CN114600774B - Atractylodes chinensis tissue culture and rapid propagation method - Google Patents
Atractylodes chinensis tissue culture and rapid propagation method Download PDFInfo
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- CN114600774B CN114600774B CN202210443388.7A CN202210443388A CN114600774B CN 114600774 B CN114600774 B CN 114600774B CN 202210443388 A CN202210443388 A CN 202210443388A CN 114600774 B CN114600774 B CN 114600774B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention relates to the technical field of plant tissue culture, and provides a method for tissue culture and rapid propagation of rhizoma atractylodis macrocephalae, which comprises the following steps: s1, explant acquisition: cutting sterile leaves of Atractylodes chinensis into blocks; s2, washing the explants, and then sterilizing; s3, inoculating the explants sterilized in the step S2 to a hormone combined culture medium for callus induction, wherein the hormone combined culture medium is MS +2.0mg/L6-BA +1mg/L NAA; s4, subculturing the callus to obtain a seedling; and S5, inducing the seedlings obtained in the S4 to root and hardening the seedlings. Through the technical scheme, the problems that a tissue culture method in the related technology is not suitable for the rhizoma atractylodis macrocephalae, the propagation coefficient is not high, the growth period is long, and the variety is degraded are solved.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for tissue culture and rapid propagation of rhizoma atractylodis.
Background
Rhizoma atractylodis is used as a large amount of traditional Chinese medicinal materials, is derived from dried rhizome of Atractylodes lancea (thunb.) DC (Atractylodes lancea) or Atractylodes chinensis Koidz (Atractylodes chinensis) of Compositae, is mainly distributed in far east areas such as China, korean peninsula, japan and the like, has great influence on the drug effect due to factors such as geographical environment and the like, and is collected by multi-national formulary. The quality of rhizoma atractylodis is evaluated by an index component atractylodin in ' pharmacopoeia of the people's republic of China ' 2020 edition.
The rhizoma atractylodis macrocephalae is used as an important source of rhizoma atractylodis, as the wild rhizoma atractylodis macrocephalae resources are exhausted day by day, but the demand of the market for rhizoma atractylodis is increased day by day, the supply and demand gaps are increased year by year, the market prospect is good, large-area planting is carried out at present, but the yield and the content of medicinal components cannot achieve the ideal effect due to the problems of various germplasm resources, non-standard planting and the like. Therefore, the problem of breaking the core germplasm resources which restrict the current development of the rhizoma atractylodis macrocephalae industry is urgently needed.
The patent with the application number of CN202110558788.8 applies for a method for quickly growing seedlings of rhizoma atractylodis and application thereof, and provides a method for quickly growing seedlings of rhizoma atractylodis, which can shorten the time limit of seedling growing and improve the seedling rate and the seedling quality.
The patent with the application number of CN202110673205.6 applies for a high-yield rhizoma atractylodis macrocephalae seedling raising and planting method, and also provides a method for improving the yield and quality of rhizoma atractylodis macrocephalae from the cultivation perspective, so that the problems of low effective components, low emergence rate, low yield and the like of rhizoma atractylodis macrocephalae are solved, but the method does not change the source diversity of the rhizoma atractylodis macrocephalae, and the consistency of planting resources is difficult to guarantee.
Due to the shortage of resources of the rhizoma atractylodis north in the current market and larger demand, the traditional propagation method has the problems of low propagation coefficient, long growth cycle, degeneration of varieties and the like, and limits the work of rapid propagation, new variety breeding and the like.
Disclosure of Invention
The invention provides a tissue culture and rapid propagation method of rhizoma atractylodis macrocephalae, which solves the problems that the tissue culture method in the related technology is not suitable for rhizoma atractylodis macrocephalae, the propagation coefficient is not high, the growth period is long, and the variety is degraded.
The technical scheme of the invention is as follows:
a method for tissue culture and rapid propagation of Atractylodes lancea comprises the following steps:
s1, explant acquisition: cutting leaves of the Atractylodes chinensis aseptic seedlings into blocks;
s2, washing the explants, and then sterilizing;
s3, inoculating the explants sterilized in the step S2 to a hormone combined culture medium for callus induction, wherein the hormone combined culture medium is MS +2.0mg/L6-BA +1mg/L NAA;
s4, subculturing the callus to obtain a seedling;
and S5, inducing the seedlings obtained in the S4 to root and hardening the seedlings.
As a further technical scheme, the step S1 is to cut leaves of the Atractylodes chinensis sterile seedling into the size of 0.3cm multiplied by 0.3 cm.
As a further technical scheme, the step S2 of sterilization and disinfection specifically comprises the steps of sterilizing with 75% alcohol for 20-30S, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5-6min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 2.5-3.5min, and washing with sterile water for 5 times.
As a further technical scheme, the step S2 of sterilization and disinfection specifically comprises the steps of washing explants and then disinfecting, sterilizing the explants for 30S with 75% alcohol, washing the explants with sterile water for 3 times, sterilizing the explants for 6min with 0.1% mercuric chloride, washing the explants with sterile water for 7 times, sterilizing the explants with 3% sodium hypochlorite for 3min, and washing the explants with the sterile water for 5 times.
In a further technical scheme, in the step S3, the culture temperature is 25 ℃, the illumination is 1500-3000lx, the illumination time is 12h/d, and the culture time is 20d when the callus is cultured.
As a further technical scheme, in the step S4, the culture temperature is 25 ℃, the illumination is 1500-3000lx, the illumination time is 12h/d, and the culture lasts 20d.
As a further technical proposal, in the step S4, the leaves are flatly laid on the surface of the culture medium downwards during inoculation.
As a further technical scheme, in the step S5, the seedling exercising specifically comprises: the tissue culture seedlings of the Atractylodes chinensis are transferred into a soil culture medium for hardening, the seedlings are placed in 12000lx incubator at the temperature of 25 ℃ and are cultured for 15 days in 12h in the light and the dark respectively.
The invention has the beneficial effects that:
1. the traditional propagation method limits the rapid propagation of the rhizoma atractylodis macrocephalae, but the problem is well solved by carrying out tissue culture on the rhizoma atractylodis macrocephalae in the invention, the rhizoma atractylodis macrocephalae used as an explant is the rhizoma atractylodis macrocephalae with consistent plant type, plant age and growth period, which is obtained by field collection in the mountain area of North Ji, and a regeneration system for inducing callus and differentiation of the rhizoma atractylodis macrocephalae seedlings is established, so that technical support is provided for artificial rejuvenation, resource protection and reasonable development and utilization of the rhizoma atractylodis macrocephalae population.
2. The invention takes the leaves of the Atractylodes chinensis as explants, and the rapid propagation of the Atractylodes chinensis can be realized only by specific sterilization conditions and hormone culture medium, thereby establishing a tissue culture regeneration system of the Atractylodes chinensis and solving the industrial problems and the technical blank.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious 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 derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.
Example 1
S1, explant acquisition: cutting leaves of the aseptic rhizoma atractylodis sinensis seedlings into 0.3cm multiplied by 0.3 cm.
S2, explant disinfection: washing with water for 15min, washing with detergent for 4 times, removing water, sterilizing with 75% ethanol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 6min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 3min, and washing with sterile water for 5 times.
S3, inducing callus: leaf explants 10 were inoculated per dish and 4 groups were cultured in parallel. During inoculation, the leaf is laid down on the surface of the culture medium and is slightly forced to fully contact with the culture medium. The culture temperature is 25 ℃, the illumination is controlled to be 2000lx, the illumination time is 12h/d, and the callus induction result is counted after 20d of culture. Wherein the hormone combination culture medium is MS +2.0mg/L6-BA +1mg/L NAA.
Callus rate = number of induced calli/total × 100%
S4, callus differentiation: selecting callus with vigorous growth and size of 0.5cm × 0.5cm, subculturing in the same hormone combined culture medium at 25 deg.C under the condition of illumination of about 2000lx for 12h/d, and culturing for 20d.
S5, inducing rooting: separating the robust seedlings one by one, transferring the robust seedlings into a rooting culture medium, controlling the culture temperature to be 25 ℃, controlling the illumination to be 1500-3000lx, controlling the illumination time to be 12h/d, and culturing for 20d.
S6, hardening seedlings. And transferring the tissue culture seedlings of the Atractylodes chinensis which are robust and consistent in state and have 5-8 rooting numbers into a soil culture medium for hardening seedlings. The soil culture medium uses common flower nutrient soil as a culture medium, the culture medium is used after being sterilized at 121 ℃ for 20min, the seedlings are placed in 12000lx incubator at the temperature of 12h in light and dark respectively, and the seedlings are cultured for 15d at the temperature of 25 ℃.
Example 2
The other steps are the same as example 1, except that the explant sterilization method: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 6min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 3.5min, and washing with sterile water for 5 times.
Example 3
The other steps are the same as example 1, except that the explant sterilization method: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5.5min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 3min, and washing with sterile water for 5 times.
Example 4
The other steps are the same as example 1, except that the explant sterilization method: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 6min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 2.5min, and washing with sterile water for 5 times.
Example 5
The other steps are the same as example 1, except that the explant sterilization method: sterilizing with 75% alcohol for 20s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 6min, washing with sterile water for 7 times, sterilizing with 3% sodium hypochlorite for 3min, and washing with sterile water for 5 times.
The results of observing the bactericidal effect after completion of sterilization during the culture in examples 1 to 5 are shown in Table 1.
TABLE 1 examples 1-5 sterility
| Examples | Sterility condition |
| Example 1 | It is preferable that |
| Example 2 | Is better |
| Example 3 | Is preferably used |
| Example 4 | Is preferably used |
| Example 5 | Is preferably used |
No colony grows on the surface-the best aseptic condition; 1-2 colonies grow on the surface-the aseptic condition is better
The sterilization method plays a decisive role in tissue culture, and bacterial colony growth can be caused by improper sterilization, so that the experimental result is influenced. The results were completely different for different explants using the same sterilization method. The sterilization method provided by the embodiment of the invention can achieve sterilization effects in different degrees, can ensure the quality of the leaves, and can cause the blackening of explants and influence the growth condition of callus if other sterilization methods are adopted. Moreover, it can be found from the examples 1 to 5 that the sterilization method in the example 1 can achieve the best sterilization effect, and it can be seen that the sterilization effect is greatly influenced by the difference of the sterilization time and the washing frequency.
Comparative example 1
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +2.0 mg/L2,4-D +1.0mg/L NAA, and the callus was found not to grow after the culture was carried out by this method.
Comparative example 2
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +4.0 mg/L2,4-D +0.4mg/L NAA +0.4mg/L KT, and the callus was found not to grow after the culture was carried out by this method.
Comparative example 3
The culture was carried out by the methods S1, S2 and S3 in example 1 except that the hormone combination medium was MS +2.0mg/L6-BA +0.5mg/L NAA +0.5mg/L KT, and the callus was not grown after the culture according to this method.
Comparative example 4
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +3.0 mg/L6-BA +0.4mg/L NAA, and the callus was not grown after the culture by this method.
Comparative example 5
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +2.0mg/L6-BA +0.25mg/L NAA, and the callus was not grown after the culture by this method.
Comparative example 6
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +2.0mg/L6-BA +0.25mg/L NAA, and the callus was not grown after the culture by this method.
Comparative example 7
The culture was carried out by the methods S1, S2 and S3 in example 1 except that the hormone combination medium was MS +0.3 mg/L6-BA +0.5mg/L NAA +1.5 mg/L2,4-D, and callus was found not to grow after the culture was carried out by this method.
Comparative example 8
The culture was carried out by the methods S1, S2 and S3 in example 1, except that the hormone combination medium was MS +0.5 mg/L6-BA +0.5mg/L IBA, and the callus was not grown after the culture according to this method.
Only the hormone combination culture medium in example 1 can induce the growth of leaves of Atractylodes lancea, and the change of the proportion of hormone or the change of the type of hormone can not induce the growth of callus. And even if the method with the best sterilization effect is adopted, the micro-adjustment of the hormone cannot lead the rhizoma atractylodis macrocephalae to be rapidly propagated.
Comparative example 9
Taking seeds as explants:
soaking in clear water for 0.5 hr, soaking in 75% ethanol for 30s, sterilizing with 0.1% mercuric chloride for 15-18min, washing for 5 times, and inoculating in 1/2MS culture medium. The sterilization method is not ideal, and bacterial colony is not controlled.
Comparative example 10
The petioles were used as explants and cultured by the methods S2 and S3 in example 1, but the callus was not grown after the culture.
The sterilization method and the hormone culture medium are only suitable for tissue culture when the leaves are used as explants, and can not induce callus and perform subsequent culture when other tissue organs are used as explants for tissue culture.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for tissue culture and rapid propagation of Atractylodes chinensis is characterized by comprising the following steps:
s1, explant acquisition: cutting leaves of the Atractylodes chinensis aseptic seedlings into blocks;
s2, washing the explants, and then sterilizing;
s3, inoculating the explants sterilized in the step S2 to a hormone combined culture medium for callus induction, wherein the hormone combined culture medium is MS +2.0mg/L6-BA +1mg/L NAA;
s4, carrying out subculture on the callus to obtain a seedling;
s5, inducing and rooting the seedlings obtained in the S4 to obtain tissue culture seedlings of the Atractylodes chinensis;
s6, hardening off the tissue culture seedlings of the Atractylodes chinensis with the root number of 5-8;
the step S2 of sterilization and disinfection specifically comprises the steps of washing explants and then disinfecting, sterilizing the explants for 30S by using 75% alcohol, washing the explants by using sterile water for 3 times, sterilizing the explants by using 0.1% mercuric chloride for 6min, washing the explants by using the sterile water for 7 times, sterilizing the explants by using 3% sodium hypochlorite for 3min, and washing the explants by using the sterile water for 5 times;
in step S4, the culture medium during subculture is the same as the hormone combination medium.
2. The method for tissue culture and rapid propagation of rhizoma atractylodis macrocephalae according to claim 1, wherein the step S1 is to cut the leaves of the rhizoma atractylodis macrocephalae aseptic seedling into 0.3cm x 0.3 cm.
3. The method for tissue culture and rapid propagation of rhizoma atractylodis macrocephalae according to claim 1, wherein in the step S3, the callus is cultured at 25 ℃, 1500-3000lx of light, 12h/d of light and 20d of culture.
4. The method for tissue culture and rapid propagation of rhizoma atractylodis macrocephalae according to claim 1, wherein in the step S4, the culture temperature is 25 ℃, the illumination is 1500-3000lx, the illumination time is 12h/d, and the culture lasts 20d.
5. The method for tissue culture and rapid propagation of Atractylodes chinensis as claimed in claim 1, wherein in step S3, the leaves are laid down on the surface of the culture medium during inoculation.
6. The method for tissue culture and rapid propagation of rhizoma atractylodis macrocephalae according to claim 1, wherein in the step S6, the hardening off specifically comprises: the tissue culture seedlings of the Atractylodes chinensis are transferred into a soil culture medium for hardening, the seedlings are placed in 12000lx incubator at the temperature of 25 ℃ and are cultured for 15 days in 12h in the light and the dark respectively.
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| CN116267620A (en) * | 2023-04-21 | 2023-06-23 | 德兴市中医研究院试验培训基地 | Method for constructing callus induction and adventitious bud regeneration system by utilizing atractylis lancea leaf stalks |
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| CN108308033A (en) * | 2018-04-24 | 2018-07-24 | 镇江市德尔生物制品研究所有限公司 | Atractylis lancea callus tissue culture base and apply its Atractylis lancea callus induction technique |
| CN114342810A (en) * | 2022-02-23 | 2022-04-15 | 内蒙古科学技术研究院 | Tissue culture method of rhizoma atractylodis with high disease resistance |
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