured in the second culture medium in a calcium-free MS culture solution containing 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution containing one stem tip into a culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose by using a sterile dropper or a pipettor, and keeping the culture solution at 25 ℃ for 15min to embed the stem tip into embedding balls, wherein each embedding ball is 4mm in size and contains one stem tip;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying for 2h in sterile air flow;
(5) After drying by sterile air flow, filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 15 embedding balls;
(6) Placing the cryovial filled with the embedded spheres on a cryovial rack, and then adding liquid nitrogen for preservation at-196 ℃.
The survival rate of the test-tube plantlet is still 100% after the test-tube plantlet is stored for 15 months, the plant grows normally, and the plant regeneration rate can reach 100%.
Example 3
A method for in vitro preservation of radix tetrastigme test-tube plantlets comprises the following steps:
(1) Selecting a radix tetrastigme stem tip as an explant, soaking the radix tetrastigme stem tip in 75% alcohol by mass concentration, washing with sterile water for 2 times, dripping mercuric chloride with the mass concentration of 0.1%, washing with sterile water for 6 times, shearing into 1.0cm long, inoculating the cut stem tip into a first culture medium, and culturing; the first medium comprises 600mg/L NH 4 NO 3 、700mg/L KNO 3 、150mg/L MgSO 4 ·7H 2 O、60mg/L KH 2 PO 4 0.1mg/L BA, 20g/L sucrose and 5g/L agar; at 20 +/-2 ℃, the illumination intensity is 1800lx, the illumination time is 16h/d, and the subculture is carried out for 1 time every 300 d;
(2) Cutting an in-vitro stem tip with the diameter of 1mm from a strong aseptic seedling under aseptic conditions to be used as a material, putting the in-vitro stem tip into a second culture medium, and culturing, wherein the second culture medium is an MS solid culture medium containing 0.3mg/L sucrose, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 1500lx, and the illumination time is 16h/d;
(3) Placing the stem tip cultured in the second culture medium in a calcium-free MS culture solution containing 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution containing one stem tip into a culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose by using a sterile dropper or a pipettor, and keeping the culture solution at 25 ℃ for 15min to embed the stem tip into embedding balls, wherein each embedding ball is 5mm in size and contains one stem tip;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying for 2h in sterile air flow;
(5) After drying by sterile air flow, filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 20 embedding balls;
(6) Placing the cryovial filled with the embedded spheres on a cryovial rack, and then adding liquid nitrogen for preservation at-196 ℃.
The survival rate of the test-tube plantlet is still 100% after the test-tube plantlet is stored for 15 months, the plant grows normally, and the plant regeneration rate can reach 100%.
Example 4
A method for in vitro preservation of radix tetrastigme test-tube plantlets comprises the following steps:
(1) Selecting a radix tetrastigme stem tip as an explant, soaking the radix tetrastigme stem tip in 75% alcohol by mass concentration, washing with sterile water for 2 times, dropwise adding 0.1% mercuric chloride by mass concentration, washing with sterile water for 7 times, dropwise adding 0.1% mercuric chloride by mass concentration, washing with sterile water for 8 times, cutting into 1.0cm long, inoculating the cut stems into a first culture medium, and culturing; the first medium comprises 500mg/L NH 4 NO 3 、650mg/L KNO 3 、100mg/L MgSO 4 ·7H 2 O、70mg/L KH 2 PO 4 0.1mg/L BA, 20g/L sucrose and 7g/L agar; at 20 +/-2 ℃, the illumination intensity is 1900lx, the illumination time is 16h/d, and the subculture is carried out for 1 time every 300 d;
(2) Cutting an in-vitro stem tip with the diameter of 1mm from a strong aseptic seedling under aseptic conditions to be used as a material, putting the in-vitro stem tip into a second culture medium for culture, wherein the second culture medium is an MS solid culture medium containing 0.3mg/L sucrose, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 1800lx, and the illumination time is 16h/d;
(3) Placing the stem tip cultured in the second culture medium in a calcium-free MS culture solution containing 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution containing one stem tip into a culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose by using a sterile dropper or a pipettor, and keeping the culture solution at 25 ℃ for 15min to embed the stem tip into embedding balls, wherein each embedding ball is 4mm in size and contains one stem tip;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying for 2h in sterile air flow;
(5) After drying by sterile air flow, filling the embedding balls into freezing pipes, wherein each freezing pipe is filled with 10 embedding balls;
(6) Placing the cryovial filled with the embedded spheres on a cryovial rack, and then adding liquid nitrogen for preservation at-196 ℃.
The survival rate of the test-tube plantlet is still 100% after the test-tube plantlet is stored for 14 months, the plant grows normally, and the plant regeneration rate can reach 99%.
Comparative example 1
The difference from example 3 is only that the amount of each component added in the first medium is halved.
The survival rate of the test-tube plantlet is 98% after 5 months of storage, the plant grows normally, and the plant regeneration rate is 90%.
Comparative example 2
The only difference from example 3 is that no embedding was carried out.
The survival rate of the test-tube plantlet stored for 4 months in the embodiment is 95%, the plant grows normally, and the regeneration rate of the plant is 85%.
Comparative example 3
The only difference from example 3 was that each sphere contained 2 stem tips. The survival rate of the test-tube plantlet in the comparative example is 90% after the test-tube plantlet is stored for 6 months, the plant grows normally, and the plant regeneration rate is 80%.
Comparative example 4
The difference from example 3 is only that the second medium does not contain calcium or calcium ions. The survival rate of the test-tube plantlet is 75% after the test-tube plantlet is stored for 5 months, the plant grows normally, and the plant regeneration rate is 65%.
The survival rate and plant regeneration rate of the examples and comparative examples of the present invention were calculated by taking out the freezing tubes of the examples and comparative examples from liquid nitrogen, thawing the freezing tubes in a water bath at 38 ℃ for 3min, and thawing the frozen embedding balls in 950mg/L KNO 3 And 825mg/L NH 4 NO 3 The MS +0.1mg/LBA +30g/L sucrose +6.5g/L agar, and the culture medium with the pH value of 5.8 are subjected to recovery culture, and after the culture is firstly carried out under dark conditions or weak light for 8 to 10 days, the culture is carried out under the same conditions as the second culture medium.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (3)
1. A method for in vitro preservation of a radix tetrastigme test-tube plantlet is characterized by comprising the following steps:
(1) Selecting radix tetrastigme stem tips as explants, soaking in 75% alcohol with mass concentration, washing with sterile water, dropwise adding 0.1% mercuric chloride with mass concentration, washing with sterile water, cutting into 0.5-1.0cm long, inoculating in a first culture medium, culturing, wherein the first culture medium comprises 500-650mg/L NH 4 NO 3 、600-700mg/L KNO 3 、100-150mg/L MgSO 4 ·7H 2 O、60-100mg/L KH 2 PO 4 0.1mg/L BA, 20-25g/L sucrose and 5-10g/L agar, and carrying out subculture for 1 time every 280-300d at the culture temperature of 20 +/-2 ℃ in a first culture medium, the illumination intensity of 1500-2000lx and the illumination time of 16h/d;
(2) Cutting an isolated stem tip with the diameter of 1-3mm from a strong aseptic seedling under aseptic conditions to be used as a material, placing the isolated stem tip into a second culture medium for culture, wherein the second culture medium is an MS solid culture medium containing 0.3-0.5mg/L sucrose, the culture temperature of the second culture medium is 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 16h/d;
(3) Placing the aseptic seedling cultured in the second culture medium in a calcium-free MS culture solution containing 25g/L sodium alginate, 2mol/L glycerol and 0.8mol/L sucrose, sucking a calcium-free MS culture solution drop containing a stem tip into the culture solution containing 0.1mol/L calcium chloride, 2mol/L glycerol and 0.8mol/L sucrose by using an aseptic dropper or a pipette, and keeping the culture solution at 25 ℃ for 2-30min to form an embedding ball;
(4) Taking out the embedding ball containing the stem tip, placing on sterile filter paper on a sterile culture dish, and drying in sterile air flow for 1-2h;
(5) After drying by sterile air flow, filling the embedding balls into freezing tubes, wherein each freezing tube is filled with 10-20 embedding balls;
(6) And putting the cryovial filled with the embedded balls into a cryovial rack or a sand bag, and then putting the cryovial into liquid nitrogen for ultralow-temperature preservation.
2. The method for in vitro preservation of tetrastigma hemsleyanum Diels et al plantlets of claim 1, wherein in step (3), each embedding sphere is 4-5mm in size and comprises a stem tip.
3. The method for in vitro preservation of tetrastigma hemsleyanum Diels et Gilg as claimed in claim 1, wherein the ultra-low temperature preservation in step (6) is preservation at-196 ℃.
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| CN103299907B (en) * | 2013-06-04 | 2014-12-03 | 浙江医药高等专科学校 | Method for conserving in-vitro body of radix tetrastigme germplasm resources and recovering growth after conservation |
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