CN108871842A - A kind of free-hand section method and its application of Chinese herbaceous peony rhizome tissue - Google Patents
A kind of free-hand section method and its application of Chinese herbaceous peony rhizome tissue Download PDFInfo
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- CN108871842A CN108871842A CN201810353453.0A CN201810353453A CN108871842A CN 108871842 A CN108871842 A CN 108871842A CN 201810353453 A CN201810353453 A CN 201810353453A CN 108871842 A CN108871842 A CN 108871842A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B26—HAND CUTTING TOOLS; CUTTING; SEVERING
- B26D—CUTTING; DETAILS COMMON TO MACHINES FOR PERFORATING, PUNCHING, CUTTING-OUT, STAMPING-OUT OR SEVERING
- B26D3/00—Cutting work characterised by the nature of the cut made; Apparatus therefor
- B26D3/28—Splitting layers from work; Mutually separating layers by cutting
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2806—Means for preparing replicas of specimens, e.g. for microscopal analysis
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Abstract
The invention discloses a kind of free-hand section method and its application of Chinese herbaceous peony rhizome tissue, free-hand section method includes, obtain clean fresh Chinese herbaceous peony rhizome tissue, dehydration to moisture content is 5-10%, rhizome tissue obtained above is cut along perpendicular to its y direction with blade, it obtains 20-30 μm of slab, above-mentioned slice is placed in dehydrated alcohol after removing excessive moisture, is placed in distilled water and is fully deployed to obtain the final product；Slice is placed under microscope and observes, takes pictures by the application of the dicing method, and completes in the 15min to take pictures after the slicing is completed.Free-hand section method provided by the present invention is easy to operate, slice efficiency is high, it is low in cost, the problems such as existing paraffin section method experiment condition is harsh, operating process is complicated, larger using time and the funds of toxic reagent and experiment investment can be thoroughly solved, used tool is simple and easy to get, cheap, without using toxic reagent, it is environmentally protective, it is widely used, theoretical and practical significance is great.
Description
Technical field
The invention belongs to plant tissue slice technical fields, and in particular to a kind of free-hand section method of Chinese herbaceous peony rhizome tissue
And its application.
Background technique
China is one of the centre of origin of world's Paeonia, has long cultivation and applicating history, flower in China
Big gorgeous beauty, mostly famous medical ornament flowers plant are deep to be liked by China and the people of the world.Fleshy root is the object of Chinese herbaceous peony platymiscium
Matter stores center, and only the normal growth of plant and development do not provide what powerful energy supply and its medicinal ingredient were extracted
Main source；The quality of the situation direct relation bloom of formation and the development of stem, is the basis that fancy points is able to be formed,
All have a direct impact to the quantity, quality and yield etc. bloomed.
Paeonia floristics is various, and Regional Distribution differs greatly, shape that is not of the same race and planting the rhizome between interior different cultivars
There are biggish differences for state and structure, thus the Rhizome structure anatomical study of Chinese herbaceous peony platymiscium is in botanical system classification, growth hair
Educate, Lycopersicum esculentum, florescence control and it is degeneration-resistant research etc. have important theoretical value and realistic meaning.
Traditionally, less to the rhizome anatomical study of Chinese herbaceous peony platymiscium, research method is mainly paraffin method.Meanwhile
The rhizome characteristic of Chinese herbaceous peony platymiscium is affected by developmental stage, while paraffin method is more harsh to experiment condition requirement,
Test process complexity is cumbersome, fix, be dehydrated from material, is transparent, waxdip, embedding, slice and dyeing etc. need to undergo it is one longer
Experimental period, wherein each link directly affects final experiment effect, it is difficult to carry out the application directly promoted on a large scale,
It needs to carry out groping for experiment condition according to material, practical application difficulty is larger；Meanwhile it needing using the toxic examination such as dimethylbenzene
Agent carries out transparent and dewaxing treatment, although related innocuous agents substitution existing at present, experimental period and cost are still higher,
Efficiency is still to be improved.
For researcher, the key of good dicing method is：1, slice energy is clear, is truly reflected reality
Object；2, method has versatility, is not limited by kind and stage of development difference；3, time and economic cost, operation letter are saved
It is single, environmentally friendly, efficient.
Existing dicing method cannot fully meet above-mentioned condition, thus show certain deficiency.In conclusion establishing
A kind of operating process is easy to learn and environmentally protective, and map obtained is clear and three-dimensional sense is strong, can be to different development stage
The free-hand section method that rhizome tissue is fast and efficiently dissected and observed is this field key technical problem urgently to be solved.
Summary of the invention
In view of the deficiencies of the prior art, it the present invention provides a kind of free-hand section method of Chinese herbaceous peony rhizome tissue and its answers
With.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of free-hand section method of Chinese herbaceous peony rhizome tissue, includes the following steps：
S1, clean fresh Chinese herbaceous peony rhizome tissue is obtained, dehydration to moisture content is 5-10%；
S2, the rhizome tissue that step S1 is obtained is cut with blade along perpendicular to its y direction, is obtained with a thickness of 20-30
μm slice；
S3, the obtained slice of step S2 is placed in dehydrated alcohol after removal excessive moisture, is placed in distilled water and opens up completely
Open to get.
Above-mentioned free-hand section method is directly operated using the fresh material of Paeonia axis, rhizome and root, experiment
Operating condition is easy to grope, and saves a large amount of conventional method material requested set times and cumbersome, harsh experiment flow, greatly
Experimental period is shortened, conventional efficient is greatly improved；And experimentation used in any conventional method without using having
Malicious reagent is suitble to operate under routine experimentation environmental condition, and method is portable strong, and operation requires lower, and applicability is extremely strong.Institute
It is clear to obtain picture, as a result reliably.
In the above-mentioned technical solutions, in step S2, the cutting is specially：Clutched up and down with left hand thumb and index finger away from
At the rhizome tissue's end cut about 0.8-1.2cm obtained from step S1, make 25-35 ° of rhizome tissue's fore-and-aft tilt, by little finger
It is placed on the experimental bench of upper left side as support, right hand thumb and index finger clutch blade end, and knife-blade abutted and is scabbled
Blade is firmly slightly pressed downward by the end face of rhizome tissue when ingressing, and then the direction from upper right side toward lower left is continuously promptly
Parallel cutting.It clutches rhizome tissue's end cut and little finger supporting bable tops is conducive to positions of materials when cutting and consolidates without position
It moves, by 25-35 ° of rhizome tissue's fore-and-aft tilt, is conducive to expand scope of sight, this inclination angle is conducive to when cutting the edge of a knife towards thumb
It is safe on the outside of index finger, guaranteeing to cut.
Preferably, in the above-mentioned technical solutions, in step S1, the dehydration is specially：When the rhizome tissue is meat
When root or tender stem, it is sequentially placed into the ethanol solution of gradient concentration and impregnates dehydration, it is preferable that the gradient concentration of the ethanol solution
For 50%-70%-85%-95%-100%-100%, the dewatering time in the ethanol solution of each gradient concentration is 0.8-1.2h；
When the rhizome tissue is full-bloom stage stalk, it is placed in dehydrated alcohol and impregnates 5-10min.Tender tissue water content is more, gradient
Dehydration is conducive to keep eucaryotic cell structure complete, and is dehydrated more abundant.Full-bloom stage stalk water content is low, and tissue fibers are flourishing, shortens de-
The water time can guarantee that water content in OK range, is conducive to cutting operation.
It is further preferred that in the above-mentioned technical solutions, in step S3, by the immersion for being sliced and being placed in dehydrated alcohol
Time is 1-2min.Be placed in dehydrated alcohol impregnate can be conducive to slice rapidly expansion, prevent slice crimp or occur fold down to
Institutional framework integrality is destroyed in subsequent operation.
Still further preferably, in the above-mentioned technical solutions, in step S3, the slice is placed in distilled water and is opened up completely
It opens specially：Clean glass slide is taken, 0.5-1ml distilled water is added dropwise, slice is placed on distilled water with microforceps, to the slice
After being fully deployed, slowly covers coverslip and the slice is completely covered.Distilled water can prevent slice transition dehydration and shrinkage, be conducive to
Histotomy reasonably adjusts position on glass slide；Bubble can be prevented and influence to observe by slowly covering coverslip.
Further preferably, in the above-mentioned technical solutions, in step S1, rhizome tissue's control is 5-7.5cm long
Segment, be conducive to finger and be directly fixed.
It is further preferred that in the above-mentioned technical solutions, the step S1 further includes, by the Sect. Rhiziridium before dehydration
The both ends edge knitted grows perpendicular direction with it and scabbles.It is conducive to both ends after scabbling while is dehydrated, can both shortens dewatering time,
Conveniently wherein one end is selected at random as needed to be cut.
It is further preferred that in the above-mentioned technical solutions, the blade is stainless steel double-sided blade, it is used as knife when being sliced
Piece half-and-half fractures.The stainless steel double-sided blade quality is thin, and good toughness is not perishable, is especially advantageous for production slice.
Still further preferably, in the above-mentioned technical solutions, in step S1, the rhizome tissue is selected from different development stage
The Chinese herbaceous peony fleshy root and stalk of normal growth, when sampling the rhizome tissue is rinsed well after be put into containing the self-styled of blotting paper
It is placed in bag and saves on ice, to guarantee that material is fresh and tender and tissue is complete.
The present invention also provides a kind of applications of free-hand section method, i.e., the slice are placed under microscope and are carried out
Observation, takes pictures, and completes in the 15min to take pictures after the slicing is completed, can be in if generating bubble because standing time is too long
A small amount of distilled water is dripped to remove it in coverslip side.
Advantages of the present invention：
Free-hand section method provided by the present invention is easy to operate, and slice efficiency is high, low in cost, can thoroughly solve existing
Paraffin section method experiment condition is harsh, operating process is complicated, larger using the time and funds of toxic reagent and experiment investment
The problems such as；And used tool is simple and easy to get, and it is cheap and do not need using toxic reagent, it is environmentally protective, it is widely used, to mention
High Efficiency and for relevant plant anatomy be sliced normalizing operation provide foundation, theoretical and practical significance is great.
Detailed description of the invention
Fig. 1 is her rattan Chinese herbaceous peony ' anatomical structure of Going banana ' stem in the embodiment of the present invention；
Fig. 2 is the anatomical structure of the tree peony tender stem in the embodiment of the present invention；
Fig. 3 is the anatomical structure of Chinese herbaceous peony ' Fenyunu ' stem in the embodiment of the present invention；
Fig. 4 is the anatomical structure of Chinese herbaceous peony ' Fenyunu ' rhizome in the embodiment of the present invention；
Fig. 5 is the anatomical structure of Chinese herbaceous peony ' Dafugui ' stem in the embodiment of the present invention；
Fig. 6 is the anatomical structure of Chinese herbaceous peony ' Dafugui ' rhizome in the embodiment of the present invention；
Fig. 7 is the anatomical structure of Chinese herbaceous peony ' Dafugui ' root in the embodiment of the present invention；
Fig. 8 is the Chinese herbaceous peony ' anatomical structure of Coral sunset ' stem in the embodiment of the present invention.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not limited to protection scope of the present invention for illustrating the present invention, and protection scope of the present invention is with claims
It is quasi-.
At the end of April to mid-May, to being planted in the national mountain flowers engineering center little Tang Germplasm Resources (Beijing, Changping
Area, the mountain little Tang town) Chinese herbaceous peony, her rattan Chinese herbaceous peony and peony plant underground fleshy root, stem and rhizome carry out observation sampling, Chinese herbaceous peony at this time
Medicine, her rattan Chinese herbaceous peony and tree peony are in florescence.
With stainless steel fork by the whole strain lifting of Chinese herbaceous peony, soil and sundries around meat root and rhizome are removed, is then chosen straight
The meat root and rhizome of diameter 0.5-1cm or so is completely cut it with scalpel, and clear water is rinsed well to be placed on to be inhaled equipped with immersion
In the plastics valve bag of water paper (water content of blotting paper, which pinches not drip with hand, to be advisable)；Tree peony, Chinese herbaceous peony are intercepted from current year raw basal part of stem
Medicine and her rattan Chinese herbaceous peony stem section, remove bud and part branches and leaves are placed in the plastics valve bag equipped with blotting paper.All material is set
It is stored in ice chest, band is spare to laboratory.
A kind of free-hand section method for present embodiments providing above-mentioned Chinese herbaceous peony rhizome tissue, specifically includes following steps：
S1, the root taken, stem and rhizome are taken out from valve bag, the section of 5cm or so is cut into after being handled again with clear water
Section, both ends is scabbled to be placed in 50% ethanol solution handles 1h, then according to 70%-85%-95%-100%-
100% concentration ethanol carries out serial dehydration processing, and every grade of gradient handles 1h, and the dissection for stem is closed according to the soft or hard degree of material
Reason arranges dewatering time, and the generally tenderer material dehydration time can be appropriately extended, and the stalk of full-bloom stage then can be directly sliced；
S2, dewatered material is taken out, left hand thumb and index finger are clutched away from notch about 1cm up and down, and root is made longitudinally to incline
Tiltedly about 30 °, little finger of toe and wrist are placed on the experimental bench of upper left side, and to support, hand thumb and index finger clutch blade end, then
Knife-blade is abutted into the root scabbled, stem section, is slightly firmly slightly pressed downward blade when ingressing, then according to from upper right side to a left side
The direction of lower section carries out continuous cutting parallel rapidly, and stock removal action need to link up, can not stop over；
In above-mentioned slicing processes, general left hand thumb and the fixed material of index finger, right hand fixed blade can also be according to a manpower
Property preference is selected, in addition, material can be suitably rotated during slice, to seek most suitable microtome angle；
S3, the thin slice cut is placed in the culture dish for fill dehydrated alcohol 1 minute or more, is then taken out clean
Glass slide drips a small amount of (0.5-1ml) distilled water, carefully the thin slice cut is placed on water droplet with microforceps, makes its full extension,
Coverslip side is first contacted into water droplet, is slowly put down after side and the water surface completely attach to, material is completely covered to get.
The embodiment of the invention also provides a kind of applications of free-hand section method, i.e.,：Gained slice is placed in
It is observed, is taken pictures under Leica Stereo microscope (Leica DFC 500), it is complete in the 15min to take pictures after the slicing is completed
At.Camera parameter setting of taking pictures can refer to：Exposure：40%；Saturation degree：1.2；Gamma：1.0, can also according to actual needs into
Row adjustment.
It is as shown in figures 1-8 respectively the dissection picture of the different rhizome tissues of variant kind, it can be clearly from figure
The structure of each rhizome tissue is found out, to further demonstrate the feasibility and reliability of free-hand section method of the present invention.
Finally, being not intended to limit the scope of the present invention the above is only preferred embodiment of the invention.It is all this
Within the spirit and principle of invention, any modification, equivalent replacement, improvement and so on should be included in protection model of the invention
Within enclosing.
Claims (10)
1. a kind of free-hand section method of Chinese herbaceous peony rhizome tissue, which is characterized in that include the following steps：
S1, clean fresh Chinese herbaceous peony rhizome tissue is obtained, dehydration to moisture content is 5-10%；
S2, the Chinese herbaceous peony rhizome tissue that step S1 is obtained is cut with blade along perpendicular to its y direction, is obtained with a thickness of 20-30
μm slice；
S3, the obtained slice of step S2 is placed in dehydrated alcohol after removal excessive moisture, is placed in distilled water and is fully deployed, i.e.,
?.
2. free-hand section method according to claim 1, which is characterized in that in step S2, the cutting is specially：With a left side
Hand thumb and index finger are clutched at rhizome tissue's end cut about 0.8-1.2cm that step S1 is obtained up and down, make Sect. Rhiziridium
25-35 ° of fore-and-aft tilt is knitted, little finger is placed on the experimental bench of upper left side as support, right hand thumb and index finger clutch blade
Knife-blade is abutted the end face for the rhizome tissue scabbled by end, is firmly slightly pressed downward blade when ingressing, then from upper right side
Toward the direction continuously promptly parallel cutting of lower left.
3. free-hand section method according to claim 1 or 2, which is characterized in that in step S1, the dehydration is specially：
When the Chinese herbaceous peony rhizome tissue is fleshy root or tender stem, it is sequentially placed into the ethanol solution of gradient concentration and impregnates dehydration, preferably
Ground, the gradient concentration of the ethanol solution are followed successively by 50%, 70%, 85%, 95%, 100%, 100%, the second of each gradient concentration
Dewatering time in alcoholic solution is 0.8-1.2h；When the rhizome tissue is full-bloom stage stalk, it is placed in dehydrated alcohol and impregnates
5-10min。
4. free-hand section method according to claim 1-3, which is characterized in that in step S3, by the slice
The soaking time being placed in dehydrated alcohol is 1-2min.
5. free-hand section method according to claim 1-4, which is characterized in that in step S3, by the slice
It is placed in distilled water and is fully deployed specially：Clean glass slide is taken, 0.5-1ml distilled water is added dropwise, is placed in slice with microforceps
On distilled water, after the slice is fully deployed, slowly covers coverslip and the slice is completely covered.
6. free-hand section method according to claim 1-5, which is characterized in that in step S1, the Sect. Rhiziridium
Knit the segment that control is 5-7.5cm long.
7. free-hand section method according to claim 1-6, which is characterized in that the step S1 further includes,
The both ends edge of the rhizome tissue perpendicular direction is grown with it before dehydration to scabble.
8. free-hand section method according to claim 1-7, which is characterized in that the blade is stainless steel double-sided
Blade half-and-half fractures blade when as slice.
9. free-hand section method according to claim 1-8, which is characterized in that in step S1, the Sect. Rhiziridium
Knit Chinese herbaceous peony fleshy root and stalk selected from different development stage normal growth, when sampling the rhizome tissue is rinsed well after put
Enter to be placed in the valve bag containing blotting paper and save on ice.
10. a kind of application of any one of claim 1-9 free-hand section method, which is characterized in that the slice to be placed in
It is observed, is taken pictures under microscope, completed in the 15min to take pictures after the slicing is completed.
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| CN103630421A (en) * | 2013-12-03 | 2014-03-12 | 沈阳农业大学 | Production method for paraffin section of paeonia lactiflora mature embryo |
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Application publication date: 20181123 |