CN103238525B - Method for breeding fritillary bulb by tissue culture technique - Google Patents
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Abstract
The invention discloses a method for breeding fritillary bulb by a tissue culture technique. The method mainly comprises the following steps: selection and disinfection of explants, material treatment, primary culture, subculture, proliferation of bulblet, rooting culture, acclimatization and transplant and the like. In different culture stages, different hormones are used for adjusting, and the formula of culture medium is accurately screened to realize the excellent effect of vigorous growth of test-tube plantlet. The purpose of improving the proliferation rate and reducing mutation is realized by combining effective sterilization technologies. The culture medium is appropriately changed in the bulblet subjected to proliferation process to facilitate quick growth of the bulblet. The optimal selection of the scale breeding material is the lower part of the inner scale by comparing different parts of the scale.
Description
Technical field
The present invention relates to a kind of method for tissue culture, be specifically related to the propagation method of a kind of fritillary bulb.
Background technology
Fritillary bulb, calls flat shellfish, northern shellfish, the bulb of fritillary, is Liliaceae perennial herb, up to 1m.The dry bulb of fritillary bulb is the traditional Chinese medicine that China commonly uses, and has the functions such as clearing heat and moistening lung, preventing phlegm from forming and stopping coughing, dissipating bind, clinically for phlegm-heat cough, the treatment coughing the disease such as sputum streaked with blood, scrofula sore swollen toxin.In recent years, owing to excessively excavating, resource reduces year by year, undersupllied commodities, and price rises steadily.
Certain scale has been had at present to the Study on tissue culture of the bulb of fritillary, but in view of the problem of technology also exists some problems always, also certain research has been had to the quick propagating technology of fritillary bulb, but the demand of fritillary bulb is increased day by day, the breeding of current fritillary bulb and plantation do not reach the demand of people far away, tissue cultures can improve the reproduction rate of fritillary bulb greatly, but tissue culturing system not yet fully sets up at present, there are some technical problems, popularizing of improved seeds not yet completes, disparities between supply and demand are increasingly sharpened, have a strong impact on the amount reproduction of fritillary bulb.
Summary of the invention
The present invention is directed to current Problems existing, a kind of method that existing resource can be utilized fast and effectively to breed fritillary bulb is provided.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is:
Utilize tissue culture technique to breed a method for fritillary bulb, it is characterized in that, comprise the following steps:
(1) selection of explant and sterilization: first select current growth healthy and strong, without the fresh fritillary bulb of the spot that goes rotten, cut off root and have and hinder scale, put into clear water after running water 30 min and soak 15min, steep 20 ~ 30 min by 50% carbendazim, 500 times of immersions again or steep 10 ~ 15min by 50% tmtd powder, 500 times of immersions, then kind of a ball scale is peeled, with ultra violet lamp 10 min, proceed to superclean bench, use 75% alcohol disinfecting 50 s again, aseptic water washing 2 ~ 3 times, to sterilize 10 min with 6% liquor natrii hypochloritis again, aseptic water washing 2 ~ 3 times.Finally to sterilize 10 min with the mercuric chloride solution of 0.2%, aseptic water washing 4 ~ 5 times.
(2) material processed: first on superclean bench, aseptically, cuts scale top 1/3, then by, lower part isolates, and scale and bulb base portion is cut into respectively the fritter of 6-8mm;
(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium is the NAA at 6-BA, the 1.2mg/L that with the addition of 0.8mg/L in 3/4MS conventional medium, the sucrose of 30-40mg/L and the ZT of 0.4-0.8mg/L; PH is 6.0, and cultivation temperature is 24 DEG C, light application time 12 h/d, intensity of illumination 2 000 Lx, agar 5 g/L, forms callus through 25-30 days;
(4) squamous subculture: the callus of Initial culture is cut and is seeded on subculture medium, this medium is the sucrose of KT and 20mg/L of IBA, the 0.3mg/L of 6-BA, the 0.3mg/L that with the addition of 0.5mg/L in MS conventional medium, PH 6.0, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, after 7-12 days time differentiated green point, namely starts next stage;
(5) propagation of clove: the callus differentiating green point is continued to cultivate, this medium is the ZT that with the addition of the sucrose of 40mg/L, KT, 0.4mg/LNAA and 0.2mg/L of 0.3mg/L in 1/2MS conventional medium, PH 6.0, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, cultivates just can differentiate the clove that grows thickly through 10-15 days;
(6) culture of rootage: differentiation clove is out inoculated in following inducing culture and carries out root induction: the active carbon of KT, 1mg/L of 1/2 MS medium, 0.3mg/L, the agar of 7-8g/L, the NAA of 0.5mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, treated that clove grew to 2.5cm through 8-12 days high, when root system is more flourishing, namely start the next stage;
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 6d, then the clove growing new root is taken out from blake bottle, shine 1-2 days in the sun, suitable shading is wanted in the process of shining, intensity of illumination is made to be 50% of natural daylight, temperature is at about 15 DEG C, after media surface becomes polygon, take out the medium that seedling cleans root attachment, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: vermiculite=2: in the mixed-matrix of 2: 1:3 (weight ratio), temperature controls at about 25 DEG C.
Beneficial effect of the present invention is mainly reflected in:
(1) establish the complete set technology of the excised cotyledon Fast-propagation of perfect fritillary bulb, reach efficient induction explant differentiation and proliferation object, and save whole merits of parent, stabilization characteristics of genetics;
(2) shorten the breeding cycle, on the basis that ensure that survival rate, improve reproductive efficiency, add economic benefit;
(3) regulate at the different hormone that utilizes of different cultivation stages, the vigorous excellent results of test-tube plantlet growth is reached to the accurate screening of culture medium prescription, and in conjunction with effective sterilization technology, reach the object improving the rate of increase and reduce variation.
(4) suitably change medium in the breeding of the clove after squamous subculture, be more conducive to the quick growth of clove.
(5) in the process of transplanting hardening, take the temperature and light of necessary controlling measurement growth according to intensity, fritillary bulb is bred at suitable temperature, improves quality and the survival rate of seedling.
Embodiment
Below preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
embodiment 1
Utilize tissue culture technique to breed a method for fritillary bulb, it is characterized in that, comprise the following steps:
(1) selection of explant and sterilization: first select current growth healthy and strong, without the fresh fritillary bulb of the spot that goes rotten, cut off root and have and hinder scale, put into clear water after running water 30 min and soak 15min, steep 20min by 50% carbendazim, 500 times of immersions again or steep 10min by 50% tmtd powder, 500 times of immersions, then kind of a ball scale is peeled, with ultra violet lamp 10 min, proceed to superclean bench, use 75% alcohol disinfecting 50 s again, aseptic water washing 2 times, to sterilize 10 min with 6% liquor natrii hypochloritis again, aseptic water washing 3 times.Finally to sterilize 10 min with the mercuric chloride solution of 0.2%, aseptic water washing 4 times.
(2) material processed: first on superclean bench, aseptically, selects the middle part of outer scale, is cut into the fritter of 6-8mm;
(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium is the NAA at 6-BA, the 1.2mg/L that with the addition of 0.8mg/L in 3/4MS conventional medium, the sucrose of 30mg/L and the ZT of 0.4mg/L; PH is 6.0, and cultivation temperature is 24 DEG C, light application time 12 h/d, intensity of illumination 2 000 Lx, agar 5 g/L, forms callus through 30 days;
(4) squamous subculture: the callus of Initial culture is cut and is seeded on subculture medium, this medium is the sucrose of KT and 20mg/L of IBA, the 0.3mg/L of 6-BA, the 0.3mg/L that with the addition of 0.5mg/L in MS conventional medium, PH 6.0, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, after 12 day time differentiated green point, namely starts next stage;
(5) propagation of clove: the callus differentiating green point is continued to cultivate, this medium is the ZT that with the addition of the sucrose of 40mg/L, KT, 0.4mg/LNAA and 0.2mg/L of 0.3mg/L in 1/2MS conventional medium, PH 6.0, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, cultivates just can differentiate the clove that grows thickly through 15 days;
(6) culture of rootage: differentiation clove is out inoculated in following inducing culture and carries out root induction: the active carbon of KT, 1mg/L of 1/2 MS medium, 0.3mg/L, the agar of 7g/L, the NAA of 0.5mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, treated that clove grew to 2.5cm through 12 days high, when root system is more flourishing, namely start the next stage;
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 6d, then the clove growing new root is taken out from blake bottle, shine 2 days in the sun, suitable shading is wanted in the process of shining, intensity of illumination is made to be 50% of natural daylight, temperature is at about 15 DEG C, after media surface becomes polygon, take out the medium that seedling cleans root attachment, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: vermiculite=2: in the mixed-matrix of 2: 1:3 (weight ratio), temperature controls at about 25 DEG C.
embodiment 2
By the bottom of the Material selec-tion outer scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 27 days in Initial culture, green point was differentiated through 9 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 12 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 11 days cloves, root system is more flourishing.
embodiment 3
By the bottom of the Material selec-tion outer scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 27 days in Initial culture, green point was differentiated through 8 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 12 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 10 days cloves, root system is more flourishing.
embodiment 4
By the middle part of the Material selec-tion middle level scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 28 days in Initial culture, green point was differentiated through 10 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 11 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 10 days cloves, root system is more flourishing.
embodiment 5
By the bottom of the Material selec-tion middle level scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 26 days in Initial culture, green point was differentiated through 9 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 13 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 10 days cloves, root system is more flourishing.
embodiment 6
By the middle part of the Material selec-tion internal layer scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 27 days in Initial culture, green point was differentiated through 10 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 12 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 9 days cloves, root system is more flourishing.
embodiment 7
By the bottom of the Material selec-tion internal layer scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 25 days in Initial culture, green point was differentiated through 7 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 10 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 8 days cloves, root system is more flourishing.
embodiment 8
By the base portion of the Material selec-tion scale in step (2) Material handling processes, all the other conditions are identical with embodiment 1, result formed callus through 26 days in Initial culture, green point was differentiated through 8 day time in squamous subculture, cultivated can differentiate the clove that grows thickly through 12 days in the breeding of clove, in process of rooting culture, grow to 2.5cm through 10 days cloves, root system is more flourishing.
Found by the contrast of embodiment 1 to embodiment 8, the bottom of internal layer scale is the optimal material that fritillary bulb carries out tissue cultures.
embodiment 9
Utilize tissue culture technique to breed a method for fritillary bulb, it is characterized in that, comprise the following steps:
(1) selection of explant and sterilization: first select current growth healthy and strong, without the fresh fritillary bulb of the spot that goes rotten, cut off root and have and hinder scale, put into clear water after running water 30 min and soak 15min, steep 30 min by 50% carbendazim, 500 times of immersions again or steep 15min by 50% tmtd powder, 500 times of immersions, then kind of a ball scale is peeled, with ultra violet lamp 10 min, proceed to superclean bench, use 75% alcohol disinfecting 50 s again, aseptic water washing 3 times, to sterilize 10 min with 6% liquor natrii hypochloritis again, aseptic water washing 3 times.Finally to sterilize 10 min with the mercuric chloride solution of 0.2%, aseptic water washing 5 times.
(2) material processed: first on superclean bench, aseptically, selects the bottom of internal layer scale to be cut into the fritter of 6-8mm;
(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium is the NAA at 6-BA, the 1.2mg/L that with the addition of 0.8mg/L in 3/4MS conventional medium, the sucrose of 40mg/L and the ZT of 0.8mg/L; Cultivation temperature is 24 DEG C, light application time 12 h/d, and intensity of illumination 2 000 Lx, PH are 6.5, agar 5 g/L, forms callus through 25 days;
(4) squamous subculture: the callus of Initial culture is cut and is seeded on subculture medium, this medium is the sucrose of KT and 20mg/L of IBA, the 0.3mg/L of 6-BA, the 0.3mg/L that with the addition of 0.5mg/L in MS conventional medium, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, after 12 day time differentiated green point, namely starts next stage;
(5) propagation of clove: the callus differentiating green point is continued to cultivate, this medium is the ZT that with the addition of the sucrose of 40mg/L, KT, 0.4mg/LNAA and 0.2mg/L of 0.3mg/L in 1/2MS conventional medium, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, cultivated through 10 days and just can differentiate the clove that grows thickly;
(6) culture of rootage: differentiation clove is out inoculated in following inducing culture and carries out root induction: the active carbon of KT, 1mg/L of 1/2 MS medium, 0.3mg/L, the agar of 8g/L, the NAA of 0.5mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, treated that clove grew to 2.5cm through 10 days high, when root system is more flourishing, namely start the next stage;
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 6d, then the clove growing new root is taken out from blake bottle, shine 2 days in the sun, suitable shading is wanted in the process of shining, intensity of illumination is made to be 60% of natural daylight, temperature is at about 18 DEG C, after media surface becomes polygon, take out the medium that seedling cleans root attachment, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: vermiculite=2: in the mixed-matrix of 2: 1:3 (weight ratio), temperature controls at about 15 DEG C.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (2)
1. utilize tissue culture technique to breed a method for fritillary bulb, it is characterized in that, comprise the following steps:
(1) selection of explant and sterilization: first select current growth healthy and strong, without the fresh fritillary bulb of the spot that goes rotten, cut off root and have and hinder scale, put into clear water after running water 30 min and soak 15min, steep 20 ~ 30 min by 50% carbendazim, 500 times of immersions again or steep 10 ~ 15min by 50% tmtd powder, 500 times of immersions, then kind of a ball scale is peeled, with ultra violet lamp 10 min, proceed to superclean bench, use 75% alcohol disinfecting 50 s again, aseptic water washing 2 ~ 3 times, to sterilize 10 min with 6% liquor natrii hypochloritis again, aseptic water washing 2 ~ 3 times, finally to sterilize 10 min with the mercuric chloride solution of 0.2%, aseptic water washing 4 ~ 5 times,
(2) material processed: first on superclean bench, aseptically, cuts scale top 1/3, then by, lower part isolates, and scale and bulb base portion is cut into respectively the fritter of 6-8mm;
(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium is the NAA of 6-BA, the 1.2mg/L that with the addition of 0.8mg/L in 3/4MS conventional medium, the sucrose of 30-40mg/L and the ZT of 0.4-0.8mg/L; Cultivation temperature is 24 DEG C, light application time 12 h/d, and intensity of illumination 2 000 Lux, pH are 6.5, agar 5 g/L, forms callus through 25-30 days;
(4) squamous subculture: the callus of Initial culture is cut and is seeded on subculture medium, this medium is the sucrose of KT and 20mg/L of IBA, the 0.3mg/L of 6-BA, the 0.3mg/L that with the addition of 0.5mg/L in MS conventional medium, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, pH 6.5, after 7-12 days time differentiated green point, namely starts next stage;
(5) propagation of clove: the callus differentiating green point is continued to cultivate, this medium is the ZT that with the addition of the sucrose of 40mg/L, KT, 0.4mg/LNAA and 0.2mg/L of 0.3mg/L in 1/2MS conventional medium, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, pH 6.5, cultivated through 10-15 days and differentiates the clove that grows thickly;
(6) culture of rootage: differentiation clove is out inoculated in following inducing culture and carries out root induction: the active carbon of KT, 1mg/L of 1/2 MS medium, 0.3mg/L, the agar of 7-8g/L, the NAA of 0.5mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, luminous intensity is 2200lux, treated that clove grew to 2.5cm through 8-12 days high, when root system is more flourishing, namely start the next stage;
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 6d, then the clove growing new root is taken out from blake bottle, shine 1-2 days in the sun, suitable shading is wanted in the process of shining, intensity of illumination is made to be 60% of natural daylight, temperature is at about 18 DEG C, after media surface becomes polygon, take out the medium that seedling cleans root attachment, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: vermiculite=2: in the mixed-matrix of 2: 1:3 weight ratio, temperature controls at about 15 DEG C.
2. a kind of method utilizing tissue culture technique to breed fritillary bulb according to claim 1, it is characterized in that, the optimal selection of scale lining material is the bottom of internal layer scale.
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| CN103430744A (en) * | 2013-08-16 | 2013-12-11 | 苏州仁成生物科技有限公司 | Method for rapid breeding of fritillaria cirrhosa by means of fritillaria cirrhosa scales |
| CN103444528A (en) * | 2013-08-16 | 2013-12-18 | 苏州仁成生物科技有限公司 | Growth promotion cultivation method of fritillary bulbs |
| CN103444526B (en) * | 2013-08-16 | 2015-11-25 | 顾祥茂 | A kind of growth-promoting cultivation method of Fritillaria unibracteata |
| CN103444527B (en) * | 2013-08-16 | 2016-04-27 | 苏州仁成生物科技有限公司 | A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb |
| CN104396746B (en) * | 2014-11-12 | 2016-05-11 | 北京林业大学 | A kind of chrysanthemum bulb of fritillary adventitious bud inducing enrichment procedure |
| CN107258539B (en) * | 2017-07-06 | 2019-06-07 | 四川省中医药科学院 | A method of control taipei fritillary bulb tissue cultures endophytic bacterial contamination |
| CN108668897A (en) * | 2018-05-18 | 2018-10-19 | 兰州市农业科技研究推广中心 | A kind of micro- numerous method of Fritillaria yuzhongensis |
| CN110100730B (en) * | 2019-05-09 | 2021-01-19 | 云南山里红生物科技有限公司 | Bulbus Fritillariae Cirrhosae tissue culture method capable of preventing browning |
| CN112673751A (en) * | 2020-12-16 | 2021-04-20 | 浙江省中药研究所有限公司 | Ex-situ over-summer method for fritillary bulb bulbs |
| CN112931210B (en) * | 2021-03-18 | 2022-06-21 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
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| JPH08112045A (en) * | 1994-10-17 | 1996-05-07 | Koa Oil Co Ltd | Method for mass production of seed and seedling of plant of genus fritillaria |
| CN101213937B (en) * | 2007-12-28 | 2010-09-08 | 浙江省农业科学院 | Method for cultivating detoxification tissue culture bulb of fritillaria thunbergii |
| CN101999317B (en) * | 2009-09-03 | 2012-08-08 | 薛永新 | Multiploid bulb induction method with firtillaria cirrhosa scale leaf as explant |
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Inventor after: Chen Yongming Inventor after: Wei Zongyou Inventor before: Chen Yongming |
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