CN115997684B - Culture medium and culture method for tissue culture and seedling raising of cercis chinensis - Google Patents
Culture medium and culture method for tissue culture and seedling raising of cercis chinensis Download PDFInfo
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- CN115997684B CN115997684B CN202211730598.0A CN202211730598A CN115997684B CN 115997684 B CN115997684 B CN 115997684B CN 202211730598 A CN202211730598 A CN 202211730598A CN 115997684 B CN115997684 B CN 115997684B
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Abstract
The invention discloses a culture medium for tissue culture and seedling raising of cercis chinensis and a culture method, wherein the culture medium comprises an induction culture medium, a proliferation culture medium and a rooting culture medium; the induction culture medium is an MS culture medium containing 1.0-3.0 mg/L6-benzyl aminopurine, 0.1-0.3 mg/L a-naphthylacetic acid, 15-25 mg/L vitamin C, 25-35 g/L sucrose and 4-8 g/L agar; the proliferation culture medium is a 3/2WPM culture medium containing 0.5-2.0 mg/L6-benzyl aminopurine, 0.05-0.15 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar; the rooting culture medium is a WPM culture medium containing 0.2-1.0 mg/La-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar. The invention provides a method for tissue culture and rapid propagation of a cercis chinensis adult superior strain, provides a proper culture medium and culture environment conditions, provides an effective way for tissue culture and rapid propagation of a cercis chinensis superior strain, is simple to operate and low in cost, can be used for large-scale production, provides technical support and reference for asexual popularization of cercis chinensis and even bauhinia plants, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for culturing cercis chinensis tissue.
Background
Cercis chinensis (Bauhinia variegata Linn.) belonging to genus Rumex (Bauhinia Linn.) of family Leguminosae, is a larch tree, and is mainly distributed in south and southwest China, india and peninsula.
The bark of cercis chinensis powder is dark brown and is almost smooth; she Jin it is a revolutionary, leaf-shaped, wide oval to nearly circular, and similar to a sheep's foot; tree form expansion, tree height can reach 7m; big flowers, like butterfly. The cercis chinensis phenotype has rich genetic diversity, especially the color, purple or pale red and white, and yellow-green and dark purple veins. The flowering period of cercis chinensis reaches full year, 3 months is full flowering period, buds and branches are full flowering period, and the cercis chinensis is a good plant for viewing flowers and viewing leaves. Cercis chinensis is planted in Guangzhou, salix and other places in large quantities, can form a landscape effect of 'flowers and plants' in the full bloom stage, has the characteristics of strong stress resistance, good adaptability, pruning resistance, huge flower quantity and the like, and is widely applied to urban and rural landscaping in southern areas of China as ornamental tree species.
At present, the propagation mode of cercis chinensis is mainly seeding and seedling raising, but the seeding and propagation have the problems of genetic differentiation, incapability of completely maintaining parent characteristics by offspring and the like, and the flower type and the flower color are not uniform, so that the landscape effect is affected.
The prior art has not been reported that the tissue culture of cercis chinensis has been successful, and the applicant has previously studied to find that the conventional tissue culture method is used for adult strains of cercis chinensis, has low induction rate, and is difficult to obtain the cercis chinensis with consistent growth vigor and color.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention provides a culture medium for tissue culture and seedling raising of cercis chinensis by using palace powder and a culture method.
The first object of the invention is to provide a culture medium for tissue culture of cercis chinensis.
The second object of the invention is to provide the application of the culture medium in tissue culture of cercis chinensis.
The third object of the invention is to provide the application of the culture medium in the cultivation of cercis chinensis seedlings.
The fourth object of the invention is to provide a method for tissue culture and seedling raising of cercis chinensis.
A fifth object of the present invention is to provide the use of the above method in the breeding and/or planting of cercis chinensis.
In order to achieve the above object, the present invention is realized by the following means:
plant cells are totipotent, i.e., each cell of a plant has the ability to develop into a complete plant. The plant asexual propagation mode can effectively preserve the excellent characteristics of the female parent, and plant tissue culture is one of the effective means of rapid asexual propagation.
A culture medium for tissue culture of cercis chinensis, comprising an induction culture medium, a proliferation culture medium and a rooting culture medium;
the induction culture medium is an MS culture medium containing 1.0-3.0 mg/L6-benzyl aminopurine, 0.1-0.3 mg/L a-naphthylacetic acid, 15-25 mg/L vitamin C, 25-35 g/L sucrose and 4-8 g/L agar;
the proliferation culture medium is a 3/2WPM culture medium containing 0.5-2.0 mg/L6-benzyl aminopurine, 0.05-0.15 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar;
the rooting culture medium is a WPM culture medium containing 0.2-1.0 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar.
The 3/2WPM medium is prepared by using 1.5 times of the formula designed in 1981 by Lloyd and McCown.
Preferably, the induction medium is MS medium comprising 2.0 mg/L6-benzylaminopurine, 0.2mg/L a-naphthylacetic acid, 20mg/L vitamin C, 30g/L sucrose and 6g/L agar.
Preferably, the proliferation medium is a 3/2WPM medium comprising 1.2 mg/L6-benzylaminopurine, 0.1mg/L a-naphthylacetic acid, 30g/L sucrose and 6g/L agar.
Preferably, the rooting medium is a WPM medium comprising 0.6mg/L a-naphthylacetic acid, 30g/L sucrose and 6g/L agar.
The application of the culture medium in tissue culture of cercis chinensis powder is also within the protection scope of the invention.
Preferably, the tissue culture of cercis chinensis comprises one or more of obtaining adventitious buds, obtaining cluster buds and/or rooting.
The application of the culture medium in raising cercis chinensis seedlings is also within the protection scope of the invention.
Preferably, the cercis chinensis seedling culture is a tissue culture seedling culture.
A tissue culture seedling method of cercis chinensis comprises the following steps:
s1, placing the sterilized explant in the induction culture medium, performing dark culture for 5-12 days, and performing light-dark alternate culture to obtain adventitious buds, wherein the length of the adventitious buds is 2-4 cm; the explant is a semi-lignified branch of cercis chinensis; the induction culture medium is an MS culture medium containing 1.0-3.0 mg/L6-benzyl aminopurine, 0.1-0.3 mg/L a-naphthylacetic acid, 15-25 mg/L vitamin C, 25-35 g/L sucrose and 4-8 g/L agar;
s2, placing the adventitious buds obtained in the step S1 in the proliferation culture medium, performing dark culture for 4-7 days, and performing light-dark alternate culture to obtain cluster buds, wherein the length of the cluster buds is 1.5-3 cm; the proliferation culture medium is a 3/2WPM culture medium containing 0.5-2.0 mg/L6-benzyl aminopurine, 0.05-0.15 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar;
s3, placing the terminal buds with the length of 1.5-2.5 cm obtained in the step S2 in the rooting culture medium, and alternately culturing in a light-dark mode to obtain tissue culture seedlings, wherein the length of roots of the tissue culture seedlings is 1.5-3 cm; the rooting culture medium is a WPM culture medium containing 0.2-1.0 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar;
s4, hardening off seedlings of the tissue culture Miao Yu obtained in the step S3 for 6-14 days at the temperature of 23-27 ℃ with the shading rate of 50% -80%;
s5, sterilizing the tissue culture seedlings after seedling hardening, transplanting the tissue culture seedlings into a substrate containing peat soil and perlite, covering a film on the surface of the substrate, and culturing for 8-12 days.
Preferably, in step S1, the explant is a stem segment with a length of 2-3 cm and 1-2 axillary buds.
Preferably, in step S1, the explants are taken from an adult elite individual of cercis pinnata.
More preferably, in step S1, the explants are harvested from an adult elite individual of cercis chinensis having a high flower mass and deep red flower color.
Preferably, in step S1, the explants are collected in the morning when the weather is clear.
More preferably, in step S1, the explants are harvested at 9 to 10 am.
Preferably, in step S1, the explant is subjected to impurity removal and then disinfection, and the impurity removal method comprises the following steps: leaves of the explant are removed, leaves stems with the length of 5-10 mm are reserved, and dirt on the explant is fully removed.
More preferably, in step S1, the leaves of the explant are removed, leaving 5mm petioles.
More preferably, in step S1, the method for substantially removing the soil on the explant is: washing with water for 5-20 min, and brushing with a soft writing brush in the detergent solution; the residual detergent solution on the explants was removed with water.
Preferably, in step S1, the method of disinfection is as follows: sterilizing with 0.8-1.2% (w/v) of benzalkonium chloride and/or 0.08-0.12% (w/v) of mercuric chloride solution.
More preferably, in step S1, the method of sterilization is: disinfecting with 1% (w/v) benzalkonium bromide and/or 0.1% (w/v) mercuric chloride solution.
Further preferably, in step S1, the method of disinfection is: washing the explant with sterile water; soaking the explant in 1% (w/v) benzalkonium chloride for 4-7 min; removing the residual benzalkonium bromide on the explant with sterile water; soaking the explant in 0.1% (w/v) mercuric chloride solution for 1-5 min; the residual mercuric solution on the explants was removed with sterile water.
Still further preferably, in step S1, the sterilization process requires continuous shaking.
Preferably, in step S2, the induction medium is MS medium comprising 2.0 mg/L6-benzylaminopurine, 0.2mg/L a-naphthylacetic acid, 20mg/L vitamin C, 30g/L sucrose and 6g/L agar.
Preferably, in step S2, the culture is performed for 7 days in dark.
Preferably, in step S2, the method for alternately culturing light and dark includes: culturing with light for 12 hr every day, and culturing in dark for the rest time.
More preferably, in step S2, the intensity of the illumination is 2000lx.
Preferably, in step S2, the temperature of the dark culture and the light-dark alternate culture is 23 to 27 ℃.
Preferably, in step S3, the propagation medium is a 3/2WPM medium comprising 1.2 mg/L6-benzylaminopurine, 0.1mg/L a-naphthylacetic acid, 30g/L sucrose and 6g/L agar.
Preferably, in step S3, the dark culture is performed for 5 days.
Preferably, in step S3, the method for alternately culturing light and dark includes: culturing with light for 12 hr every day, and culturing in dark for the rest time.
More preferably, in step S3, the intensity of the illumination is 2000lx.
Preferably, in step S3, the temperature of the dark culture and the light-dark alternate culture is 23 to 27 ℃.
Preferably, in step S4, the rooting medium is WPM medium comprising 0.6mg/L a-naphthylacetic acid, 30g/L sucrose and 6g/L agar.
Preferably, in step S5, the tissue culture Miao Zheguang obtained in step S4 is subjected to seedling hardening at a rate of 70%.
Preferably, in the step S5, the seedlings are acclimatized for 7 to 10 days.
Preferably, in the step S5, the tissue culture Miao Yuzu is cultured in a culture bottle, and after hardening off the seedlings for 6-9 days, the tissue culture bottle cap is screwed to be half-opened and placed for 1-2 days.
More preferably, in step S5, the tissue culture Miao Yuzu is cultured in a flask, and after hardening the seedling for 6 days, the tissue culture flask is closed to half open for 2 days.
Preferably, in step S6, the method for disinfecting the root is: removing the culture medium on the roots, and soaking in 600 times carbendazim solution for 15-20 min.
Preferably, in step S6, the substrate is formed by peat soil and perlite according to the volume ratio (8-12): (0.8-1.2) and evenly mixing.
More preferably, in step S6, the matrix is formed by peat soil and perlite in a volume ratio of 10:1, uniformly mixing to obtain the product.
Preferably, in step S6, the substrate is a potassium permanganate sterilization substrate.
More preferably, in step S6, the method for disinfecting potassium permanganate is as follows: spraying potassium permanganate solution with mass fraction of 0.1-0.2% to penetrate the matrix, standing for 24-48 h, and removing the potassium permanganate solution by tap water.
Preferably, in step S6, the transplanting depth is the depth at which the soil is just covered by the root.
Preferably, in step S6, the method further comprises compacting the matrix to stabilize the nursery stock, spraying root fixing water, and spraying 600 times carbendazim solution.
Preferably, in step S6, the surface of the substrate is covered with a film, covered with a sunshade net, and cultured for 10 days, after which the film is uncovered for conventional seedling management.
The application of any of the above methods in the breeding and/or planting of cercis chinensis is also within the scope of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a tissue culture and rapid propagation seedling method for an adult high-quality plant of cercis chinensis, which provides a culture medium and culture environment conditions with proper plant growth regulator components and concentration level ratios in each stage of tissue culture according to the physiological state of the adult high-quality plant of cercis chinensis, reduces the pollution rate and browning rate in the sterile system establishment process, improves the induction rate and proliferation coefficient, has strong cluster buds, rapid elongation and growth, multiple effective buds, rapid rooting speed, high rooting rate, developed root system and high survival rate of tissue culture bud transplantation, and finally can obtain healthy seedlings with regular growth and consistent phenotype in batches.
The invention provides a tissue culture rapid propagation technology using the half lignified branches of the adult superior plant of cercis chinensis as explants, provides an effective way for tissue culture rapid propagation of superior germplasm of cercis chinensis, has simple operation and low cost, can be used for large-scale production, provides technical support and reference for asexual popularization of the cercis chinensis and even the bauhinia plants, and has wide application prospect.
Drawings
FIG. 1 is a photograph of adventitious buds obtained after induction culture using the medium of example 1.
FIG. 2 is a photograph of a proliferation seedling obtained after proliferation culture using the medium of example 1.
FIG. 3 is a photograph of root systems of rooted seedlings obtained after rooting culture using the medium of example 1.
FIG. 4 is a photograph of individual seedlings after rooting culture using the medium of example 1.
FIG. 5 is a photograph showing a rooting seedling cultured using the medium of example 1 after transplanting for one month.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The MS medium used in the examples of the present invention was a formulation designed in 1962 by Murashige and Skoog, and the WPM medium used was a formulation designed in 1981 by Lloyd and McCown.
The method for collecting and processing the explant used in the embodiment of the invention is a conventional method in the field, and comprises the following steps:
(1) Acquisition of
In the morning of sunny weather, collecting semi-lignified branches with 4-6 axillary buds of adult fine single plants (large flower quantity and deep purple red) of cercis chinensis as explants;
(2) Pretreatment of
Cutting off leaves of the explant, reserving a 5-8 mm leaf stalk, and flushing for 5-20 min by running water; placing the explant in a detergent solution, carefully brushing the explant with a soft hair pen, and cleaning the places where dirt is easy to be stored in the stem sections and the leaf axilla; brushing the explant with deionized water, and cleaning off the detergent solution and the residual dirt; washing the residual detergent solution on the explant with deionized water;
(3) Sterilization
Cutting the explant into stem segments with 2-3 cm and 1 or 2 axillary buds in a sterile environment, and cleaning the explant with sterile water; placing the explant into 1% (w/v) of benzalkonium bromide for soaking and disinfecting for 4-7 min, pouring out the benzalkonium bromide, and flushing with sterile water for 2-4 times; soaking and sterilizing the explant in 0.1% (w/v) mercuric chloride solution for 1-5 min, pouring out the mercuric chloride solution, and flushing with sterile water for 5-6 times; the sterilization with benzalkonium bromide, mercuric chloride and aseptic water cleaning processes all require continuous shaking.
The transplanting method used in the embodiment of the invention is a conventional method in the field, and comprises the following steps:
2-4 d before transplanting, peat soil and perlite are mixed according to the volume ratio of 10:1, uniformly mixing to obtain a matrix, spraying and leaching with a potassium permanganate solution with the mass fraction of 0.1-0.2%, standing for 24-48 h, spraying and leaching with tap water, washing the potassium permanganate clean, and using after 1-3 d;
carefully taking out the tissue culture seedlings from the tissue culture bottles, cleaning a culture medium on the roots of the tissue culture seedlings, soaking the tissue culture seedlings in 600 times carbendazim solution for 10-20 min, and then transplanting the tissue culture seedlings into a matrix, wherein the transplanting depth is that the roots are just covered by covering soil. Compacting the matrix to stabilize the seedlings;
after transplanting, sprinkling root fixing water, spraying 600 times carbendazim solution, covering a film on the surface of the soil for moisturizing, and covering a sunshade net for shading;
after 10d, the film was uncovered and managed as conventional seedlings.
Example 1A culture Medium for tissue culture and seedling of Cercis chinensis
The invention provides a culture medium combination for tissue culture and seedling raising of cercis chinensis, which comprises an induction culture medium, a proliferation culture medium and a rooting culture medium:
the induction culture medium is an MS culture medium containing 1.0-3.0 mg/L6-benzyl aminopurine (6-BA), 0.1-0.3 mg/L a-naphthylacetic acid (NAA), 15-25 mg/L Vitamin C (VC), 25-35 g/L sucrose and 4-8 g/L agar;
the proliferation culture medium is a 3/2WPM culture medium containing 0.5-2.0 mg/L6-benzyl aminopurine, 0.05-0.15 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar, wherein the 3/2WPM culture medium is prepared by using 1.5 times of a formula designed in 1981 by Lloyd and McCown;
the rooting culture medium is a WPM culture medium containing 0.2-1.0 mg/L a-naphthylacetic acid, 25-35 g/L sucrose and 4-8 g/L agar.
Three specific medium combinations are given below for the following cercis palustris tissue culture seedling experiments:
(1) Culture medium combination 1 for tissue culture and seedling raising of cercis chinensis palace powder:
the induction culture medium is an MS culture medium containing 2.0 mg/L6-BA, 0.2mg/L NAA, 20mg/L VC, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the proliferation culture medium is a 3/2WPM culture medium containing 1.2 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the rooting culture medium is a WPM culture medium containing 0.6mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
(2) Culture medium combination 2 for tissue culture and seedling raising of cercis chinensis palace powder:
the induction culture medium is an MS culture medium containing 1.0 mg/L6-BA, 0.1mg/L NAA, 15mg/L VC, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the proliferation culture medium is a 3/2WPM culture medium containing 1.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the rooting culture medium is a WPM culture medium containing 1.0mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
(3) Culture medium combination 3 for tissue culture and seedling raising of cercis chinensis palace powder:
the induction culture medium is an MS culture medium containing 3.0 mg/L6-BA, 0.2mg/L NAA, 25mg/L VC, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the proliferation culture medium is a 3/2WPM culture medium containing 0.8 mg/L6-BA, 0.08mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0;
the rooting culture medium is a WPM culture medium containing 0.3mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
Example 2A method for tissue culture and seedling raising of Cercis chinensis
Using the medium combination 1 of example 1, tissue culture seedling raising of Cercis chinensis was performed according to the following procedure: 1. collecting and processing explants; 2. induction culture; 3. proliferation culture; 4. rooting culture; 5. hardening seedlings; 6. transplanting. Steps 1 and 6 are conventional in the art, and specific operation methods are given below for reference.
1. Acquisition and processing of explants
(1) Acquisition of
At 10 am when the weather is clear: about 00 hours, collecting semi-lignified branches with 4-6 axillary buds of adult fine single plants (large flower quantity and deep purple red) of cercis chinensis as explants.
(2) Pretreatment of
Cutting off leaves of the explant, reserving a 5mm petiole, and cleaning the explant by running water, detergent solution and deionized water.
(3) Sterilization
In a sterile environment, cutting the explant into stem segments with 2-3 cm and 1 or 2 axillary buds, cleaning the explant with sterile water, putting into 1% (w/v) benzalkonium bromide for soaking and sterilizing for 5min, pouring out the benzalkonium bromide, and flushing with sterile water for 3 times; the explant was then soaked in 0.1% (w/v) mercuric chloride solution for 5min and sterilized, after which the mercuric chloride solution was poured off and rinsed 5 times with sterile water.
2. Induction culture
Cutting wound parts of the explants contacted with the sterilizing solution by using a sterile blade, transferring the wound parts into tissue culture bottles filled with an induction culture medium, and inducing the growth of axillary buds by using 1 explant per bottle; the induction culture medium is MS culture medium containing 2.0 mg/L6-BA, 0.2mg/L NAA, 20mg/L VC, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark-cultured for 7d at the initial stage of transfer, then the culture is carried out for 12h by illumination every day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ for the rest of the dark culture.
3. Proliferation culture
Cutting axillary buds with the length of more than 2cm on an explant by using a sterile blade in a sterile environment, transferring the axillary buds into a tissue culture bottle filled with a proliferation culture medium, and culturing the proliferation buds; the proliferation culture medium is 3/2WPM culture medium containing 1.2 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark cultured for 5 days at the initial stage of transfer, then the culture is carried out under illumination for 12 hours every day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ during the rest of the dark culture.
The period of multiplication culture is about 30d, and after 2-3 times of subculture under the same condition, single buds form bud clusters (namely multiplication buds).
4. Rooting culture
Cutting the terminal buds with the length of 1.5-2.0 cm on the proliferation buds which grow robustly by using a sterile blade in a sterile environment, and inoculating the terminal buds into a tissue culture bottle filled with a rooting culture medium for rooting culture; the rooting culture medium is WPM culture medium containing 0.6mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
The culture is carried out for 12 hours under the illumination of about 1500lx every day, and the culture is carried out in dark for the rest time, wherein the culture temperature is 25+/-2 ℃.
5. Hardening off seedlings
After rooting culture for 20d, transferring the tissue culture seedlings after rooting culture to a greenhouse with a sunshade rate of 70% to smelt seedlings, wherein the temperature is 25+/-2 ℃.
After hardening seedlings for 6d, the tissue culture bottle cap is screwed to a half-open state for 2d.
6. Transplanting
2d before transplanting, peat soil and perlite are mixed according to the volume ratio of 10:1, uniformly mixing to obtain a matrix, spraying and leaching the matrix by using a potassium permanganate solution with the mass fraction of 0.1%, standing for 24 hours, spraying and leaching the matrix by using tap water, washing the potassium permanganate, and using the matrix after 2 days.
Carefully taking out the tissue culture seedlings from the tissue culture bottles, cleaning a culture medium on the roots of the tissue culture seedlings, soaking the tissue culture seedlings in a 600-time carbendazim solution for 15min, and transplanting the tissue culture seedlings into a matrix, wherein the transplanting depth is that the roots are just covered by covering soil. Compacting the matrix to stabilize the nursery stock.
After transplanting, the root fixing water is sprayed, 600 times carbendazim solution is sprayed, the surface of the soil is covered with a film for moisturizing, and a sunshade net is covered for shading.
After 10d, the film was uncovered and managed as conventional seedlings.
Example 3A method for tissue culture and seedling raising of Cercis chinensis
Using the medium combination 2 of example 1, tissue culture seedling raising of Cercis chinensis was performed according to the following procedure: 1. collecting and processing explants; 2. induction culture; 3. proliferation culture; 4. rooting culture; 5. hardening seedlings; 6. transplanting. Steps 1 and 6 are conventional in the art, and specific operation methods are given below for reference.
1. Acquisition and processing of explants
(1) Acquisition of
And collecting semi-lignified branches with about 4-6 axillary buds of adult fine single plants (large flower quantity and deep red flower) of cercis chinensis as explants in the morning of sunny weather about 9.
(2) Pretreatment of
Cutting off leaves of the explant, reserving 7-8 mm leaf stalks, and cleaning the explant by running water, detergent solution and deionized water.
(3) Sterilization
In a sterile environment, cutting the explant into stem segments with 2-3 cm and 1 or 2 axillary buds, cleaning the explant with sterile water, putting into 1% (w/v) benzalkonium bromide for soaking and sterilizing for 6min, pouring out the benzalkonium bromide, and flushing with sterile water for 4 times; the explant was soaked in 0.1% (w/v) mercuric chloride solution for 3min and sterilized, and after pouring out the mercuric chloride solution, rinsed 6 times with sterile water.
2. Induction culture
Cutting wound parts of the explants contacted with the sterilizing solution by using a sterile blade, transferring the wound parts into tissue culture bottles filled with an induction culture medium, and inducing the growth of axillary buds by using 1 explant per bottle; the induction culture medium is MS culture medium containing 1.0 mg/L6-BA, 0.1mg/L NAA, 15mg/L VC, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark-cultured for 6 days at the initial stage of transfer, then the culture is carried out under illumination for 12 hours every day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ during the rest of the dark culture.
3. Proliferation culture
Cutting axillary buds with the length of more than 2cm on an explant by using a sterile blade in a sterile environment, transferring the axillary buds into a tissue culture bottle filled with a proliferation culture medium, and culturing the proliferation buds; the proliferation culture medium is 3/2WPM culture medium containing 1.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark-cultured for 6 days at the initial stage of transfer, then the culture is carried out under illumination for 12 hours every day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ during the rest of the dark culture.
The period of multiplication culture is about 30d, and after 2-3 times of subculture under the same condition, single buds form bud clusters (namely multiplication buds).
4. Rooting culture
Cutting the terminal buds with the length of 2.0-2.5 cm on the proliferation buds which grow robustly by using a sterile blade in a sterile environment, and inoculating the terminal buds into a tissue culture bottle filled with a rooting culture medium for rooting culture; the rooting culture medium is WPM culture medium containing 1.0mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
The culture is carried out for 12 hours under the illumination of about 1500lx every day, and the culture is carried out in dark for the rest time, wherein the culture temperature is 25+/-2 ℃.
5. Hardening off seedlings
After rooting culture for 25d, the tissue culture seedlings after rooting culture are transferred to a greenhouse with a sun shading rate of 60% with a bottle for seedling hardening, and the temperature is 25+/-2 ℃.
After hardening the seedlings for 7d, the tissue culture bottle cap is screwed to a half-open state for 1d.
6. Transplanting
3d before transplanting, peat soil and perlite are mixed according to the volume ratio of 10:1, uniformly mixing to obtain a matrix, spraying and spraying the matrix by using a potassium permanganate solution with the mass fraction of 0.2%, standing for 24 hours, spraying and spraying by using tap water, washing the potassium permanganate, and standing for 2 days for use.
Carefully taking out the tissue culture seedlings from the tissue culture bottles, cleaning a culture medium on the roots of the tissue culture seedlings, soaking the tissue culture seedlings in a 600-time carbendazim solution for 20min, and transplanting the tissue culture seedlings into a matrix, wherein the transplanting depth is that the roots are just covered by covering soil. Compacting the matrix to stabilize the nursery stock.
After transplanting, the root fixing water is sprayed, 600 times carbendazim solution is sprayed, the surface of the soil is covered with a film for moisturizing, and a sunshade net is covered for shading.
After 10d, the film was uncovered and managed as conventional seedlings.
Example 4A method for tissue culture and seedling raising of Cercis chinensis
Using the medium combination 3 of example 1, tissue culture seedling raising of Cercis chinensis was performed according to the following procedure: 1. collecting and processing explants; 2. induction culture; 3. proliferation culture; 4. rooting culture; 5. hardening seedlings; 6. transplanting. Steps 1 and 6 are conventional in the art, and specific operation methods are given below for reference.
1. Acquisition and processing of explants
(1) Acquisition of
And collecting semi-lignified branches with about 6 axillary buds of adult fine single plants (large flower quantity and deep red flower) of cercis chinensis as explants in the morning of sunny weather about 10 a.m.
(2) Pretreatment of
Cutting off leaves of the explant, reserving 6mm petioles, and cleaning the explant by running water, detergent solution and deionized water.
(3) Sterilization
In a sterile environment, cutting the explant into stem segments with 2-3 cm and 1 or 2 axillary buds, cleaning the explant with sterile water, putting into 1% (w/v) benzalkonium bromide for soaking and sterilizing for 7min, pouring out the benzalkonium bromide, and flushing with sterile water for 2 times; the explant was soaked in 0.1% (w/v) mercuric chloride solution for 5min and sterilized, and after pouring out the mercuric chloride solution, rinsed 5 times with sterile water.
The sterilization with benzalkonium bromide, mercuric chloride and aseptic water cleaning processes all require continuous shaking.
2. Induction culture
Cutting wound parts of the explants contacted with the sterilizing solution by using a sterile blade, transferring the wound parts into tissue culture bottles filled with an induction culture medium, and inducing the growth of axillary buds by using 1 explant per bottle; the induction culture medium is MS culture medium containing 3.0 mg/L6-BA, 0.2mg/L NAA, 25mg/L VC, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark-cultured for 9d at the initial stage of transfer, then the culture is carried out under illumination for 12h each day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ during the rest of the dark culture.
3. Proliferation culture
Cutting axillary buds with the length of more than 2cm on an explant by using a sterile blade in a sterile environment, transferring the axillary buds into a tissue culture bottle filled with a proliferation culture medium, and culturing the proliferation buds; the proliferation culture medium is 3/2WPM culture medium containing 0.8 mg/L6-BA, 0.08mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
The culture is completely dark-cultured for 6 days at the initial stage of transfer, then the culture is carried out under illumination for 12 hours every day, the illumination intensity is about 2000lx, and the culture temperature is 25+/-2 ℃ during the rest of the dark culture.
The period of the multiplication culture is about 30d, and after 2-3 times of subculture under the same conditions, single buds form bud clusters (multiplication buds).
4. Rooting culture
Cutting the terminal buds with the length of 1.5-2.0 cm on the proliferation buds which grow robustly by using a sterile blade in a sterile environment, and inoculating the terminal buds into a tissue culture bottle filled with a rooting culture medium for rooting culture; the rooting culture medium is WPM culture medium containing 0.3mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
The culture is carried out for 12 hours under the illumination of about 1500lx every day, and the culture is carried out in dark for the rest time, wherein the culture temperature is 25+/-2 ℃.
5. Hardening off seedlings
After rooting culture for 30d, the tissue culture seedlings after rooting culture are transferred to a greenhouse with a sunshade rate of 70% with a bottle for hardening seedlings, and the temperature is 25+/-2 ℃.
After hardening the seedlings for 9d, the tissue culture bottle cap is screwed to a half-open state for 2d.
6. Transplanting
4d before transplanting, peat soil and perlite are mixed according to the volume ratio of 10:1, uniformly mixing to obtain a matrix, spraying and leaching the matrix by using a potassium permanganate solution with the mass fraction of 0.1%, standing for 48 hours, spraying and leaching the matrix by using tap water, washing the potassium permanganate, and using the matrix after 3 days.
Carefully taking out the tissue culture seedlings from the tissue culture bottles, cleaning a culture medium on the roots of the tissue culture seedlings, soaking the tissue culture seedlings in a 600-time carbendazim solution for 10min, and transplanting the tissue culture seedlings into a matrix, wherein the transplanting depth is that the roots are just covered by covering soil. Compacting the matrix to stabilize the nursery stock.
After transplanting, the root fixing water is sprayed, 600 times carbendazim solution is sprayed, the surface of the soil is covered with a film for moisturizing, and a sunshade net is covered for shading.
After 10d, the film was uncovered and managed as conventional seedlings.
Comparative example 1A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the induction culture medium is MS culture medium containing 2.0 mg/L6-benzylaminopurine (6-BA), 0.2mg/L a-Naphthalene Acetic Acid (NAA), 30g/L sucrose and 6g/L agar, and the pH value is 5.8-6.0.
Comparative example 2A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the induction culture medium is an MS culture medium containing 2.0mg/L of 6-BA, 0.2mg/L of NAA, 0.5mg/L of silver nitrate, 30g/L of sucrose and 6g/L of agar, and the pH value is 5.8-6.0.
Comparative example 3A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the induction culture medium is MS culture medium containing 2.0 mg/L6-BA, 0.2mg/L NAA, 0.5mg/L indole-3-butyric acid (IBA), 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 4A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the induction culture medium is MS culture medium containing 2.0 mg/L6-BA, 0.2mg/L NAA, 20mg/L VC, 0.5mg/L IBA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 5A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the proliferation culture medium is WPM culture medium containing 1.2 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 6A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the proliferation culture medium is 3/2WPM culture medium containing 1.2 mg/L6-BA, 0.1mg/L NAA, 0.5mg/L IBA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 7A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: the rooting culture medium is as follows: MS culture medium containing 0.1mg/L NAA, 1.0mg/L IBA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 8A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that:
the induction culture medium is an MS culture medium containing 2.0 mg/L6-BA, 0.2mg/L NAA, 0.5mg/L indole-3-butyric acid (IBA), 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0; the proliferation culture medium is WPM culture medium containing 1.2 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0; the rooting culture medium is as follows: MS culture medium containing 0.1mg/L NAA, 1.0mg/L IBA, 30g/L sucrose and 6g/L agar, and the pH is 5.8-6.0.
Comparative example 9A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: seedling hardening is not carried out, and the seedlings are directly transplanted after rooting.
Comparative example 10A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: and 5, in seedling hardening, transferring the tissue culture seedlings subjected to rooting culture to a greenhouse with a sun-shading rate of 90% with a bottle.
Comparative example 11A method for tissue culture and seedling raising of Cercis chinensis
The same procedure as in example 2 is followed, with the only difference that: and 5, in seedling hardening, transferring the tissue culture seedlings subjected to rooting culture to a greenhouse with 40% sun-shading rate with a bottle.
Application example
1. Experimental method
Tissue culture seedling raising is carried out on Cercis chinensis in Cercis chinensis landscaping areas of agricultural university of south China according to the methods of examples 2-4 and comparative examples 1-11.
The change condition of the tissue culture seedlings is monitored and recorded in the whole process, and the induction rate, proliferation coefficient, the number of effective buds (the bud height is more than or equal to 1.5 cm), rooting rate, average single plant rooting rate and transplanting survival rate are counted.
The calculation formula is as follows:
inductivity (%) = number of aseptic explants inducing axillary buds/number of uncontaminated explants x 100%;
multiplication coefficient (times) =total bud number/inoculated bud number (effective bud ≡1.5cm is used as data source);
rooting rate (%) =number of rooted plants/number of vaccinations×100%;
average individual root number (bars) =total root number/root number (excluding seedlings without roots);
transplanting survival rate (%) =number of surviving plants/total transplanting number×100%.
2. Experimental results
TABLE 1 Induction culture conditions
As can be seen from Table 1, the induction medium containing VC of example 1 is effective in improving the induction rate of axillary buds of Cercis chinensis.
TABLE 2 proliferation culture conditions
As can be seen from Table 2, the use of WPM minimal medium alone is not beneficial to proliferation culture of Cercis chinensis, and example 1 greatly increases the proliferation factor of proliferation culture after increasing the content of WPM minimal medium to 1.5 times.
TABLE 3 rooting culture conditions
As can be seen from Table 3, the culture medium of example 1 facilitates rooting of Cercis chinensis tissue culture seedlings.
TABLE 4 survival after transplanting
As can be seen from Table 4, the seedling hardening time, the seedling hardening method, the sun-shading rate and other steps in the transplanting process need to be strictly controlled to obtain the container seedlings with high survival rate.
As shown in FIG. 1, using the medium of example 1, robust adventitious buds were induced after induction culture; as shown in fig. 2, a proliferation seedling with vigorous growth is obtained after proliferation culture; as shown in fig. 3, the tissue culture seedlings obtained after rooting culture have developed root systems; as shown in FIG. 4, the single plant rooting seedling obtained after rooting culture has excellent growth vigor; as shown in FIG. 5, the rooted seedlings successfully survived after one month of transplantation.
The results show that the method for tissue culture and rapid propagation of the adult superior plant of cercis chinensis provided by the invention can cultivate strong clump buds, not only has fast elongation and growth and more effective buds, but also has fast rooting speed, high rooting rate, developed root system and high transplanting survival rate, and finally can obtain healthy seedlings with regular growth and consistent phenotype in batches.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. A culture medium combination for tissue culture of cercis chinensis, which is characterized by comprising an induction culture medium, a proliferation culture medium and a rooting culture medium;
the induction culture medium consists of 1.0-3.0 mg/L6-benzyl aminopurine, 0.1-0.3 mg/L a-naphthylacetic acid, 15-25 mg/L vitamin C, 25-35 g/L sucrose, 4-8 g/L agar and MS culture medium;
the proliferation culture medium consists of 0.5-2.0 mg/L6-benzylaminopurine, 0.05-0.15 mg/L a-naphthylacetic acid, 25-35 g/L sucrose, 4-8 g/L agar and 3/2WPM culture medium;
the rooting culture medium consists of 0.2-1.0 mg/L a-naphthylacetic acid, 25-35 g/L sucrose, 4-8 g/L agar and WPM culture medium.
2. Use of the combination of media of claim 1 in tissue culture of cercis chinensis.
3. The use of the combination of culture media according to claim 1 for raising seedlings of cercis chinensis.
4. A method for tissue culture seedling raising of cercis chinensis in palace powder is characterized by comprising the following steps:
s1, placing the sterilized explant in the induction culture medium of claim 1, culturing in dark for 5-12 days, and culturing in light-dark alternation to obtain adventitious buds, wherein the length of the adventitious buds is 2-4 cm; the explant is a semi-lignified branch of cercis chinensis, and is a stem segment with the length of 2-3 cm and 1-2 axillary buds;
s2, placing the adventitious buds obtained in the step S1 in the proliferation culture medium of claim 1, culturing in dark for 4-7 days, and culturing in light-dark alternation to obtain cluster buds, wherein the length of the cluster buds is 1.5-3 cm;
s3, placing the terminal buds with the length of 1.5-2.5 cm of the cluster buds obtained in the step S2 in the rooting culture medium according to claim 1, and alternately culturing the terminal buds in a light-dark manner to obtain tissue culture seedlings, wherein the length of roots of the tissue culture seedlings is 1.5-3 cm;
s4, hardening off seedlings of the tissue culture Miao Yu obtained in the step S3 for 6-14 days at the temperature of 23-27 ℃ with the shading rate of 50% -80%;
s5, transplanting the tissue culture seedlings after seedling hardening after disinfection into a matrix containing peat soil and perlite, covering the surface of the matrix with a film, and culturing for 8-12 days.
5. The method according to claim 4, wherein in step S1, the disinfection is performed with 0.8-1.2% (w/v) of benzalkonium chloride and/or 0.08-0.12% (w/v) of a mercuric chloride solution.
6. The method of claim 4, wherein in step S1, the explant is obtained from an adult elite individual of cercis chinensis.
7. The method according to claim 4, wherein in step S5, the tissue culture Miao Yuzu is cultured in a flask, and after hardening off the seedlings for 6-9 days, the flask is closed and left open for 1-2 days.
8. The method according to claim 4, wherein in step S5, the matrix is formed by peat soil and perlite in a volume ratio (8-12): (0.8-1.2) and evenly mixing.
9. Use of the method according to any one of claims 4 to 8 for breeding and/or planting cercis chinensis.
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