CN103843567A - Method for rapidly propagating Chinese cymbidium by using root induced protocorm and culture medium thereof - Google Patents
Method for rapidly propagating Chinese cymbidium by using root induced protocorm and culture medium thereof Download PDFInfo
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- CN103843567A CN103843567A CN201410085648.3A CN201410085648A CN103843567A CN 103843567 A CN103843567 A CN 103843567A CN 201410085648 A CN201410085648 A CN 201410085648A CN 103843567 A CN103843567 A CN 103843567A
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Abstract
The invention discloses a method for rapidly propagating Chinese cymbidium by using root induced protocorm and a culture medium thereof. According to the method and the culture medium thereof, protocorm induction is carried out by adopting an improved MS (Murashige and Skoog) culture medium which contains plant growth regulators (6-BA (6-benzylaminopurine) and PIC (Picloram)) and hydrolyzed casein, a specific culture method is adopted for a Chinese cymbidium root explant, Chinese cymbidium roots are enabled to induce a protocorm, the induction ratio reaches over 75%, the number of protocorms of the root explant is greater than 15 after culturing for 45 days, the survival rate of the protocorms is over 80%, and plants grow robustly and root directly.
Description
Technical field
The invention belongs to field of plant cell engineering technology, relate to a kind of method and medium thereof that utilizes root induction protocorm Fast-propagation state orchid.
Background technology
State orchid is often referred to the non-hibernating eggs partially in Cymbidium (Cymbidium) plant, as Chunlan (Cymbidiumgoeringii), orchid (Cymbidium/aberi), sword-leaved cymbidium (Cymbidium ensiJol), Chinese cymbidium (Cymbidiumsinensis), cold blue (Cymbidiumkanran) and their mutation spring sword (Cymbidiumlongibracteatym), lotus lobe orchid (Cymbidiumlianpan) etc., one of China's ten great tradition famous flowers, apart from the cultivation history of modern existing more than 2500 year.It hides delicate fragrance, and pattern is simple and elegant, have " spending middle gentleman " laudatory title, has very high ornamental value and economic worth.
The seed that the blue crossbreed pollination of most of state obtains is after planting, and proterties can separate, and is difficult to keep the merit of maternal plant.And traditional division propagation coefficient is low, speed is slow, the cycle is long, be difficult to meet the needs of large-scale production, some crossbreed also may be due to the highly sterile seed that can not get.Therefore the application of tissue culture technique the most effective modes of reproduction beyond doubt.It is to further develop and form in the technical foundation of cultivating at cattleya tissue that the blue tissue of state is cultivated, to 20 end of the centurys, Chunlan, orchid, sword-leaved cymbidium, Chinese cymbidium, cold orchid, spring sword, lotus lobe orchid and part originate in the aerial orchid kind of China, all succeed with method for tissue culture breeding.
The successful explant of the blue tissue cultivation of state is at present mainly seed, stem apex and lateral bud, and blade and bud are successfully little.In theory, every robust growth, the cell with strong activity, tissue, organ (as seed, lateral bud, stem apex, root, blade, pollen, ovary, petal etc.) can be served as explant, but different orchid kind explant position used is also not quite similar.Especially the tissue of state orchid is cultivated and restricted by the factor such as technology, parent quantity, and the explant kind of application is relatively less.
The tip of a root is also the position that meristematic tissue exists, but in tissue culture procedures, botanists think that to change into the merismatic possibility of bud from root meristematic tissue very little.Although the blue organization of root tips of state is cultivated, also someone attempts, and there is not yet successful report.Plum is red also points out that the organization of root tips cultivation of state orchid is more difficult, need to carry out research and inquirement to many condition of culture.
The general procedure that state's orchid tissue is cultivated is:---induction forms middle brood body (root-like stock, protocorm, embryoid), and---middle brood body is expanded numerous---bud differentiation---root induction---strong seedling culture---test-tube seedling transplanting to explant.
Up to now, the process that the blue tissue of state is cultivated is more loaded down with trivial details, cost is high, want to raise the efficiency, reduce costs and carry out Fast-propagation, research emphasis from now on should be to simplify reproductive process, and 4 stages of avoiding induction, subculture as far as possible, grow seedlings and take root are with different medium, discussion can reduce variation can improve again growth coefficient, and the China blue method for tissue culture of regeneration plant robust growth.
Summary of the invention
The object of the present invention is to provide a kind of cultural method and medium thereof that utilizes the blue root induction protocorm of state, make the inductivity of the blue root induction protocorm of state reach 100%, cultivating 45 days each explant protocorm numbers reaches more than 15, protocorm survival rate reaches more than 80%, plant strain growth stalwartness, directly takes root.
The method of utilizing root induction protocorm Fast-propagation state orchid of the present invention, comprises the following steps:
1) choose the root of the blue aseptic seedling of state, be inoculated in medium, secretly cultivate, obtain protocorm, culturing room's temperature is 25 ± 2 DEG C; Described nutrient media components is: MS minimal medium+sucrose+agar powder+caseinhydrolysate, and the plant growth regulator being made up of 6-benzyl aminopurine+picloram;
2) protocorm of secretly cultivating acquisition is forwarded under light and cultivated, illumination 10-16 hour every day, temperature is 25 ± 2 DEG C, it is green that protocorm turns gradually;
3) be transferred to by turing green protocorm the medium that is placed in blake bottle, develop into gradually normal plant, take root simultaneously; Described medium is the medium that does not contain 6-benzyl aminopurine+picloram+caseinhydrolysate in step 1);
4) room temperature, opens bottle cap hardening, plants after 1 week in the dish of cave, greenhouse.
Preferably, described in step 1) nutrient media components consumption be: MS minimal medium+sucrose 20000-30000mg/L+ agar powder 5000-7000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being formed by 6-benzyl aminopurine 0.5-2mg/L+ picloram 0.5-2mg/L.
Preferably, the nutrient media components content described in step 1) is: MS minimal medium+sucrose 30000mg/L+ agar powder 5000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being made up of 6-benzyl aminopurine 0.5mg/L+ picloram 0.5mg/L.
A kind of medium for root induction protocorm Fast-propagation state orchid of the present invention, its component is: MS minimal medium+sucrose+agar powder+caseinhydrolysate, and the plant growth regulator being made up of 6-benzyl aminopurine+picloram.
Preferably, described nutrient media components content is: MS minimal medium+sucrose 20000-30000mg/L+ agar powder 5000-7000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being made up of 6-benzyl aminopurine 0.5-2mg/L+ picloram 0.5-2mg/L.
Wherein, described MS minimal medium formula is as shown in table 1 below, and mg/L represents the milligram number containing each composition in every liter of MS medium.
Table 1
The 6-benzyl aminopurine using in plant growth regulator of the present invention, English name is that 6-Benzylaminopurine(is abbreviated as 6-BA, below repeats no more), chemical formula is C
12h
11n
5, belong to broad spectrum activity plant growth regulator, can promote plant cell growth, suppress the degraded of plant chlorophyll, improve amino acid whose content, Delaying Leaf-Senescence etc.Extensive use in Plant Tissue Breeding, can promote the differentiation of cell.
The picloram using in plant growth regulator of the present invention, chemical name is 4-amino-3,5,6-trichloropyridine-2-acid, English name is that Picloram(is abbreviated as PIC, below repeats no more), chemical formula is C
6h
3c
l3n
2o
2, 1963 is now DowElanco by DowChemical() and company's exploitation, E.R.Laning reports activity of weeding, sets it as weed killer herbicide and uses, and finds afterwards that it had plant growth regulator effect, as increased crop yield.Found in the last few years that it had the effect that good promotion somatic embryo occurs in Plant Tissue Breeding.The present invention is applied to PIC in the blue tissue cultivation of state first, generation taking root as explant induction protocorm, obtain induction cultivation effect rapidly and efficiently, not only numerous soon to state orchid, breeding, biotechnology, transgenic technology, the preservation of elite germplasm etc. has meaning, is also the orchid family, and the plant of other well developed root system carries out correlative study foundation is provided.
The caseinhydrolysate that medium of the present invention uses is taking crude milk albumen as raw material, through hydrochloric acid hydrolysis, decolouring, desalination, the dry product forming of spraying, contains 18 kinds of free amino acids, and nitrogenous and protein content is all greater than 80%.Amino acid is the basis of synthetic protein, is one of the most basic nutrient component of people, animals and plants, microorganism.Caseinhydrolysate has the effect of long-acting benefit nitrogen to plant growth, can Ao close trace element, promotes plant absorption, and in Plant Tissue Breeding, application is mainly that inductor cell stage occurs.
The present invention contains plant growth adjustment (6-BA+PIC) and caseinhydrolysate MS medium after improveing by employing carries out the induction of protocorm, for the blue root explant of state, with the specific cultural method of the present invention, make the blue root induction protocorm of state, inductivity reaches more than 75%, and it is more than 15 cultivating root explant protocorm number after 45 days, and protocorm survival rate is more than 80%, plant strain growth stalwartness, directly takes root.
The present invention adopts dark condition of culture in the time that induction protocorm produces, in tissue is cultivated, dark cultivation is conducive to the generation of inductor cell stage, it is exactly that somatic embryo occurs that the protocorm of orchid produces, and with respect to illumination cultivation, under dark condition of culture, is more conducive to produce protocorm.
With respect to prior art, the present invention has the following advantages:
1. utilize root induction protocorm breeding state orchid, root obtains easily as explant, and induction starts early, and within 15 days, root expands, and within 30 days, produces protocorm.
2. adopt technical scheme of the present invention to carry out root induction, induction frequency is high, and every section of long root of 1.5cm can produce more than 15 protocorm, and inductivity reaches more than 75%.
3. induction only needs two step incubation with seedling, inducing culture is simple, the induction of first step protocorm is cultivated and is only added plant growth regulator (6-BA+PIC) and caseinhydrolysate, do not need to add the additive such as banana puree, active carbon, reduce expense, this step only needs dark culturing, saves again the energy; Second step is illumination cultivation on the medium that does not add 6-benzyl aminopurine+picloram+caseinhydrolysate, directly takes root, and obtains complete regenerated plant, and cost is low.
4. because plant growth regulating substance consumption is low, in Induction Process, do not produce browning, accelerated cultivation process.
5. the regeneration plant obtaining comes from root, and its rootability is extremely strong, just can produce many sturdy roots, plant strain growth stalwartness without root-growing agent.Plant in cave, greenhouse dish, 100% survives.
6. incubation is simple, has reduced opportunities for contamination.
7. acquisition protocorm that can be a large amount of, quick, lasting has been realized the circulation of protocorm and has been bred under aseptic condition after this Establishing.
8. incubation step is simple, and inductivity is high, and cost is low, and plantation survival rate is high, is beneficial to batch production and produces.
Brief description of the drawings
Fig. 1 is that swordleaf cymbidium root tip of a root on inducing culture of the embodiment of the present invention 1 expands.
Fig. 2 is that the embodiment of the present invention 1 is cultivated 15 days each explants and started to expand.
Fig. 3 is that the embodiment of the present invention 1 produces protocorm on inducing culture.
Fig. 4 is that the embodiment of the present invention 1 protocorm grows up to regeneration plant under light.
Fig. 5 is the whole plant of the embodiment of the present invention 1 long root.
Fig. 6 is the plant of the embodiment of the present invention 2 upgrowth situation on the medium containing.
Embodiment
Below in conjunction with specific embodiments and the drawings, technical scheme of the present invention is described in further detail, but described embodiment does not limit the scope of the invention.
Embodiment 1
1) preparation medium, that is: MS minimal medium+sucrose 30000mg/L+ agar powder 5000mg/L+ caseinhydrolysate 500mg/L+ plant growth regulator (6-BA0.5mg/L+PIC0.5mg/L).After preparation, adjust pH=5.4-5.6,121 ± 2 DEG C, 15-20 pound sterilizing 20 minutes.
2) choose the root of sword-leaved cymbidium aseptic seedling, be inoculated in the medium of step 1) preparation and secretly cultivate, 25 ± 2 DEG C of temperature, 15 days roots expand (seeing Fig. 1, Fig. 2), about 1 month, produce protocorm (see figure 3), place it under light and cultivate, 25 ± 2 DEG C of temperature, illumination every day 16 hours, it is green that protocorm turns gradually.
3) (the long root of average every section of 1.5cm produces 30 protocorms to cut the protocorm that turns green, inductivity 100%, protocorm survival rate 95%) turn and be inoculated in step 1) in the medium containing plant growth regulator (6-BA+PIC) and caseinhydrolysate, grow up to the normal plant (seeing Fig. 4, Fig. 5) of taking root through cultivation in 1 month.
4) open bottle cap, hardening, after one week, washes away agar, plants in the dish of cave, greenhouse robust growth, survival rate 100%.
Embodiment 2
1) preparation medium, that is: MS minimal medium+sucrose 30000mg/L+ agar powder 5000mg/L+ caseinhydrolysate 500mg/L+ plant growth regulator (6-BA2mg/L+PIC2mg/L).After preparation, adjust pH=5.4-5.6,121 ± 2 DEG C, 15-20 pound sterilizing 20 minutes.
2) choose the root of sword-leaved cymbidium aseptic seedling, be inoculated in the medium of step 1) preparation and secretly cultivate, 25 ± 2 DEG C of temperature, within 15 days, root expands, and about 1 month, produces protocorm, places it under light and cultivates, 25 ± 2 DEG C of temperature, illumination every day 16 hours, protocorm turns green (see figure 6) gradually.
3) (the long root of average every section of 1.5cm produces 15 protocorms to cut the protocorm that turns green, inductivity 75%, protocorm survival rate 80%) turn and be inoculated in step 1) in the medium containing plant growth regulator (6-BA+PIC) and caseinhydrolysate, grow up to the normal plant of taking root through cultivation in 1 month.
4) open bottle cap, hardening, after one week, washes away agar, plants in the dish of cave, greenhouse robust growth, survival rate 100%.
Finally should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
Claims (5)
1. a method of utilizing root induction protocorm Fast-propagation state orchid, comprises the steps,
1) choose the root of the blue aseptic seedling of state, be inoculated in medium, secretly cultivate, obtain protocorm, culturing room's temperature is 25 ± 2 DEG C; Described nutrient media components is: MS minimal medium+sucrose+agar powder+caseinhydrolysate, and the plant growth regulator being made up of 6-benzyl aminopurine+picloram;
2) protocorm of secretly cultivating acquisition is forwarded under light and cultivated, illumination 10-16 hour every day, temperature is 25 ± 2 DEG C, it is green that protocorm turns gradually;
3) be transferred to by turing green protocorm the medium that is placed in blake bottle, develop into gradually normal plant, take root simultaneously; Described medium is the medium that does not contain 6-benzyl aminopurine+picloram+caseinhydrolysate in step 1);
4) room temperature, opens bottle cap hardening, plants after one week in the dish of cave, greenhouse.
2. method according to claim 1, it is characterized in that, nutrient media components content described in step 1) is: MS minimal medium+sucrose 20000-30000mg/L+ agar powder 5000-7000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being made up of 6-benzyl aminopurine 0.5-2mg/L+ picloram 0.5-2mg/L.
3. method according to claim 1, it is characterized in that, nutrient media components content described in step 1) is: MS minimal medium+sucrose 30000mg/L+ agar powder 5000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being made up of 6-benzyl aminopurine 0.5mg/L+ picloram 0.5mg/L.
4. for a medium for root induction protocorm Fast-propagation state orchid, its component is: MS minimal medium+sucrose+agar powder+caseinhydrolysate, and the plant growth regulator being made up of 6-benzyl aminopurine+picloram.
5. medium according to claim 4, it is characterized in that, described nutrient media components content is: MS minimal medium+sucrose 20000-30000mg/L+ agar powder 5000-7000mg/L+ caseinhydrolysate 500mg/L, and the plant growth regulator being made up of 6-benzyl aminopurine 0.5-2mg/L+ picloram 0.5-2mg/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115281094A (en) * | 2022-09-15 | 2022-11-04 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for improving proliferation coefficient of wild Chinese orchid protocorm |
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| CN101889550A (en) * | 2010-08-04 | 2010-11-24 | 扬州大学 | Culture Medium and Culture Method of Chinese Cymbidium Tissue Culture |
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| CN102754599A (en) * | 2012-07-16 | 2012-10-31 | 上海市农业科学院 | Method for quickly breeding cymbidium hybridium by use of root inducing protocorm |
| CN103004603A (en) * | 2012-12-27 | 2013-04-03 | 福建农林大学 | Plant regeneration method for butterfly orchid |
| CN103329807A (en) * | 2013-07-16 | 2013-10-02 | 上海市农业科学院 | Method for rapidly breeding hybrid orchid by root, as well as culture medium |
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Patent Citations (5)
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| CN101983554A (en) * | 2010-03-19 | 2011-03-09 | 西南林学院 | Reproduction method of Chinese cymbidium seedlings |
| CN101889550A (en) * | 2010-08-04 | 2010-11-24 | 扬州大学 | Culture Medium and Culture Method of Chinese Cymbidium Tissue Culture |
| CN102754599A (en) * | 2012-07-16 | 2012-10-31 | 上海市农业科学院 | Method for quickly breeding cymbidium hybridium by use of root inducing protocorm |
| CN103004603A (en) * | 2012-12-27 | 2013-04-03 | 福建农林大学 | Plant regeneration method for butterfly orchid |
| CN103329807A (en) * | 2013-07-16 | 2013-10-02 | 上海市农业科学院 | Method for rapidly breeding hybrid orchid by root, as well as culture medium |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115281094A (en) * | 2022-09-15 | 2022-11-04 | 烟台市森林资源监测保护服务中心(烟台沿海防护林省级自然保护区管理服务中心、烟台市林业科学研究所) | Method for improving proliferation coefficient of wild Chinese orchid protocorm |
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Application publication date: 20140611 |