CN103238525A - 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 is called flat shellfish, northern shellfish, the bulb of fritillary, for the Liliaceae perennial herb, up to 1m.The dry bulb of fritillary bulb is China's traditional Chinese medicine commonly used, has functions such as clearing heat and moistening lung, preventing phlegm from forming and stopping coughing, dissipating bind, is used for phlegm-heat cough clinically, coughs treatment of diseases such as sputum streaked with blood, scrofula sore swollen toxin.In recent years, owing to excessively excavate, resource reduces year by year, undersupllied commodities, and price rises steadily.
Study on tissue culture to the bulb of fritillary has had certain scale at present, but in view of the problem of technology exists some problems always, quick propagating technology to fritillary bulb has also had certain research, but the demand to fritillary bulb increases day by day, the breeding of fritillary bulb at present and plantation do not reach people's demand far away, tissue is cultivated can improve the reproduction rate of fritillary bulb greatly, but tissue culturing system does not fully set up as yet at present, there are some technical problems, popularizing as yet of improved seeds do not finished, disparities between supply and demand are increasingly sharpened, and have had a strong impact on a large amount of breedings of fritillary bulb.
Summary of the invention
The present invention is directed to the problem of present existence, a kind of method that can utilize existing resource breeding fritillary bulb fast and effectively is provided.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is:
A kind of method of utilizing tissue culture technique breeding fritillary bulb is characterized in that, may further comprise the steps:
(1) selection of explant and sterilization: the fresh fritillary bulb of at first selecting current growth stalwartness, the mildew and rot spot of nothing, cut off root and the scale of wound is arranged, flowing water washes to be put into clear water behind 30 min and soaks 15min, soak 20 ~ 30 min or soak 10 ~ 15min with 500 times of liquid of 50% tmtd powder with 500 times of liquid of 50% carbendazim again, to plant the ball scale then peels, with ultra violet lamp 10 min, change superclean bench over to, use 75% alcohol disinfecting, 50 s again, aseptic water washing 2 ~ 3 times, again with 6% liquor natrii hypochloritis, 10 min that sterilize, aseptic water washing 2 ~ 3 times.At last with 0.2% mercuric chloride solution, 10 min that sterilize, aseptic water washing 4 ~ 5 times.
(2) material processed: at first on superclean bench, under aseptic condition, cut scale top 1/3, then will in, the lower part isolates, and scale and bulb base portion is cut into the fritter of 6-8mm respectively;
(3) it is foster to be commissioned to train at the beginning of: on the explant that under the aseptic condition step (2) is obtained is seeded in just for medium, this medium is to add the 6-BA of 0.8mg/L, the NAA of 1.2mg/L, the sucrose of 30-40mg/L and the ZT of 0.4-0.8mg/L in the conventional medium of 3/4MS; PH is 6.0, and cultivation temperature is 24 ℃, light application time 12 h/d, and intensity of illumination 2 000 Lx, agar 5 g/L were through 25-30 days formation callus;
(4) subculture is cultivated: the callus that will just be commissioned to train foster downcuts and is seeded on the subculture medium, this medium is to have added the KT of IBA, 0.3mg/L of 6-BA, 0.3mg/L of 0.5mg/L and the sucrose of 20mg/L in the conventional medium of MS, PH 6.0, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, and the 7-12 days time of process namely begins next stage after differentiating green point;
(5) propagation of clove: the callus that will differentiate green point continues to cultivate, this medium is to have added the sucrose of 40mg/L, KT, the 0.4mg/LNAA of 0.3mg/L and the ZT of 0.2mg/L in the conventional medium of 1/2MS, PH 6.0, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, cultivates through 10-15 days and just can differentiate the clove that grows thickly;
(6) culture of rootage: the clove that differentiation is come out is inoculated in and carries out root induction in the following inducing culture: the active carbon of KT, the 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 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, treat that through 8-12 days clove grows to the 2.5cm height, when root system is relatively more flourishing, namely begin the next stage;
(7) acclimatization and transplants: earlier blake bottle is moved to the greenhouse, natural conditions lower refining seedling 6d, the clove that will grow new root is taken out from blake bottle then, shone in the sun 1-2 days, in the process of shining, want suitable shading, making intensity of illumination is 50% of natural daylight, temperature is about 15 ℃, after media surface becomes polygon, take out seedling and clean the medium that root adheres to, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: in the mixed-matrix of vermiculite=2: 2: 1:3 (weight ratio), temperature is controlled about 25 ℃.
Beneficial effect of the present invention is mainly reflected in:
(1) in vitro tissue of having set up perfect fritillary bulb is cultivated the complete set technology of breeding fast, has reached and has efficiently induced explant differentiation and proliferation purpose, and preserved whole merits of parent, stabilization characteristics of genetics;
(2) shorten the breeding cycle, on the basis that has guaranteed survival rate, improved reproductive efficiency, increased economic benefit;
(3) regulate at the different hormone of utilizing of different cultivation stages, the accurate screening of culture medium prescription is reached the vigorous excellent results of test-tube plantlet growth, and in conjunction with effective sterilization technology, reach the purpose that improves the rate of increase and reduce variation.
(4) suitably change medium in the breeding of the clove after subculture is cultivated, more be conducive to the quick growth of clove.
(5) in the process of transplanting hardening, take measures necessary to control temperature and the intensity of illumination of growth, fritillary bulb is bred under suitable temperature, improved quality and the survival rate of seedling.
Embodiment
Below preferred embodiment of the present invention is described in detail, thereby so that advantages and features of the invention can be easier to be it will be appreciated by those skilled in the art that protection scope of the present invention is made more explicit defining.
Embodiment 1
A kind of method of utilizing tissue culture technique breeding fritillary bulb is characterized in that, may further comprise the steps:
(1) selection of explant and sterilization: the fresh fritillary bulb of at first selecting current growth stalwartness, the mildew and rot spot of nothing, cut off root and the scale of wound is arranged, flowing water washes to be put into clear water behind 30 min and soaks 15min, soak 20min or soak 10min with 500 times of liquid of 50% tmtd powder with 500 times of liquid of 50% carbendazim again, to plant the ball scale then peels, with ultra violet lamp 10 min, change superclean bench over to, use 75% alcohol disinfecting, 50 s again, aseptic water washing 2 times, again with 6% liquor natrii hypochloritis, 10 min that sterilize, aseptic water washing 3 times.At last with 0.2% mercuric chloride solution, 10 min that sterilize, aseptic water washing 4 times.
(2) material processed: at first on superclean bench, under aseptic condition, select the middle part of outer scale, be cut into the fritter of 6-8mm;
(3) it is foster to be commissioned to train at the beginning of: on the explant that under the aseptic condition step (2) is obtained is seeded in just for medium, this medium is to add the 6-BA of 0.8mg/L, the NAA of 1.2mg/L, the sucrose of 30mg/L and the ZT of 0.4mg/L in the conventional medium of 3/4MS; PH is 6.0, and cultivation temperature is 24 ℃, light application time 12 h/d, and intensity of illumination 2 000 Lx, agar 5 g/L were through 30 days formation callus;
(4) subculture is cultivated: the callus that will just be commissioned to train foster downcuts and is seeded on the subculture medium, this medium is to have added the KT of IBA, 0.3mg/L of 6-BA, 0.3mg/L of 0.5mg/L and the sucrose of 20mg/L in the conventional medium of MS, PH 6.0, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, differentiated green point through 12 day time after, namely begin next stage;
(5) propagation of clove: the callus that will differentiate green point continues to cultivate, this medium is to have added the sucrose of 40mg/L, KT, the 0.4mg/LNAA of 0.3mg/L and the ZT of 0.2mg/L in the conventional medium of 1/2MS, PH 6.0, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, cultivates through 15 days and just can differentiate the clove that grows thickly;
(6) culture of rootage: the clove that differentiation is come out is inoculated in and carries out root induction in the following inducing culture: the active carbon of KT, the 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 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, treat that through 12 days clove grows to the 2.5cm height, when root system is relatively more flourishing, namely begin the next stage;
(7) acclimatization and transplants: earlier blake bottle is moved to the greenhouse, natural conditions lower refining seedling 6d, the clove that will grow new root is taken out from blake bottle then, shone in the sun 2 days, in the process of shining, want suitable shading, making intensity of illumination is 50% of natural daylight, temperature is about 15 ℃, after media surface becomes polygon, take out seedling and clean the medium that root adheres to, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: in the mixed-matrix of vermiculite=2: 2: 1:3 (weight ratio), temperature is controlled about 25 ℃.
Embodiment 2
Material in step (2) the material processed process is selected the bottom of outer scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 27 days, in cultivating, subculture differentiated green point through 9 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 12 days, grow to 2.5cm through 11 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 3
Material in step (2) the material processed process is selected the bottom of outer scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 27 days, in cultivating, subculture differentiated green point through 8 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 12 days, grow to 2.5cm through 10 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 4
Material in step (2) the material processed process is selected the middle part of middle level scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 28 days, in cultivating, subculture differentiated green point through 10 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 11 days, grow to 2.5cm through 10 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 5
Material in step (2) the material processed process is selected the bottom of middle level scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 26 days, in cultivating, subculture differentiated green point through 9 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 13 days, grow to 2.5cm through 10 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 6
Material in step (2) the material processed process is selected the middle part of internal layer scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 27 days, in cultivating, subculture differentiated green point through 10 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 12 days, grow to 2.5cm through 9 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 7
Material in step (2) the material processed process is selected the bottom of internal layer scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 25 days, in cultivating, subculture differentiated green point through 7 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 10 days, grow to 2.5cm through 8 days cloves in process of rooting culture, root system is relatively more flourishing.
Embodiment 8
Material in step (2) the material processed process is selected the base portion of scale, all the other conditions are identical with embodiment 1, the result just be commissioned to train support in through forming callus in 26 days, in cultivating, subculture differentiated green point through 8 day time, in the breeding of clove, can differentiate the clove that grows thickly through cultivation in 12 days, grow to 2.5cm through 10 days cloves in process of rooting culture, root system is relatively more flourishing.
Contrast by embodiment 1 to embodiment 8 finds that the bottom of internal layer scale is the optimal material that fritillary bulb is organized cultivation.
Embodiment 9
A kind of method of utilizing tissue culture technique breeding fritillary bulb is characterized in that, may further comprise the steps:
(1) selection of explant and sterilization: the fresh fritillary bulb of at first selecting current growth stalwartness, the mildew and rot spot of nothing, cut off root and the scale of wound is arranged, flowing water washes to be put into clear water behind 30 min and soaks 15min, soak 30 min or soak 15min with 500 times of liquid of 50% tmtd powder with 500 times of liquid of 50% carbendazim again, to plant the ball scale then peels, with ultra violet lamp 10 min, change superclean bench over to, use 75% alcohol disinfecting, 50 s again, aseptic water washing 3 times, again with 6% liquor natrii hypochloritis, 10 min that sterilize, aseptic water washing 3 times.At last with 0.2% mercuric chloride solution, 10 min that sterilize, aseptic water washing 5 times.
(2) material processed: at first on superclean bench, under aseptic condition, the fritter of 6-8mm is cut in the bottom of selection internal layer scale;
(3) it is foster to be commissioned to train at the beginning of: on the explant that under the aseptic condition step (2) is obtained is seeded in just for medium, this medium is to add the 6-BA of 0.8mg/L, the NAA of 1.2mg/L, the sucrose of 40mg/L and the ZT of 0.8mg/L in the conventional medium of 3/4MS; Cultivation temperature is 24 ℃, light application time 12 h/d, and intensity of illumination 2 000 Lx, PH are 6.5, agar 5 g/L were through 25 days formation callus;
(4) subculture is cultivated: the callus that will just be commissioned to train foster downcuts and is seeded on the subculture medium, this medium is to have added the KT of IBA, 0.3mg/L of 6-BA, 0.3mg/L of 0.5mg/L and the sucrose of 20mg/L in the conventional medium of MS, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, differentiated green point through 12 day time after, namely begin next stage;
(5) propagation of clove: the callus that will differentiate green point continues to cultivate, this medium is to have added the sucrose of 40mg/L, KT, the 0.4mg/LNAA of 0.3mg/L and the ZT of 0.2mg/L in the conventional medium of 1/2MS, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, cultivate through 10 days and just can differentiate the clove that grows thickly;
(6) culture of rootage: the clove that differentiation is come out is inoculated in and carries out root induction in the following inducing culture: the active carbon of KT, the 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 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, treat that through 10 days clove grows to the 2.5cm height, when root system is relatively more flourishing, namely begin the next stage;
(7) acclimatization and transplants: earlier blake bottle is moved to the greenhouse, natural conditions lower refining seedling 6d, the clove that will grow new root is taken out from blake bottle then, shone in the sun 2 days, in the process of shining, want suitable shading, making intensity of illumination is 60% of natural daylight, temperature is about 18 ℃, after media surface becomes polygon, take out seedling and clean the medium that root adheres to, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: in the mixed-matrix of vermiculite=2: 2: 1:3 (weight ratio), temperature is controlled about 15 ℃.
The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes description of the present invention to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (2)
1. a method of utilizing tissue culture technique breeding fritillary bulb is characterized in that, may further comprise the steps:
(1) selection of explant and sterilization: at first select the current growth stalwartness, the fresh fritillary bulb that does not have the spot that goes rotten, cut off root and the scale of wound is arranged, flowing water washes to be put into clear water behind 30 min and soaks 15min, soak 20 ~ 30 min or soak 10 ~ 15min with 500 times of liquid of 50% tmtd powder with 500 times of liquid of 50% carbendazim again, to plant the ball scale then peels, with ultra violet lamp 10 min, change superclean bench over to, use 75% alcohol disinfecting, 50 s again, aseptic water washing 2 ~ 3 times is again with 6% liquor natrii hypochloritis, 10 min that sterilize, aseptic water washing 2 ~ 3 times, at last with 0.2% mercuric chloride solution, 10 min that sterilize, aseptic water washing 4 ~ 5 times;
(2) material processed: at first on superclean bench, under aseptic condition, cut scale top 1/3, then will in, the lower part isolates, and scale and bulb base portion is cut into the fritter of 6-8mm respectively;
(3) it is foster to be commissioned to train at the beginning of: on the explant that under the aseptic condition step (2) is obtained is seeded in just for medium, this medium is to add the 6-BA of 0.8mg/L, the NAA of 1.2mg/L, the sucrose of 30-40mg/L and the ZT of 0.4-0.8mg/L in the conventional medium of 3/4MS; Cultivation temperature is 24 ℃, light application time 12 h/d, and intensity of illumination 2 000 Lx, PH are 6.5, agar 5 g/L were through 25-30 days formation callus;
(4) subculture is cultivated: the callus that will just be commissioned to train foster downcuts and is seeded on the subculture medium, this medium is to have added the KT of IBA, 0.3mg/L of 6-BA, 0.3mg/L of 0.5mg/L and the sucrose of 20mg/L in the conventional medium of MS, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, and the 7-12 days time of process namely begins next stage after differentiating green point;
(5) propagation of clove: the callus that will differentiate green point continues to cultivate, this medium is to have added the sucrose of 40mg/L, KT, the 0.4mg/LNAA of 0.3mg/L and the ZT of 0.2mg/L in the conventional medium of 1/2MS, cultivation temperature is 24 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, PH 6.5, cultivate through 10-15 days and just can differentiate the clove that grows thickly;
(6) culture of rootage: the clove that differentiation is come out is inoculated in and carries out root induction in the following inducing culture: the active carbon of KT, the 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 ℃, periodicity of illumination 13h/d, luminous intensity is 2200lux, treat that through 8-12 days clove grows to the 2.5cm height, when root system is relatively more flourishing, namely begin the next stage;
(7) acclimatization and transplants: earlier blake bottle is moved to the greenhouse, natural conditions lower refining seedling 6d, the clove that will grow new root is taken out from blake bottle then, shone in the sun 1-2 days, in the process of shining, want suitable shading, making intensity of illumination is 60% of natural daylight, temperature is about 18 ℃, after media surface becomes polygon, take out seedling and clean the medium that root adheres to, be transplanted to the humus soil through sterilization: the peat composed of rotten mosses: perlite: in the mixed-matrix of vermiculite=2: 2: 1:3 (weight ratio), temperature is controlled about 15 ℃.
2. a kind of method of utilizing tissue culture technique breeding fritillary bulb according to claim 1 is characterized in that the optimal selection of described scale propagating materials is the bottom of internal layer scale.
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| CN104396746A (en) * | 2014-11-12 | 2015-03-11 | 北京林业大学 | Fritillaria verticillata adventitious bud induced propagation method |
| CN107258539A (en) * | 2017-07-06 | 2017-10-20 | 四川省中医药科学院 | A kind of method for controlling taipei fritillary bulb tissue cultures endophytic bacterial contamination |
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| 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 |
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| CN103444526B (en) * | 2013-08-16 | 2015-11-25 | 顾祥茂 | A kind of growth-promoting cultivation method of Fritillaria unibracteata |
| 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 |
| 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 |
| CN104396746A (en) * | 2014-11-12 | 2015-03-11 | 北京林业大学 | Fritillaria verticillata adventitious bud induced propagation method |
| 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 |
| CN107258539A (en) * | 2017-07-06 | 2017-10-20 | 四川省中医药科学院 | A kind of method for controlling 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 |
| CN110100730A (en) * | 2019-05-09 | 2019-08-09 | 云南山里红生物科技有限公司 | A kind of bulbus fritillariae cirrhosae method for tissue culture that can prevent browning |
| 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 |
| CN112931210A (en) * | 2021-03-18 | 2021-06-11 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
| 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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