CN110317825B - Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method - Google Patents
Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method Download PDFInfo
- Publication number
- CN110317825B CN110317825B CN201910352893.9A CN201910352893A CN110317825B CN 110317825 B CN110317825 B CN 110317825B CN 201910352893 A CN201910352893 A CN 201910352893A CN 110317825 B CN110317825 B CN 110317825B
- Authority
- CN
- China
- Prior art keywords
- agrobacterium rhizogenes
- soybean
- transformation
- hairy roots
- soybean hairy
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a soybean hairy root induced transformation method mediated by agrobacterium rhizogenes, which comprises the following steps: selecting soybean seedlings after sowing and about to expand true leaves, transversely cutting hypocotyls 0.2-0.8cm below cotyledon nodes, dipping agrobacterium rhizogenes bacterial solution at the hypocotyl cuts of the seedlings with cotyledon, cutting, and pouring rooting induction nutrient solution containing the agrobacterium rhizogenes bacterial solution for culture. The invention has the advantages that the soybean planting matrix and the space are saved; the time for hairy root generation is shortened, the operation steps and time are saved, and especially when the interaction between rhizobia and soybean is researched, the pollution of other rhizobia is reduced.
Description
Technical Field
The present invention belongs to the field of gene engineering technology. In particular to a hairy root induced transformation method mediated by agrobacterium rhizogenes, in particular to a soybean hairy root transformation method.
Background
At present, the soybean genetic transformation technology mainly comprises the genetic transformation mediated by agrobacterium tumefaciens (Ti) to obtain stable transgenic plants and the hairy root transformation mediated by agrobacterium rhizogenes (Ri). The disadvantage of Agrobacterium tumefaciens (Ti) mediated genetic transformation is that the transformation rate is extremely low and it is difficult to screen transformants. The agrobacterium rhizogenes (Ri) mediated hairy root transformation is a method for quickly obtaining a transgenic chimeric plant, and is widely applied to researches on symbiotic nitrogen fixation between soybean and rhizobia, specific expression of soybean root genes and the like.
Agrobacterium rhizogenes (Ri) mediated hairy root transformation is a method for rapidly obtaining transgenic chimera plants. Currently, in the transformation process of soybean hairy roots, a commonly used high-efficiency transformation strain is K599, and the transformation method adopts a hypocotyl puncture method (kerezt a, li D et al (2007) Agrobacterium rhizogenes-mediated transformation of soybean to root biology. Nat 2, 948-952), wherein the disclosed method is as follows: (1) Sowing soybean seeds in vermiculite, culturing under illumination, and performing puncture inoculation after 5 days. (2) The Agrobacterium rhizogenes bacterial solution with the target vector is cultured in a solid LB culture medium containing a vector screening antibiotic and streptomycin (concentration 50mg/L, the same below) at 28 ℃ for 2 days, a single clone is selected and added into a liquid LB culture medium containing the antibiotic for 500ul overnight, 200ul of bacterial solution is taken and coated on the LB solid culture medium for overnight culture, and a coater is used for collecting the bacteria for puncture. (3) When the soybean cotyledon has not been developed, the soybean hypocotyl is punctured with a 1ml syringe (FIG. 1.a), and then the cultivation is performed by moisturizing and illuminating. (4) After the transgenic hairy roots grow out, main roots are cut off (figure 1.b), transplanting is carried out, and related researches such as inoculation and the like are carried out after seedling recovery (figure 1.c). However, the hypocotyl puncture method has the following disadvantages: (1) hairy root transformation efficiency is affected by genotype: some soybean varieties are only 50% efficient at producing hairy roots. (2) long period of hairy root formation: in general, hairy root generation starts to be seen after 14 days of puncture, and basically no new hairy root is generated after 30 days. The time for most varieties to generate hairy roots is about 15-30 days. (3) After the hairy roots are formed, main roots need to be cut off and transplanted again, and in the process, the soybeans need to be re-planted and are easy to cause artificial pollution. There are also studies on inducing hairy roots by cotyledons, that is, cutting soybean cotyledons cultured for 7-10 days, deeply cutting several cotyledons at cotyledonary nodes, and soaking the cut cotyledons in activated agrobacterium rhizogenes bacterial solution to induce hairy roots (Zong Xiaoqiu, etc., recommendation of agrobacterium rhizogenes to induce soybean hairy root system, proceedings of university of agriculture in huazhong, volume 31, phase 6, pages 699-703 in 2012).
Therefore, there is a need to find or develop more efficient, easier to manipulate methods for agrobacterium rhizogenes-mediated transformation of soybean hairy roots.
Disclosure of Invention
Aiming at the defects of the hypocotyl puncture method, the invention carries out technical improvement to overcome the defects, and solves the technical problems that: shortening the period of hairy root transformation and improving the efficiency of producing hairy roots; the operation steps are reduced, and the artificial pollution is reduced.
The method is characterized in that a hypocotyl of soybean seedlings is transversely cut, agrobacterium rhizogenes is smeared at a cut position and is cut into vermiculite, agrobacterium rhizogenes bacterial liquid is poured, and the cut position can be expanded and hairy roots can grow after cultivation, and the method is called as a one-step cutting method.
In order to ensure the success of the method, firstly, the problem that the hypocotyl of the soybean seedling does not survive after the cross cutting is solved, and for the purpose, sufficient humidity needs to be ensured after the cross cutting is adopted, and secondly, the hairy root can be effectively induced to grow, on one hand, the soybean fine seedling in a specific growth stage needs to be selected, and simultaneously, the cross cutting position needs to be selected.
In view of the above, the present invention therefore provides a method for agrobacterium rhizogenes-mediated soybean hairy root induced transformation, comprising the steps of: selecting soybean, sowing, transversely cutting seedling at hypocotyl position of 0.2-0.8cm under cotyledonary node, dipping Agrobacterium rhizogenes thallus at hypocotyl incision of seedling with cotyledons, cutting, pouring rooting induction nutrient solution containing said Agrobacterium rhizogenes bacterial solution, culturing, preferably containing 0.5mM MgSO 4 in the nutrient solution 4 ；1mM CaCl 2 ；0.7mM KH 2 PO 4 ；0.5mM Na 2 HPO 4 ；0.05mM Fe-EDTA；0.5mM NH 4 NO 3 ；1×Hougland Minor Salt。
Preferably, wherein the temperature during the cultivation is 20-24 ℃ and the relative humidity at the cultivation temperature is above 80%, preferably above 85%, such as 85-90%. In order to maintain humidity, the following is adopted: the fresh-keeping bag is covered by a material with light transmittance of more than 80%, preferably 90%, such as high-light-transmittance fresh-keeping bag to maintain humidity, and placed on a culture shelf to be cultured under normal illumination (such as 16 h illumination, 24 ℃, 8 h dark, 20 ℃).
Preferably, the transected position is 0.4-0.6cm, most preferably 0.45-0.55cm, below the cotyledonary node.
Preferably, the cross cut is cut at an angle of 40-50 degrees, preferably 45 degrees, to the hypocotyl.
In one embodiment, the cuttage is that after the hypocotyl cuts of the seedlings with cotyledons are dipped with agrobacterium rhizogenes thalli, the agrobacterium rhizogenes thalli are placed into vermiculite in a pre-inserting hole, and the vermiculite on one side is compacted.
Preferably, the agrobacterium rhizogenes is agrobacterium rhizogenes K599.
Preferably, the incision of the hypocotyl is dipped with agrobacterium rhizogenes, namely agrobacterium rhizogenes thalli cultured on a solid LB culture medium; then pouring a bacterial liquid containing the agrobacterium rhizogenes with the concentration of OD 600 The concentration of the bacterial suspension is preferably 0.05 or more, preferably 0.1 or more, for example, 0.1 to 0.15.
In a relative embodiment, the method of the invention operates as follows:
(1) The first day, soybean seeding: sowing seeds with sterilized surfaces in the sterilized vermiculite, wherein the sowing depth is 1.5cm, and sufficiently irrigating with sterile water;
(2) And fourthly, activating and culturing agrobacterium: carrying the target vector of Agrobacterium rhizogenes K599 (with streptomycin resistance, str +) preservation bacterial liquid in solid LB culture medium (containing target vector antibiotic and streptomycin, the concentration of streptomycin is 25 mg/L) 28 ℃ overnight culture;
(3) On the sixth day, agrobacterium is mass cultured: inoculating 2-3 monoclonal bacteria to antibiotic-free solid LB culture medium for overnight culture;
(4) And on the seventh day, cutting transformation: after the seventh day, the true leaves are unfolded, the cotyledon is pinched by the left hand, a sterile/sharp dissecting blade is used at the position 0.5cm below the cotyledon node of the soybean, the dissecting blade inclines at an angle of 45 degrees with the hypocotyl, the cut of the hypocotyl is dipped with K599 thalli, the hypocotyl is placed into vermiculite which is inserted in the holes in advance, and the vermiculite on one side is compacted;
(5) Using 5ml of OD 600 K599 bacterial liquid rooting induction nutrient solution (containing 0.5mM MgSO. RTM.) of =0.1 4 ；1mM CaCl 2 ；0.7mM KH 2 PO 4 ；0.5mM Na 2 HPO 4 ；0.05mM Fe-EDTA；0.5mM NH 4 NO 3 (ii) a 1 × Hougland Minor Salt), covering a high-pass light fresh-keeping bag, and putting the fresh-keeping bag on a culture shelf for illumination culture (16 h for illumination, 24 ℃;8 h dark, 20 ℃);
(6) Culturing until new hairy roots grow: new roots grow out at the cut part, and rhizobia can be inoculated or other related experiments can be carried out.
The method adopts a mode of transversely cutting the hypocotyl, and adopts the above mentioned measures to smear the agrobacterium rhizogenes on the cut part, so as to ensure the survival of cuttage, pour the agrobacterium rhizogenes bacterial liquid, directly generate hairy roots on the cut part, and realize the rapid, efficient and one-step completion of the transformation of the hairy roots. Therefore, the invention has the following obvious beneficial effects: (1) soybean planting matrix and space are saved: and (3) culturing transformed soybean seedlings, wherein 20 soybeans can be planted in each box (8cm x 111cm x 9cm), and the cultivation is suitable for large-scale culture. (2) shortening of the time for hairy root production: the time for producing the hairy roots by adopting the transverse cutting-cuttage method is 5-7 days faster than that of the puncturing method. (3) not affected by genotype: the method can be used for producing transgenic hairy roots of soybean with a ratio of 90-100%, and is not affected by genotype. (4) After cuttage, the main roots do not need to be cut off and then transplanted like a puncturing method, operation steps and time are saved, and especially when interaction between rhizobia and soybeans is researched, pollution of other rhizobia is reduced.
Drawings
FIG. 1 shows a method for transforming soybean hairy root into hypocotyl puncture in the prior art
a. And (c) puncturing soybean hypocotyls by using an injector, b, cutting off main roots after transgenic hairy roots grow out, and c, transplanting.
FIG. 2. Example A one-step Process for transformation and cuttage of soybean variety Williams 82 hairy root
a. B, dipping agrobacterium rhizogenes at a cut of an hypocotyl of a seedling with a true leaf to be unfolded, c, cutting into vermiculite, d, growing a hairy root at the 10 th day, and e, growing a hairy root at the 17 th day.
FIG. 3 example two soybean hairy root transformation cutting method for No. 13 yellow soybean at day 17 root growth.
Detailed Description
The present invention will be further described with reference to specific embodiments for better understanding, but the present invention is not limited thereto. Operations such as culture medium and the like which are not specifically described therein are performed in a conventional manner.
Example one
(1) The first day, soybean variety Williams 82 (soybean genomic sequencing open variety) was sown: the surface sterilized seeds were sown in sterilized vermiculite at a sowing depth of 1.5cm and sufficiently watered with sterile water.
(2) And fourthly, activating and culturing agrobacterium: agrobacterium rhizogenes K599 (with streptomycin resistance, str +) preserved bacterial liquid carrying the target vector is cultured in a solid LB culture medium (containing target vector antibiotics and streptomycin, the concentration of the streptomycin is 25 mg/L) at 28 ℃ overnight.
(3) And sixthly, culturing the agrobacterium rhizogenes K599 in a large scale: 2-3 monoclonal bacteria were inoculated to antibiotic-free solid LB medium overnight.
(4) And on the seventh day, cutting transformation: after soybean seeding for 7 days, true leaves are unfolded (2.a), cotyledons are pinched by the left hand, the part 0.5cm below the cotyledonary node of the soybean is cut off by a sterilized sharp dissecting blade inclined at an angle of 45 degrees with respect to the hypocotyl, the cut part of the hypocotyl is dipped in agrobacterium rhizogenes K599 cultured on a solid LB culture medium, namely, thallus collected from the solid culture medium by a coater (2.b), is put into vermiculite in a pre-inserting hole, and is compacted by vermiculite on one side (2.c).
(5) 5ml of K599-containing bacterial solution (OD) was used 600 = 0.1) hair root inducing nutrient solution, the specific composition of the nutrient solution is 0.5mM MgSO 4 ；1mM CaCl 2 ；0.7mM KH 2 PO 4 ；0.5mM Na 2 HPO 4 ；0.05mM Fe-EDTA；0.5mM NH 4 NO 3 (ii) a 1 XHougland Minor Salt, covering a high-pressure PE high-light-transmission fresh-keeping bag (the thickness is 0.03mm, the light transmission rate is 90 percent), and putting the fresh-keeping bag into a culture shelf for illumination culture;
(6) New hairy roots begin to grow on the 10 th day after cuttage: there was a new root growing at the hypocotyl cut (FIG. 2.d). On day 15 after cuttage, the average length of Mao Xinsheng root is about 5cm, and on day 17 after cuttage, the average number of new roots of each plant is about 15, and the average root length can reach 9cm (figure 2.e). Rhizobia can be inoculated at this time or other related experiments can be performed.
Example two
The soybean variety used in this example was Zhonghuang No. 13, and the detailed operation thereof was carried out in the manner of example one, as shown in FIG. 3, in the case of root growth at the cut site after day 17.
Claims (10)
1.A soybean hairy root induction transformation method mediated by agrobacterium rhizogenes comprises the following steps: selecting soybean seedlings after sowing, transversely cutting the seedlings with true leaves to be unfolded at hypocotyls 0.2-0.8cm below cotyledonary nodes, dipping agrobacterium rhizogenes bacterial liquid at the hypocotyl cuts of the seedlings with cotyledons, cutting, pouring a rooting induction nutrient solution containing the agrobacterium rhizogenes bacterial liquid for culture, wherein the concentration of the agrobacterium rhizogenes bacterial liquid isIs OD 600 =0.05 or more, relative humidity at the time of the culture is 80% or more, light irradiation culture; the culture medium composition of the hairy root induction nutrient solution is 0.5mM MgSO 4 ；1mM CaCl 2 ；0.7mM KH 2 PO 4 ；0.5mM Na 2 HPO 4 ；0.05mM Fe-EDTA；0.5mM NH 4 NO 3 ；1×Hoagland Minor Salt。
2. The method for inducing transformation of soybean hairy roots of claim 1, wherein the transecting position is 0.4-0.6cm under cotyledonary node.
3. The method for inducing transformation of soybean hairy roots according to claim 1, wherein said transverse cutting is inclined at an angle of 40 to 50 degrees with respect to the hypocotyl.
4. The method for inducing transformation of soybean hairy roots according to claim 1, wherein the relative humidity at the time of cultivation is 85% or more.
5. The method for inducing transformation of soybean hairy roots according to claim 4, wherein the soybean hairy roots are cultivated by exposure to light on a cultivation shelf by covering a high-pass light fresh-keeping bag.
6. The method for inducing transformation of soybean hairy roots according to claim 5, wherein the light conditions are 16 h light, 24 degrees; 8 h dark, 20 degrees.
7. The method for inducing the transformation of the hairy roots of the soybeans as claimed in claim 5, wherein the cuttage is that the hypocotyl cut of the seedling with cotyledons is dipped with the agrobacterium rhizogenes solution, then the seedling is placed into vermiculite in a pre-inserting hole, and the vermiculite on one side is compacted.
8. The method for inducing transformation of soybean hairy roots according to claim 5, wherein the concentration of the bacterial liquid containing the Agrobacterium rhizogenes is OD 600 And =0.1 or more.
9. The method for inducible transformation of soybean hairy roots according to claim 8, wherein the concentration of the solution containing said Agrobacterium rhizogenes is OD 600 = 0.1-0.15。
10. The method for the induced transformation of soybean hairy roots according to any one of claims 1 to 9, wherein said agrobacterium rhizogenes is agrobacterium rhizogenes K599.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910352893.9A CN110317825B (en) | 2019-04-29 | 2019-04-29 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910352893.9A CN110317825B (en) | 2019-04-29 | 2019-04-29 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110317825A CN110317825A (en) | 2019-10-11 |
| CN110317825B true CN110317825B (en) | 2023-03-31 |
Family
ID=68113066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910352893.9A Active CN110317825B (en) | 2019-04-29 | 2019-04-29 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110317825B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113832178A (en) * | 2021-09-08 | 2021-12-24 | 浙江省农业科学院 | Method for establishing agrobacterium rhizogenes-mediated vegetable pea genetic transformation system |
| CN113652446B (en) * | 2021-09-22 | 2023-07-07 | 内蒙古农业大学 | Agrobacterium rhizogenes mediated one-step transformation method for hairy roots of Caragana carryover |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705243A (en) * | 2009-11-13 | 2010-05-12 | 华南农业大学 | Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation |
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102766650A (en) * | 2012-07-03 | 2012-11-07 | 吉林大学 | Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
-
2019
- 2019-04-29 CN CN201910352893.9A patent/CN110317825B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705243A (en) * | 2009-11-13 | 2010-05-12 | 华南农业大学 | Application of method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation |
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102766650A (en) * | 2012-07-03 | 2012-11-07 | 吉林大学 | Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110317825A (en) | 2019-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104542299B (en) | A kind of method improving common wild-rice filial generation Anther Culture Efficiency | |
| Vijayakumar et al. | In vitro propagation of Bacopa monnieri L.-a multipurpose medicinal plant | |
| CN100581352C (en) | Method for high frequency plant regenerating of tallow tree tissue culture adventitious bud | |
| CN103966258A (en) | Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method | |
| CN103858770A (en) | Rapid hosta plantagineu propagation method | |
| CN110317825B (en) | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN106258976B (en) | A kind of tissue culturing fast seedling-cultivating method of mustard type rape | |
| CN102715089B (en) | Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant | |
| CN106755084A (en) | A kind of quick transgenic method of tea tree | |
| CN101248762B (en) | Golden leaf carex shoot-tip culture quick propagation method | |
| CN113973658B (en) | Efficient genetic transformation and plant regeneration method for capsicum | |
| CN112493126B (en) | Method for induction of lagerstroemia indica somatic embryo and plant regeneration | |
| KR900017463A (en) | Transgenic Plants and Methods for Making the Same | |
| CN110771512B (en) | Efficient induction method of rabdosia lophanthide callus | |
| CN112042532B (en) | Culture medium for inducing calluses to generate and application of culture medium in establishment of calluses regeneration system | |
| CN102719475A (en) | Genetic transformation method by utilization of cotton meristematic tissue | |
| CN107667859A (en) | A kind of rhizome of cyrtomium tissue culture and rapid propagation method | |
| CN114150000A (en) | Application of rape BnLAC2 gene in improving cold resistance early blossoming | |
| Kumar Jha et al. | Production of adventitious shoots and plantlets from the hypocotyl explants of Sesbania rostrata (Bremek & obrem) | |
| CN110301335B (en) | Culture method for lateral bud breeding corm of saffron | |
| Stephan et al. | Propagation of Coriandrum sativum L. through somatic embryogenesis | |
| CN113215191A (en) | Agrobacterium-mediated genetic transformation method for toona sinensis | |
| CN106856998B (en) | Method for researching plant stress tolerance molecular mechanism by using grafting system of Thellungiella halophila and Arabidopsis thaliana | |
| CN101669446B (en) | Method using hypocotyl of catharanthus roseus for cultivating regenerated plants |
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 |