CN119422881A - Method for high-efficiency mutagenesis of EMS by using dendrobium seeds - Google Patents
Method for high-efficiency mutagenesis of EMS by using dendrobium seeds Download PDFInfo
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- CN119422881A CN119422881A CN202510018908.3A CN202510018908A CN119422881A CN 119422881 A CN119422881 A CN 119422881A CN 202510018908 A CN202510018908 A CN 202510018908A CN 119422881 A CN119422881 A CN 119422881A
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- 231100000350 mutagenesis Toxicity 0.000 title claims abstract description 52
- 238000002703 mutagenesis Methods 0.000 title claims abstract description 52
- 241001523681 Dendrobium Species 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000009331 sowing Methods 0.000 claims abstract description 15
- 230000001954 sterilising effect Effects 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 230000035784 germination Effects 0.000 claims abstract description 8
- 238000002791 soaking Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000012153 distilled water Substances 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 10
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 7
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000012869 germination medium Substances 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 240000004638 Dendrobium nobile Species 0.000 claims description 4
- 238000005286 illumination Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000010899 nucleation Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 229920000298 Cellophane Polymers 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 231100000111 LD50 Toxicity 0.000 abstract description 13
- 239000000463 material Substances 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000011521 glass Substances 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 40
- 238000009395 breeding Methods 0.000 description 8
- 230000001488 breeding effect Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 231100000219 mutagenic Toxicity 0.000 description 4
- 230000003505 mutagenic effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000011086 glassine Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000000123 paper Substances 0.000 description 2
- 206010068051 Chimerism Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
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- 239000002699 waste material Substances 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
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- Pretreatment Of Seeds And Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application relates to a method for performing EMS efficient mutagenesis by using dendrobium seeds, which relates to the technical field of botanic technology. The method comprises the steps of (1) sterilizing dendrobium seeds, (2) sowing the dendrobium seeds on a germination culture medium with sterile glass paper paved on the surface, (3) culturing the seeds after sowing until the seeds just sprout, (4) carrying out seed mutagenesis, namely soaking the seeds just sprouted in an EMS buffer solution with the volume concentration of 0.4%, carrying out mutagenesis for 4 h under the conditions of 25+/-2 ℃ and shaking, and then terminating the mutagenesis. Compared with the prior art, the method uses dendrobe protocorm and protocorm-like as the semi-lethal dose of the mutagenesis material, and uses the just-germinated seeds as the mutagenesis material to achieve the EMS dose of the semi-lethal dose to be obviously reduced.
Description
Technical Field
The application relates to the technical field of botanic, in particular to a method for efficiently mutagenizing EMS by using dendrobium seeds.
Background
Dendrobium (Dendrobium) is an important ornamental flower and medicinal plant in orchid and is deeply favored by people in China. At present, the breeding of the dendrobium is mainly hybridization, but because the dendrobium seeds have special biological characteristics, do not have endosperm, are very fine, have low reproduction rate under natural conditions and have long growth period. Therefore, the traditional breeding mode has the defects of long growth period, low efficiency and the like. Compared with the traditional breeding method, the chemical mutation breeding is a rapid and effective breeding method, and has a particularly important significance for plant variety improvement. Ethyl methylsulfonate (ETHYL METHANE sulfonate, EMS) is an alkylating agent, can cause gene mutation by changing base pairs in DNA, induces plant phenotype to generate mutation, and is the chemical mutagen which is most widely applied and has the best effect in crop mutation breeding at present. The semi-lethal dose (MEDIAN LETHAL dose, LD 50) is the optimal dose for mutagenic breeding.
Dendrobium plants have no endosperm, so that seeds are tiny like dust, and the seeds need to be attached to a culture medium to obtain nutrition in the sterile germination process, but are difficult to collect again. At present, the mutation materials for the EMS mutation breeding of dendrobium are mainly protocorms and protocorm-like. In the prior art, no report on direct EMS mutagenesis by using dendrobium seeds is known. The protocorm and protocorm-like are used as mutagenesis materials, and because the tissue volume is large, the mutagenesis agent is difficult to permeate into cells, so that the mutagenesis efficiency is low and chimerism is easy to generate. When using protocorm-like as a mutagenesis material for EMS mutagenesis, LD 50 is 0.8% EMS treatment 90 min or LD 50 is 1.8% EMS treatment 4h, whereas when using protocorm as a mutagenesis material for EMS mutagenesis, LD 50 is 1.0% EMS treatment 6 h.
Disclosure of Invention
In order to solve or partially solve the problems existing in the related art, the application provides a method for efficiently mutagenizing EMS by using dendrobe seeds, wherein the dendrobe seeds are directly used as EMS mutagenic materials, EMS mutagenesis is performed when the seeds just sprout, the result shows that LD 50 is that the volume concentration of EMS is 0.4%, the treatment time is 4. 4h, and compared with the prior art, the semi-lethal dose of the dendrobe protocorm and protocorm-like materials is used as the mutagenic materials, and the EMS dose of the semi-lethal dose is obviously reduced by using the seeds just sprouted as the mutagenic materials.
The application discloses a method for high-efficiency mutagenesis of EMS by using dendrobium seeds, which comprises the following steps:
(1) Dendrobium seed disinfection
Sterilizing dendrobe pods;
(2) Sowing seeds
Cutting open the sterilized dendrobe pods, and sowing seeds on a germination culture medium with sterile cellophane laid on the surface;
(3) Seed culture
Culturing the sown seeds until the seeds just sprout;
(4) Seed mutagenesis
Soaking the just germinated seeds in EMS solution with the volume concentration of 0.4%, mutagenizing for 4 hours at the temperature of 25+/-2 ℃ under the vibration condition, then adding sodium thiosulfate solution with the mass concentration of 25% into the EMS solution to terminate the mutagenesis, wherein the volume ratio of the sodium thiosulfate solution with the mass concentration of 25% to the EMS solution is 1:4, uniformly mixing, standing for 10 min, removing the supernatant, and cleaning the seeds subjected to the termination of the mutagenesis by using sterilized distilled water to obtain the mutagenized dendrobium seeds.
Further, the step (1) is specifically performed by immersing dendrobe pods in 75% alcohol for 3min times, washing with sterile distilled water for 2 times, immersing and sterilizing with 0.1% mercuric chloride for 8 min times, washing with sterile distilled water for 3 times, immersing and sterilizing with 1% sodium hypochlorite for 5min times, and washing with sterile distilled water for 10 times.
Further, the specific operation of washing the seeds after termination of the reaction with sterilized distilled water is that 40 mL distilled water is added into a centrifuge tube each time, the mixture is inverted and mixed for 10 times, then the mixture is stood, after the seeds are settled to the bottom of the tube, the supernatant is sucked, and the operation is repeated for 10 times.
Further, the method also comprises the steps of sowing and culturing the dendrobium seeds after mutagenesis, wherein the sowing operation is that the dendrobium seeds after mutagenesis are resuspended by sterile distilled water, the resuspension is paved in a culture bottle filled with a solid sowing culture medium, and the culturing operation is that the culture bottle is cultured under the conditions of 25+/-2 ℃ of temperature, 12 h/d of illumination and 2000-3000 lx of illumination intensity.
Further, the germination medium has the formula of 1/2 MS, 1 mg/L6-BA, 0.1 mg/L NAA, 20 g/L sucrose, 0.3% active carbon, 7 g/L agar and pH of 5.8.
The beneficial effects of the application are as follows:
According to the application, the dendrobium seeds are used as EMS mutagenesis materials for the first time, the method is used for carrying out EMS mutagenesis on the dendrobium seeds, the result shows that LD 50 is that the volume concentration of EMS is 0.4%, the treatment time is 4. 4 h, and the result shows that the method can reach LD 50 only by using lower EMS dosage, namely, the mutagenesis method is more efficient.
Drawings
The foregoing and other objects, features and advantages of the application will be apparent from the following more particular descriptions of exemplary embodiments of the application as illustrated in the accompanying drawings wherein like reference numbers generally represent like parts throughout the exemplary embodiments of the application.
FIG. 1 is a schematic view of example 1 of the present application after sowing seeds on germination medium;
FIG. 2 is a schematic diagram showing germination process of Dendrobium nobile in application example 1 from sowing to mutagenesis;
FIG. 3 is a schematic representation of phenotypes of the time-mutagenized dendrobe of example 1 according to the present application at different EMS concentrations;
FIG. 4 is a statistical diagram of the seedling rates of the time-induced dendrobe with different EMS concentrations in application example 1;
FIG. 5 is a schematic diagram of seedlings obtained by EMS mutagenesis of dendrobe seeds in example 2 and seedlings obtained by EMS mutagenesis of dendrobe seeds in example 1.
Detailed Description
Embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While embodiments of the present application are illustrated in the drawings, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.
Example 1
In this embodiment, the mutagenesis of the dendrobium seeds by using EMS includes the following operations:
(1) Dendrobium seed disinfection
Soaking herba Dendrobii pod in 75% alcohol for 3 min times in sterile distilled water for 2 times, soaking and sterilizing with 0.1% mercuric chloride for 8 min times, soaking and sterilizing with 1% sodium hypochlorite for 5min times, washing with sterile distilled water for 10 times, taking out pod, and absorbing surface water with sterile absorbent paper;
(2) Sowing seeds
Pod was cut with a sterile blade, seeds were sown on germination medium with sterile glassine paper laid on the surface, a small amount of sterile distilled water was added, and the dish was shaken slightly obliquely to uniformly distribute the seeds on the glassine paper, and fig. 1 is a schematic view showing after sowing seeds on germination medium. Each pod was spread on 5 dishes.
The germination culture medium formula comprises 1/2 MS +1 mg/L6-BA+0.1 mg/L NAA+20 g/L sucrose+0.3% active carbon+7 g/L agar, regulating pH to 5.8, specifically, sterilizing the prepared culture medium at 121deg.C for 15min under high pressure, cooling to 55deg.C, pouring the culture medium into a culture dish with diameter of 90mm and height of 20mm, and spreading sterilized cellophane with diameter of 95mm on the culture medium after the culture medium is solidified.
(3) Seed culture
The culture dish with the seeds sown is placed in a dark culture room at 25 ℃, taken out every monday, the germination condition is observed under a microscope, the seeds germinate at 14 d, and the seeds germinate at 21 d. FIG. 2 is a schematic diagram showing germination process of dendrobium seeds from sowing to mutagenesis.
(4) Mutagenesis of freshly germinated dendrobe seeds:
① Phosphate buffer, pH 7.5, was prepared at 100 mM.
100ML of 1M K 2HPO4 solution and 100mL of 1M KH 2PO4 solution were prepared, 70 mL of 1M K 2HPO4 solution and 20mL of 1M KH 2PO4 solution were respectively taken, the pH was adjusted to 7.5 with 1M KH 2PO4 solution, and 1M phosphate buffer was diluted to 100 mM concentration with water.
② EMS preparation with different concentrations.
The phosphate buffer of 40 mL was placed in a sterile centrifuge tube of 50 mL, and EMS solution sterilized by suction filtration through a 0.22/um filter was added to the buffer in a fume hood to a final concentration (V/V) of 0%, 0.2%, 0.4%, 0.6%, respectively, and mixed upside down for use, wherein the 0% treated group was the control group. 3 replicates were prepared for each concentration and 3 replicates were set.
③ The mutagenesis condition is that in a sterile super clean bench, the seeds which are just germinated on the glass paper are taken by a sterilizing medicine spoon and are approximately and uniformly divided into 12 parts and respectively placed in centrifuge tubes containing EMS with different concentrations, and each treatment group contains about 800 seeds. The tubes were placed in shaking incubators at 25℃and 50 rpm culture conditions for mutagenesis reactions.
④ The reaction is stopped, 25% sodium thiosulfate solution is prepared, and the solution is filtered and sterilized by a filter of 0.22 um for later use. The sample solutions of 13 mL were taken into new 50 mL sterile centrifuge tubes at 4h, 8h and 12 h of mutagenesis, 3.2 mL of 25% sodium thiosulfate at 25℃was added, the reaction was stopped at 50 rpm, 10min were left to stand, after the seeds were settled to the bottom of the tubes, the supernatant was sucked dry, and the supernatant was sucked into a waste liquid bottle containing 50% sodium thiosulfate solution.
⑤ Washing with sterilized distilled water to remove residual EMS, adding 40 mL distilled water each time, mixing for 10 times, standing, settling the seed to the bottom of the tube, sucking the supernatant, and repeating for 10 times to obtain herba Dendrobii seed after mutagenesis.
(5) Seed sowing of dendrobium after mutagenesis
Adding 15 mL sterile distilled water into each centrifuge tube, re-suspending the mutagenized dendrobium seeds, uniformly mixing, uniformly dividing the seed suspension into 5 parts, and paving in culture flasks containing 100 mL solid seeding medium, wherein 150 seeds are calculated in each culture flask in consideration of partial loss of the seeds in the operation process. Culturing the culture flask at 25+ -2 deg.C, 12 h/d under light intensity of 2000-3000 lx, and culturing for 60 d, and observing seedling rate.
FIG. 3 is a schematic diagram showing phenotypes of different EMS concentrations and time-induced dendrobe;
FIG. 4 is a statistical diagram of the seedling rate of the time-induced dendrobe with different EMS concentrations.
Example 2
In example 2, no EMS mutagenesis was performed on the dendrobe seeds, as compared to the control group for the mutagenesis performed in example 1.
The results of comparing seedlings obtained after EMS mutagenesis in example 2 and EMS mutagenesis in example 1 are shown in FIG. 5. As can be seen from FIG. 5, after the EMS mutagenesis treatment in example 1, part of plants showed a variation of widening leaves, and part of plants showed yellowing, no leaves growth, etc.
The foregoing description of embodiments of the application has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (5)
1. The method for high-efficiency mutagenesis of EMS by using dendrobium seeds is characterized by comprising the following steps:
(1) Dendrobium seed disinfection
Sterilizing dendrobe pods;
(2) Sowing seeds
Cutting open the sterilized dendrobe pods, and sowing seeds on a germination culture medium with sterile cellophane laid on the surface;
(3) Seed culture
Culturing the sown seeds until the seeds just sprout;
(4) Seed mutagenesis
Soaking the just germinated seeds in EMS solution with the volume concentration of 0.4%, mutagenizing for 4 hours at the temperature of 25+/-2 ℃ under the vibration condition, then adding sodium thiosulfate solution with the mass concentration of 25% into the EMS solution to terminate the mutagenesis, wherein the volume ratio of the sodium thiosulfate solution with the mass concentration of 25% to the EMS solution is 1:4, uniformly mixing, standing for 10 min, removing the supernatant, and cleaning the seeds subjected to the termination of the mutagenesis by using sterilized distilled water to obtain the mutagenized dendrobium seeds.
2. The method for performing EMS efficient mutagenesis by using dendrobium nobile seeds according to claim 1, wherein the step (1) is specifically performed by immersing dendrobium nobile pods in 75% alcohol for 3 min times, washing with sterile distilled water for 2 times, immersing and sterilizing with 0.1% mercuric chloride for 8 min times, washing with sterile distilled water for 3 times, immersing and sterilizing with 1% sodium hypochlorite for 5min times, and washing with sterile distilled water for 10 times.
3. The method for performing EMS efficient mutagenesis by using dendrobium nobile seeds according to claim 1, wherein the specific operation of washing the seeds after termination of the reaction with sterilized distilled water is that 40 mL distilled water is added into a centrifuge tube each time, the mixture is inverted and mixed uniformly for 10 times, then the mixture is left stand, after the seeds settle to the bottom of the tube, the supernatant is sucked, and the above operation is repeated for 10 times.
4. The method for high-efficiency mutagenesis by using dendrobium seeds according to claim 1, further comprising the steps of seeding and culturing the dendrobium seeds after mutagenesis, wherein the seeding specific operation is to resuspend the dendrobium seeds after mutagenesis by using sterile distilled water, then spread the resuspension in a culture flask filled with a solid seeding medium, and the culturing specific operation is to culture the culture flask under the conditions of 25+/-2 ℃ of temperature, 12 h/d of illumination and 2000-3000 lx of illumination intensity.
5. The method for high-efficiency mutagenesis of EMS using dendrobe seeds according to claim 1, wherein the germination medium has a formula of 1/2MS, 1 mg/L6-BA, 0.1 mg/L NAA, 20g/L sucrose, 0.3% activated carbon, 7 g/L agar, and pH5.8.
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| CN202510018908.3A CN119422881A (en) | 2025-01-07 | 2025-01-07 | Method for high-efficiency mutagenesis of EMS by using dendrobium seeds |
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| CN202510018908.3A CN119422881A (en) | 2025-01-07 | 2025-01-07 | Method for high-efficiency mutagenesis of EMS by using dendrobium seeds |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213940A (en) * | 2008-01-18 | 2008-07-09 | 中国科学院昆明植物研究所 | Rapid Propagation Method of Dendrobium |
| WO2016119703A1 (en) * | 2015-01-27 | 2016-08-04 | 中国科学院遗传与发育生物学研究所 | Method for conducting site-specific modification on entire plant via gene transient expression |
| CN106922526A (en) * | 2017-03-01 | 2017-07-07 | 广州中医药大学 | A kind of breeding method of the dendrobium candidum of anti-colletotrichum gloeosporioides Penz |
| CN109418158A (en) * | 2017-08-23 | 2019-03-05 | 北京林业大学 | A kind of method and its used medium and application for promoting dendrobium candidum axenic to sprout |
-
2025
- 2025-01-07 CN CN202510018908.3A patent/CN119422881A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213940A (en) * | 2008-01-18 | 2008-07-09 | 中国科学院昆明植物研究所 | Rapid Propagation Method of Dendrobium |
| WO2016119703A1 (en) * | 2015-01-27 | 2016-08-04 | 中国科学院遗传与发育生物学研究所 | Method for conducting site-specific modification on entire plant via gene transient expression |
| CN106922526A (en) * | 2017-03-01 | 2017-07-07 | 广州中医药大学 | A kind of breeding method of the dendrobium candidum of anti-colletotrichum gloeosporioides Penz |
| CN109418158A (en) * | 2017-08-23 | 2019-03-05 | 北京林业大学 | A kind of method and its used medium and application for promoting dendrobium candidum axenic to sprout |
Non-Patent Citations (1)
| Title |
|---|
| 俞涵曦;王晶晶;林蔚;丁金涛;王雪妮;李菁;何碧珠;: "串珠石斛组培快繁技术", 亚热带农业研究, no. 01, 25 February 2020 (2020-02-25), pages 1 * |
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