CN105638458A - Tissue cultivation method of Fritillaria cirrhosa D.Don - Google Patents
Tissue cultivation method of Fritillaria cirrhosa D.Don Download PDFInfo
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- CN105638458A CN105638458A CN201511018871.7A CN201511018871A CN105638458A CN 105638458 A CN105638458 A CN 105638458A CN 201511018871 A CN201511018871 A CN 201511018871A CN 105638458 A CN105638458 A CN 105638458A
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- 241001547127 Fritillaria cirrhosa Species 0.000 title abstract 2
- 238000012364 cultivation method Methods 0.000 title abstract 2
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 23
- 230000006698 induction Effects 0.000 claims abstract description 22
- 230000001954 sterilising effect Effects 0.000 claims abstract description 22
- 230000004069 differentiation Effects 0.000 claims abstract description 12
- 238000009395 breeding Methods 0.000 claims abstract description 8
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 5
- 239000007943 implant Substances 0.000 claims description 24
- 238000005286 illumination Methods 0.000 claims description 23
- 244000223014 Syzygium aromaticum Species 0.000 claims description 22
- 235000016639 Syzygium aromaticum Nutrition 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 18
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 239000005720 sucrose Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 230000001939 inductive effect Effects 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 8
- 239000008399 tap water Substances 0.000 claims description 7
- 235000020679 tap water Nutrition 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000010408 film Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000012879 subculture medium Substances 0.000 claims description 4
- 230000002745 absorbent Effects 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000003044 adaptive effect Effects 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000013039 cover film Substances 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 235000016709 nutrition Nutrition 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
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- 239000003643 water by type Substances 0.000 claims description 3
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- 238000007598 dipping method Methods 0.000 claims 1
- 239000011435 rock Substances 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 5
- 206010011224 Cough Diseases 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 230000001850 reproductive effect Effects 0.000 abstract description 3
- 241000605372 Fritillaria Species 0.000 abstract description 2
- 206010062717 Increased upper airway secretion Diseases 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 210000004072 lung Anatomy 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 208000026435 phlegm Diseases 0.000 abstract description 2
- 230000007812 deficiency Effects 0.000 abstract 1
- 238000003306 harvesting Methods 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 230000000644 propagated effect Effects 0.000 abstract 1
- 238000002054 transplantation Methods 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 3
- 241001644290 Fritillaria unibracteata Species 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- 238000012090 tissue culture technique Methods 0.000 description 2
- 241001644297 Fritillaria delavayi Species 0.000 description 1
- 241000935235 Fritillaria meleagris Species 0.000 description 1
- 241001644294 Fritillaria pallidiflora Species 0.000 description 1
- 241000827798 Hemerocallis citrina Species 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Biotechnology (AREA)
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Abstract
The invention discloses a tissue cultivation method of bulbus fritillariae cirrhosae. Fritillaria cirrhosa D.Don is a Liliaceae Fritillaria perennial medicinal herb, underground bulbs are used as medicine, the bulbus fritillariae cirrhosae has the effects of clearing away heat and moistening the lungs, preventing phlegm from forming and stopping coughing, and is one of the main sources for the famous traditional Chinese medicinal material bulbus fritilariae specified in the Chinese Pharmacopoeia (Year 2005). The bulbus fritillariae cirrhosae is distributed in high altitude localities, is propagated through seeds, and is low in reproductive rate and long in growth cycle; due to long-term harvesting and wild resource deficiency, the problem of large market demand needs to be solved urgently. Bulbs of the bulbus fritillariae cirrhosae are used as explants, bulbus fritillariae cirrhosae in-vitro regenerated plants are obtained through the processes of explant sterilization, induction of callus tissues in two different states, bulb differentiation, multiplication, rooting culture, acclimatization and transplantation and the like, an effective tissue culture rapid propagation system is established, a large number of seedlings can be produced in a short period of time, the breeding efficiency is improved, and the propagation and popularization of the bulbus fritillariae cirrhosae are facilitated.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae (FritillariacirrhosaD.Don).
Background technology
Bulbus Fritillariae Cirrhosae (FritillariacirrhosaD.Don) is Liliaceae Fritillaria perennial herb medicinal plants, and medicinal part is underground bulb, has effect of clearing heat and moistening lung, preventing phlegm from forming and stopping coughing, is the important good medicine for the treatment of cough. Bulbus Fritillariae Cirrhosae and Fritillaria unibracteata (FritillariaunibracteataHsiaoetK.C.Hsia), Gansu Bulbus Fritillariae Uninbracteatae (FritillariaprzewalskiiMaxim), Bulbus Fritillariae cirrhosae (FritillariadelavayiFranch.) etc. are all in " Chinese Pharmacopoeia " (2005) main source of the famous Chinese crude drug Bulbus Fritillariae Cirrhosae specified. Bulbus Fritillariae Cirrhosae main product in Sichuan, Tibet, the provinces and regions such as Yunnan, like cold and cool weather conditions, have cold-resistant, happiness is wet, be afraid of the characteristic that high temperature, Xi Yin cover, temperature reaches 30 DEG C or ground temperature more than 25 DEG C, and plant will wither, low at height above sea level, can not survive in the area that temperature is high. Bulbus Fritillariae Cirrhosae is by seminal propagation under natural environment, and growth cycle is long, and general 4-5 just can gather, and because of the characteristic of its seed existing forms and physiology after-ripening, breeding coefficient is low, and yield is also low. Manually introduce a fine variety Bulbus Fritillariae Cirrhosae to carry out producing mainly by bulb breeding, bulb is by screening, big bulb is used as medicine, clove is planted, vary in size because of it after clove plantation and need 2-3 to gather in the crops, wasting the bulb resource of preciousness so on the one hand, adding again production cost on the other hand, and yield is not high, it is impossible to meet the market demand of Bulbus Fritillariae Cirrhosae. At present, Bulbus Fritillariae Cirrhosae medication is largely dependent upon wild resource, and through excavating for many years, wild resource destroys serious, and yield reduces, and resource is very in short supply, and it is very urgent for therefore finding new approach to solve its resource problem.
Plant tissue culture technique can fundamentally solve the seedling problem of Bulbus Fritillariae Cirrhosae, and its cost is low, instant effect, and breeding potential is high, can keep again the merit of kind. It addition, the drug effect of Bulbus Fritillariae Cirrhosae callus can be studied by organizing training, utilize callus to produce effective substance and be likely to become effective substitute technology. Therefore, the research of Bulbus Fritillariae Cirrhosae tissue culture method all had important practice significance in medicinal plants develops and produces. In existing patent, the tissue culture about Bulbus Fritillariae Uninbracteatae has been reported, but quantity is few, the kind related to has Fritillaria unibracteata, Hupeh Fritillary Bulb, Fritillaria pallidiflora Schrenk, Bulbus Fritillariae Ussuriensis, Hemerocallis citrina Baroni Bulbus Fritillariae Uninbracteatae, Anhui Bulbus Fritillariae Uninbracteatae, Gansu Bulbus Fritillariae Uninbracteatae etc., wherein, report about Bulbus Fritillariae Cirrhosae (FritillariacirrhosaD.Don) is less, and Wang Yuehua etc. has carried out the induction research of callus with the root of Bulbus Fritillariae Cirrhosae for outer implant.Xue Gang etc. report the method that directly induction produces Bulbus Fritillariae Uninbracteatae bulb from Bulbus Fritillariae Cirrhosae carnification spire, and induce Bulbus Fritillariae Cirrhosae callus and embryoid to produce with young tender leaf sheath for outer implant. The present invention utilizes tissue culture technique with the bulb of Bulbus Fritillariae Cirrhosae for outer implant, is obtained the regeneration of clove by two kinds of approach (i.e. the callus of different conditions), and realizes amount reproduction. Compared with the method for tissue culture of other Bulbus Fritillariae Uninbracteataes, the present invention has the higher rate of increase, it is possible to from the reproductive speed largely improving Bulbus Fritillariae Cirrhosae.
Summary of the invention
The technical problem that present invention mainly solves is to provide the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae (FritillariacirrhosaD.Don), this method utilizes tissue culture technique, a large amount of seedlings can be produced in short time, improve reproductive efficiency, amount reproduction Bulbus Fritillariae Cirrhosae, is conducive to the breeding accelerating Bulbus Fritillariae Cirrhosae to promote.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows:
The method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae, comprises the following steps:
(1) selection of outer implant and process: choose healthy fresh Bulbus Fritillariae Cirrhosae bulb, rinse under tap water, earth is cleaned with banister brush, and remove the root of bulb base portion, dip a small amount of liquid detergent dilute solution (1g liquid detergent is dissolved in 100ml distilled water) again and clean outer implant, and clean with tap water, about rinse 30 minutes; To cuiller and the more close bulb of squama hearty cord composition and division in a proportion, whole bulb surface clean is clean, if cuiller and squama have gap in the heart, for the earth inside wash clean, cuiller and the squama heart need to be broken into two with one's hands and clean, clean standby;
(2) outer implant sterilization: clean bulb is put in small beaker, go on superclean bench, pour 75% alcoholic solution into and jiggle, soak 8-10s, outwell ethanol, with aseptic washing 2 times, and bulb is relayed to through the aseptic bottle of high temperature sterilize from beaker, add 0.1% mercuric chloride solution (1gHgcl2 is dissolved in 1000ml distilled water) to soak, shake, sterilizing 8-10min, outwells mercuric chloride, with aseptic washing 6 times, each 2min, finally stays part sterilized water to soak outer implant standby;
(3) callus induction: with aseptic absorbent paper, the outer implant disinfected is blotted surface moisture, aseptically cuts into the fritter of 0.5-0.8cm, is inoculated in just for, in callus inducing medium, inducing Bulbus Fritillariae Uninbracteatae callus. The cultivation temperature of culturing room is 23 �� 1 DEG C, relative air humidity 70%-80%, light application time 12 hour/day, intensity of illumination 2000Lx. Initially forming callus through 15 days, this callus is fine and close bulk (Bulbus Fritillariae Cirrhosae callus has two kinds: a kind of block callus for densification, and one is loose particles shape callus).
(4) propagation of block callus and differentiation: Bulbus Fritillariae Cirrhosae bulb dice, after (3) induction, forms block callus, and this tissue line is closely, in light green color, faint yellow. This callus is cut into the fritter of about 0.6cm, is inoculated in subculture medium, it may be achieved the propagation of callus, and the differentiation of Bulbus Fritillariae Uninbracteatae clove occurs simultaneously. Condition of culture: temperature 23 �� 1 DEG C, relative air humidity 70%-80%, light application time 12 hour/day, intensity of illumination 2000Lx. 30 days subcultures of this process once, can subculture repeatedly, obtain more clove;
(5) induction of loose particles shape callus and propagation: the block dense callus after step (4) subculture, can by just producing loose graininess callus for calli induction media induction, this callus cell height breaks up, growth rate is fast, is inoculated in proliferated culture medium and can realize short time amount reproduction. Condition of culture: illumination 500-1200Lx, temperature about 21 DEG C, humidity 70%-80%, light application time 12 hour/day, in breeding, best 25 days of Subculture Time;
(6) differentiation of embryoid and growth: in step (5), the callus of gained mostly is embryo callus, it can be cultivated in MS culture medium through the regular hour under illumination condition and obtain Bulbus Fritillariae Cirrhosae clove embryoid, and this embryoid continues to cultivate the clove that can grow up to health. Condition of culture: intensity of illumination 1500-2000LX, light application time 10-15 hour/day, cultivation temperature 19-22 DEG C, humidity 70%-80%.
(7) root culture: cut one by one by the clove obtained in step (4) and step (6), be inoculated in root media, can root induction. The cultivation temperature of culturing room is 23 �� 1 DEG C, relative air humidity 70%-80%, light application time 12 hour/day, intensity of illumination 2000Lx. Treating that clove length is high to 5cm, root system is more healthy and stronger, and when growing 6-10 bar root, can carry out the next stage;
(8) acclimatization and transplants: what step (7) obtained takes root aseptic seedling in bottle outlet forward to greenhouse seedbed, keep ambient temperature 20-25 DEG C, humidity 80%-85%, about intensity of illumination 5000Lx, cultivate 1 week, it is gradually opened bottleneck, cultivates 3-4 days, strengthen the adaptive capacity of environment to external world. Seedling of taking root is taken out from bottle the culture medium cleaning base portion again, is then transplanted to preprepared by fertile soil: perlite: in the substrate that Vermiculitum=4:2:1 forms, regularly waters and execute MS nutritional solution, cover film, take off film after one week, take off 20 days statistics survival rates of film.
Above-mentioned steps (3) described inducing culture is: MS+0.5-1mg/L6-BA+0.5-1.5mg/LNAA+1-2mg/L2,4-D+30g/L sucrose+4.5-5g/L agar (pH5.8,121 DEG C sterilizing 25 minutes).
The described proliferation and differentiation culture medium of above-mentioned steps (4) is: MS+0.3-2mg/L6-BA+0.1-1mg/L2,4-D+30g/L sucrose+4.5-5g/L agar (pH5.8,121 DEG C sterilizing 25 minutes).
The proliferated culture medium of the described graininess callus of above-mentioned steps (5) is: MS+0-1.5mg/L6-BA+0-0.5mg/L2,4-D+0-0.5mg/LNAA+30g/L sucrose+4.5-5g/L agar (pH5.8,121 DEG C sterilizing 25 minutes).
The differentiation of above-mentioned steps (6) described embryonic callus induction clove embryoid and growth medium be: MS+0-0.5mg/L6-BA+30-40g/L sucrose+4.5-6g/L agar (pH5.8,121 DEG C sterilizing 25 minutes).
Above-mentioned steps (7) described root media is: MS+0.2-0.5mg/LIBA+0-0.2mg/LKT+30mg/L sucrose+5g/L agar (pH5.8,121 DEG C sterilizing 25 minutes).
It is an advantage of the invention that with Bulbus Fritillariae Cirrhosae bulb for outer implant, obtain Bulbus Fritillariae Cirrhosae Regeneration in Vitro plant by processes such as the sterilization of outer implant, the induction of the callus of two states, propagation and differentiation, bulb root culture, acclimatization and transplantses, establish effective Bulbus Fritillariae Cirrhosae tissue-culturing rapid propagation system.
Accompanying drawing explanation
Fig. 1 is block and loose particles shape Bulbus Fritillariae Cirrhosae callus photo;
Fig. 2 is block and loose particles shape Bulbus Fritillariae Cirrhosae clove differentiation photo;
Fig. 3 is the root culture photo of Bulbus Fritillariae Cirrhosae clove.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing 1-3, the invention will be further described, it will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention. Unreceipted concrete technology or condition person in embodiment, technology or condition described by the document in this area or carry out according to product description. Agents useful for same or the unreceipted production firm person of instrument, be and can pass through to buy the conventional products obtained.
Embodiment 1
(1) selection of outer implant and process: select fresh, healthy Bulbus Fritillariae Cirrhosae bulb is outer implant, under tap water, earth is cleaned up with banister brush, remove bulb fibrous root, dip a small amount of liquid detergent dilute solution (1g is dissolved in 100ml distilled water) again and clean outer planting surface, clean with tap water, about rinse 30 minutes;Heal more close bulb to cuiller and the squama heart, and whole bulb surface clean is clean, if the cuiller of bulb and squama have gap in the heart, in order to clean the earth of the inside, cuiller and the squama heart need to be broken into two with one's hands and clean, clean standby;
(2) outer implant sterilization: with Bulbus Fritillariae Cirrhosae bulb for outer implant, with tap water Bulbus Fritillariae Uninbracteatae bulb, cleans earth, removes fibrous root, on superclean bench, with the soak with ethanol 10s of 75%, aseptic water washing 2 times, 0.1% mercuric chloride solution immersion 8min, aseptic water washing 5 times, each 2 minutes. Finally, part sterilized water is stayed to soak outer implant standby;
(3) callus induction: with aseptic absorbent paper, the bulb disinfected in step (2) is blotted surface moisture, aseptically cuts into the fritter of about 0.6cm, is inoculated in just in inducing culture. Inoculation is placed on 24 DEG C of cultivations, relative air humidity 80%, intensity of illumination 2000Lx, illumination every day 12 hours. Described for inducing culture it is just: MS+0.5mg/L6-BA+1mg/LNAA+1.5mg/L2,4-D+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes. Observing after 30 days, callus induction rate reaches 83.5%;
(4) differentiation of the propagation of block callus and clove: the block callus obtained with step (3) induction, for material, is cut into the fritter of 0.8cm, is inoculated in subculture medium. Inoculation is placed on temperature 24 DEG C, relative air humidity 80%, intensity of illumination 2000Lx, cultivates when illumination every day 12 hours. After cultivating 30 days, it is seen that become big to block callus volume, having the little bulbil of part to be formed on callus surface, now sprout is less, after more than 40 day, it is seen that occur to more little bulbil, and many little bulbil cluster clusters raw. Described propagation and be divided into same culture medium: MS+0.3mg/L6-BA+0.6mg/L2,4-D+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes;
(5) induction of loose particles shape callus and propagation: the block dense callus in step (4) is cut, it is inoculated in the callus inducing medium described in step (3), cultivates and obtain loose particles shape callus. This graininess callus is being inoculated in proliferated culture medium, it may be achieved a large amount of propagation of this callus. Described proliferated culture medium is: MS+0.25mg/L6-BA+0.5mg/L2,4-D+0.5mg/LNAA+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes. Condition of culture: illumination 500-1200Lx, temperature about 21 DEG C, humidity 70%-80%, light application time 12 hour/day.
(6) induction of embryoid and growth: in step (5), the callus of gained mostly is embryo callus, it can be cultivated in MS culture medium through the regular hour under illumination condition and obtain Bulbus Fritillariae Cirrhosae clove embryoid, and this embryoid continues to cultivate the clove that can grow up to health. Described embryoid induction and growth medium be: MS+0.2mg/L6-BA+40g/L sucrose+5g agar, pH5.8,121 DEG C of sterilizings 25 minutes. Condition of culture: intensity of illumination 1500-2000LX, light application time 10-15 hour/day, cultivation temperature 19-22 DEG C, humidity 70%-80%.
(7) root culture: the healthy and strong clove obtained in step (4) and step (6) is proceeded to root media, root induction. Inoculation is placed on temperature 24 DEG C, relative air humidity 80%, 12 hours every days of illumination, cultivates, observe rooting rate, up to 93% after 30 days when intensity of illumination 2000LX.Described root media is MS+0.5mg/LIBA+0.1mg/LKT+30mg/L sucrose+5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes;
(8) acclimatization and transplants: what step (7) obtained takes root aseptic seedling in bottle outlet forward to greenhouse seedbed, keep ambient temperature 20-25 DEG C, humidity 80%-85%, about intensity of illumination 5000Lx, cultivate 1 week, it is gradually opened bottleneck, cultivates 3-4 days, strengthen the adaptive capacity of environment to external world. Again Seedling of taking root is taken out the culture medium washing out base portion from bottle, then it is transplanted to preprepared by fertile soil: perlite: in the substrate that Vermiculitum=4:2:1 forms, regularly waters and execute MS nutritional solution, cover film, take off film after one week, take off 20 days statistics survival rate 91% of film.
The present invention is not restricted to the described embodiments; described in above-described embodiment and description is that principles of the invention is described; without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these changes and improvements both fall within the claimed scope of the invention. Claimed scope is defined by appending claims and equivalent thereof.
Claims (7)
1. the method for tissue culture of a Bulbus Fritillariae Cirrhosae, it is characterised in that comprise the following steps:
(1) selection of outer implant and process: choosing healthy fresh Bulbus Fritillariae Cirrhosae bulb is outer implant, rinse under tap water, earth is cleaned with banister brush, and remove the fibrous root of bulb base portion, dip the liquid detergent dilute solution cleaning outer planting surface being dissolved in 100ml distilled water by 1g liquid detergent again, and with tap water 25-30 minute, rinse well, standby;
(2) outer implant sterilization: outer for clean bulb implant is put in small beaker, go on superclean bench, pour concentration 75% alcohol solution dipping into and rock 8-10s, take out and use aseptic water washing 2 times, and outer for bulb implant is placed in the aseptic bottle of high temperature sterilize, add and be dissolved in sterilizing 8-10min under the concentration 0.1% mercuric chloride solution immersion also earthquake that 1000ml distilled water is made by 1gHgcl2, outwell mercuric chloride, with aseptic washing 6 times, each 2min, part sterilized water is finally stayed to soak outer implant, standby;
(3) callus induction: the outer implant sterilized through previous step blots surface moisture with aseptic absorbent paper, aseptically cuts into the blockage of 0.5-0.8cm, is inoculated in just for inducing Bulbus Fritillariae Uninbracteatae callus in callus inducing medium; The cultivation temperature of culturing room is 23 �� 1 DEG C, relative air humidity 70%-80%, intensity of illumination 2000Lx, light application time 12 hour/day, initially forms callus through 15 days, and this callus is fine and close bulk;
(4) propagation of block callus and differentiation: obtain after step (3) is induced arrangement closely, in light green color or flaxen block callus, this bulk callus is cut into the blockage of 0.5-0.7cm, it is inoculated in subculture medium, realize the propagation of block callus, and the differentiation of Bulbus Fritillariae Cirrhosae clove occurs simultaneously, this subculture process once through one or many subculture, obtains more clove in 30 days; Condition of culture: cultivation temperature is 23 �� 1 DEG C, relative air humidity 70%-80%, intensity of illumination 2000Lx, light application time 12 hour/day;
(5) induction of loose particles shape callus and propagation: the block dense callus obtained after step (4) subculture, being inoculated in and just produce loose graininess callus for callus inducing medium induction, this loose graininess callus is inoculated in proliferated culture medium and realizes short time amount reproduction; Condition of culture: cultivation temperature 21 �� 1 DEG C, relative air humidity 70%-80%, intensity of illumination 500-1200Lx, light application time 12 hour/day, in breeding, Subculture Time is 24-26 days;
(6) differentiation of embryoid and growth: the loose particles shape callus breeding gained through step (5) mostly is embryo callus, cultivating in MS culture medium and obtain Bulbus Fritillariae Cirrhosae clove embryoid, this embryoid continues to cultivate the Bulbus Fritillariae Cirrhosae clove growing up to health in MS culture medium; Condition of culture: intensity of illumination 1500-2000LX, light application time 10-15 hour/day, cultivation temperature 19-22 DEG C, relative air humidity 70%-80%;
(7) root culture: cut by the Bulbus Fritillariae Cirrhosae clove obtained in step (4) and step (6), is inoculated in root media root induction; Condition of culture: cultivation temperature is 23 �� 1 DEG C, relative air humidity 70%-80%, light application time 12 hour/day, intensity of illumination 2000Lx, treats that Bulbus Fritillariae Cirrhosae clove length is high to 5cm, and when growing 6-10 bar root system, can carry out next step;
(8) acclimatization and transplants: what step (7) obtained takes root aseptic seedling in bottle outlet forward to greenhouse seedbed, keep ambient temperature 20-25 DEG C, humidity 80%-85%, intensity of illumination 5000Lx, cultivate 1 week, it is gradually opened bottleneck, cultivates 3-4 days, strengthen the adaptive capacity of environment to external world; Aseptic seedling is taken out from bottle the culture medium cleaning base portion again, is then transplanted to by weight proportion by fertile soil: perlite: in the substrate that Vermiculitum=4:2:1 forms, regularly waters and execute MS nutritional solution, cover film, take off film after one week, take off 20 days statistics survival rates of film.
2. the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae according to claim 1, it is characterized in that, step (3) and step (5) is described for callus inducing medium is just: MS+0.5-1mg/L6-BA+0.5-1.5mg/LNAA+1-2mg/L2,4-D+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes.
3. a kind of Bulbus Fritillariae Cirrhosae method for tissue culture according to claim 1, it is characterized in that, step (4) described subculture medium is: MS+0.3-2mg/L6-BA+0.1-1mg/L2,4-D+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes.
4. the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae according to claims 1, it is characterized in that, step (5) described proliferated culture medium is: MS+0-1.5mg/L6-BA+0-0.5mg/L2,4-D+0-0.5mg/LNAA+30g/L sucrose+4.5-5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes.
5. a kind of Bulbus Fritillariae Cirrhosae method for tissue culture according to claims 1, it is characterised in that the MS culture medium described in step (6) is: MS+0-0.5mg/L6-BA+30-40g/L sucrose+4.5-6g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes.
6. the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae according to claim 1, it is characterized in that, root media described in step (7) is: MS+0.2-0.5mg/LIBA+0-0.2mg/LKT+30mg/L sucrose+5g/L agar, pH5.8,121 DEG C of sterilizings 25 minutes.
7. the method for tissue culture of a kind of Bulbus Fritillariae Cirrhosae according to claim 1, it is characterised in that the process of the described outer implant of step (1), when the cuiller of the outer implant of bulb has gap in the heart with squama, breaks into two with one's hands cuiller and the squama heart and cleans.
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| 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 |
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| 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 |
| CN112970589A (en) * | 2021-05-12 | 2021-06-18 | 北京欣康研医药科技有限公司 | Tissue culture method and application of fritillaria cirrhosa |
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