CN1194605C - Maka seedling reproducing method - Google Patents
Maka seedling reproducing method Download PDFInfo
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- CN1194605C CN1194605C CNB031282636A CN03128263A CN1194605C CN 1194605 C CN1194605 C CN 1194605C CN B031282636 A CNB031282636 A CN B031282636A CN 03128263 A CN03128263 A CN 03128263A CN 1194605 C CN1194605 C CN 1194605C
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- agate coffee
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- seedling
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- explant
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Abstract
The present invention relates to a maca seedling propagating method which belongs to a biological engineering and biological technique for plants. The propagating method is used for realizing the fast propagation of maca seedlings. The propagating method comprises the steps: (1) explant obtaining, (2) callus inducing, (3) the differentiation of indefinite buds, (4) rooting culturing, (5) rooting after-treatment and transplantation after seedling refining. The propagating method adopts an MS culture medium or an improved MS culture medium, and the proportion of 6-benzoylaminopurine (6BA) and naphthalene acetic acid (NAA) with different concentrations is used in different culture stages. The present invention has the advantages of short time required for seedling breeding, good quality, high seedling-producing rate and large seedling-producing amount, and can provide high-quality test-tube seedlings for large-scale planting of maca which is an important economic crop.
Description
Technical field
The invention belongs to plant biological engineering and biotechnology, particularly the plant seedling propagation method.
Background technology
Agate coffee (Maca, Lepidium meyenii Walp) is Cruciferae (Cruciferae) separate row Vegetable spp (Lepidium) plant.Common first names Maca, have another name called Peruvian ginseng, maka, maca-maca, maino, ayak chinchira, ayak willku etc., originate in the mountain area, Andean, South America of 3500～4500 meters of height above sea level, mainly be distributed in Puna ecotope and Peru the southeastern city of Puno at Peru middle part now.The agate coffee is a kind of medicine food dual purpose plant, and the edible history in South America has had more than 5800 year, is used for strengthening body traditionally, improves fertility, improves sexual function, antidepression, anti-anaemia etc.Existing researchs such as the chemical composition evaluation of agate coffee, active component.Current agate coffee improve fertility, improve sexual function, effects such as anticancer, anti-leukocythemia, treatment climacteric metancholia become the research focus.As the medicinal crucifer of uniqueness, the agate coffee is in also not plantation of China, and its research is also at the early-stage.At present, be not reported both at home and abroad, do not have the patent documentation of this respect yet about the quick breeding of agate coffee tissue culture.Therefore, explore agate coffee method for quickly breeding, be a problem that urgency is to be solved with suiting measures to local conditions.
Summary of the invention
The invention provides agate coffee sapling multiplication method, be used to realize the fast breeding of agate coffee, reach indoor large-scale breeding kind bacterium, for large-scale development agate coffee provides high-quality kind bacterium in enormous quantities, to advance of the application of agate coffee in China.
A kind of agate coffee sapling multiplication method of the present invention, comprise the steps: (1) root in regular turn with agate coffee live plant, the tip of a root, stem, stem apex or blade are as explant, (2) explant induction is cultivated the formation callus, medium is MS or its improved culture medium, wherein be added with agar or carragheen, (3) well-grown callus is carried out the bud differentiation culture, differentiation and bud formation, its medium is MS or its improved culture medium, (4) will have the bud differentiated tissues and carry out culture of rootage, the plantlet that formation is taken root, its medium is 1/2MS or its improved culture medium, (5) described inducing culture and bud differentiation culture are at intensity of illumination 800-1600LX, at light application time 10-16 hour every day, carry out under the temperature 18-26 degree Celsius condition.
Described agate coffee sapling multiplication method, it is further characterized in that: be added with the agar of percentage by weight 0.7-1.0% or the carragheen of 0.8-1.0% in (1) described inducing culture, the 6-benzylaminopurine (6BA) of additional 0.1～1.0mg/L and the methyl (NAA) of 0.1～1.0mg/L, (2) 6 benzylaminopurines (6BA) of additional 1-4mg/L and the methyl (NAA) of 0.05～0.8mg/L in the described bud differential medium, the methyl (NAA) of additional 0.05～0.5mg/L in (3) described root media.
Described agate coffee sapling multiplication method, described explant can derive from the live plant of the open environment that carries disease germs, the sterile-processed explant that is re-used as, detoxicating handle be meant clean repeatedly with running water after, in super-clean environment, handle with the Ethanol Treatment of concentration 70% or at the hydrogen peroxide of 5-10% concentration earlier, after washing repeatedly with sterile water again, adopt the liquor natrii hypochloritis of 5-10% or the mercuric chloride solution of 0.1-1% to handle, wash repeatedly only with sterile water at last.
Described agate coffee sapling multiplication method, Ethanol Treatment time 1-30 second, in 1-5 minute hydrogen peroxide processing time, liquor natrii hypochloritis or mercuric chloride solution processing time are 4-10 minute.
Described agate coffee sapling multiplication method, described explant also can derive from the live body aseptic seedling, and agate coffee seed is clean, the immersion preliminary treatment of described live body aseptic seedling system is disinfected then, inoculates cultivation, sprouts to form aseptic seedling.
Described disinfect be meant clean repeatedly with running water after, in super-clean environment, handle with the Ethanol Treatment of concentration 70% or at the hydrogen peroxide of 5-10% concentration earlier, after washing repeatedly with sterile water again, adopt the liquor natrii hypochloritis of 5-10% or the mercuric chloride solution of 0.1-1% to handle, rinse well repeatedly with sterile water at last.
The red toxin of 0-10mg/L is adopted in described immersion preliminary treatment, and inoculated and cultured is MS or its improved culture medium, directly adds inert solid particle in the medium in the carragheen of the agar of adding 0.7-1.0% or 0.8-1.0% or the liquid medium within.
The method for quickly breeding of agate coffee tissue culture provided by the invention, can not be subjected to the influence in season, increase substantially the plant propagation coefficient, obtain a large amount of high-quality agate coffee seedlings at short notice, shortened the time of field sapling multiplication greatly, distinguishing features such as the while has been saved the required place of sapling multiplication greatly, has the efficient height, and cost is low.
Agate coffee test-tube plantlet provided by the invention is effectively sloughed the infection of disease such as fungi and bacterium in the agate coffee seedling process of growth, can also adopt the stem apex and the tip of a root to cultivate the test-tube plantlet that obtains virus removal, and the test-tube plantlet breediness is good.
Embodiment
Embodiment 1
Choose the blade of the good live plant of growing way, after cleaning repeatedly with running water, in super-clean environment, selected 70% Ethanol Treatment for use 20 seconds or handled 3 minutes, after sterile water washes repeatedly, adopt 8% hypochlorite solutions to carry out 10 minutes disinfecting at 5% hydrogen peroxide.After washing repeatedly with sterile water again, cut into the segment of 1cm, be used for inoculated and cultured as explant, carry out the differentiation and the culture of rootage of callus induction and indefinite bud, condition of culture is intensity of illumination 1500LX, and every day, light application time was 16 hours, and cultivation temperature is at 22 ± 2 degree.At first carry out callus induction: (keep trace element, organic element in the MS medium to remain unchanged, the content of macroelement is KNO to adopt the MS improved culture medium
31250mg/L, NH
4NO
31650mg/L, KH
2PO
4370mg/L, MgSO
47H
2O 370mg/L, CaCl
2440mg/L), add 0.7% agar, 6BA concentration is 0.5mg/L, and NAA concentration is 0.5mg/L, but through the generation of 25 days cultivation evoked callus.Carry out the differentiation of indefinite bud then: adopt above-mentioned MS improved culture medium, 6BA concentration is 4mg/L, and NAA concentration is 0.5mg/L, through forming indefinite bud by callus after 30 days the cultivation.Carry out culture of rootage again: adopt 1/2MS improved culture medium (macroelement reduces by half in the MS improved culture medium, and other elements remain unchanged), the concentration of NAA is 0.5mg/L, takes root through 20 days cultivation, so far develops into complete plant.Can body weight be newly carried out callus induction, breaking up, taking root forms new plant again as planting with this plant.
Embodiment 2
Choose the stem section of the good live plant of growing way, after cleaning repeatedly with running water, in super-clean environment, handled 5 minutes with 70% Ethanol Treatment 10 seconds or at 5% hydrogen peroxide earlier, after sterile water washes repeatedly, adopt 10% liquor natrii hypochloritis to carry out 8 minutes disinfecting.After washing repeatedly with sterile water again, cut into the segment of 1cm, be used for inoculated and cultured as explant, carry out the differentiation and the culture of rootage of callus induction and indefinite bud, condition of culture is that intensity of illumination is 1500LX, and every day, light application time was 16 hours, and cultivation temperature is at 22 ± 2 degree.At first carry out callus induction: (keep trace element, organic element in the MS medium to remain unchanged, the content of macroelement is KNO to adopt the MS improved culture medium
32500mg/L, NH
4NO
3150mg/L, KH
2PO
4150mg/L, MgSO
47H
2O 200mg/L, CaCl
2110mg/L), add 1.0% agar, 6BA concentration is 0.5mg/L, and NAA concentration is 0.5mg/L, but through 25 days the just generation of evoked callus of cultivation.Carry out the differentiation of indefinite bud then: adopt above-mentioned MS improved culture medium, and to add 6BA concentration be 3.5mg/L, NAA concentration is 0.4mg/L, through producing indefinite bud by the callus differentiation after 30 days the cultivation.Carry out culture of rootage again: adopt 1/2MS improved culture medium (macroelement reduces by half in the MS improved culture medium, and other elements remain unchanged), the concentration of NAA is 0.45mg/L, through taking root after 20 days the cultivation, so far develops into complete plant.Can with this plant as explant carry out callus induction again, breaking up, taking root forms new plant again.
Embodiment 3
The full agate coffee seed of selecting is cleaned with running water, used the gibberellin (GA of 10mg/L again
3) immersion treatment, sterilization then, sterilization method is with embodiment 2, seed after the sterilization is inserted the MS medium, do not contain exogenous hormone, add and directly add inert solid particle in 0.9% agar or 1.0% carragheen or the liquid medium within, as bead, perlite, vermiculite.After 3 weeks, treat that seedling grows to the 4-5cm height, obtains aseptic seedling.Choose the aseptic seedling that grows fine, the clip blade is as explant, and the segment that cuts into 1cm carries out the differentiation and the culture of rootage of the inducing of callus, indefinite bud, and condition of culture is that intensity of illumination is 1500LX, every day, light application time was 16 hours, and cultivation temperature is at 24 ± 2 degree.At first carry out callus induction: (keep trace element, organic element in the MS medium to remain unchanged, the content of macroelement is KNO to adopt the MS improved culture medium
31900mg/L, NH
4NO
31250mg/L, KH
2PO
4300mg/L, MgSO
47H
2O 250mg/L, CaCl
2220mg/L), add 1.0% carragheen, 6BA concentration is 0.5mg/L, and NAA concentration is 0.25mg/L, but through the generation of 25 days cultivation evoked callus.Carry out the differentiation of indefinite bud then: adopt above-mentioned MS improved culture medium, adding 6BA concentration is 3.0mg/L, and NAA concentration is 0.1mg/L, through the differentiation of 30 days cultivation indefinite buds.Carry out culture of rootage again: adopt 1/2MS solid culture medium (macroelement reduces by half in the above-mentioned MS improved culture medium, and other elements remain unchanged), the concentration of NAA is 0.35mg/L, through taking root after 20 days the cultivation, so far develops into complete plant.Can with this plant as explant carry out callus induction again, breaking up, taking root forms new plant again.
Embodiment 4
The full agate coffee seed of selecting is cleaned with running water, used the gibberellin (GA of 5mg/L again
3) soak preliminary treatment, sterilization then, sterilization method inserts the MS medium with embodiment 2 with the seed after the sterilization, does not contain exogenous hormone, directly adds solid particle in the agar of adding 0.8% or the liquid medium within, as bead, perlite, vermiculite.After 3 weeks, treat that seedling grows to the 4-5cm height, obtains aseptic seedling.Choose the aseptic seedling that grows fine, clip stem section is as explant, and the segment that cuts into 1cm carries out the differentiation and the culture of rootage of the inducing of callus, indefinite bud, and condition of culture is that intensity of illumination is 1500LX, every day, light application time was 16 hours, and cultivation temperature is at 24 ± 2 degree.At first carry out callus induction: (keep trace element, organic element in the MS medium to remain unchanged, the content of macroelement is KNO to adopt the MS improved culture medium
32000mg/L, NH
4NO
31250mg/L, KH
2PO
4170mg/L, MgSO
47H
2O 200mg/L, CaCl
2200mg/L), add 0.8% carragheen, adding 6BA concentration is 0.5mg/L, and NAA concentration is 0.3mg/L, but through the generation of 25 days cultivation evoked callus.Carry out the differentiation of indefinite bud then: adopt above-mentioned MS improved culture medium, and to add 6BA concentration be 2.0mg/L, NAA concentration is 0.05mg/L, through the differentiation of 30 days cultivation indefinite buds.Carry out culture of rootage again: adopt 1/2MS improved culture medium (macroelement reduces by half in the above-mentioned MS improved culture medium, and other elements remain unchanged), the concentration of NAA is 0.25mg/L, takes root through 20 days cultivation, so far develops into complete plant.This plant can be carried out inducing, break up, take root and forming new plant again of callus again as explant.
Claims (7)
1. an agate coffee sapling multiplication method comprises the steps: in regular turn
(1) with root, the tip of a root, stem, stem apex or the blade of agate coffee live plant as explant,
(2) explant induction is cultivated the formation callus, medium is MS or its improved culture medium, wherein is added with agar or carragheen,
(3) well-grown callus is carried out the bud differentiation culture, differentiation and bud formation, its medium are MS or its improved culture medium,
(4) will have the bud differentiated tissues and carry out culture of rootage, and form the plantlet of taking root, its medium is 1/2MS or its improved culture medium,
(5) described inducing culture and bud differentiation culture carried out under the temperature 18-26 degree Celsius condition intensity of illumination 800-1600LX, every day light application time 10-16 hour.
2. agate coffee sapling multiplication method as claimed in claim 1 is characterized in that:
(1) be added with the agar of percentage by weight 0.7-1.0% or the carragheen of 0.8-1.0% in the described inducing culture, the 6--base aminopurine (6BA) of additional 0.1～1.0mg/L and the methyl (NAA) of 0.1～1.0mg/L,
(2) 6 benzylaminopurines (6BA) of additional 1-4mg/L and the methyl (NAA) of 0.05～0.8mg/L in the described bud differential medium,
(3) methyl (NAA) of additional 0.05～0.5mg/L in the described root media.
3. agate coffee sapling multiplication method as claimed in claim 1 or 2, it is characterized in that described explant derives from the live plant of the open environment that carries disease germs, the sterile-processed explant that is re-used as, detoxicating handle be meant clean repeatedly with running water after, in super-clean environment, handle with the Ethanol Treatment of concentration 70% or at the hydrogen peroxide of 5-10% concentration earlier, after washing repeatedly with sterile water again, adopt the liquor natrii hypochloritis of 5-10% or the mercuric chloride solution of 0.1-1% to handle, wash repeatedly only with sterile water at last.
4. agate coffee sapling multiplication method as claimed in claim 3 is characterized in that Ethanol Treatment time 1-30 second, and in 1-5 minute hydrogen peroxide processing time, liquor natrii hypochloritis or mercuric chloride solution processing time are 4-10 minute.
5. agate coffee sapling multiplication method as claimed in claim 1 or 2 is characterized in that described explant derives from the live body aseptic seedling, and agate coffee seed is clean, the immersion preliminary treatment of described live body aseptic seedling system is disinfected then, inoculates cultivation, sprouts to form aseptic seedling.
6. agate coffee sapling multiplication method as claimed in claim 5, it is characterized in that described disinfect be meant clean repeatedly with running water after, in super-clean environment, handle with the Ethanol Treatment of concentration 70% or at the hydrogen peroxide of 5-10% concentration earlier, after washing repeatedly with sterile water again, adopt the liquor natrii hypochloritis of 5-10% or the mercuric chloride solution of 0.1-1% to handle, rinse well repeatedly with sterile water at last.
7. agate coffee sapling multiplication method as claimed in claim 6, the red toxin that it is characterized in that described immersion preliminary treatment employing 0-10mg/L, inoculated and cultured is MS or its improved culture medium, directly adds inert solid particle in the medium in the carragheen of the agar of adding 0.7-1.0% or 0.8-1.0% or the liquid medium within.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031282636A CN1194605C (en) | 2003-07-01 | 2003-07-01 | Maka seedling reproducing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031282636A CN1194605C (en) | 2003-07-01 | 2003-07-01 | Maka seedling reproducing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1473465A CN1473465A (en) | 2004-02-11 |
| CN1194605C true CN1194605C (en) | 2005-03-30 |
Family
ID=34153302
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031282636A Expired - Fee Related CN1194605C (en) | 2003-07-01 | 2003-07-01 | Maka seedling reproducing method |
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| Country | Link |
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| CN (1) | CN1194605C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455367B (en) * | 2008-12-31 | 2012-09-05 | 云南省农业科学院高山经济植物研究所 | Lepidium meyenii Walp. health products |
| CN102511396B (en) * | 2011-10-14 | 2013-07-17 | 新疆维吾尔自治区中药民族药研究所 | In vitro rapid breeding method of medicinal plant Lipidium meyenii |
| CN102812874B (en) * | 2012-08-08 | 2014-04-02 | 曹诗红 | Green control method of citrus diseases and insects |
| CN102884935A (en) * | 2012-11-02 | 2013-01-23 | 昆明滇然生物科技有限公司 | Plant maca growing method in lower altitude areas |
| CN104686370B (en) * | 2015-04-03 | 2017-04-26 | 会泽枢康中草药种植有限公司 | Method for cultivating maca polyploid plants |
| CN105191801A (en) * | 2015-10-24 | 2015-12-30 | 大理白族自治州农业科学推广研究院药用植物及农业新技术研究所 | Healthcare medicinal material Maca tissue cultured seedling vitrification control method |
| CN105494102A (en) * | 2016-01-12 | 2016-04-20 | 丽水学院 | Method for culture of efficient maca virus-free tissue |
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2003
- 2003-07-01 CN CNB031282636A patent/CN1194605C/en not_active Expired - Fee Related
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| CN1473465A (en) | 2004-02-11 |
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