CN103688854B - The tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud - Google Patents
The tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud Download PDFInfo
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- CN103688854B CN103688854B CN201310655251.9A CN201310655251A CN103688854B CN 103688854 B CN103688854 B CN 103688854B CN 201310655251 A CN201310655251 A CN 201310655251A CN 103688854 B CN103688854 B CN 103688854B
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Abstract
The invention discloses one and can improve transplanting survival rate and growth coefficient, and the roxburgh anoectochilus terminal bud tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud merit and the property of medicine can be kept, belong to field of plant tissue culture technique.The method first chooses the wild high eyebrow roxburgh anoectochilus terminal bud of growth in 5 ~ October, make explant and disinfection, then by the measure that the induction of Multiple Buds, squamous subculture, Rooting and hardening-off culture and intermediate house booth are cultivated, roxburgh anoectochilus terminal bud Fast-propagation is made by hormone induction.The inventive method, can keep the merit of high eyebrow roxburgh anoectochilus terminal bud, keep the property of medicine that it is excellent, and method is simple, cost is low, can realize factorial seedling growth.Employing this method induces Multiple Buds and the ratio of taking root reaches more than 90%, and transplanting survival rate, more than 95%, can reach 8-10 doubly at 4 months internal breeding coefficients.By the high eyebrow roxburgh anoectochilus terminal bud that the inventive method is produced, do not make a variation, output is high, and cost is low, and the cycle is short, the market competitiveness that tool is stronger.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to one and can improve transplanting survival rate and growth coefficient, and the roxburgh anoectochilus terminal bud tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud merit and the property of medicine can be kept.
Background technology
High eyebrow roxburgh anoectochilus terminal bud (AnoectochilusemeiensisK.Y.Lang.) is the orchid family (Orchidaceae), Anoectochilus Blume (Anoectochilus) herbaceos perennial, and same Anoectochilus Roxburghii, anoectochilus formosanus, Anoectochilus roxburghii etc. are referred to as roxburgh anoectochilus terminal bud.Often do medicinal among the people, its amino acid composition, composition, content and vitamin, polysaccharide equal size are all higher than American Ginseng and wild ginseng.The flat taste of its property is sweet, has heat-clearing, cool blood, expelling wind and removing dampness, cardiac stimulant diuresis, reinforce the kidney flat liver and the effect such as hypotensive, in treatment hepatopathy, hypertension, diabetes and tumour etc., have good effect.In addition, roxburgh anoectochilus terminal bud is also the high ornamental plants of ornamental value.
But because the seed of roxburgh anoectochilus terminal bud platymiscium is small, do not have an endosperm, without germinating capacity, only have and could sprout with when mycosymbiosis, therefore, germination rate is low, field grown condition is harsh, natural propagation and poor growth.In addition due to high eyebrow roxburgh anoectochilus terminal bud habitat destroy serious, wild high eyebrow roxburgh anoectochilus terminal bud resource is very rare, now endangered.
Study the artificial culture technique of high eyebrow roxburgh anoectochilus terminal bud for this reason, set up the tissue culture quick breeding system of a set of seedling, to reasonable protective development high eyebrow roxburgh anoectochilus terminal bud resource and realize industrialization, scale has very important effect.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of high eyebrow roxburgh anoectochilus terminal bud tissue culture and rapid propagation method improving transplanting survival rate and growth coefficient, makes seeling industry can meet the needs of large-scale production.
The technical solution adopted for the present invention to solve the technical problems is: the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud, comprises the steps:
A, choose growth at the wild roxburgh anoectochilus terminal bud of high eyebrow in 5 ~ October, removing root and leaf, stem is cut into the section shape of band axillary-bud or top-bud as explant material, explant disinfection;
The induction of b, Multiple Buds: the explant after sterilization is inoculated into inducing culture and cultivates; Inducing culture is: MS medium+sucrose 25 ~ 35g/L+ agar 6 ~ 10g/L+ active carbon 1.5 ~ 2.5g/L+1 ~ 2mg/L6-BA+0.3 ~ 0.7mg/LNAA, and pH value is 5.6 ~ 6.0; Cultivation cycle is 30 ~ 40 days;
C, squamous subculture;
D, Rooting and hardening-off culture;
E, intermediate house booth are cultivated.
Wherein, in the specific implementation, in step a, the length of explant is that 0.5cm is proper.Subculture times 5 ~ 10 times is proper.
Wherein, the medium that in said method step c, squamous subculture adopts is identical with inducing culture described in step b.
Wherein, in said method steps d, the medium of Rooting and hardening-off culture is: 1/2MS medium+sucrose 25 ~ 35g/L+ agar 6 ~ 10g/L+ active carbon 1.5 ~ 2.5g/L+0.3 ~ 0.7mg/LNAA+0.15 ~ 0.25mg/L6-BA, pH value is 5.6 ~ 6.0.
Wherein, in said method steps d, the medium of Rooting and hardening-off culture is: 1/2MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+0.5mg/LNAA+0.2mg/L6-BA, pH value is 5.8.
Wherein, in said method step b, inducing culture is: MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+1.5mg/L6-BA+0.5mg/LNAA, pH value is 5.8.
Wherein, after said method step e intermediate house booth is cultivated and referred to Rooting and hardening-off culture, hardening 7 days, transplant to artificial greenhouse when plantlet in vitro grows to 4 ~ 5cm height, transplanting medium is that peat soil adds a small amount of perlitic mixture.
Wherein, disinfect described in said method step a and refer to explant tap water 2 ~ 3 hours, then clean with banister brush, with distilled water flushing, put in superclean bench, 75% alcohol-pickled 30 seconds, sterile water wash is clean, surface sterilization 15 minutes in the mercuric chloride solution of 0.1%, sterile water wash 4-5 time, each 2 minutes.
Wherein, said method step b, c and d cultivate in culturing room, and culturing room's condition is: intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22 ~ 25 DEG C; Culturing room was regularly sterilized in every two months.
Wherein, green house described in said method step e adopts light transmittance 50% shading screen to shade, and in green house, temperature controls at 20 ~ 32 DEG C, and relative moisture controls 65 ~ 75%.
Wherein, roxburgh anoectochilus terminal bud described in said method step a refers to the high eyebrow roxburgh anoectochilus terminal bud of growth at Emeishan.
The invention has the beneficial effects as follows: the present invention produces Multiple Buds by the axillary-bud or top-bud of the high eyebrow roxburgh anoectochilus terminal bud of hormone induction to carry out Fast-propagation to realize, by the screening to medium, find the medium being particularly suitable for high eyebrow roxburgh anoectochilus terminal bud seedling fostering, growth coefficient (i.e. proliferation times) reaches 8-10 doubly, transplanting survival rate, more than 95%, can realize commercial scale plantation with this.
The method of quick breeding by group culture of the present invention, can keep the merit of high eyebrow roxburgh anoectochilus terminal bud, keep the property of medicine that it is excellent, and method is simple, production cost is low, free from environmental pollution, can realize factorial seedling growth.Employing this method induces Multiple Buds and the ratio of taking root reaches more than 90%, and transplanting survival rate, more than 95%, can reach 8-10 doubly at 4 months internal breeding coefficients.By the high eyebrow roxburgh anoectochilus terminal bud that the inventive method is produced, do not make a variation, output is high, and cost is low, and the cycle is short, the market competitiveness that tool is stronger.
Embodiment
The present invention specifically can implement in such a way, and the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud, comprises the steps:
A, choose growth at the wild high eyebrow roxburgh anoectochilus terminal bud in 5 ~ October, removing root and leaf, is cut into the section shape of band axillary-bud or top-bud as explant material using stem, explant tap water 2 ~ 3 hours, then clean with banister brush, with distilled water flushing, put in superclean bench, 75% alcohol-pickled 30 seconds, sterile water wash is clean, surface sterilization 15 minutes in the mercuric chloride solution of 0.1%, sterile water wash 4-5 time, each 2 minutes;
The induction of b, Multiple Buds: the explant after sterilization is inoculated into inducing culture and cultivates; Inducing culture is: MS medium+sucrose 25 ~ 35g/L+ agar 6 ~ 10g/L+ active carbon 1.5 ~ 2.5g/L+1 ~ 2mg/L6-BA+0.3 ~ 0.7mg/LNAA, and pH value is 5.6 ~ 6.0; Cultivation cycle is 30 ~ 40 days;
C, squamous subculture 5 ~ 10 times; Medium is: MS medium+sucrose 25 ~ 35g/L+ agar 6 ~ 10g/L+ active carbon 1.5 ~ 2.5g/L+1 ~ 2mg/L6-BA+0.3 ~ 0.7mg/LNAA, and pH value is 5.6 ~ 6.0; Cultivation cycle is 30 ~ 40 days;
D, Rooting and hardening-off culture; Medium is: 1/2MS medium+sucrose 25 ~ 35g/L+ agar 6 ~ 10g/L+ active carbon 1.5 ~ 2.5g/L+0.3 ~ 0.7mg/LNAA+0.15 ~ 0.25mg/L6-BA, and pH value is 5.6 ~ 6.0;
E, intermediate house booth are cultivated: after Rooting and hardening-off culture, hardening 7 days, and transplant to artificial greenhouse when plantlet in vitro grows to 4 ~ 5cm height, transplanting medium is that peat soil adds a small amount of perlitic mixture.
MS medium of the present invention is the MS medium of conventional MS medium or conventional method preparation.1/2MS medium: mineral salt are the half of MS medium.It will be understood by those skilled in the art that, the medium that step b of the present invention, c and d adopt is all obtained with addition of preparations such as sucrose, agar, active carbons by basal medium (MS medium or 1/2MS medium), and the content of above-mentioned each material is all the content having prepared cumulative volume shared by rear each material mass.Wherein, agar is the glue product made with the Gelidium of algae (Gelidium) and Gracilaria (Gracilaria), is the curing agent of medium.Namely 6-BA is 6-benzyl aminoadenine, molecular formula: C12H11N5.Namely NAA is methyl α-naphthyl acetate, English common name 1-naphthlceticacid, other title a-methyl α-naphthyl acetates.
Preferably, in said method steps d, the medium of Rooting and hardening-off culture is: 1/2MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+0.5mg/LNAA+0.2mg/L6-BA, pH value is 5.8.
Preferably, the medium that said method step b and c adopts is: MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+1.5mg/L6-BA+0.5mg/LNAA, pH value is 5.8.
Wherein, said method step b, c and d cultivate in culturing room, and preferred culturing room condition is: intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22 ~ 25 DEG C; Culturing room was regularly sterilized in every two months.
Preferably, green house described in said method step e adopts light transmittance 50% shading screen to shade, and in green house, temperature controls at 20 ~ 32 DEG C, and relative moisture controls 65 ~ 75%.
Below by embodiment, the specific embodiment of the present invention is described further, but not therefore by protection scope of the present invention restriction in one embodiment.
Embodiment one
The high eyebrow roxburgh anoectochilus terminal bud of material, picks up from Mount Emei, sichuan, China.Breed high eyebrow roxburgh anoectochilus terminal bud according to the following steps.
1, get wild high eyebrow roxburgh anoectochilus terminal bud plant, cut the segment that long 0.5cm contains an axillalry bud.
2, tap water 2-3 hour, banister brush cleans up, and distilled water flushing is clean, puts in superclean bench, 75% alcohol-pickled 30 seconds, and sterile water wash is clean, surface sterilization 15 minutes in the mercuric chloride solution of 0.1%, sterile water wash 4-5 time, each 2 minutes.
3, the explant after sterilization is inoculated into inducing culture and cultivates; Inducing culture is: MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+1.5mg/L6-BA+0.5mg/LNAA, and pH value is 5.8; Cultivation cycle is 30-40 days; Condition of culture is: intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22-25 DEG C.Cultivate and within about 7 days, start to sprout, when Multiple Buds is sprouted long to 0.5cm, cut open, be forwarded on subculture medium, squamous subculture based component and the same inducing clumping bud of condition of culture, after 2 months, growth coefficient is 3-4.
4, when Multiple Buds grow to 1cm long time, individual plant is transferred to Rooting and hardening-off culture base and cultivates, and Rooting and hardening-off culture based component is: 1/2MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+0.5mg/LNAA+0.2mg/L6-BA, and pH value is 5.8; The same inducing clumping bud of condition of culture (intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22-25 DEG C).Culture of rootage 50-60 days, rooting rate can reach more than 95%, long about 4-5cm.
5, hardening was transplanted to artificial greenhouse after 7 days, and booth 50% shading screen shades, and culture matrix is that peat soil adds a small amount of perlitic mixture.After transplanting, every day waters 3 times, water to culture matrix is drenched (making plant ambient humidity, light and temperature about 70%), every first quarter moon sealing fertilizer is (Vegetables special fertilizer: nitrogen 15%, phosphorus pentoxide 7% and potassium oxide 8%) once, and attention control damage by disease and insect.
This breeds the high eyebrow roxburgh anoectochilus terminal bud that obtains in 4-5 month by the induction of Multiple Buds and the step such as subculture, strong plantlets and rootage, induce Multiple Buds and the ratio of taking root reaches more than 90%, after transplanting, survival rate is more than 95%, can 8-10 be reached, long about 5cm at internal breeding more than four months coefficient, and by high eyebrow roxburgh anoectochilus terminal bud that the present embodiment is produced, do not make a variation, output is high, and cost is low, substantially increases the quantity of high eyebrow roxburgh anoectochilus terminal bud.
Embodiment two
The high eyebrow roxburgh anoectochilus terminal bud of material, picks up from Mount Emei, sichuan, China.Breed high eyebrow roxburgh anoectochilus terminal bud according to the following steps.
1, get wild high eyebrow roxburgh anoectochilus terminal bud plant, cut the segment that long 0.5cm contains a terminal bud.
2, tap water 2-3 hour, banister brush cleans up, and distilled water flushing is clean, puts in superclean bench, 75% alcohol-pickled 30 seconds, and sterile water wash is clean, surface sterilization 15 minutes in the mercuric chloride solution of 0.1%, sterile water wash 4-5 time, each 2 minutes.
3, the explant after sterilization is inoculated into inducing culture and cultivates; Inducing culture is: MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+1.5mg/L6-BA+0.5mg/LNAA, and pH value is 5.8; Cultivation cycle is 30-40 days; Condition of culture is: intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22-25 DEG C.Cultivate and within about 7 days, start to sprout, when Multiple Buds sprouts the sprouting inducing generation about 2-4 also about to have 0.5cm long, cut open, be forwarded on subculture medium, squamous subculture based component and the same inducing clumping bud of condition of culture, after 2 months, growth coefficient is 3-4.
4, when Multiple Buds grow to 1cm long time, individual plant is transferred to Rooting and hardening-off culture base and cultivates, and Rooting and hardening-off culture based component is: 1/2MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+0.5mg/LNAA+0.2mg/L6-BA, and pH value is 5.8; The same inducing clumping bud of condition of culture (intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22-25 DEG C).Culture of rootage 50-60 days, rooting rate can reach more than 95%, long about 4-5cm.
5, hardening was transplanted to artificial greenhouse after 7 days, and booth 50% shading screen shades, and culture matrix is that peat soil adds a small amount of perlitic mixture.After transplanting, every day waters 3 times, water to culture matrix is drenched (making plant ambient humidity, light and temperature about 70%), every first quarter moon sealing fertilizer is (Vegetables special fertilizer: nitrogen 15%, phosphorus pentoxide 7% and potassium oxide 8%) once, and attention control damage by disease and insect.
This breeds the high eyebrow roxburgh anoectochilus terminal bud that obtains in 4-5 month by the induction of Multiple Buds and the step such as subculture, strong plantlets and rootage, induce Multiple Buds and the ratio of taking root reaches more than 90%, after transplanting, survival rate is more than 95%, can reach 8-10 doubly at a 4-5 month internal breeding coefficient.And by the high eyebrow roxburgh anoectochilus terminal bud that the present embodiment is produced, do not make a variation, output is high, and cost is low, substantially increases the nursery production capacity of high eyebrow roxburgh anoectochilus terminal bud.
Claims (4)
1. the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud, is characterized in that comprising the steps:
A, choose growth at the wild high eyebrow roxburgh anoectochilus terminal bud in 5 ~ October, removing root and leaf, stem is cut into the section shape of band axillary-bud or top-bud as explant material, explant disinfection;
The induction of b, Multiple Buds: the explant after sterilization is inoculated into inducing culture and cultivates; Inducing culture is: MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+1.5mg/L6-BA+0.5mg/LNAA, and pH value is 5.8; Cultivation cycle is 30 ~ 40 days;
C, squamous subculture; The medium that squamous subculture adopts is identical with inducing culture described in step b;
D, Rooting and hardening-off culture; The medium of described Rooting and hardening-off culture is: 1/2MS medium+sucrose 30g/L+ agar 8g/L+ active carbon 2.0g/L+0.5mg/LNAA+0.2mg/L6-BA, and pH value is 5.8;
E, intermediate house booth are cultivated;
Described step b, c and d cultivate in culturing room, and culturing room's condition is: intensity of illumination is 2000lux, 16h/d, and cultivation temperature is 22 ~ 25 DEG C; Culturing room was regularly sterilized in every two months.
2. the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud according to claim 1, it is characterized in that: after step e intermediate house booth is cultivated and referred to Rooting and hardening-off culture, hardening 7 days, transplant to artificial greenhouse when plantlet in vitro grows to 4 ~ 5cm height, transplanting medium is that peat soil adds a small amount of perlitic mixture.
3. the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud according to claim 1, it is characterized in that: disinfect described in step a and refer to explant tap water 2 ~ 3 hours, then clean with banister brush, with distilled water flushing, put in superclean bench, 75% alcohol-pickled 30 seconds, sterile water wash is clean, surface sterilization 15 minutes in the mercuric chloride solution of 0.1%, sterile water wash 4-5 time, each 2 minutes.
4. the tissue culture and rapid propagation method of high eyebrow roxburgh anoectochilus terminal bud according to any one of claim 1 to 3, it is characterized in that: green house described in step e adopts light transmittance 50% shading screen to shade, in green house, temperature controls at 20 ~ 32 DEG C, and relative moisture controls 65 ~ 75%.
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| CN104904600A (en) * | 2015-06-03 | 2015-09-16 | 吴华球 | Tissue-culturing and rapid-propagating method of anoectochilus formosanus |
| CN105010145B (en) * | 2015-08-12 | 2017-04-26 | 江苏凤谷生物有限公司 | Anoectochilus roxburghii seedling propagation expanding method |
| CN105660400B (en) * | 2016-01-25 | 2018-02-13 | 宁波易中禾生物技术有限公司 | A kind of strong sprout weight increasing method of roxburgh anoectochilus terminal bud tissue-cultured seedling |
| CN105660401B (en) * | 2016-01-27 | 2018-02-13 | 漳州市萌得尔农业科技有限公司 | A kind of tissue culture roxburgh anoectochilus terminal bud culture medium and its preparation method and application |
| CN106386487B (en) * | 2016-09-08 | 2018-04-03 | 福建虎伯寮生物集团有限公司 | A kind of roxburgh anoectochilus terminal bud tissue culture method |
| CN106973788A (en) * | 2017-03-21 | 2017-07-25 | 华南农业大学 | A kind of tissue culture production method of roxburgh anoectochilus terminal bud tufted seedling |
| CN108012923A (en) * | 2017-12-05 | 2018-05-11 | 罗来辉 | A kind of cultural method of selenium-rich roxburgh anoectochilus terminal bud |
| CN109042328A (en) * | 2018-09-07 | 2018-12-21 | 天津大学 | A kind of method of roxburgh anoectochilus terminal bud tissue-culturing quick-propagation |
| CN110100738A (en) * | 2019-06-21 | 2019-08-09 | 台州市椒江草之堂生物科技有限公司 | A kind of breeding method of roxburgh anoectochilus terminal bud tissue-cultured seedling |
| CN116250480B (en) * | 2023-02-15 | 2024-06-07 | 河北工业大学 | Sterile rapid propagation method of trollius chinensis and planting method of trollius chinensis |
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