CN101773069B - Tissue culture rapid propagation method of Guangdong anoectochilus roxburghii - Google Patents
Tissue culture rapid propagation method of Guangdong anoectochilus roxburghii Download PDFInfo
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- CN101773069B CN101773069B CN 200910214353 CN200910214353A CN101773069B CN 101773069 B CN101773069 B CN 101773069B CN 200910214353 CN200910214353 CN 200910214353 CN 200910214353 A CN200910214353 A CN 200910214353A CN 101773069 B CN101773069 B CN 101773069B
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Abstract
The invention discloses a tissue culture rapid propagation method of Guangdong anoectochilus roxburghii, which selects an axillary bud or a terminal bud of anoectochilus roxburghii in the Guangdong area in China as an explant and obtains a mass of vegetative propagated progeny plants by inducing the adventitious buds, carrying out proliferation and carrying out rooting cultivation. By adopting the method of the invention, one axillary bud can be induced to generate about 2 to 4 sprouts, after two months, the proliferation coefficient is between 3 and 4, and the rooting rate can reach over 90 percent. Therefore, the method greatly improves the seedling production capacity of Guangdong anoectochilus roxburghii, shortens the seedling culture time, saves the planting cost and provides the technical guarantee for the storage of the genetic resources of Guangdong anoectochilus roxburghii.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of tissue culture and rapid propagation method of Guangdong anoectochilus roxburghii.
Background technology
Roxburgh anoectochilus terminal bud (Anoectochitus formosanus) is the traditional rare medicinal herbs of China, belongs to the orchid family and opens lip Cymbidium herbaceos perennial, calls golden silkworm, Shorthairy Antenoron, metal and stone pine, gold thread taiwan anetochilus herb, gold thread and disappears to the marrow etc.Roxburgh anoectochilus terminal bud also can be used as the high ornamental plants of ornamental value.Roxburgh anoectochilus terminal bud rich vitamins and polysaccharide, its micronutrient levels, amino acid content are high, have antibiotic, anticancer, cardiac stimulant, pain relieving, effect such as hypoglycemic.Because the good effects of roxburgh anoectochilus terminal bud aspect treatment hepatopathy, hypertension, diabetes and tumour causes the attention of medical circle day by day.
But the roxburgh anoectochilus terminal bud seed is small, and germination rate is low, and its growth is strict to ecotope, natural propagation and poor growth.Along with the ecological resources environment damage, wild roxburgh anoectochilus terminal bud growing environment has received destruction in recent years, and wild roxburgh anoectochilus terminal bud resource is very rare, endangered at present.At present, the propagation method of roxburgh anoectochilus terminal bud has seminal propagation, cottage propagation and tissue culture propagation.But the embryo prematurity of the seed of roxburgh anoectochilus terminal bud is difficult for germinateing; Adopt the reproduction coefficient of cuttage low, be difficult to form the plantation of commercialization scale.
If can set up the tissue culture rapid propagation technique of a cover roxburgh anoectochilus terminal bud seedling, will and rationally utilize the Guangdong anoectochilus roxburghii resource to make significant contribution to protection, and the planting technology of make greater efforts to promote roxburgh anoectochilus terminal bud commercialization, scale promoted.
Summary of the invention
The objective of the invention is to overcome the especially deficiency of roxburgh anoectochilus terminal bud propagation technique of existing roxburgh anoectochilus terminal bud, set up a kind of tissue culture and rapid propagation method of Guangdong anoectochilus roxburghii, the reproduction coefficient that improves Guangdong anoectochilus roxburghii makes seedling production can satisfy the production needs.
The object of the invention specifically is to produce indefinite bud through the terminal bud of hormone induction Guangdong anoectochilus roxburghii or axillalry bud to breed the commercialization scale plantation that realizes roxburgh anoectochilus terminal bud fast.
The concrete technical scheme of the present invention is:
A kind of tissue culture and rapid propagation method of Guangdong anoectochilus roxburghii is provided, comprises that sterilization, adventitious bud inducing, the culture of rootage of roxburgh anoectochilus terminal bud explant obtains tissue cultivating seedling and tissue cultivating seedling transplant step.
The axillary-bud or top-bud of the preferred wild Guangdong anoectochilus roxburghii of the present invention is as explant material.
The sterilization of said explant is that to adopt mass volume ratio be that 0.1% mercuric chloride solution (0.1g mercuric chloride/100ml water) carries out surface sterilization, and disinfecting time is 10min, sterile water wash three times, and each scavenging period is 2min.
Abductive approach is: explant is cut to the segment that has a bud, is seeded on the inducing culture.
The medium of said adventitious bud inducing is: MS+1.0mg/l 6-BA+0.1mg/l IBA, pH value are 5.8.
Said root media is: MS+0.5mg/l NAA+0.3mg/l IBA, pH value are 5.8.
The illumination condition of culture of rootage preferably adopts artificial lighting, luminous intensity 2000Lx, and cultivation temperature is 25 ℃.
Transplant after emerging.The present invention preferably cultivated in root media after 1 month, and refining seedling 7 days is transplanted to artificial greenhouse, transplanted and used matrix to be peat soil and the garden mould mixture with 3: 1 volume ratio mixing.
Method is particularly: the axillary-bud or top-bud of choosing wild Guangdong anoectochilus roxburghii is an explant, 0.1% mercuric chloride solution sterilization 10min, sterile water wash three times; Each 2min is inoculated into MS+1.0mg/l 6-BA+0.1mg/l IBA, pH value and is in 5.8 the inducing culture on superclean bench, carry out artificial lighting and cultivate; Luminous intensity 2000Lx, cultivation temperature is 25 ℃, treats that adventitious bud inducing is forwarded to same medium propagation after coming out; Subculture 5～10 times; Root media is MS+0.5mg/lNAA+0.3mg/l IBA, and PH is 5.8, the same adventitious bud inducing of condition of culture.Culture of rootage 1 month, refining seedling after 7 days is transplanted to artificial greenhouse, and matrix is peat soil and the volume ratio mixing of garden mould with 3: 1.
With existing Guangdong anoectochilus roxburghii sapling multiplication compared with techniques, beneficial effect of the present invention is:
Adopt tissue culture and rapid propagation method breeding Guangdong anoectochilus roxburghii of the present invention can reach 7～8, greatly improved the seedling quantity of Guangdong anoectochilus roxburghii, be the technical guarantee that provides of factorial seedling growth and Guangdong anoectochilus roxburghii industrialized development at 3 months internal breeding coefficients.Can obtain parent's hereditary information fully through vegetative seedling, maternal fine quality is able to genetic stability.Seedling has the advantage that seedling is strong, growth is consistent, is convenient to unified management, can save cost greatly.
The present invention has set up the tissue culture quick propagation culturing method of the simple Guangdong anoectochilus roxburghii of a cover; Value-added coefficient improves than existing cottage propagation and division propagation greatly; And can the anniversary produce seedling; For the implant mass of roxburgh anoectochilus terminal bud provides abundant seedling resource, effectively solve the technical barrier and the technical bottleneck that limit the roxburgh anoectochilus terminal bud industry development at present because of the seedling wretched insufficiency.
Adopt the inventive method to carry out manual work and keep, technologic material and technical guarantee are provided for carrying out correlative study from now on to the wild germplasm resource of Guangdong anoectochilus roxburghii.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to set forth in further detail.
Embodiment 1
The material that adopts is wild Guangdong anoectochilus roxburghii, takes from Guangdong Nanning-Kunming mountain.
Breed Guangdong anoectochilus roxburghii according to following steps:
1, gets wild Guangdong anoectochilus roxburghii plant, be cut into every section segment that contains an axillalry bud.
2, the stem section being put into mass volume ratio is the 0.1% mercuric chloride solution 10min that sterilizes, sterile water wash three times, each 2min.
3, the stem section after will sterilizing on the superclean bench is inoculated into inducing culture, and inducing culture consists of MS+1.0mg/l 6-BA+0.1mg/l IBA, and pH value is 5.8.Artificial lighting, intensity 2000Lx, cultivation temperature is 25 ℃.
4,1 axillalry bud can be induced and produced about 2～4 sprouting.Treat that adventitious bud inducing is forwarded to same medium (consist of MS+1.0mg/l 6-BA+0.1mg/l IBA, pH value is 5.8) subculture with bud after having 2～4, propagation.Growth coefficient is 3～4 after 2 months.
5, get the indefinite bud that is about about 1cm, individual plant is received root media and is cultivated, and root media is MS+0.5mg/l NAA+0.3mg/l IBA, and pH value is 5.8; The same adventitious bud inducing of condition of culture (cultivation temperature is 25 ℃ for artificial lighting, intensity 2000Lx).Culture of rootage is the time about 1 month, and rooting rate can reach more than 90%.
6, refining seedling after 7 days transplants tissue cultivating seedling to artificial greenhouse, and matrix is peat soil and garden mould with 3: 1 volume ratio mixture.
7, carry out daily management after the transplanting.Water every day 3 times, water to matrix drenched till, every first quarter moon sealing fertilizer 1 time, and attention control damage by disease and insect.Said composite fertilizer adopts fertilizer special for vegetable material (nitrogen 15%, phosphorus pentoxide 7% and potassium oxide 8%).
Through the inducing of indefinite bud, growth coefficient can reach 7～8 to the Guangdong anoectochilus roxburghii that obtains of breeding, has greatly improved the seedling quantity of Guangdong anoectochilus roxburghii in 3 months.
Embodiment 2
The material that adopts is wild Guangdong anoectochilus roxburghii, takes from Guangdong Nanning-Kunming mountain.
Breed Guangdong anoectochilus roxburghii according to following steps:
1, gets wild Guangdong anoectochilus roxburghii plant, downcut the segment that contains a terminal bud.
2, the stem section being put into mass and size is 0.1% mercuric chloride solution sterilization 10min, sterile water wash three times, each 2min.
3, the stem section after will sterilizing on the superclean bench is inoculated into inducing culture, and inducing culture consists of MS+1.0mg/l 6-BA+0.1mg/l IBA, and pH value is 5.8.Artificial lighting, intensity 2000Lx, cultivation temperature is 25 ℃.
4,1 axillalry bud can be induced and produced about 2～4 sprouting.Treat that adventitious bud inducing is forwarded to same medium (consist of MS+1.0mg/l 6-BA+0.1mg/l IBA, pH value is 5.8) subculture with bud after coming out, propagation.Growth coefficient is 3～4 after 2 months.
5, get the indefinite bud that is about about 1cm, individual plant is received root media and is cultivated, and root media is MS+0.5mg/l NAA+0.3mg/l IBA, and pH value is 5.8; The same adventitious bud inducing of condition of culture (cultivation temperature is 25 ℃ for artificial lighting, intensity 2000Lx).Culture of rootage is the time about 1 month, and rooting rate can reach more than 90%.
6, refining seedling after 7 days transplants tissue cultivating seedling to artificial greenhouse, and matrix is peat soil and garden mould with 3: 1 volume ratio mixture.
7, carry out daily management after the transplanting.Water every day 3 times, water to matrix drenched till, every first quarter moon sealing fertilizer 1 time, and attention control damage by disease and insect.Said composite fertilizer adopts fertilizer special for vegetable material (nitrogen 15%, phosphorus pentoxide 7% and potassium oxide 8%).
1 axillalry bud of the inventive method can be induced and produced about 2～4 sprouting; Growth coefficient is 3～4 after 2 months; Rooting rate can reach more than 90%, has improved the production capacity of growing seedlings of Guangdong anoectochilus roxburghii greatly, shortens seedling raise period; Save planting cost, for the preservation of Guangdong anoectochilus roxburghii genetic resources provides technical guarantee.
Claims (2)
1. the method for the tissue-culturing rapid propagation of a Guangdong anoectochilus roxburghii is characterized in that it being that terminal bud or axillalry bud through the hormone induction Guangdong anoectochilus roxburghii produces indefinite bud and breed fast; May further comprise the steps:
(1) axillary-bud or top-bud of getting wild Guangdong anoectochilus roxburghii is as explant material, and explant carries out disinfection;
(2) adventitious bud inducing;
(3) treat that adventitious bud inducing is forwarded to proliferated culture medium, subculture 5～10 times after coming out;
(4) culture of rootage obtains tissue cultivating seedling;
(5) tissue cultivating seedling is transplanted;
Wherein, the said adventitious bud inducing of step (2) is the explant after the sterilization to be inoculated into inducing culture cultivate; It is MS+1.0mg/l 6-BA+0.1mg/lIBA that inducing culture is formed, and the pH value is 5.8; The adventitious bud inducing condition is that intensity is the artificial lighting of 2000Lx, and cultivation temperature is 25 ℃;
The composition of the said proliferated culture medium of step (3) is MS+1.0mg/l 6-BA+0.1mg/lIBA, and the pH value is 5.8;
The root media of the said culture of rootage of step (4) is MS+0.5mg/1NAA+0.3mg/lIBA, and the pH value is 5.8; The illumination condition of culture of rootage is the employing artificial lighting, luminous intensity 2000Lx, and cultivation temperature is 25 ℃;
The said transplanting of step (5) is that tissue cultivating seedling was cultivated in root media after 1 month, refining seedling 7 days, and tissue cultivating seedling is long transplants to artificial greenhouse when high to 2cm, transplants and uses matrix to be peat soil and the garden mould mixture with 3: 1 volume ratio mixing.
2. according to the method for the tissue-culturing rapid propagation of the said Guangdong anoectochilus roxburghii of claim 1, it is characterized in that step (1) sterilization is that to adopt mass volume ratio be that 0.1% mercuric chloride solution carries out explant surface sterilization 10min.
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| CN 200910214353 CN101773069B (en) | 2009-12-29 | 2009-12-29 | Tissue culture rapid propagation method of Guangdong anoectochilus roxburghii |
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| CN 200910214353 CN101773069B (en) | 2009-12-29 | 2009-12-29 | Tissue culture rapid propagation method of Guangdong anoectochilus roxburghii |
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| CN101773069B true CN101773069B (en) | 2012-12-26 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102100171B (en) * | 2010-12-30 | 2012-02-22 | 福建永安天奇健金线莲生态实业有限公司 | Aqueous culture method of anoectochilus |
| CN102499088B (en) * | 2011-11-03 | 2013-04-10 | 广西壮族自治区中国科学院广西植物研究所 | Method for quickly breeding seedlings of Guangxi anoectochilus roxburghii capsules by utilizing Guangxi anoectochilus roxburghii capsules |
| CN103688854B (en) * | 2013-12-06 | 2016-03-30 | 四川省自然资源科学研究院 | The tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud |
| CN105123512B (en) * | 2015-07-15 | 2017-11-14 | 河源市裕森农林发展有限公司 | A kind of breeding method of roxburgh anoectochilus terminal bud |
| CN105557514B (en) * | 2015-12-14 | 2018-11-13 | 李操 | A kind of Cryopreservation of bud germ plasm resource |
| CN105766647B (en) * | 2016-04-05 | 2018-01-12 | 福建农林大学 | A kind of hectolitre pine method for tissue culture |
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2009
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Non-Patent Citations (3)
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| 吴坤林.金线莲快繁及工厂化生产中间试验.《中药材》.1997,(第12期),第595-597页. * |
| 段玉云等.台湾金线莲的组织培养与快速繁殖.《植物生理学通讯》.2005,第41卷(第2期),第198页. * |
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