CN105340746A - Method for cultivating octoploid lowland type switchgrass - Google Patents
Method for cultivating octoploid lowland type switchgrass Download PDFInfo
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention provides a method for cultivating octoploid lowland type switchgrass. The method comprises the following steps: (1) induction of tissue culture; (2) micropropagation of the tissue culture; (3) chromosome doubling induction; (4) bud differentiation induction; (5) rooting induction; (6) transplanting; and (7) ploidy identification. The young panicle of the switchgrass serves as an explant to induce the tissue culture, so that the regeneration rate of the young panicle is high; colchicine is used for carrying out artificial chromosome doubling treatment, the doubling effect of the colchicine is stable, and chimera plants are few; and ploidy identification is carried out after the induction of germination and rooting so as to guarantee that all screened plants are the octoploid lowland type switchgrass, and a foundation is laid for future selection of varieties of the octoploid lowland type switchgrass.
Description
Technical field
The invention belongs to field of plant breeding, be specifically related to a kind of method of cultivating octoploid low ground type switchgrass.
Background technology
Switchgrass (PanicumvrigatumL.), grass family Panicum is perennial herb C4 plant, originates in the east of the rockies of North America, prairie on the south north latitude 55 °, its plant is tall and big, well developed root system, be normally used for herding, water and soil conservation, ecological construction and afforestation etc.Due to switchgrass strong adaptability, there is higher yield potentiality and stronger drought-enduring resistance to ridge ability, and wood fibre cellulose content is high, be one of the most potential energy crop, carried out commercial development and utilization in the U.S. by the pattern energy-source plant as development cellulosic ethanol.
Switchgrass is introduced into China the eighties in 20th century, in Beijing, Ningxia, Shanxi, the ground such as Shaanxi carried out experimental study for many years, to the evaluation that its annidation, ecological functions, biological yield, quality etc. are carried out, find that this plant has stronger annidation and higher biological yield, soil can be improved, increase soil organic carbon, reduce hillside fields water and soil and loss of soil nutrient, sulfur dioxide absorption and stagnantly fall the functions such as dust, shows good ecological benefits and living beings productive potential.But as advantitious plant, switchgrass at home germ plasm resource is very limited, and varieties of plant is comparatively single, therefore, the germplasm innovation and the breed breeding that the basis of existing resource are carried out switchgrass seem particularly important.And low ground type switchgrass kind mostly is tetraploid, the acquisition of the low ground type switchgrass plant of octoploid, lays the foundation the seed selection for octoploid low ground type switchgrass kind from now on.
Summary of the invention
The object of the present invention is to provide a kind of method of cultivating octoploid low ground type switchgrass.
A kind of method of cultivating octoploid low ground type switchgrass of the present invention, comprises the steps:
1) evoked callus: by tetraploid low ground type switchgrass containing internal layer bract young fringe segment sterilization after rip cutting be two halves, be placed in evoked callus on inducing culture;
2) callus expands numerous: cut step 1) callus that induces, be inoculated into proliferated culture medium expands numerous;
3) chromosome doubling induction: by step 2) callus that obtains is inoculated into induction in colchicine liquid nutrient medium and doubles;
4) induced bud differentiation: by step 3) in process after callus clean, blot excessive moisture, be seeded on differential medium, induced bud break up;
5) root induction: after bud grows to 1-2 centimetre, cut bud, is inoculated into root induction on root media;
6) transplant: treat Seedling Height 6-8 centimetre, the long 2-4 cm thick of root, be transplanted in matrix after hardening and plant, obtain switchgrass seedling;
7) Ploidy Identification: get step 6) blade of gained switchgrass seedling, carries out pore and/or flow cytometry qualification, screens the seedling that wherein stoma number reduces and/or blade cell nuclear dna content doubles, obtain octoploid low ground type switchgrass.
Described inducing culture is for minimal medium with MS medium, maltose mass percentage 3%, 2, the medium of 4-dichlorphenoxyacetic acid (2,4-D) content 5mg/L, 6-benzyl aminopurine (6-BA) content 1.2mg/L, plant gel mass percentage 0.8%, pH value 5.8.
Described proliferated culture medium is for minimal medium with MS medium, maltose mass percentage 3%, 2, the medium of 4-dichlorphenoxyacetic acid (2,4-D) content 5mg/L, 6-benzyl aminopurine (6-BA) content 1.2mg/L, proline content 2g/L, plant gel mass percentage 0.8%, pH value 5.8.
Described colchicine liquid nutrient medium is for minimal medium with MS medium, the liquid nutrient medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, mannitol content 20g/L, colchicine mass percentage 0.08%-0.12%, pH value 5.8.
Wherein, described chromosome doubling induction, for rotating speed being the concussion process 48-96 hour of 120rpm/min in colchicine liquid nutrient medium.
Described differential medium is with MS medium for minimal medium, the medium of maltose mass percentage 3%, gibberellin (GA3) content 0.5mg/L, plant gel mass percentage 0.8%, pH value 5.8.
Described root media is with 1/2MS medium for minimal medium, the medium of plant gel mass percentage 0.6%, pH value 5.8.
Wherein, step 1) the described young fringe segment containing internal layer bract, its procurement process is as follows: in booting stage, get switchgrass prematurity children in bud fringe, peel off outer bract and leaf sheath, the young fringe containing internal layer bract is cut into 1.8-2.2 centimetre of segment, obtain the young fringe segment containing internal layer bract.
Wherein, step 1) described sterilization, concrete grammar is: with the ethanol surface sterilization 30 seconds of volumn concentration 75%, aseptic water washing 2 times, the NaClO of volumn concentration 20% soaks 10 minutes, and aseptic water washing 3 ~ 4 times, blots surface moisture.
Wherein, described evoked callus, the Initial culture time is 10 days, and every 2 weeks afterwards subcultures once.Described evoked callus, 4 weeks-6 weeks of Dual culture.
Wherein, described callus expands numerous, and 2 weeks subcultures once.Described callus expands numerous, and Dual culture cultivates 8 weeks-10 weeks.
Wherein, described evoked callus, callus expand numerous, chromosome doubling induction, and its environmental condition is carry out under dark condition, and temperature is 25 ± 2 DEG C.
Wherein, described induced bud differentiation, root induction, its environmental condition is temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx.
Wherein, described hardening is uncork hardening 1-2 days, and environmental condition is temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx.
Wherein, described transplanting, for washing away root medium, being transplanted into and spending soil: perlite volume ratio is in the matrix of 1:1, cultivates in booth or greenhouse.
The present invention also provides the method for described cultivation octoploid low ground type switchgrass, the application in switchgrass breeding.
Beneficial effect of the present invention is:
The method of octoploid low ground of the present invention type switchgrass is ripe, stable, and the regeneration culture technique system of switchgrass of the present invention is comparatively ripe, and with the young fringe of switchgrass for sugarcane explants through callus induction, its regeneration rate is high; Carry out chromosome doubling induction with colchicine process switchgrass callus, it doubles effect stability, and chimera plant is few.
Accompanying drawing explanation
Fig. 1 is the plant leaf pore optical microscope photograph of switchgrass kind Alamo after method of the present invention is cultivated in embodiment 1; Wherein left side is tetraploid (contrast), and right side is octoploid.
Fig. 2 is the flow cytometry qualification result of switchgrass kind Alamo after method of the present invention is cultivated in embodiment 1; Wherein left side is tetraploid (contrast), and right side is octoploid.
Fig. 3 is the plant leaf pore optical microscope photograph of switchgrass kind Kanlow after method of the present invention is cultivated in embodiment 2; Wherein left side is tetraploid (contrast), and right side is octoploid.
Fig. 4 is the flow cytometry qualification result of switchgrass kind Kanlow after method of the present invention is cultivated in embodiment 2; Wherein left side is tetraploid (contrast), and right side is octoploid.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
Embodiment 1
With tetraploid low ground type switchgrass kind Alamo for research object, in booting stage, the switchgrass prematurity children in bud fringe of clip robust growth, peels off outer bract and leaf sheath, young fringe containing internal layer bract is cut into 1.8-2.2 centimetre of segment, obtains the young fringe segment containing internal layer bract.On superclean bench, first carry out surface sterilization 30 seconds, after sterile distilled water rinses 2 times with the ethanol of volumn concentration 75%, use volumn concentration 20%NaClO sterilizing 10 minutes again, finally with sterile distilled water washing 3-4 time, be placed on sterilizing filter paper, blot surface moisture.
1): by the young fringe segment rip cutting containing internal layer bract of sterilizing into two, then inducing culture (MS minimal medium+3% maltose+5mg/L2 is inoculated in, 4-D+1.2mg/L6-BA+0.8% plant gel, pH value 5.8) in, evoked callus under 25 ± 2 DEG C of dark conditions, in 10 days follow-up generations, later 2 weeks subcultures once, are cultivated 4 weeks;
2): by step 1), well-grown callus is forwarded to proliferated culture medium (MS minimal medium+3% maltose+5mg/L2,4-D+1.2mg/L6-BA+2g/L proline+0.8% plant gel, pH value 5.8) in, under 25 ± 2 DEG C of dark conditions, carry out callus expands numerous, 2 weeks subcultures once, are cultivated 10 weeks;
3) expand numerous callus: by step 2) and be switched in the colchicine liquid nutrient medium of colchicine mass percentage 0-0.12% that (described colchicine liquid nutrient medium is for minimal medium with MS medium, the liquid nutrient medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, mannitol content 20g/L, colchicine mass percentage 0-0.12%, pH value 5.8.), in temperature 25 ± 2 DEG C of dark condition concussion process 72 hours, rotating speed was 120rpm/min.
4) callus be disposed: by step 3) rinses 2-3 time repeatedly with sterile water respectively, then with aseptic filter paper, water is blotted, be forwarded to differential medium (MS minimal medium+3% maltose+0.5mg/LGA3+0.8% plant gel again, pH value 5.8) on, (light application time is 16h/d, intensity of illumination 1500-2000lx) induced bud differentiation under 25 ± 2 DEG C of illumination conditions;
5): when step 4), induced bud grows to 1-2 centimetre, cut with scalpel and be forwarded to root media (1/2MS minimal medium+0.6% plant gel, pH value 5.8) in, root induction under temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx condition;
6) seedling: when step 5) grows to 6-8 centimetre, during root 2-4 centimetre, open sealed membrane, temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx condition lower refining seedling 1-2 days, then plantlet in vitro is taken out from medium, rinse out the residual medium of root with clear water, be transplanted to be equipped with to spend in soil and the pot for growing seedlings of vermiculite (volume ratio 1:1) and cultivate in plastic greenhouse;
7): get step 6), the tender blade of plantlet in vitro children carries out pore (see Fig. 1) and flow cytometry qualification (see Fig. 2) respectively, screen the seedling that wherein stoma number reduces and/or blade cell nuclear dna content doubles, to filter out real octoploid switchgrass plant.
Table 1 colchicine concentration is on the impact of process 72 hours inductivities
| Colchicine mass percentage (%) | Inductivity (%) |
| 0.00 | 0 |
| 0.04 | 7.2 |
| 0.08 | 26.3 |
| 0.12 | 14.3 |
Induction the results are shown in Table 1, the process adding colchicine all has certain inducing effect, wherein the process inductivity of colchicine mass percentage 0.08%-0.12% is more than 10%, and with the best results of 0.08% process, the inductivity obtaining octoploid low ground type switchgrass Alamo is 26.3%.
Embodiment 2
With tetraploid low ground type switchgrass kind Kanlow for research object, in booting stage, the switchgrass prematurity children in bud fringe of clip robust growth, peels off outer bract and leaf sheath, young fringe containing internal layer bract is cut into 1.8-2.2 centimetre of segment, obtains the young fringe segment containing internal layer bract.On superclean bench, first carry out surface sterilization 30 seconds, after sterile distilled water rinses 2 times with the ethanol of volumn concentration 75%, use volumn concentration 20%NaClO sterilizing 10 minutes again, finally with sterile distilled water washing 3-4 time, be placed on sterilizing filter paper, blot surface moisture.
1): by the young fringe segment rip cutting containing internal layer bract of sterilizing into two, then inducing culture (MS minimal medium+3% maltose+5mg/L2 is inoculated in, 4-D+1.2mg/L6-BA+0.8% plant gel, pH value 5.8) in, evoked callus under 25 ± 2 DEG C of dark conditions, in 10 days follow-up generations, later 2 weeks subcultures once, are cultivated 6 weeks;
2): by step 1), well-grown callus is forwarded to proliferated culture medium (MS minimal medium+3% maltose+5mg/L2,4-D+1.2mg/L6-BA+2g/L proline+0.8% plant gel, pH value 5.8) in, under 25 ± 2 DEG C of dark conditions, carry out callus expands numerous, 2 weeks subcultures once, are cultivated 8 weeks;
3) expand numerous callus: by step 2) and be switched in the colchicine liquid nutrient medium of colchicine mass percentage 0-0.12% that (described colchicine liquid nutrient medium is for minimal medium with MS liquid nutrient medium, the liquid nutrient medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, mannitol content 20g/L, colchicine mass percentage 0-0.12%, pH value 5.8.), in temperature 25 ± 2 DEG C of dark condition concussion process 48 hours, rotating speed was 120rpm/min.
4) callus be disposed: by step 3) rinses 2-3 time repeatedly with sterile water respectively, then with aseptic filter paper, water is blotted, be forwarded to differential medium (MS minimal medium+3% maltose+0.5mg/LGA3+0.8% plant gel again, pH value 5.8) on, (light application time is 16h/d, intensity of illumination 1500-2000lx) induced bud differentiation under 25 ± 2 DEG C of illumination conditions;
5): when step 4), induced bud grows to 1-2 centimetre, cut with scalpel and be forwarded to root media (1/2MS minimal medium+0.6% plant gel, pH value 5.8) in, root induction under temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx condition;
6) seedling: when step 5) grows to 6-8 centimetre, during root 2-4 centimetre, open sealed membrane, temperature 25 ± 2 DEG C, light application time 16 hours/day, intensity of illumination 1500-2000lx condition lower refining seedling 1-2 days, then plantlet in vitro is taken out from medium, rinse out the residual medium of root with clear water, be transplanted to be equipped with to spend in soil and the pot for growing seedlings of vermiculite (volume ratio 1:1) and cultivate in plastic greenhouse;
7): get step 6), the tender blade of plantlet in vitro children carries out pore (see Fig. 3) and flow cytometry qualification (see Fig. 4) respectively, screen the seedling that wherein stoma number reduces and/or blade cell nuclear dna content doubles, to filter out real octoploid switchgrass plant.
Table 2 colchicine concentration is on the impact of process 48 hours inductivities
| Colchicine mass percentage (%) | Inductivity (%) |
| 0.00 | 0 |
| 0.04 | 3.1 |
| 0.08 | 15.4 |
| 0.12 | 19.6 |
The results are shown in Table 2, the process adding colchicine all has certain inducing effect, wherein the process inductivity of colchicine mass percentage 0.08%-0.12% is more than 15%, with best results during colchicine mass percentage 0.12%, the inductivity of octoploid low ground type switchgrass Kanlow is 19.6%.
Claims (10)
1. cultivate a method for octoploid low ground type switchgrass, it is characterized in that, comprise the steps:
1) evoked callus: by tetraploid low ground type switchgrass containing internal layer bract young fringe segment sterilization after rip cutting be two halves, be placed in evoked callus on inducing culture;
2) callus expands numerous: cut step 1) callus that induces, be inoculated into proliferated culture medium expands numerous;
3) chromosome doubling induction: by step 2) callus that obtains is inoculated into induction in colchicine liquid nutrient medium and doubles;
4) induced bud differentiation: by step 3) in process after callus clean, blot excessive moisture, be seeded on differential medium, induced bud break up;
5) root induction: after bud grows to 1-2 centimetre, cut bud, is inoculated into root induction on root media;
6) transplant: treat Seedling Height 6-8 centimetre, the long 2-4 cm thick of root, be transplanted in matrix after hardening and plant, obtain switchgrass seedling;
7) Ploidy Identification: get step 6) blade of gained switchgrass seedling, carries out pore and/or flow cytometry qualification, screens the seedling that wherein stoma number reduces and/or blade cell nuclear dna content doubles, obtain octoploid low ground type switchgrass.
2. the method for claim 1, it is characterized in that, described inducing culture is for minimal medium with MS medium, the medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, plant gel mass percentage 0.8%, pH value 5.8.
3. the method for claim 1, it is characterized in that, described proliferated culture medium is for minimal medium with MS medium, the medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, proline content 2g/L, plant gel mass percentage 0.8%, pH value 5.8.
4. the method for claim 1, it is characterized in that, described colchicine liquid nutrient medium is for minimal medium with MS medium, the liquid nutrient medium of maltose mass percentage 3%, 2,4-dichlorphenoxyacetic acid content 5mg/L, 6-benzyl aminopurine content 1.2mg/L, mannitol content 20g/L, colchicine mass percentage 0.08%-0.12%, pH value 5.8.
5. method as claimed in claim 4, is characterized in that, described chromosome doubling induction, for rotating speed being the concussion process 48-96 hour of 120rpm/min in colchicine liquid nutrient medium.
6. the method for claim 1, it is characterized in that, described differential medium is with MS medium for minimal medium, the medium of maltose mass percentage 3%, GA content 0.5mg/L, plant gel mass percentage 0.8%, pH value 5.8.
7. the method for claim 1, is characterized in that, described root media is with 1/2MS medium for minimal medium, the medium of plant gel mass percentage 0.6%, pH value 5.8.
8. the method for claim 1, it is characterized in that, step 1) the described young fringe segment containing internal layer bract, its procurement process is as follows: in booting stage, get switchgrass prematurity children in bud fringe, peel off outer bract and leaf sheath, the young fringe containing internal layer bract is cut into 1.8-2.2cm segment, obtain the young fringe segment containing internal layer bract.
9. the method as described in any one of claim 1-8, is characterized in that, described evoked callus, callus expand numerous, chromosome doubling induction, and its environmental condition is carry out under dark condition, and temperature is 25 ± 2 DEG C.
10. the method for the cultivation octoploid low ground type switchgrass described in any one of claim 1-9, the application in switchgrass breeding.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106305421A (en) * | 2016-08-23 | 2017-01-11 | 北京市农林科学院 | Method for breeding salt tolerent switchgrass |
| CN110122332A (en) * | 2019-06-06 | 2019-08-16 | 西北农林科技大学 | A kind of method of broom corn millet spire in vitro culture and plant regeneration |
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| US20120042569A1 (en) * | 2010-08-19 | 2012-02-23 | The Institute For Advanced Learning And Research | Methods and media formulations for large-scale and efficient micropropagation of bio-energy grasses |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20120042569A1 (en) * | 2010-08-19 | 2012-02-23 | The Institute For Advanced Learning And Research | Methods and media formulations for large-scale and efficient micropropagation of bio-energy grasses |
Non-Patent Citations (2)
| Title |
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| ZHI YONG YANG ET AL.: "Production of Autopolyploid Lowland Switchgrass Lines Through In Vitro Chromosome Doubling", 《BIOENERGY RESEARCH》 * |
| 孟敏等: "柳枝稷的组织培养技术研究", 《安徽农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106305421A (en) * | 2016-08-23 | 2017-01-11 | 北京市农林科学院 | Method for breeding salt tolerent switchgrass |
| CN106305421B (en) * | 2016-08-23 | 2019-02-12 | 北京市农林科学院 | A method of cultivating salt tolerant switchgrass |
| CN110122332A (en) * | 2019-06-06 | 2019-08-16 | 西北农林科技大学 | A kind of method of broom corn millet spire in vitro culture and plant regeneration |
| CN110122332B (en) * | 2019-06-06 | 2022-08-05 | 西北农林科技大学 | Method for in-vitro culture and plant regeneration of broom corn millet young leaves |
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Application publication date: 20160224 |