CN115176704A - Method for tissue culture of poplar variety' Senhai No. 2 - Google Patents
Method for tissue culture of poplar variety' Senhai No. 2 Download PDFInfo
- Publication number
- CN115176704A CN115176704A CN202210854816.5A CN202210854816A CN115176704A CN 115176704 A CN115176704 A CN 115176704A CN 202210854816 A CN202210854816 A CN 202210854816A CN 115176704 A CN115176704 A CN 115176704A
- Authority
- CN
- China
- Prior art keywords
- culture
- culture medium
- medium
- senhai
- rooting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the field of plant tissue culture, and particularly discloses a method for tissue culture of a poplar variety 'Senhai No. 2', which comprises the following steps: s1, dividing the sterile leaf blade of 'Senhai No. 2' into two sections along the direction vertical to a main vein, taking a near petiole section, cutting off the edge as an explant, inoculating the explant into an induction culture medium, and performing induction culture to obtain callus, thereby obtaining the leaf blade with the callus; s2, transferring the leaves with the callus tissues in the step S1 to a differentiation medium, and carrying out differentiation culture to obtain cluster seedlings; s3, transferring the clump seedlings growing to 1.0-1.5cm high to a rooting culture medium for rooting culture to obtain the tissue culture seedlings of 'Senhai No. 2'. The invention mainly aims at the improved variety 'Senhai No. 2' of the poplar, comprehensively considers each link in the tissue culture process, optimizes the tissue culture condition and establishes a set of efficient tissue culture system suitable for 'Senhai No. 2'.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for tissue culture of a poplar variety 'Senhai No. 2'.
Background
The improved variety of poplar 'Senhai No. 2' is obtained by artificial pollination and breeding controlled by Hu Jianjun of the institute of forestry of the Chinese academy of forestry and the like, and the variety which passes the improved variety approval is obtained in 2021 (the number is national S-SV-PS-001-2021). The male parent is populus euphratica and the female parent is populus deltoides. The related relationship between the populus tremuloides and the populus nigra is relatively close, the hybridization compatibility is high, and the populus tremuloides and the populus nigra are very excellent in hybridization combination (Hu Bin, fan Junfeng, high construction, and the like). 'Senhai No. 2' has excellent fast growing property and material property, but has poor saline-alkali resistance and disease and insect resistance. The tissue culture regeneration system is the foundation of germplasm resource preservation and utilization and gene transformation research, and the establishment of the efficient tissue culture regeneration system of the poplar, senhai No. 2, can lay the foundation for introducing the haloduric gene or the insect-resistant gene into the Senhai No. 2 group by an agrobacterium tumefaciens mediated method to obtain an excellent new variety.
Disclosure of Invention
The invention aims to solve the technical problem of how to efficiently perform tissue culture on improved poplar seeds 'Senhai No. 2'.
In order to solve the technical problems, the invention provides a method for tissue culture (tissue culture) of a poplar variety 'Senhai No. 2', which comprises the following steps:
s1, dividing the sterile leaf blade of 'Senhai No. 2' into two sections along the direction vertical to a main vein, taking a near petiole section, cutting off the edge as an explant, inoculating the explant into an induction culture medium, and performing induction culture to obtain callus, thereby obtaining the leaf blade with the callus;
s2, transferring the leaves with the callus in the step S1 to a differentiation medium, and carrying out differentiation culture to obtain cluster seedlings;
s3, transferring the clump seedlings growing to the height of 1.0-1.5cm to a rooting culture medium for rooting culture to obtain tissue culture seedlings of 'Senhai No. 2';
the induction culture medium is a solid culture medium which is obtained by taking an MS minimal medium as a basal medium and adding 6-BA, ZT, IBA and 2,4-D into the basal medium, and the concentration of the 6-BA in the induction culture medium is 0.5 mg.L -1 ZT concentration is 0.25 mg.L -1 The concentration of IBA was 0.25 mg.L -1 2,4-D concentration of 1.0 mg. L -1 ；
The differentiation medium is based on MSThe culture medium is basic culture medium, and solid culture medium obtained by adding 6-BA, ZT and IBA into the basic culture medium, wherein the concentration of 6-BA in differentiation culture medium is 0.5 mg.L -1 ZT concentration is 0.25 mg. L -1 The concentration of IBA was 0.25 mg.L -1 ；
The rooting culture medium is a solid culture medium obtained by taking a 1/2MS basic culture medium as a basic culture medium and adding IBA and NAA into the basic culture medium, wherein the concentration of the IBA in the rooting culture medium is 0.05 mg.L -1 The concentration of NAA was 0.02 mg.L -1 。
In the above method, the explant is preferably (0.8 to 1.2) cm x (0.8 to 1.2) cm in size.
In the method, the culture condition of the induction culture is 24-25 ℃ and darkness.
In the above method, the induction culture is subcultured every 2 weeks.
In the above method, the induction culture is carried out for 3 to 4 weeks.
In the method, the culture conditions of the differentiation culture and the rooting culture are that the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
In the above method, the differentiation culture is performed for 3 to 4 weeks.
In the above method, the rooting culture is carried out for 15-21 days.
In the method, the acquisition process of the 'Senhai No. 2' sterile leaf comprises the following steps:
a1, taking a new twig of 'Senhai No. 2', cleaning, disinfecting, cutting into stem sections containing 1-2 bud points, vertically placing the stem sections on a rooting culture medium, and culturing until the bud length of a lateral bud is 1-2cm;
a2, taking down the lateral buds obtained by culturing in the step A1, and putting the lateral buds into a rooting culture medium for culturing until seedlings are obtained;
and A3, putting the terminal bud of the plantlet in the step A2 into a rooting culture medium for culturing for 25-30d, and taking the leaves on the terminal bud, namely the sterile leaves of the 'Senhai No. 2'.
In the method, the culture conditions of the culture in the steps A1-A3 are all 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
In the above method, the culture of A2 to obtain the plantlet needs 30 days.
The method also comprises a seedling hardening and transplanting step.
The invention mainly aims at the improved variety 'Senhai No. 2' of the poplar, comprehensively considers each link in the tissue culture process, optimizes the tissue culture condition and establishes a set of efficient tissue culture system suitable for 'Senhai No. 2'.
Drawings
FIG. 1 is a photograph of a clustered seedling differentiated from an improved variety of poplar of "Senhai No. 2" in example 1 of the present invention.
FIG. 2 is a photograph showing the root growth of 30 days of the improved poplar seed "Senhai No. 2" cultured on a rooting medium in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The improved variety of poplar ` Senhai No. 2 ` used in this example was a variety approved by the improved variety obtained in 2021 (national S-SV-PS-001-2021).
The media used in this example were as follows:
induction medium: MS +6-BA 0.5 mg.L -1 +ZT 0.25mg·L -1 +IBA 0.25mg·L -1 +2,4-D 1.0mg·L -1 . The induction culture medium is MS minimal medium as basic culture medium, and 6-BA (6-benzylaminopurine), ZT (zeatin), IBA (indolebutyric acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are added into the basic culture medium to obtain the final productThe culture medium of (1), wherein the concentration of 6-BA in the induction medium is 0.5 mg.L -1 ZT concentration is 0.25 mg.L -1 The concentration of IBA was 0.25 mg.L -1 2,4-D concentration of 1.0 mg. L -1 。
Differentiation medium: MS +6-BA 0.5 mg.L -1 +ZT 0.25mg·L -1 +IBA 0.25mg·L -1 . The differentiation culture medium is obtained by adding 6-BA (6-benzylaminopurine), ZT (zeatin) and IBA (indolebutyric acid) into MS minimal medium as basic culture medium, wherein the concentration of 6-BA in the differentiation culture medium is 0.5 mg.L -1 ZT concentration is 0.25 mg.L -1 The concentration of IBA was 0.25 mg.L -1 。
Rooting culture medium: 1/2MS + IBA 0.05mg. L -1 +NAA 0.02mg·L -1 . The rooting culture medium is obtained by adding IBA (indolebutyric acid) and NAA (naphthylacetic acid) into 1/2MS minimal medium as basic culture medium, wherein the concentration of IBA in the rooting culture medium is 0.05 mg.L -1 The concentration of NAA was 0.02 mg.L -1 。
1. Preparation of the Material
A1, cutting seedlings of improved varieties of poplar, namely annual seedlings of No. 2 Senhai, cutting off new tender branches from the cutting seedlings within 5 months, and flushing the cut seedlings for 2 hours in running mode under tap water. Soaking in 75% ethanol for 30s in an ultra-clean workbench, washing with sterile water for 1 time, soaking with 10% sodium hypochlorite for 30min, washing with sterile water for 4-5 times, cutting into stem segments with length of about 2cm, wherein each stem segment contains 1-2 bud points, and vertically placing on rooting medium for culturing until the bud length of lateral bud (obtained by bud point development) is 1-2cm.
And A2, taking down the lateral buds obtained by the culture in the step A1, putting the lateral buds into a rooting culture medium, and culturing for 30d to obtain robust seedlings.
And A3, putting the terminal bud of the plantlet obtained in the step A2 into a rooting culture medium, and culturing for 25-30d to obtain a leaf with a good leaf state (fresh green, strong and good leaf growth) suitable for being used as an explant.
The culture conditions of A1-A3 are as follows: the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
2. Callus induction and differentiation
S1, dividing a leaf blade perpendicular to a main leaf vein into two parts, reserving a clear upper section of the main vein, cutting a circle along the edge of the leaf blade to remove the leaf edge, placing the cut leaf blade serving as an explant (with the size of (0.8-1.2) cm multiplied by (0.8-1.2) cm) on an induction culture medium (repeating for 3 times and inoculating 30 leaf blades each time), culturing at 24-25 ℃ in the dark for 3-4 weeks, subculturing every 2 weeks, and counting the induction rate on the 28 th day of induction culture.
Induction rate = number of leaves from which callus was induced/total number of leaves
The result shows that the induction rate reaches 100%.
S2, transferring the explants induced with the callus to a differentiation medium (repeating for 3 times, inoculating 30 leaves each time), carrying out differentiation culture for 3-4 weeks at the temperature of 24-25 ℃, with the illumination intensity of 1600-2000lux and the illumination of 16 h/dark 8h, and differentiating to obtain the plantlets. Statistical differentiation rate on day 28 of differentiation culture:
differentiation rate = number of differentiated clumpy seedlings/total number of leaves
The differentiation rate reaches more than 90 percent. The picture of the differentiated clumped seedlings is shown in figure 1.
3. Rooting culture
When the clump seedlings grow to be 1.0-1.5cm high, transferring the clump seedlings to a rooting culture medium (repeating for 3 times, about 27 clump seedlings are transferred each time), starting rooting after 24-25 ℃, the illumination intensity is 1600-2000lux, the illumination is 16 h/dark 8h, rooting begins after 7-15d, and the rooting rate is counted on the 21 st day of rooting culture:
rooting rate = number of rooted plants/total number of plants
The total number of plants is the number of plants placed on the rooting medium.
The result shows that the rooting rate reaches more than 80%. A photograph of rooting after 30 days of culture on rooting medium is shown in FIG. 2.
4. Transplanting
Rooting and culturing for 15 days, after strong roots grow, hardening seedlings at normal temperature (or room temperature) for 2-3 days: the rubber band is loosened firstly on the first day, the cover is opened in a shade place on the second day, and sunlight can be directly irradiated on the third day. After hardening the seedlings for 2-3d, when new roots grow to 1cm, the culture medium of the rooted seedlings is washed clean and transplanted into soil. And (5) covering a preservative film on the transplanted seedling 5 to 7 days, and watering frequently.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (10)
1. A method for tissue culture of a poplar variety 'Senhai No. 2', which is characterized by comprising the following steps:
s1, dividing the sterile leaf blade of 'Senhai No. 2' into two sections along the direction vertical to a main vein, taking a near petiole section, cutting off the edge as an explant, inoculating the explant into an induction culture medium, and performing induction culture to obtain callus, thereby obtaining the leaf blade with the callus;
s2, transferring the leaves with the callus tissues in the step S1 to a differentiation medium, and carrying out differentiation culture to obtain cluster seedlings;
s3, transferring the clump seedlings growing to 1.0-1.5cm high to a rooting culture medium for rooting culture to obtain tissue culture seedlings of 'Senhai No. 2';
the induction culture medium is a solid culture medium which is obtained by taking an MS minimal medium as a basal medium and adding 6-BA, ZT, IBA and 2,4-D into the basal medium, and the concentration of the 6-BA in the induction culture medium is 0.5 mg.L -1 ZT concentration is 0.25 mg.L -1 The concentration of IBA was 0.25 mg.L -1 2,4-D concentration of 1.0 mg. L -1 ；
The differentiation culture medium is a solid culture medium obtained by taking an MS minimal medium as a basal medium and adding 6-BA, ZT and IBA into the basal medium, and the concentration of the 6-BA in the differentiation culture medium is 0.5 mg.L -1 ZT concentration is 0.25 mg.L -1 The concentration of IBA was 0.25 mg.L -1 ；
The rooting culture medium is a solid culture medium obtained by taking a 1/2MS basic culture medium as a basic culture medium and adding IBA and NAA into the basic culture medium, wherein the concentration of the IBA in the rooting culture medium is 0.05 mg.L -1 The concentration of NAA was 0.02 mg.L -1 。
2. The method of claim 1, wherein: the culture condition of the induction culture is 24-25 ℃ and darkness.
3. The method according to claim 1 or 2, characterized in that: the induction culture was subcultured every 2 weeks.
4. A method according to any one of claims 1 to 3, wherein: the induction culture is carried out for 3-4 weeks.
5. The method according to any one of claims 1 to 4, wherein: the culture conditions of the differentiation culture and the rooting culture are that the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
6. The method according to any one of claims 1 to 5, wherein: the differentiation culture is carried out for 3-4 weeks.
7. The method according to any one of claims 1-6, wherein: the rooting culture is carried out for 15-21 days.
8. The method according to any one of claims 1-7, wherein: the acquisition process of the 'Senhai No. 2' sterile leaf comprises the following steps:
a1, taking a new twig of 'Senhai No. 2', cleaning, disinfecting, cutting into stem sections containing 1-2 bud points, vertically placing the stem sections on a rooting culture medium, and culturing until the bud length of a lateral bud is 1-2cm;
a2, taking down the lateral buds obtained by culturing in the step A1, and putting the lateral buds into a rooting culture medium for culturing to obtain seedlings;
and A3, putting the terminal bud of the plantlet in the step A2 into a rooting culture medium for culturing for 25-30d, and taking the leaves on the terminal bud, namely the sterile leaves of the 'Senhai No. 2'.
9. The method of claim 8, wherein: and A2, culturing for 30 days until seedlings are obtained.
10. The method according to any one of claims 1-9, wherein: the method also comprises a step of hardening off and transplanting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210854816.5A CN115176704B (en) | 2022-07-18 | 2022-07-18 | Method for tissue culture of poplar variety' Senhai No. 2 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210854816.5A CN115176704B (en) | 2022-07-18 | 2022-07-18 | Method for tissue culture of poplar variety' Senhai No. 2 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115176704A true CN115176704A (en) | 2022-10-14 |
| CN115176704B CN115176704B (en) | 2023-04-25 |
Family
ID=83519870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210854816.5A Active CN115176704B (en) | 2022-07-18 | 2022-07-18 | Method for tissue culture of poplar variety' Senhai No. 2 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115176704B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5354943A (en) * | 1988-05-04 | 1994-10-11 | The United States Of America As Represented By The Secretary Of The Agriculture | Methods of high frequency tissue regeneration, regeneration of herbicide-tolerant populus plants therewith, and the herbicide-tolerant plants made thereby |
| CN112335550A (en) * | 2020-12-23 | 2021-02-09 | 江苏省中国科学院植物研究所 | Method for establishing regeneration system of populus colourianus by taking petioles as explants |
| CN112715363A (en) * | 2021-01-26 | 2021-04-30 | 北京林业大学 | Optimization method of poplar callus budding regeneration system |
| CN113317200A (en) * | 2021-06-25 | 2021-08-31 | 四川大学 | Tissue culture medium for male populus diversifolia plants and application of tissue culture medium |
-
2022
- 2022-07-18 CN CN202210854816.5A patent/CN115176704B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5354943A (en) * | 1988-05-04 | 1994-10-11 | The United States Of America As Represented By The Secretary Of The Agriculture | Methods of high frequency tissue regeneration, regeneration of herbicide-tolerant populus plants therewith, and the herbicide-tolerant plants made thereby |
| CN112335550A (en) * | 2020-12-23 | 2021-02-09 | 江苏省中国科学院植物研究所 | Method for establishing regeneration system of populus colourianus by taking petioles as explants |
| CN112715363A (en) * | 2021-01-26 | 2021-04-30 | 北京林业大学 | Optimization method of poplar callus budding regeneration system |
| CN113317200A (en) * | 2021-06-25 | 2021-08-31 | 四川大学 | Tissue culture medium for male populus diversifolia plants and application of tissue culture medium |
Non-Patent Citations (5)
| Title |
|---|
| 周熙莹等: "一种84K杨再生体系的建立", 《广东农业科学》 * |
| 李科友等: "84K杨再生和遗传转化体系的优化", 《西北农林科技大学学报（自然科学版）》 * |
| 沈周高等: "3个杨树品种叶片再生体系的建立", 《中国农学通报》 * |
| 许继飞等: "欧美杨108离体叶片和茎段再生体系的优化", 《安徽农业科学》 * |
| 黄海杰等: "欧美杨热杨1号离体叶片快繁体系的研究", 《安徽农业科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115176704B (en) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110476812B (en) | Method for rapid breeding of Maranta americana | |
| CN111280056A (en) | Subculture breeding method of stingless pepper tissue culture seedlings | |
| CN113661924B (en) | Tissue culture rapid propagation method of Baotihua | |
| CN105379624B (en) | A kind of tissue culture and rapid propagation method of Eucalyptus pellita | |
| CN114375840B (en) | Culture medium for recovering growth vigor of hybrid offspring of hydrangea and method for recovering growth | |
| CN106538382B (en) | Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants | |
| CN110583488A (en) | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink | |
| CN109452170B (en) | Method for callus culture induced by cordyceps sobolifera roots | |
| CN110278871B (en) | Tissue culture method for one-step planting of tissue-cultured cluster seedlings by using pinellia ternata | |
| CN109601388B (en) | Tissue culture rapid propagation method of hybrid clematis | |
| CN109479721B (en) | A kind of peanut plant regeneration method | |
| CN110024688B (en) | Method for caragana germ proliferation and plant regeneration and culture medium thereof | |
| CN109526746B (en) | Tissue culture method for petioles of hairyvein agrimony | |
| CN114600772B (en) | Tissue culture method and rapid propagation method of michelia figo in remote mountains | |
| CN115413578B (en) | Method for cultivating new camellia seedling by utilizing immature hybrid seeds | |
| CN109673514A (en) | A kind of method of south yampi type Chinese yam tissue-culturing quick-propagation | |
| CN111480574B (en) | Tissue culture method for rapid seedling formation of sweet cherry intraspecific hybridization F1 generation | |
| CN109042322A (en) | A kind of rapid propagation method of gold heart A Erta Chinese ilex | |
| CN115176704B (en) | Method for tissue culture of poplar variety' Senhai No. 2 | |
| CN100407901C (en) | Asexual generation method for salt tolerant reed | |
| CN106613973A (en) | Method for quickly breeding rhododendron molle by approach of regenerating adventitious buds by utilizing tissue culture seedling leaves | |
| CN112293252A (en) | Artificial efficient clonal propagation method of dendrobium santalinum | |
| CN111194695A (en) | Tissue culture rapid propagation method of Populus deltoides Lu Lin No. 1 | |
| CN111528089A (en) | Method for quickly breeding new rice line by combining anther culture and in-vitro chromosome doubling | |
| CN112042532A (en) | Culture medium for inducing calluses to generate and application of culture medium in establishment of calluses regeneration system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |