CN107258539B - A method of control taipei fritillary bulb tissue cultures endophytic bacterial contamination - Google Patents
A method of control taipei fritillary bulb tissue cultures endophytic bacterial contamination Download PDFInfo
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- CN107258539B CN107258539B CN201710547593.7A CN201710547593A CN107258539B CN 107258539 B CN107258539 B CN 107258539B CN 201710547593 A CN201710547593 A CN 201710547593A CN 107258539 B CN107258539 B CN 107258539B
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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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- 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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Abstract
The present invention relates to field of biotechnology, and in particular to a method of control taipei fritillary bulb tissue cultures endophytic bacterial contamination.Control method of the present invention includes: S1, explant selection and pretreatment: selection taipei fritillary bulb Fresh Bulbs are impregnated in washing powder solution, are wiped, rinse, filter paper blots, spare；S2, explant disinfection treatment: pretreated explant is immersed in mercuric chloride solution, stirring, in superclean bench, is rinsed, dry, spare；S3, Bulblet induction differentiation: the explant scale that S2 processing obtains is broken into two with one's hands, 2~4mm wide fritter is slit longitudinally into, is inoculated on the culture medium for being added to penicillin and carbendazim and cultivates；S4, squamous subculture: every 20 days squamous subcultures are primary；S5 clove Multiplying culture: the healthy clove that S4 is obtained is transferred on the culture medium for not adding any bactericidal agent and cultivates.The method that the present invention controls taipei fritillary bulb tissue cultures endophytic bacterial contamination, endophytic bacterial contamination control rate can reach 96.5%.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to endophyte is dirty in a kind of control taipei fritillary bulb tissue culture procedures
The method of dye.
Background technique
The bulbus fritillariae cirrhosae that the Pharmacopoeia of the People's Republic of China records is liliaceous plant bulbus fritillariae cirrhosae Fritillaria
Cirrhosa D.Don, Fritillaria unibracteata Fritillaria unibracteata Hsiao et K.C.Hsia, Gansu fritillaria
Fritillaria przewalskii Maxim. Bulbus Fritillariae cirrhosae Fritillaria delavayi Franch. taipei fritillary bulb
Fritillaria taipaiensis P.Y.Li or watt cloth fritillaria Fritillaria unibracteata Hsiao et
K.C.Hsia var wabuensis (Y.Tang et S.C.Yue) Z.D.Liu, the dry bulb of S.C.Chen.It is rare river
Birth canal ground one of bulk medicinal materials, are mainly used for lung-heat type cough, dry cough lacks phlegm, and deficiency of yin and overstrain cough coughs up the illnesss such as sputum streaked with blood, clinical demand
It measures huge.
Taipei fritillary bulb is included in 2010 editions " Chinese Pharmacopoeias " as newly-increased source, has few, adaptable, the low sea of wild resource
Pull out also can the characteristics such as normal growth and development, the best kind for family's kind cultivation that be in bulbus fritillariae cirrhosae be suitable for.Taipei fritillary bulb planting industry welcomes
New opportunity to develop.
And existing bulbus fritillariae cirrhosae yield wretched insufficiency, demand but increase year by year, there are serious disparities between supply and demand.Its medicinal material comes
There are 4 kinds to be included in state three level protecting plant register in source and plantation is difficult.Within its growth period biomass accumulation slowly, plant
The death rate is high, and so as to cause low in economic efficiency, peasant planting enthusiasm is not high, this is always the bottle for restricting planting bulbus fritilariae development
Neck.
It has recorded " the quick reproduction test of taipei fritillary bulb " " CHINA JOURNAL OF CHINESE MATERIA MEDICA " 1996 01 is interim, has been reported in text
The test of taipei fritillary bulb tissue-culturing quick-propagation.Research shows that: MS+NAA1.0mg/L+6~BA3.0mg/L culture medium is used, is lured
It leads callus and bulb bearing frequency is 93% again；In callus and regeneration bulb containing 15 kinds of constants needed by human and
Microelement, wherein the content of most elements is significantly more than cultivation bulb.But it is cultivated in the prior art by the method
The probability that obtained bulb infects endophyte in cultivating process is very big, can go on smoothly and turn low for the ratio for continuing culture.
Therefore, very crucial to the pollution of control taipei fritillary bulb tissue culture endophyte.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of control taipei fritillary bulb tissue cultures endophytes
The high control method of pollution rate.
The control method of endophyte, includes the following steps: in taipei fritillary bulb tissue culture procedures of the present invention
S1, explant are selected and are pre-processed: using the taipei fritillary bulb Fresh Bulbs taken for 3~April as explant, removing outer layer
It rots, the scale of browning, cuts off root system；Bulb surface is cleaned, impregnates 4~6min in washing powder solution, wipes bulb surface, stream
Water rinses 0.8~1.2h, then is rinsed 4~6 times with deionized water, and filter paper blots, spare；
S2, explant disinfection treatment: by S1 treated explant, it is immersed in the mercury chloride that concentration is 0.08~0.12%
In solution, stirring rinses in superclean bench, dry, spare；
S3, Bulblet induction differentiation: the aseptic explant that S2 is obtained is broken into two with one's hands its scale with sterilized tweezers, then
Fritter, is inoculated in the culture for being added to penicillin and carbendazim by the strip fritter that 2~4mm wide is obtained with the longitudinal sectional scale of sterile razor blade
It is cultivated on base；
S4, clove squamous subculture: by the clove of the induction differentiation culture of S3 step in same medium, every 20 days
Squamous subculture is primary, altogether squamous subculture 3 times；
S5, clove Multiplying culture: the eugonic healthy clove that S4 is obtained is transferred to and does not add any sterilizing
85~95d of Multiplying culture on the culture medium of agent.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein washing powder solution described in S1 step be
It is configured by washing powder and water according to 0.8~1.2g:1000mL of w/v.
Further, the method for above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, is wherein washed described in S1 step
Clothing powder solution is to be configured by washing powder and water according to w/v 1.0g:1000mL.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein stirring uses magnetic force in the S2 step
Blender stirring, mixing time are 6~8min, and mixing speed is 150~200r/min.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein rinsing is with sterile in the S2 step
Water rinses 3~5 times, and each rinsing time is 3~5min；Dry is to be blotted with aseptic filter paper.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein cultivation temperature is 18 in the S3 step
~22 DEG C, relative humidity is 75~85%, and intensity of illumination is 1000~1500Lx, and light application time is 11~13h.
Further, the method for above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein being trained in the S3 step
Supporting temperature is 20 DEG C, relative humidity 80%, and intensity of illumination is 1000~1500Lx, light application time 12h.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein the pH value of the culture medium be 5.6~
5.8；Be by+0.8~1.2mg/L of 6- benzyl aminoadenine of+3.0~5.0mg/L of MS culture medium α-naphthylacetic acid+4.0~
+ 3% sucrose+9g/L agar of the penicillin+0.8 of 6.0mg/L~1.2g/L carbendazim composition.
Further, the method for above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein the induced medium
It is by the penicillin+1.0g/L of the methyl α-naphthyl acetate+5.0mg/L of the 6- benzyl aminoadenine+1.0mg/L of MS culture medium+4.0mg/L
+ 3% sucrose+9g/L agar of carbendazim composition.
The method of above-mentioned control taipei fritillary bulb tissue cultures endophytic bacterial contamination, wherein culture medium described in S5 step is by MS
+ 3% sucrose+9g/L agar of methyl α-naphthyl acetate of+0.8~1.2mg/L of minimal medium+3.0~5.0mg/L 6- benzyl aminoadenine
Composition.
The beneficial effects of the present invention are:
1, taipei fritillary bulb tissue cultures endophytic bacterial contamination is controlled, contamination control rate reaches 96.5%, and explant usage amount subtracts
It is few, reduce production cost；
2, taipei fritillary bulb tissue cultures endophytic bacterial contamination is controlled, direct induced synthesis clove is more, produces to peasant household, enterprise
Provenance is in short supply is of great importance for plantation；
3, pollution rate is controlled, and bulb flourish is fast, is provided a large amount of bulb medicinal materials for market, is alleviated imbalance between supply and demand.
Specific embodiment
Technical solution of the present invention is described in further detail combined with specific embodiments below, but protection scope of the present invention is not
It is confined to as described below.
Embodiment 1
1, explant selection and pretreatment:
The taipei fritillary bulb Fresh Bulbs in 3~April of excavation make explant, remove outer layer rot, the scale of browning, cut off root
System；Bulb surface soil is cleaned, 5min is impregnated in the washing powder solution that concentration is 1g/L, with flowing after brush gently brushing surface
Water rinses 1h, then is rinsed 5 times with aseptic deionized water, and filter paper blots, spare；
2, explant is disinfected: by treated, explant is placed in wide-mouth bottle, under room temperature, pours into concentration 0.1%
Mercuric chloride solution is to flooding explant.6~8min of explant, 75% alcohol wipe wide-mouth bottle table are stirred at low speed with magnetic stirring apparatus
It is put into superclean bench behind face, under aseptic condition, with rinsed with sterile water bulb 3~5 times, 3~5min, finally uses sterilizing every time
Filter paper blots, spare；
3, the induction differentiation of clove: under aseptic condition, taipei fritillary bulb scale is broken into two with one's hands with tweezers, with sterile razor blade by meat
Matter scale is slit longitudinally into the fritter of about 3mm wide, is inoculated on Bulblet induction culture medium, the culture medium are as follows: and MS minimal medium+
6-BA (6- benzyl aminoadenine) 4.0mg/L+NAA (α-naphthylacetic acid) 1.0mg/L+ penicillin 5.0mg/L+ carbendazim 1.0g/L+
Sucrose 3%+9g/L agar 1.0%, pH are 5.6~5.8.After inoculation, it is placed in 20 DEG C of temperature, relative humidity 80%, illumination 12h,
Intensity is cultivated under the conditions of being 1000~1500Lx；
4, squamous subculture: it is primary to the every 20 days squamous subcultures of clove progress after induction differentiation in same medium,
Squamous subculture 3 times altogether；
5, clove Multiplying culture: aging and undergrowth bulb is removed, and selects the small of eugonic health
Bulb is cut, and removes base portion residue, is transferred to culture medium MS+6-BA (6- benzyl aminoadenine) 4.0mg/L+NAA (α-naphthalene
Acetic acid) 1.0mg/L+ sucrose 3%+9g/L agar 1.0%, pH is 5.6~5.8 progress shoot proliferation cultures, is counted after cultivating 90d
Clove survival rate.
In order to reduce endophytic bacterial contamination during taipei fritillary bulb bulb tissue culture, the proliferation rate and survival rate of bulb are improved.Examination
It tests design and adds two kinds of bactericidal agents of carbendazim and penicillin in culture medium MS+6-BA4.0mg/L+NAA1.0mg/L, in it
Raw bacterium pollution is controlled.Concentration is shown in Table 1.Two factor completely randoms, each processing are shown in Table 2.
With culture medium: MS+6-BA 4.0mg/L+NAA 1.0mg/L is control.Under aseptic condition, with sterile razor blade by meat
Matter scale is slit longitudinally into the fritter of about 3mm wide, is inoculated in each processing culture medium, 5 repetitions/processing, and 5 bottles/repeat, 3 pieces of bulbs/
Bottle.Other conditions are consistent.It directly removes, is not counted if any pollution scale in 7d.After 7d, endophytic bacterial contamination situation is monitored daily,
Such as pollution, untainted scale is shifted immediately, and pollution scale continues to observe.And observe and record Bulblet induction differentiation situation.60d
Count endophytic bacterial contamination rate.
1 experimental factor of table and concentration
Culture medium is managed everywhere in endophyte growth during the control taipei fritillary bulb bulb tissue culture of table 2
| Processing | Culture medium |
| 1 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 5.0mg/L+ carbendazim 0.5g/L |
| 2 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 5.0mg/L+ carbendazim 1.0g/L |
| 3 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 5.0mg/L+ carbendazim 1.5g/L |
| 4 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 10.0mg/L+ carbendazim 0.5g/L |
| 5 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 10.0mg/L+ carbendazim 1.0g/L |
| 6 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 10.0mg/L+ carbendazim 1.5g/L |
| 7 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 20.0mg/L+ carbendazim 0.5g/L |
| 8 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 20.0mg/L+ carbendazim 1.0g/L |
| 9 | MS+6-BA4.0mg/L+NAA1.0mg/L+ penicillin 20.0mg/L+ carbendazim 1.5g/L |
| CK | MS+6-BA4.0mg/L+NAA1.0mg/L |
3 taipei fritillary bulb bulb tissue culture endophytic bacterial contamination application form of table
Note:
As a result:
1, the culture medium (CK) of penicillin and carbendazim is not added, endophytic bacterial contamination is serious, and pollution rate is up to 72.3%；
2, penicillin and carbendazim concentration are too low (processing 1), and pollution rate is higher (23.2%), are differentiated to form clove, after
Phase growth is preferable；
3, increase with penicillin and carbendazim concentration, endophytic bacterial contamination reduces, but scale is undifferentiated does not induce small squama
Stem.
It is comprehensively compared, handles 2 penicillin and carbendazim concentration proportion is most suitable, pollution rate is low (2.5%), and scale breaks up shape
At clove it is more, and grow it is vigorous.Compared with the control, endophytic bacterial contamination control rate reaches 96.5%.
The above is only a preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form should not be regarded as an exclusion of other examples, and can be used for other combinations, modifications, and environments, and can be at this
In the text contemplated scope, modifications can be made through the above teachings or related fields of technology or knowledge.And those skilled in the art institute into
Capable modifications and changes do not depart from the spirit and scope of the present invention, then all should be in the protection scope of appended claims of the present invention
It is interior.
Claims (8)
1. the method for controlling taipei fritillary bulb tissue cultures endophytic bacterial contamination, which comprises the steps of:
S1, explant are selected and are pre-processed: using the taipei fritillary bulb Fresh Bulbs taken for 3~April as explant, removing outer layer corruption
Rotten, browning scale cuts off root system；Bulb surface is cleaned, 4~6min is impregnated in washing powder solution, wipes bulb surface, flowing water
0.8~1.2h is rinsed, then is rinsed 4~6 times with deionized water, filter paper blots, spare；
S2, explant disinfection treatment: by S1 treated explant, it is immersed in the mercuric chloride solution that concentration is 0.08~0.12%
In, stirring rinses in superclean bench, and it is dry, it is spare；
S3, Bulblet induction differentiation: its scale is broken into two with one's hands with sterilized tweezers, then uses nothing by the aseptic explant that S2 is obtained
The longitudinal sectional scale of bacterium blade obtains the strip fritter of 2~4mm wide, and fritter is inoculated on culture medium and is cultivated；
S4, squamous subculture: by the clove of the culture of S3 step in same medium, every 20 days squamous subcultures are primary, altogether after
It is commissioned to train feeding 3 times；
S5, clove Multiplying culture: the eugonic healthy clove that S4 is obtained is transferred to and does not add any bactericidal agent
85~95d of Multiplying culture on culture medium；
Cultivation temperature is 18~22 DEG C in the S3 step, and relative humidity is 75~85%, and intensity of illumination is 1000~1500Lx,
Light application time is 11~13h；
The pH value of culture medium described in S3, S4 step is 5.6~5.8；It is by the 6- benzyl of+3.0~5.0mg/L of MS minimal medium
Aminoadenine+0.8~1.2mg/L α-naphthylacetic acid+4.0~6.0mg/L penicillin+0.8~1.2g/L carbendazim+3%
Sucrose+9g/L agar composition.
2. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that S1 step
Described in washing powder solution be to be configured by washing powder and water according to 0.8~1.2g:1000mL of w/v.
3. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 2, which is characterized in that S1 step
Described in washing powder solution be to be configured by washing powder and water according to w/v 1.0g:1000mL.
4. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that the S2
Stirring uses magnetic stirrer in step, and mixing time is 6~8min, and mixing speed is 150~200r/min.
5. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that the S2
Rinsing is, with rinsed with sterile water 3~5 times, each rinsing time is 3~5min in step；Dry is to be blotted with aseptic filter paper.
6. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that the S3
Cultivation temperature is 20 DEG C in step, relative humidity 80%, and intensity of illumination is 1000~1500Lx, light application time 12h.
7. the method for control taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that described
Induced medium is by the α-naphthylacetic acid+5.0mg/L of the 6- benzyl aminoadenine+1.0mg/L of MS minimal medium+4.0mg/L
Penicillin+1.0g/L+3% sucrose+9g/L agar of carbendazim composition.
8. controlling the method for taipei fritillary bulb tissue cultures endophytic bacterial contamination according to claim 1, which is characterized in that S5 step
Described in culture medium be by α-naphthalene of+0.8~1.2mg/L of 6- benzyl aminoadenine of+3.0~5.0mg/L of MS minimal medium
+ 3% sucrose+9g/L agar of acetic acid composition.
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