CN104782488A - Tissue culture method of Fritillaria unibracteata Hsiao - Google Patents
Tissue culture method of Fritillaria unibracteata Hsiao Download PDFInfo
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- CN104782488A CN104782488A CN201510216173.1A CN201510216173A CN104782488A CN 104782488 A CN104782488 A CN 104782488A CN 201510216173 A CN201510216173 A CN 201510216173A CN 104782488 A CN104782488 A CN 104782488A
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Abstract
The invention discloses a tissue culture method of Fritillaria unibracteata Hsiao. Fritillaria unibracteata Hsiao is a liliaceous fritillary medicinal plant, and the underground bulb of the Fritillaria unibracteata Hsiao is used as medicine and has the effects of clearing heat and removing stasis, preventing phlegm from forming and stopping coughing, and the like. As wild Fritillaria unibracteata Hsiao mostly grows in high-altitude regions, and the growing period is long, shortage of the resource is caused. As a result, the medicinal Fritillaria unibracteata Hsiao has the problem of short supply in the long term. According to the tissue culture method, the scale of the Fritillaria unibracteata Hsiao is taken as an explant, a Fritillaria unibracteata Hsiao in-vitro regenerated plant is obtained by virtue of the processes such as explant sterilization, adventitious bud induction, multiplication culture, rooting culture, and acclimatization and transplanting, and an effective tissue culture rapid propagation technical system of the Fritillaria unibracteata Hsiao is established; as a result, a large quantity of seedlings can be produced in short time, the multiplication efficiency is improved, the production cost can be reduced, and then the breeding and popularization of the Fritillaria unibracteata Hsiao can be accelerated favorably.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of Fritillaria unibracteata method for tissue culture.
Background technology
Fritillaria unibracteata is the distinctive rare traditional Chinese medicine of China, is Liliaceae Fritillaria herbaceous plant, is used as medicine with underground bulb.There are clearing heat and moistening lung, preventing phlegm from forming and stopping coughing effect, are used for the treatment of the symptoms such as cold cough, lung-heat type cough, the few phlegm of dry cough.Fritillaria unibracteata primary growth is on the plateau and mountains meadow of height above sea level 2800 ~ 4500 meters, and growth cycle is slow, and very harsh to requirement for environmental conditions, it is expensive, and its wild resource is on the verge of exhaustion.Therefore, the Fritillaria unibracteata long-term existence problem that supply falls short of demand.
At present, Fritillaria unibracteata mainly adopts bulb and seminal propagation, and bulb breeding method sowing quantity is large and reproduction coefficient is low, and general 1 bulb can only receive 1.5 ~ 1.6 bulbs.Seminal propagation planting percent is low, and speed is slow, needs the size that just can develop into commodity bulb for 5 ~ 6 years.And the reproduction speed of Fritillaria unibracteata can be improved by tissue culture technique, expanding propagation coefficient, substantially reduce the formation time limit of bulb, as long as the time of about 6 months just can obtain the bulb Gong being used for medicinal purpose.Therefore, the present invention with Fritillaria unibracteata scale for explant, the in vitro plant again of Fritillaria unibracteata is obtained by processes such as explant sterilization, adventitious bud inducing, Multiplying culture, culture of rootage, acclimatization and transplantses, establish effective Fritillaria unibracteata tissue-culturing rapid propagation plantation technology system, a large amount of seedling can be produced in short-term, improve reproductive efficiency, reduce production cost, favourable quickening its breed and promote.
Summary of the invention
The object of the present invention is to provide out a kind of Fritillaria unibracteata method for tissue culture, the present invention with Fritillaria unibracteata scale for explant, the in vitro plant again of Fritillaria unibracteata is obtained by processes such as explant sterilization, adventitious bud inducing, Multiplying culture, culture of rootage, acclimatization and transplantses, establish effective Fritillaria unibracteata tissue-culturing rapid propagation plantation technology system, thus achieve object of the present invention.
A kind of Fritillaria unibracteata method for tissue culture of the present invention, comprises the following steps:
(1) explant sterilization: with Fritillaria unibracteata bulb for explant, first 75% alcohol immersion 15 ~ 30s,
Aseptic water washing 3 ~ 5 times, then soak 20 ~ 30min with 10% ~ 20% liquor natrii hypochloritis, for subsequent use after aseptic water washing 3 ~ 5 times.
(2) Fiber differentiation: the Fritillaria unibracteata bulb that step (1) is handled well be cut into the stripping and slicing of about 0.5cm and be inoculated in inducing culture and carry out adventitious bud inducing.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 1000 ~ 2000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% after 30 days to add up inductivity.
(3) Multiplying culture: obtain indefinite bud as material using step (2) induction, aging is removed with dysgonic indefinite bud, and indefinite bud good for growth conditions is cut into simple bud and proceeds to proliferated culture medium to carry out expansion numerous.Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% after 30 days to add up proliferative conditions.
(4) culture of rootage: the comparatively consistent and aseptic seedling individual plant of not taking root of stalwartness cuts and proceeds to root media induction by the growth obtained after step (3).Inoculation is placed on illumination every day 12 ~ 14 hours, and intensity of illumination is 2000 ~ 3000lx, and being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% to add up situation of taking root afterwards in 30 days.
(5) acclimatization and transplants: the aseptic seedling of taking root is taken out from culturing room and is placed on outdoor hardening after 30 days, again seedling of taking root is taken out the medium washing away base portion from bottle, then be transplanted in the matrix (peat soil: perlite: vermiculite=3:1:1) of high-temperature sterilization, regularly water 1/2MS macro-element nutrients liquid, cover film, take off film after 10 days, within after taking off film 20 days, add up survival rate.
Inducing culture described in above-mentioned steps (2) is: MS+1.0 ~ 5.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+0.5 ~ 1mg/L AC+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+0.1 ~ 1.0mg/L 6-BA+0.1 ~ 1.0mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (4) is: 1/2MS+0.1 ~ 0.5mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Advantage of the present invention is for explant with Fritillaria unibracteata scale, obtain the in vitro plant again of Fritillaria unibracteata by processes such as explant sterilization, adventitious bud inducing, Multiplying culture, culture of rootage, acclimatization and transplantses, establish effective Fritillaria unibracteata tissue-culturing rapid propagation plantation technology system.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
embodiment 1
(1) explant sterilization: with Fritillaria unibracteata bulb for explant, first 75% alcohol immersion 15s,
Aseptic water washing 3 times, then soak 20min with 10% liquor natrii hypochloritis, for subsequent use after aseptic water washing 3 times.
(2) Fiber differentiation: the Fritillaria unibracteata bulb that step (1) is handled well be cut into the stripping and slicing of about 0.5cm and be inoculated in inducing culture and carry out adventitious bud inducing.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivate inductivity after 30 days under the condition of 75% to reach 85.7%.Described inducing culture is: MS+1.0mg/L NAA+0.1mg/L 6-BA+0.5mg/L AC+15g/L sucrose+3.5g/L agar, pH is 5.4.
(3) Multiplying culture: obtain indefinite bud as material using step (2) induction, aging is removed with dysgonic indefinite bud, and indefinite bud good for growth conditions is cut into simple bud and proceeds to proliferated culture medium to carry out expansion numerous.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivate growth coefficient after 30 days under the condition of 75% to reach 5.86.Described proliferated culture medium is: MS+0.1mg/L 6-BA+0.1mg/LNAA+15g/L sucrose+3.5g/L agar, pH is 5.4.
(4) culture of rootage: the comparatively consistent and aseptic seedling individual plant of not taking root of stalwartness cuts and proceeds to root media induction by the growth obtained after step (3).Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivate rooting rate after 30 days under the condition of 75% to reach 91.8%.Described root media is: 1/2MS+0.1mg/LNAA+15g/L sucrose+3.5g/L agar, pH is 5.4.
(5) acclimatization and transplants: the aseptic seedling of taking root is taken out from culturing room and is placed on outdoor hardening after 30 days, again seedling of taking root is taken out the medium washing away base portion from bottle, then be transplanted in the matrix (peat soil: perlite: vermiculite=3:1:1) of high-temperature sterilization, regularly water 1/2MS macro-element nutrients liquid, cover film, take off film after 10 days, after taking off film, 20 days survival rate are to 93.4%.
embodiment 2
(1) explant sterilization: with Fritillaria unibracteata bulb for explant, first 75% alcohol immersion 25s,
Aseptic water washing 5 times, then soak 25min with 15% liquor natrii hypochloritis, for subsequent use after aseptic water washing 4 times.
(2) Fiber differentiation: the Fritillaria unibracteata bulb that step (1) is handled well be cut into the stripping and slicing of about 0.5cm and be inoculated in inducing culture and carry out adventitious bud inducing.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is cultivate inductivity after 30 days under the condition of 75% to reach 93.7%.Described inducing culture is: MS+3.0mg/L NAA+0.5mg/L 6-BA+0.5mg/L AC+25g/L sucrose+3.5g/L agar, pH is 5.4.
(3) Multiplying culture: obtain indefinite bud as material using step (2) induction, aging is removed with dysgonic indefinite bud, and indefinite bud good for growth conditions is cut into simple bud and proceeds to proliferated culture medium to carry out expansion numerous.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 3000lx, and being placed in cultivation temperature is 27 DEG C, and relative air humidity is cultivate growth coefficient after 30 days under the condition of 75% to reach 6.92.Described proliferated culture medium is: MS+0.5mg/L 6-BA+0.2mg/LNAA+15g/L sucrose+4.5g/L agar, pH is 5.4.
(4) culture of rootage: the comparatively consistent and aseptic seedling individual plant of not taking root of stalwartness cuts and proceeds to root media induction by the growth obtained after step (3).Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2000lx, and being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivate rooting rate after 30 days under the condition of 75% to reach 94.1%.Described root media is: 1/2MS+0.3mg/LNAA+18g/L sucrose+4.0g/L agar, pH is 5.4.
(5) acclimatization and transplants: the aseptic seedling of taking root is taken out from culturing room and is placed on outdoor hardening after 30 days, again seedling of taking root is taken out the medium washing away base portion from bottle, then be transplanted in the matrix (peat soil: perlite: vermiculite=3:1:1) of high-temperature sterilization, regularly water 1/2MS macro-element nutrients liquid, cover film, take off film after 10 days, after taking off film, 20 days survival rate are to 91.8%.
Claims (4)
1. a Fritillaria unibracteata method for tissue culture, is characterized in that comprising the following steps:
(1) explant sterilization: with Fritillaria unibracteata bulb for explant, first 75% alcohol immersion 15 ~ 30s,
Aseptic water washing 3 ~ 5 times, then soak 20 ~ 30min with 10% ~ 20% liquor natrii hypochloritis, for subsequent use after aseptic water washing 3 ~ 5 times;
(2) Fiber differentiation: the Fritillaria unibracteata bulb that step (1) is handled well be cut into the stripping and slicing of about 0.5cm and be inoculated in inducing culture and carry out adventitious bud inducing, inoculation is placed on illumination every day 12 ~ 14 hours, intensity of illumination is 1000 ~ 2000lx, being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% after 30 days to add up inductivity;
(3) Multiplying culture: obtain indefinite bud as material using step (2) induction, aging is removed with dysgonic indefinite bud, and indefinite bud good for growth conditions is cut into simple bud and proceeds to proliferated culture medium to carry out expansion numerous, inoculation is placed on illumination every day 12 ~ 14 hours, intensity of illumination is 2000 ~ 3000lx, being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% after 30 days to add up proliferative conditions;
(4) culture of rootage: the comparatively consistent and aseptic seedling individual plant of not taking root of stalwartness cuts and proceeds to root media induction by the growth obtained after step (3), inoculation is placed on illumination every day 12 ~ 14 hours, intensity of illumination is 2000 ~ 3000lx, being placed in cultivation temperature is 25 ~ 28 DEG C, and relative air humidity is cultivate under the condition of 75% to add up situation of taking root afterwards in 30 days;
(5) acclimatization and transplants: the aseptic seedling of taking root is taken out from culturing room and is placed on outdoor hardening after 30 days, again seedling of taking root is taken out the medium washing away base portion from bottle, then be transplanted in the matrix (peat soil: perlite: vermiculite=3:1:1) of high-temperature sterilization, regularly water 1/2MS macro-element nutrients liquid, cover film, take off film after 10 days, within after taking off film 20 days, add up survival rate.
2. a kind of Fritillaria unibracteata method for tissue culture according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+1.0 ~ 5.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+0.5 ~ 1mg/L AC+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. a kind of Fritillaria unibracteata method for tissue culture according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+0.1 ~ 1.0mg/L 6-BA+0.1 ~ 1.0mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. a kind of Fritillaria unibracteata method for tissue culture according to claim 1, is characterized in that the root media described in step (4) is: 1/2MS+0.1 ~ 0.5mg/LNAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105265314A (en) * | 2015-10-20 | 2016-01-27 | 韦丽 | Rapid propagation method for tendrilleaf fritillary bulbs |
| CN107258535A (en) * | 2017-06-10 | 2017-10-20 | 内江师范学院 | A kind of wild bulb rapid propagation method of Fritillaria unibracteata |
| CN107318655A (en) * | 2017-08-11 | 2017-11-07 | 云南青谷生物科技有限公司 | A kind of bulbus fritillariae cirrhosae seedling fostering method |
| 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105265314A (en) * | 2015-10-20 | 2016-01-27 | 韦丽 | Rapid propagation method for tendrilleaf fritillary bulbs |
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| CN107318655A (en) * | 2017-08-11 | 2017-11-07 | 云南青谷生物科技有限公司 | A kind of bulbus fritillariae cirrhosae seedling fostering method |
| 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 |
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