CN106212289A - A kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system - Google Patents
A kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system Download PDFInfo
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- CN106212289A CN106212289A CN201610743823.2A CN201610743823A CN106212289A CN 106212289 A CN106212289 A CN 106212289A CN 201610743823 A CN201610743823 A CN 201610743823A CN 106212289 A CN106212289 A CN 106212289A
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- bulbil
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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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- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to field of plant tissue culture, a kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system of concrete offer, comprise the steps: 1) gather, 2) stem-segment with single bud cleans, 3) sprout peels off, and 4) sterilization of outer implant, 5) inoculation.The method of the present invention is outer implant with oil with the bulbil of Paeonia suffruticosa, through strict cleaning, peels off and sterilizes, the efficient aseptic culture system of structure after starting surrounding, pollution rate≤10%；Melting brown rate≤20%；Inductivity >=70%, solves current Paeonia suffruticosa tissue culture technology and is applied to during production practices set up the problem that aseptic culture system efficiency is low.
Description
Technical field
The invention belongs to field of plant tissue culture, be specifically related to a kind of foundation oil Paeonia suffruticosa bulbil efficient aseptic culture body
The method of system.
Background technology
The current oil promoted in China essentially from Paeonia ostii (Paeonia ostii) population, is from medicinal pellet of uniting with Paeonia suffruticosa
Paeonia ostii that skin produces, view and admire with Paeonia ostii and be used as the Paeonia ostii population of ornamental peonies stock and select.The biography of Paeonia ostii breeding
System method is sowing, and owing to seed is the family half sibs that free pollination obtains, its genotypic variation is relatively big, and the seedling obtained exists
Plant height, adaptability, resistance, aspect of blossoming and bearing fruit vary, and are not suitable for scale oilseed and produce.Therefore, from 2006
Since Nian, after within particularly 2011, Ministry of Public Health approval peony seed oil is country's new resource food, many scientific research institutions and colleges and universities
And agricultural business unit puts into a large amount of manpower and materials, it is engaged in oil and uses Paeonia suffruticosa good variety selection, release some successively through examining
Fixed specific breed.But, breeding of these improved seeds, obtain the strong sprout that hereditary quality is consistent, it is clear that can not be by sexual
The mode of breeding obtains, and the main way grafting nourished and generated, and breeding coefficient is low, and cost is high, by woody material source and
Season limits, so tissue-culturing quick-propagation becomes the one preferred technique approach of large-scale production tank oil Paeonia suffruticosa seedling.Lose
Regret, from till now 32 years in 1984, although including that the Paeonia tissue culture document of each Paeonia suffruticosa population is more than 400
, but Section Moutan training does not but have successful case the most together for what commercial seedling produced, and therefore Paeonia suffruticosa tissue culture technology walks out laboratory
And conscientiously in producing the bottleneck always restricting Paeonia suffruticosa industry development.
Cause Paeonia suffruticosa tissue culture technology can not form the four problems that has of actual productivity:
First, set up aseptic culture system efficiency low, from technological layer particularly, current existing Paeonia suffruticosa aseptic culture
The outer implant body pollution rate of system high (40-70%), melting brown rate are high (50-100%), inductivity low (less than 30%).
Second, Shoot propagation rate is low, subculture multiplication bud is of poor quality, and vitrification physiological abnormalities is serious.
3rd, rooting rate is low, and functional root system is not enough.
4th, Section Moutan seedlings cultivating transplanting survival rate is low, less than 42%.
Summary of the invention
It is an object of the invention to provide a kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system.
A kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system of the present invention, comprises the steps:
1) gather: contain the stem section of 2-3 full bulbil in healthy and strong oil with Paeonia suffruticosa branch bottom clip, and be cut into long 2.5-4
Centimetre stem-segment with single bud；
2) stem-segment with single bud clean: by stem-segment with single bud with the sodium dichloro cyanurate solution soaking of mass percentage concentration 5% after,
Flowing water cleans sodium dichloro cyanurate solution residual, then removes petiole with liquid detergent after scrubbing, and carefully scrubs at petiole disengaging, stream
Water rinses, and divests the phloem of stem-segment with single bud, alcohol-pickled with percent by volume 70%；
3) sprout peel off: take out step 2) process after stem-segment with single bud, blot surface liquid, with the blade after alcohol disinfecting
Divest the scale of bulbil, only retain one layer of scale of innermost layer, after blade is dipped 50% lotion of compound flavescent sophora root, excise sprout base
Light green vascular tissue is exposed by cortical tissue of portion, is chosen by sprout from stem-segment with single bud, and sprout base portion retains a small amount of xylem, will
Sprout is put in the culture dish filling a thin layer 100mg/L penicillin sodium；
4) outer implant sterilization: step 3) gained sprout is with ultraviolet disinfection, then the mercuric chloride solution with mass percentage concentration 0.1%
Sterilization, with aseptic water washing more than 5 times；
5) inoculation: the sprout after sterilization is wiped out the scale of innermost layer, is inoculated on Primary culture base and cultivates, it is thus achieved that
Oil Paeonia suffruticosa bulbil efficient aseptic culture system.
Step 1) described collection, the time is preferably the sunny noon of mid or late January.
Step 1) described stem-segment with single bud is the stem section pointing to a bulbil.Preferably the upper end clip of stem-segment with single bud away from
1-1.5 centimetre from bulbil center, 1.5-2.5 centimetre, bulbil center of lower end clip distance.Described upper end refers to relatively
The port slightly held close to branch, lower end refers to be relatively close to the port of branch base portion.
Step 2) time of described sodium dichloro cyanurate solution soaking is 30 minutes.
Step 2) described flowing water clean sodium dichloro cyanurate solution residual, the time used is 30 minutes.
Step 2) described in scrub, for scrubbing with small brushes, the preferred toothbrush of described small brushes.
Step 2) flushing of described flowing water, the time used is 10-20 minute.
Step 2) described alcohol-pickled with percent by volume 70%, the time used is 60 seconds.
Step 3) described in divest the scale of bulbil, method particularly includes: it is upper and lower that left index finger, thumb pinch stem-segment with single bud respectively
Two ends, sprout, in the face of operator, withstands sprout reverse side stem section xylem with middle finger first knuckle；The right hand is held and was dipped 70% ethanol
Single-edge blade, with point of a knife Boli scale.
Step 3) described sprout is chosen from stem-segment with single bud, method particularly includes: in each deep partial application in sprout base portion left and right
To the xylem of stem-segment with single bud, with point of a knife on the downside of sprout base portion on choose, sprout is chosen from stem-segment with single bud.
Step 3) described a small amount of xylem, preferably size be about the xylem that 1mm is square, 0.5mm is thick.
Step 4) described ultraviolet disinfection, for ultra violet lamp 20-30min.
Step 4) sterilization of described mercuric chloride solution, detailed step is: every 5 pieces of sprouts are put into through autoclaved aseptic empty three
In the bottle of angle, to entering the mercuric chloride solution of mass percentage concentration 0.1%, instill a polysorbas20, shake 8min, slow rotary triangle bottle
Turn solution in making bottle and flow through triangle bottleneck everywhere, solution is poured out.
Step 5) described inoculation, the xylem of sprout submerges culture medium.
Step 5) described Primary culture base is with modified MS medium as minimal medium, PVP (PVP)
Content 1.0g/L, activated carbon (AC) content 0.5g/L, 6-benzyl aminopurine (6-BA) content 0.5g/L, Fructus Citri Limoniae juice (Lemon) are (new
Lemon extruding obtain) content 1g/L, caseinhydrolysate (CH) content 0.5g/L, gibberellins (GA3) content 0.3mg/L, sucrose
Content 30g/L, agar content 6g/L, the culture medium of pH value 5.8.
Step 5) described cultivation, its environmental condition is temperature 22 ± 2 DEG C, relative humidity 30%, and first week is dark, the most then
50μmol photon·m-2·s-1Illumination.
The present invention also provides for the described method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system, cultivates in peony tissue
In application.
The beneficial effects of the present invention is:
The oil set up with the inventive method Paeonia suffruticosa bulbil efficient aseptic culture system, after starting surrounding, pollution rate
≤ 10%；Melting brown rate≤20%；Inductivity >=70%.
Accompanying drawing explanation
Fig. 1 is embodiment 1 step 1) in the photo of stem-segment with single bud.
Fig. 2 is embodiment 1 step 2) in sprout peel off under phloem photo.
Fig. 3 is embodiment 1 step 3) middle with the photo of point of a knife Boli scale.
Fig. 4 is embodiment 1 step 3) in point of a knife on the downside of sprout base portion on the schematic picture chosen.
Fig. 5 is embodiment 1 step 3) in the photo of sprout peeled off.
Fig. 6 is embodiment 1 step 3) in the sprout of stripping put into the photograph in the culture dish filling a thin layer penicillin sodium
Sheet.
Fig. 7 is embodiment 1 step 5) the middle photo inoculating outer implant.
Fig. 8 is embodiment 1 step 5) in inoculation after sealing photo.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
The part chemical reagent that following example relate to is as follows: 50% lotion of compound flavescent sophora root (traditional Chinese medicines quasi-word B20020433,
Zhejiang Sino-french Pharmaceutical Co., Ltd), 100mg/L penicillin sodium (traditional Chinese medicines Zhun Zi H13020657 Huabei Pharmaceutic Co., Ltd).
Remaining chemical reagent is conventional chemical reagent, the most commercially available.
Embodiment 1
1) gather: the sunny noon of mid or late January, in healthy and strong oil Paeonia suffruticosa branch bottom, select the full person of sprout, from base
Portion shears branch；Choose bottom 2-3 bud, during broken strip, be cut into 2.5-4 centimetre of stem-segment with single bud, note upper end clip distance bud centre bit
Put 1-1.5 centimetre, 1.5-2.5 centimetre, bud center of lower end clip distance.Load immediately and seal bag, sealing, take back reality as early as possible
Test room.
2) stem-segment with single bud (Fig. 1 is shown in by photo) cleans: laboratory taken back by material, and stem-segment with single bud immerses 5% dichloro isocyanuric urine
Acid sodium solution, soaks 30min；Flowing water rinses 30min；Dip in liquid detergent with toothbrush and scrub stem-segment with single bud, remove petiole, carefully scrub
Petiole base position；Flowing water rinses 10min；Divest stem section phloem (phloem being stripped out is shown in Fig. 2), then stem section is thrown
Enter immersion 60s in 70% ethanol.
3) sprout peels off: taking out rapidly stem-segment with single bud from ethanol, clean filter paper blots surface solution；Left index finger, thumb
Referring to pinch the upper and lower two ends of stem-segment with single bud respectively, sprout, in the face of operator, withstands sprout reverse side stem section with middle finger first knuckle wooden
Portion；The right hand holds the single-edge blade dipping 70% ethanol, with point of a knife Boli scale (see Fig. 3), until last layer of scale；Pass through
The blade of alcohol disinfecting, dips 50% lotion of compound flavescent sophora root (traditional Chinese medicines quasi-word B20020433, Zhejiang Sino-french Pharmaceutical Co., Ltd),
With blade, sprout base portion cortical tissue is excised gently, expose light green vascular tissue；Deep partial application on the upside of sprout base portion, must
Must be as deep as stem section xylem, each partial application in left and right, then with point of a knife on the downside of sprout base portion gently on choose that (concrete operations see figure
4), retain the xylem that about 1mm is square, 0.5mm is thick, the sprout (Fig. 5 is shown in by photo) peeled off is put into and fills a thin layer
(see figure in the clean culture dish of 100mg/L penicillin sodium (traditional Chinese medicines Zhun Zi H13020657 Huabei Pharmaceutic Co., Ltd) solution
6)。
4) outer implant sterilization: culture dish moves into super-clean bench, opens ultra violet lamp 20min；Blow in machine, turn off uviol lamp；
With the tweezers through sterilization, sprout is put in autoclaved aseptic empty triangular flask (150ml), tweezers and three before inoculation
Angle bottle bottleneck calcination sterilization, every bottle of 5 pieces of sprout (outer implant), pour 0.1% mercuric chloride into, instill a polysorbas20, shake 8min；Right
Bottom hand-held triangular flask, left hand prop up triangular flask cervical region with control height, slow rotary triangle bottle, disinfectant solution is poured into gently useless
In liquid bucket, note must by rotation make sterilization liquid stream flow through triangle bottleneck everywhere, there is no blind spot.Then with aseptic water washing 5
More than Bian.
5) inoculation: horizontal the holding of left hand fills the aseptic triangular flask of outer implant (sterilization bottle), and bottleneck is positioned at alcohol burner flame top, right
Hand-held through autoclaved elbow shears, wipe out interior one layer of perula；Then the sterilization bottle of outer implant is filled with horizontal the holding of left hand
With fill the culture bottle of Primary culture base, bottleneck is positioned at alcohol burner flame top, and the right hand is that beak shape is armed with thumb and forefinger
Shape tweezers, move into outer implant culture bottle from sterilization bottle, and the xylem of outer implant base portion submerges and (sees Fig. 7) in culture medium,
After inoculation, bottleneck puts sealing (seeing Fig. 8) on alcohol burner flame.
Primary culture base: improvement MS+1.0g/L PVP+0.5g/L AC+0.5g/L 6-BA+1g/L Lemon+0.5g/L
CH+0.3mg/L GA3Every liter of culture medium is containing sucrose 30 grams, 6 grams of agar, pH value 5.8.
Improvement MS minimal medium formula is shown in Table 1:
Table 1: improvement MS minimal medium formula
Condition of culture: temperature 22 ± 2 DEG C, relative humidity 30%, first week dark, the most then 50 μm ol photon m-2·s-1Illumination.
Cultivation results: after starting surrounding, pollution rate≤10%；Melting brown rate≤20%；Inductivity >=70%.
Claims (10)
1. the method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system, it is characterised in that comprise the steps:
1) gather: contain the stem section of 2-3 full bulbil in healthy and strong oil with Paeonia suffruticosa branch bottom clip, and be cut into long 2.5-4 centimetre
Stem-segment with single bud；
2) stem-segment with single bud clean: by stem-segment with single bud with the sodium dichloro cyanurate solution soaking of mass percentage concentration 5% after, flowing water
Cleaning sodium dichloro cyanurate solution residual, then remove petiole after scrubbing with liquid detergent, carefully scrub at petiole disengaging, flowing water rushes
Wash, divest the phloem of stem-segment with single bud, alcohol-pickled with percent by volume 70%；
3) sprout peel off: take out step 2) process after stem-segment with single bud, blot surface liquid, divest with the blade after alcohol disinfecting
The scale of bulbil, only retains one layer of scale of innermost layer, after blade is dipped 50% lotion of compound flavescent sophora root, excises sprout base portion skin
Layer tissue exposes light green vascular tissue, is chosen by sprout from stem-segment with single bud, and sprout base portion retains a small amount of xylem, by sprout
Put in the culture dish filling a thin layer 100mg/L penicillin sodium；
4) outer implant sterilization: step 3) gained sprout is with ultraviolet disinfection, then disappear with the mercuric chloride solution of mass percentage concentration 0.1%
Poison, with aseptic water washing more than 5 times；
5) inoculation: the sprout after sterilization is wiped out the scale of innermost layer, is inoculated on Primary culture base and cultivates, it is thus achieved that oil is used
Paeonia suffruticosa bulbil efficient aseptic culture system.
2. the method for claim 1, it is characterised in that step 1) described collection, the time is preferably the fine of mid or late January
Bright noon.
3. the method for claim 1, it is characterised in that step 1) described stem-segment with single bud upper end clip distance bulbil in
1-1.5 centimetre, heart position, 1.5-2.5 centimetre, bulbil center of lower end clip distance.
4. the method for claim 1, it is characterised in that step 2) time of described sodium dichloro cyanurate solution soaking
It it is 30 minutes.
5. the method for claim 1, it is characterised in that step 2) to clean sodium dichloro cyanurate solution residual for described flowing water
Staying, the time used is 30 minutes.
6. the method for claim 1, it is characterised in that step 3) described a small amount of xylem, square for 1mm, 0.5mm is thick
Xylem.
7. the method for claim 1, it is characterised in that step 4) described ultraviolet disinfection, for ultra violet lamp
20min。
8. the method for claim 1, it is characterised in that step 4) sterilization of described mercuric chloride solution, detailed step is: every 5
Piece sprout is put in autoclaved aseptic empty triangular flask, to entering the mercuric chloride solution of mass percentage concentration 0.1%, instills one
Drip polysorbas20, shake 8min, slow rotary triangle bottle turn make bottle in solution flow through triangle bottleneck everywhere, solution is poured out.
9. the method for claim 1, it is characterised in that step 5) described Primary culture base is to be with modified MS medium
Minimal medium, PVP content 1.0g/L, activated carbon content 0.5g/L, 6-benzyl aminopurine content 0.5g/L,
Fructus Citri Limoniae juice content 1g/L, caseinhydrolysate content 0.5g/L, GA content 0.3mg/L, cane sugar content 30g/L, agar content
6g/L, the culture medium of pH value 5.8.
10. the method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system described in any one of claim 1-9, in peony tissue
Application in cultivation.
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Cited By (1)
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| CN111602594A (en) * | 2020-04-20 | 2020-09-01 | 四川农业大学 | Method for reducing explant pollution in peony tissue culture |
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Application publication date: 20161214 |