CN109738249A - A kind of production method of paraffin section that observing Process of Flower Bud Differentiation - Google Patents
A kind of production method of paraffin section that observing Process of Flower Bud Differentiation Download PDFInfo
- Publication number
- CN109738249A CN109738249A CN201811595700.4A CN201811595700A CN109738249A CN 109738249 A CN109738249 A CN 109738249A CN 201811595700 A CN201811595700 A CN 201811595700A CN 109738249 A CN109738249 A CN 109738249A
- Authority
- CN
- China
- Prior art keywords
- ethyl alcohol
- alcohol
- tert
- dehydrated
- paraffin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Sampling And Sample Adjustment (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to a kind of paraffin section production methods for observing Process of Flower Bud Differentiation, including plant sample materials to fix, be dehydrated transparent, saturating wax embedding, wax stone amendment slice, bonding die exhibition piece, dye sealing；The fixed plant sample materials include cutting plant stem apex central tissue, the plant stem apex central tissue that cuts includes using the raw line of the base of stem apex central point and blade as axis, it is 4-6mm being parallel to the raw line direction of blade base to cut width, it is 2-4mm cutting width perpendicular to the raw line direction of blade base, cuts 5-7mm in short transverse.The present invention uses special method of drawing material, can really and accurately reflect the growth conditions of plant stem apex, provide cytology visual angle for observation of plant Process of Flower Bud Differentiation；More traditional paraffin section fixing means, fixing means of the invention reduce the case where becoming fragile after the tender material of children is fixed.Method of the invention ensure that the accuracy for observing and measuring shoot tip meristem angle, more more efficient than prior paraffin slice, and obtained slice accuracy is higher.
Description
Technical field
The present invention relates to plant sample observational technique fields, and are more particularly related to a kind of observation of plant bud point
The production method of the paraffin section of change process.
Background technique
Blooming is important stage in the higher plant history of life.In recent years, with to model plant arabidopsis at flower molecule
The parsing of mechanism, people have more deep understanding to the formation and development mechanism of annual herb plant flower；And it is perennial
There is huge differences with annual herb plant breeding habit for herbaceous plant, although utilizing the research of arabidopsis flowering transition
Method also makes some progress, but up to the present, we to herbaceos perennial at flower molecular mechanism understanding also
Need further to be explored.
In perennial plant flower-shape in the correlative study with development, bud anatomical structure is as the weight for identifying bud differentiation
Will according to one of, frequently as the important means for judging bud differentiation degree, mainly by plant tissue slice under the microscope
It observes the development degree of its structure and is achieved.Therefore, it explores efficient, low toxicity and obtains the method for slice in identification bud differentiation
There is important application value when development degree, is a kind of scientific and effective identification method.
For the histological section research of plant bud differentiation, traditional method has very much, including prior paraffin tissue is cut
Piece method, freezing method.Traditional paraffin tissue sections are transparent using dimethylbenzene, but the volatile toxicity of dimethylbenzene is very big, is adding
In the process of heat, it will be distributed in air, cause researcher uncomfortable；Fabrication cycle is very long simultaneously；Frozen section rule
Above-mentioned two problems are avoided, but the piece minor structure of frozen section excision is not easy completely, piece is thicker, and piece of excision can not be grown
Kubo is deposited, and frozen section requires that fresh sample is taken to be fixed and be sliced every time, is limited by sample time, ineffective
It is living.Can not show the variation of plant tissue cell well, at the same can not for deeper into the molecular biology such as in situ hybridization grind
Offer plant section is provided.
For the rhizome of meat shape, bud is resulted from stem apex.During making slice, biopsy tissues include that children is tender
Stem apex, the harder rhizome tissue of meat shape, the two adhesion together, considerably increases the difficulty of production.Therefore it explores and is suitble to
Paraffin section technical application and tawny daylily tissue anatomical structure and cytology research in be an important problem.Though there is it in the past
The report of his plant tissue paraffin section, but different types of plant tissue has different characteristics.Though previous correlated results has
Reference but cannot completely be applied to the slice research of tawny daylily bud differentiated tissue.
In conclusion exploring a kind of paraffin section technology quickly, less toxic, it is applied to stem apex cytology and bud differentiation is seen
Research is examined, is an important technical problem.
Summary of the invention
The present invention is in view of the above-mentioned problems, be designed to provide a kind of production side of paraffin section for observing Process of Flower Bud Differentiation
Method, can solve prior art toxicity is big or slice is imperfect, sample time it is limited/technical problems such as manufacture difficulty is big.
In order to achieve the above objectives, the present invention adopts the following technical scheme:
Embodiment of the invention discloses a kind of paraffin section production methods for observing Process of Flower Bud Differentiation, including plant sample
This materials is fixed, is dehydrated transparent, saturating wax embedding, wax stone amendment slice, bonding die exhibition piece, dyeing sealing；
Wherein, it includes cutting plant stem apex central tissue that the plant sample materials are fixed, described to cut in plant stem apex
Heart tissue includes using the raw line of the base of stem apex central point and blade as axis, is 4- being parallel to the raw line direction of blade base to cut width
6mm is 2-4mm cutting width perpendicular to the raw line direction of blade base, cuts 5-7mm in short transverse.Preferably, it is being parallel to
It is 5mm that the raw line direction of blade base, which cuts width, is 3mm cutting width perpendicular to the raw line direction of blade base, cuts in short transverse
Take 6mm.
Further, the plant sample materials fixation includes: to cut open from base life after the blade on plant rhizomes, is cut
Plant stem apex central tissue is dipped in the container equipped with FPA fixer, and the container is placed in vacuum desiccator, makes sample
Product are sunk to the bottom, and obtain fixed sample.
Wherein, the test tube with cover of 10ml can be selected in container, and sample sinks to the bottom, when open test tube cap, vacuumize 1-2 hours, directly
It is all sunk to the bottom to sample.Fixer uses FPA fixer (formalin-acetone-ethanol fixer), configured by following reagent and
At: 50% ethyl alcohol: acetone: formalin=90ml:7ml:3ml；In addition, every 100ml FPA fixer adds 5ml glycerol.Material
Material fixer volume ratio is 1:40.
Further, it includes: after pouring out the liquid in the fixed sample that the dehydration is transparent, and displacement is different thereto
The dewaterer and clarifier of gradient be dehydrated transparent.
It is specifically as follows, after fixed, invisible spectro fixer is poured out, is placed on rack for test tube.Successively in test tube
The dehydrating agents of different gradients is replaced, is dehydrated in clarifier, is transparent, operation all carries out in shaking table, and shaking table temperature is 25
DEG C, revolving speed 100r/h can effectively accelerate the displacement efficiency of Intracellular solution, improve and be dehydrated transparent quality.May include
Following steps: 50% ethyl alcohol → 60% ethyl alcohol → 70% ethyl alcohol → 75% ethyl alcohol and tert-butyl alcohol mixed liquor (13:7, v/v) → 85%
Ethyl alcohol tert-butyl alcohol mixed liquor (9:11, v/v) → 95% ethyl alcohol tert-butyl alcohol mixed liquor (1:3, v/v, above step impregnate 20min)
→ pure tert-butyl alcohol A → pure tert-butyl alcohol B (above step impregnates 1h).I.e. the dewaterer and clarifier of the different gradients of the displacement into
Transparent row dehydration includes: 50% ethyl alcohol immersion treatment, 60% ethyl alcohol immersion treatment, 70% ethyl alcohol immersion treatment, volume ratio 13:
7 75% ethyl alcohol and tert-butyl alcohol mixed liquid dipping is handled, volume ratio is handled for the 85% ethyl alcohol tert-butyl alcohol mixed liquid dipping of 9:11,
Volume ratio is the 95% ethyl alcohol tert-butyl alcohol mixed liquid dipping processing of 1:3, pure tert-butyl alcohol A immersion treatment, at pure tert-butyl alcohol B immersion
Reason.The 50% ethyl alcohol immersion treatment, 60% ethyl alcohol immersion treatment, 70% ethyl alcohol immersion treatment, 75% that volume ratio is 13:7
Ethyl alcohol and the processing of tert-butyl alcohol mixed liquid dipping, the 85% ethyl alcohol tert-butyl alcohol mixed liquid dipping processing that volume ratio is 9:11, volume ratio
The processing time handled for the 95% ethyl alcohol tert-butyl alcohol mixed liquid dipping of 1:3 is 20 minutes；At the pure tert-butyl alcohol A immersion
It manages, the processing time of pure tert-butyl alcohol B immersion treatment is 1 hour.
Increase a kind of dehydrating agent tert-butyl alcohol；Ethyl alcohol is used alone to be easy to make the too fast dehydration of tender tissue, the tert-butyl alcohol is more warm
With, the situation that can effectively avoid excessive material from shrinking, raising dehydration quality.Using the tert-butyl alcohol as clarifier.The tert-butyl alcohol is low
Poison can effectively mitigate during transparent, waxdip, because dimethylbenzene constantly volatilizees caused by heating, seriously affect scientific research people
The case where health of member；Meanwhile dimethylbenzene is easy that tender tissue is made to shrink, become fragile, the tert-butyl alcohol is more mild, can be effective
It avoids being sliced fragmentary situation, improves safety and the transparent effect of operation.
Further, the wax include: will be dehydrated it is transparent after after obtained material pre-processed, wrapped
It buries, the pretreatment includes that the material that the dehydration obtains after transparent is sequentially placed into the paraffin and the tert-butyl alcohol that volume ratio is 1:6
Mixed liquor, volume ratio be 3:8 paraffin and tert-butyl alcohol mixed liquor, the paraffin refined wax A of fusing, the paraffin refined wax B of fusing, fusing pure stone
Wax C.
Specifically, can by be dehydrated it is transparent after material be sequentially placed into the first paraffin and tert-butyl alcohol mixed liquor (1:6, v/v,
50 DEG C, 3h) → the second paraffin and tert-butyl alcohol mixed liquor (3:8, v/v, 55 DEG C, 3h) → melted in advance paraffin refined wax A (60 DEG C,
5h) → paraffin refined wax B (60 DEG C, 5h) → paraffin refined wax C (60 DEG C, 5h).Then, it can be embedded.When embedding, with dissecting needle by sample
Product are adjusted to the middle part of embedded block as far as possible.
Then the wax stone after embedding is subjected to slicing treatment, determined between the raw line of the base of plant stem apex central point and blade
Axis；It is cemented in wax stone is parallel with axis on small wood on one side, as section；Meanwhile being inverted plant tissue, stem apex hangs down
Directly downward, root tissue is upward, is cut, and wax disk(-sc) thickness can be 10 μm.Judge whether shoot tip meristem is divided into observation
When inflorescence meristem structure, the angle of slice is extremely important, has decided on whether to cut out with complete shoot tip meristem structure
Piece.Therefore, the axis between the raw line of the base of stem apex central point and blade is determined when slice first, then by wax stone and axis
Parallel cements on one side on small wood, as section.The structure of shoot tip meristem can be completely presented in piece after optimization,
It is ideal piece for judging plant bud differentiation degree；Meanwhile plant tissue is inverted, stem apex (tender tissue) vertically downward,
Root tissue is upward, and for this method when handling the tissue of two kinds of different structures, reduction that can be very big is sliced broken situation,
It can get the slice of high quality.
Further, the bonding die exhibition piece includes: to be sticked to wax disk(-sc) on glass slide with gelatin alite paste, is placed on exhibition piece platform
It is heated to full extension, exhibition piece platform temperature is set as 42 DEG C.The glass slide pasted can then be dried, baked piece seed cell temperature
It saves.
Further, the dyeing sealing includes: successively to use dimethylbenzene, volume ratio for the dehydrated alcohol of 1:2 and diformazan
Benzene mixed liquor, the dehydrated alcohol and dimethylbenzene mixed liquor, dehydrated alcohol, 95% ethyl alcohol, 90% ethyl alcohol, 80% that volume ratio is 1:1
Ethyl alcohol, 70% ethyl alcohol, 60% ethyl alcohol impregnate respectively；
After dyeing 3-5 hours using 1% aniline sarranine dye liquor dark situation, 60% ethyl alcohol, 70% ethyl alcohol, 80% are successively used
Ethyl alcohol, 90% ethyl alcohol, 95% ethyl alcohol impregnate 20-30 seconds；
After the fast green dye liquor dyeing of 0.5% aniline, successively use 95% ethyl alcohol, dehydrated alcohol, volume ratio for the nothing of 1:1
Water-ethanol and dimethylbenzene mixed liquor, pure dimethylbenzene carry out immersion treatment；
Using the diluted canada balsam of dimethylbenzene as mounting medium, covered sealing.
Further, the solvent of the 1% aniline sarranine dye liquor is 50% ethanol solution；
The solvent of the fast green dye liquor of 0.5% aniline is 95% ethanol solution, and dyeing time is 30 seconds；
After the fast green dye liquor dyeing of 0.5% aniline, the processing time of 95% ethyl alcohol is 3-5 seconds, the anhydrous second
The processing time of alcohol, the dehydrated alcohol that volume ratio is 1:1 and dimethylbenzene mixed liquor is 20-30 seconds, the place of the pure dimethylbenzene
Managing the time is 2 minutes.
In one embodiment of the invention, shown dyeing sealing is specifically includes the following steps: dimethylbenzene (1h) → dehydrated alcohol+two
Toluene (1:2, v/v) → dehydrated alcohol+dimethylbenzene (1:1, v/v) → dehydrated alcohol → 95%, 90%, 80%, 70%, 60% second
Alcohol (the equal 2min of above step) → 1% aniline sarranine dye liquor (prepare by 50% ethanol solution；Dark situation dyes 3-5h) → 60%,
70%, 80%, 90%, 95% ethyl alcohol (the equal 20-30s of the above step) → fast green dye liquor of 0.5% aniline (match by 95% ethanol solution
System dyes 30s) → 95% ethyl alcohol (3-5s) → (1:1, v/v, above step are each for dehydrated alcohol → dehydrated alcohol+dimethylbenzene
20-30s) → pure dimethylbenzene (2min).Finally use the diluted canada balsam of dimethylbenzene as mounting medium, covered envelope
Gu.Shady place is put in dry.
OLYMPUS CH20 optical microphotograph sem observation slice can be used in microexamination.With OLYMPUS BX51 digital micrograph
Sem observation is simultaneously taken pictures.
The beneficial effects of the present invention are:
It using special method of drawing material, can really and accurately reflect the growth conditions of plant stem apex, be observation of plant
Process of Flower Bud Differentiation provides cytology visual angle；More traditional paraffin section fixing means, fixing means of the invention reduce
The case where becoming fragile after the tender material of children is fixed.Method of the invention ensure that the accuracy for observing and measuring shoot tip meristem angle, than
Prior paraffin slice is more efficient, and obtained slice accuracy is higher.
Detailed description of the invention
Fig. 1 a- Fig. 1 d is the paraffin section figure of the embodiment of the present invention 1；
Fig. 2 a- Fig. 2 b is the paraffin section figure of the embodiment of the present invention 2；
Fig. 3 a- Fig. 3 b is the paraffin section figure of the embodiment of the present invention 3；
Fig. 4 a- Fig. 4 b is that the paraffin section result of different biopsy method angle compares；
Fig. 5 a- Fig. 5 b is that the paraffin section result in different section directions compares；
Fig. 6 a- Fig. 6 c is the paraffin section figure of comparative example 1.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
Embodiment 1: the microstructure of trophophase tawny daylily stem apex
The fixed pumping of materials: cut open it is raw from base after the blade on tawny daylily rhizomes, with tawny daylily stem apex central point and blade
The raw line of base is axis, and it is (high carefully to cut 5mm (being parallel to the raw line of blade base) × 3mm (perpendicular to the raw line of blade base) × 5mm at once
Degree) tawny daylily stem apex central tissue, be dipped in the 10ml test tube (with cover) equipped with FPA fixer fixed preservation, material fixer
Volume ratio is 1:40.Then, test tube is put into vacuum desiccator, opens test tube cap, vacuumizes 1-2 hours, until whole samples
Product are sunk to the bottom.
It is dehydrated transparent: after fixed, invisible spectro fixer being poured out, is placed on rack for test tube.Successively toward built in test tube
The dehydrating agents of different gradients is changed, is dehydrated in clarifier, is transparent, operation all carries out in shaking table.The following steps are included:
50% ethyl alcohol → 60% ethyl alcohol → 70% ethyl alcohol → 75% ethyl alcohol and the tertiary fourth of tert-butyl alcohol mixed liquor (13:7, v/v) → 85% ethyl alcohol
Alcohol mixed liquor (9:11, v/v) → 95% ethyl alcohol tert-butyl alcohol mixed liquor (1:3, v/v, above step impregnate 20min) → pure tertiary fourth
Alcohol A → pure tert-butyl alcohol B (above step impregnates 1h).Shaking table temperature is set as 25 DEG C, and revolving speed is set as 100r/h.
Saturating wax embedding: by upper step treated material successively puts the first paraffin and tert-butyl alcohol mixed liquor (1:6, v/v, 50 DEG C,
3h) → second the paraffin refined wax A (60 DEG C, 5h) for paraffin and tert-butyl alcohol mixed liquor (3:8, v/v, 55 DEG C, 3h) → melted in advance →
Paraffin refined wax B (60 DEG C, 5h) → paraffin refined wax C (60 DEG C, 5h).Then, it can be embedded.When embedding, sample is use up with dissecting needle
Amount is adjusted to the middle part of embedded block.
It repairs wax stone, slice: when finishing wax stone, determining the axis between the raw line of the base of tawny daylily stem apex central point and blade；It will
Wax stone is parallel with axis to be cemented on one side on small wood, as section；Meanwhile plant tissue is inverted, stem apex vertically downward,
Root tissue is upward, is cut.Wax disk(-sc) is with a thickness of 10 μm.
Bonding die and exhibition piece: wax disk(-sc) is sticked on glass slide with gelatin alite paste, is placed on exhibition piece platform and is heated to full extension,
Exhibition piece platform temperature is set as 42 DEG C.Then the glass slide pasted is dried, baked piece room temperature preservation.
Dyeing and sealing: include the following steps operation: dimethylbenzene (1h) → dehydrated alcohol+dimethylbenzene (1:2, v/v) → nothing
Water-ethanol+dimethylbenzene (1:1, v/v) → dehydrated alcohol → 95%, (above step is equal for 90%, 80%, 70%, 60% ethyl alcohol
2min) → 1% aniline sarranine dye liquor (prepare by 50% ethanol solution；Dark situation, dye 3-5h) → 60%, 70%, 80%,
90%, 95% ethyl alcohol (the equal 20-30s of above step) → fast green dye liquor of 0.5% aniline (95% ethanol solution is prepared, and 30s is dyed)
→ 95% ethyl alcohol (3-5s) → dehydrated alcohol → dehydrated alcohol+dimethylbenzene (1:1, v/v, each 20-30s of above step) → pure two
Toluene (2min).Finally use the diluted canada balsam of dimethylbenzene as mounting medium, covered sealing.Shady place is put in dry in the air
It is dry.
Microexamination: it is sliced using OLYMPUS CH20 optical microphotograph sem observation.With OLYMPUS BX51 digit microscope
It observes and takes pictures, see Fig. 1, wherein Fig. 1 a, Fig. 1 c is object lens magnification 10X；Fig. 1 b, Fig. 1 d is object lens magnification 20X.
Embodiment 2: the microstructure of flower bud differentiation period tawny daylily bud
In the fixed air aspiration stage of the materials of embodiment 1: cuing open from base life after the blade on tawny daylily rhizomes, with tawny daylily stem apex
The raw line of the base of central point and blade is axis, carefully cuts 5mm (being parallel to the raw line of blade base) × 3mm at once (perpendicular to blade
Base gives birth to line) the tawny daylily stem apex central tissue of × 5mm (height), it is dipped in the 10ml test tube (with cover) equipped with FPA fixer fixation
It saves, material fixer volume ratio is 1:40.Then, test tube is put into vacuum desiccator, opens test tube cap, vacuumizes 1-2
Hour, until whole samples are sunk to the bottom.
In the dehydration of the embodiment 1 transparent stage: after fixed, invisible spectro fixer being poured out, is placed on rack for test tube.
The dehydrating agents of different gradients is successively replaced in test tube, is dehydrated in clarifier, is transparent, and operation all carries out in shaking table.
The following steps are included: 50% ethyl alcohol → 60% ethyl alcohol → 70% ethyl alcohol → 75% ethyl alcohol and tert-butyl alcohol mixed liquor (13:7, v/v) →
85% ethyl alcohol tert-butyl alcohol mixed liquor (9:11, v/v) → 95% ethyl alcohol tert-butyl alcohol mixed liquor (impregnate by 1:3, v/v, above step
20min) → pure tert-butyl alcohol A → pure tert-butyl alcohol B (above step impregnates 1h).Shaking table temperature is set as 25 DEG C, and revolving speed is set as 100r/
h。
The stage is embedded in the saturating wax of embodiment 1: upper step treated material is sequentially placed into the first paraffin and the tert-butyl alcohol are mixed
Liquid (1:6, v/v, 50 DEG C, 3h) → second paraffin and tert-butyl alcohol mixed liquor (3:8, v/v, 55 DEG C, 3h) → melted in advance are pure
Paraffin A (60 DEG C, 5h) → paraffin refined wax B (60 DEG C, 5h) → paraffin refined wax C (60 DEG C, 5h).Then, it can be embedded.When embedding,
Sample is adjusted to the middle part of embedded block as far as possible with dissecting needle.
Repaired the block sections stage in embodiment 1: when finishing wax stone, determine the raw line of the base of tawny daylily stem apex central point and blade it
Between axis；It is cemented in wax stone is parallel with axis on small wood on one side, as section；Meanwhile plant tissue being inverted, stem
Vertically downward, root tissue is upward, is cut for point.Wax disk(-sc) is with a thickness of 10 μm.As a result as shown in Figure 2, wherein Fig. 2 a is object lens
Amplification factor 10X；Fig. 2 b is object lens magnification 20X.
Embodiment 3: the microstructure of small bud formation phase tawny daylily bud
In the fixed air aspiration stage of the materials of embodiment 1: cuing open from base life after the blade on tawny daylily rhizomes, with tawny daylily stem apex
The raw line of the base of central point and blade is axis, carefully cuts 5mm (being parallel to the raw line of blade base) × 3mm at once (perpendicular to blade
Base gives birth to line) the tawny daylily stem apex central tissue of × 5mm (height), it is dipped in the 10ml test tube (with cover) equipped with FPA fixer fixation
It saves, material fixer volume ratio is 1:40.Then, test tube is put into vacuum desiccator, opens test tube cap, vacuumizes 1-2
Hour, until whole samples are sunk to the bottom.
In the dehydration of the embodiment 1 transparent stage: after fixed, invisible spectro fixer being poured out, is placed on rack for test tube.
The dehydrating agents of different gradients is successively replaced in test tube, is dehydrated in clarifier, is transparent, and operation all carries out in shaking table.
The following steps are included: 50% ethyl alcohol → 60% ethyl alcohol → 70% ethyl alcohol → 75% ethyl alcohol and tert-butyl alcohol mixed liquor (13:7, v/v) →
85% ethyl alcohol tert-butyl alcohol mixed liquor (9:11, v/v) → 95% ethyl alcohol tert-butyl alcohol mixed liquor (impregnate by 1:3, v/v, above step
20min) → pure tert-butyl alcohol A → pure tert-butyl alcohol B (above step impregnates 1h).Shaking table temperature is set as 25 DEG C, and revolving speed is set as 100r/
h。
The stage is embedded in the saturating wax of embodiment 1: upper step treated material is sequentially placed into the first paraffin and the tert-butyl alcohol are mixed
Liquid (1:6, v/v, 50 DEG C, 3h) → second paraffin and tert-butyl alcohol mixed liquor (3:8, v/v, 55 DEG C, 3h) → melted in advance are pure
Paraffin A (60 DEG C, 5h) → paraffin refined wax B (60 DEG C, 5h) → paraffin refined wax C (60 DEG C, 5h).Then, it can be embedded.When embedding,
Sample is adjusted to the middle part of embedded block as far as possible with dissecting needle.
Repaired the block sections stage in embodiment 1: when finishing wax stone, determine the raw line of the base of tawny daylily stem apex central point and blade it
Between axis；It is cemented in wax stone is parallel with axis on small wood on one side, as section；Meanwhile plant tissue being inverted, stem
Vertically downward, root tissue is upward, is cut for point.Wax disk(-sc) is with a thickness of 10 μm.As a result as shown in figure 3, wherein Fig. 3 a is object lens
Amplification factor 10X；Fig. 3 b is object lens magnification 20X.
Comparative example 1
Dehydration clearing method in comparative example 1 is that conventional ethanol dimethylbenzene is dehydrated transillumination, and concrete operations are, after fixation
Material be sequentially placed into 50%, 60%, 70%, 80%, 90%, 95% ethyl alcohol and be dehydrated, 1.5 hours every time；It places into
It is dehydrated in dehydrated alcohol twice, 1 hour every time；Then, the material after dehydration is respectively placed in dimethylbenzene+dehydrated alcohol
Carried out in (1:1, v/v), pure dimethylbenzene A, pure dimethylbenzene B it is transparent, 2 hours every time.Then it is dehydrated transparent completion.Again place the material in
Waxdip 10 hours in isometric dimethylbenzene+paraffin, 40 DEG C of constant temperature.Hereafter it is embedded, is sliced, mounting and etc..See Fig. 6,
Wherein, Fig. 6 a, the paraffin section structure of trophophase tawny daylily stem apex；Fig. 6 b, the paraffin section structure of flower bud differentiation period tawny daylily bud；
Fig. 6 c, the paraffin section structure of small bud idiophase tawny daylily bud；Fig. 6 a, object lens magnification 5X；Fig. 6 b-Fig. 6 c: object lens are put
Big multiple 10X.
The present invention also compares the tawny daylily bud paraffin section result of different biopsy method method, is illustrated in figure 4 small bud point
The materials of change phase, wherein Fig. 4 a is paraffin method of drawing material of the invention；Fig. 4 b is common paraffin method of drawing material；Object lens times magnification
Number 10X.The present invention also compares the tawny daylily bud paraffin section result in different section directions, is illustrated in figure 5 taking for trophophase
Material, wherein Fig. 5 a is section method of the invention；Fig. 5 b is traditional section direction；Object lens magnification 10X.
In order to further prove that production method of the invention also can produce effect same as tawny daylily for other plant,
Paraffin section has also been made to herbaceos perennial Chinese herbaceous peony, lily, dahlia etc., and has observed its Process of Flower Bud Differentiation, has been tested
It proves, the production method of the paraffin section of observation of plant Process of Flower Bud Differentiation provided by the present invention passes through the plant roots of acquisition
Through drawing materials, FPA fixer is fixed to be saved stem end material；Ethyl alcohol, ethyl alcohol tert-butyl alcohol mixed liquor serial dehydration；The tert-butyl alcohol is transparent；Stone
Wax embedding；Modify wax stone, slice, exhibition piece, bonding die, drying, mounting and etc. after, the paraffin section produced is for bud differentiation
The observational study of process.Compared with prior paraffin dicing method, method of drawing material of the invention can really and accurately reflect stem
The growth conditions of point provide cytology visual angle for observation Process of Flower Bud Differentiation；More traditional paraffin section fixing means, this hair
Bright fixing means reduces the case where becoming fragile after the tender material of children is fixed.Meanwhile above-mentioned dehydrating agent and clarifier are selected, it can obtain
The paraffin section of high quality is obtained, reduces and adds thermogenetic toxicity in manufacturing process.And the dehydration clearing method that the present invention optimizes,
The Production Time using paraffin section observation Process of Flower Bud Differentiation is substantially reduced, time efficiency is improved.
The foregoing is merely presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention；If do not taken off
It from the spirit and scope of the present invention, modifies or equivalently replaces the present invention, should all cover in the claims in the present invention
In protection scope.
Claims (10)
1. a kind of paraffin section production method for observing Process of Flower Bud Differentiation, which is characterized in that fixed including plant sample materials,
It is dehydrated transparent, saturating wax embedding, wax stone amendment slice, bonding die exhibition piece, dyeing sealing；
Wherein, it includes cutting plant stem apex central tissue that the plant sample materials are fixed, described to cut plant stem apex central set
It knits including giving birth to line as axis using the base of stem apex central point and blade, is 4-6mm being parallel to the raw line direction of blade base to cut width,
Cutting width perpendicular to the raw line direction of blade base is 2-4mm, cuts 5-7mm in short transverse.
2. the method according to claim 1, wherein plant sample materials fixation includes: to cut open to exist from base life
After blade on plant rhizomes, plant stem apex central tissue is cut, is dipped in the container equipped with FPA fixer, and will be described
Container is placed in vacuum desiccator, sinks to the bottom sample, obtains fixed sample.
3. according to the method described in claim 2, it is characterized in that, it is described dehydration it is transparent include: will be in the fixed sample
After liquid is poured out, the dewaterer and clarifier for replacing different gradients thereto be dehydrated transparent.
4. the method according to claim 1, wherein the wax include: will be dehydrated it is transparent after obtain
It after material is pre-processed, is embedded, the pretreatment includes that the material that the dehydration obtains after transparent is sequentially placed into body
The paraffin refined wax A of paraffin and tert-butyl alcohol mixed liquor, fusing that the paraffin and tert-butyl alcohol mixed liquor, volume ratio that product ratio is 1:6 are 3:8,
The paraffin refined wax B of fusing, fusing paraffin refined wax C.
5. according to the method described in claim 3, it is characterized in that, the dewaterer and clarifier of the different gradients of the displacement carry out
Being dehydrated transparent includes: 50% ethyl alcohol immersion treatment, 60% ethyl alcohol immersion treatment, 70% ethyl alcohol immersion treatment, volume ratio 13:7
75% ethyl alcohol and the tert-butyl alcohol mixed liquid dipping processing, volume ratio be 9:11 the 85% ethyl alcohol tert-butyl alcohol mixed liquid dipping processing,
Volume ratio is the 95% ethyl alcohol tert-butyl alcohol mixed liquid dipping processing of 1:3, pure tert-butyl alcohol A immersion treatment, at pure tert-butyl alcohol B immersion
Reason.
6. according to the method described in claim 5, it is characterized in that, at the 50% ethyl alcohol immersion treatment, 60% ethyl alcohol immersion
Reason, 70% ethyl alcohol immersion treatment, 75% ethyl alcohol that volume ratio is 13:7 and the processing of tert-butyl alcohol mixed liquid dipping, volume ratio 9:11
The 85% ethyl alcohol tert-butyl alcohol mixed liquid dipping processing, volume ratio be 1:3 95% ethyl alcohol tert-butyl alcohol mixed liquid dipping handle place
Managing the time is 20 minutes；The pure tert-butyl alcohol A immersion treatment, pure tert-butyl alcohol B immersion treatment the processing time be 1 hour.
7. according to the method described in claim 6, it is characterized in that, the dewaterer and clarifier of the different gradients of the displacement carry out
Be dehydrated it is transparent carried out in shaking table, shaking table temperature be 25 DEG C, revolving speed 100r/h.
8. the method according to claim 1, wherein bonding die exhibition piece includes: by wax disk(-sc) gelatin alite paste
It is sticked on glass slide, is placed on exhibition piece platform and is heated to full extension, exhibition piece platform temperature is set as 42 DEG C.
9. the method according to claim 1, wherein the dyeing sealing includes: successively using dimethylbenzene, volume
Than for 1:2 dehydrated alcohol and dimethylbenzene mixed liquor, volume ratio be 1:1 dehydrated alcohol and dimethylbenzene mixed liquor, dehydrated alcohol,
95% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, 70% ethyl alcohol, 60% ethyl alcohol impregnate respectively；
After dyeing 3-5 hours using 1% aniline sarranine dye liquor dark situation, 60% ethyl alcohol, 70% ethyl alcohol, 80% second are successively used
Alcohol, 90% ethyl alcohol, 95% ethyl alcohol impregnate 20-30 seconds；
After the fast green dye liquor dyeing of 0.5% aniline, successively use 95% ethyl alcohol, dehydrated alcohol, volume ratio for the anhydrous second of 1:1
Pure and mild dimethylbenzene mixed liquor, pure dimethylbenzene carry out immersion treatment；
Using the diluted canada balsam of dimethylbenzene as mounting medium, covered sealing.
10. according to the method described in claim 9, it is characterized in that, the solvent of the 1% aniline sarranine dye liquor is 50% ethyl alcohol
Solution；
The solvent of the fast green dye liquor of 0.5% aniline is 95% ethanol solution, and dyeing time is 30 seconds；
After the fast green dye liquor dyeing of 0.5% aniline, the processing time of 95% ethyl alcohol is 3-5 seconds, the dehydrated alcohol, body
Product is 20-30 seconds than the processing time of dehydrated alcohol and dimethylbenzene mixed liquor for 1:1, the processing time of the pure dimethylbenzene
It is 2 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811595700.4A CN109738249A (en) | 2018-12-25 | 2018-12-25 | A kind of production method of paraffin section that observing Process of Flower Bud Differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811595700.4A CN109738249A (en) | 2018-12-25 | 2018-12-25 | A kind of production method of paraffin section that observing Process of Flower Bud Differentiation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109738249A true CN109738249A (en) | 2019-05-10 |
Family
ID=66359842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811595700.4A Pending CN109738249A (en) | 2018-12-25 | 2018-12-25 | A kind of production method of paraffin section that observing Process of Flower Bud Differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109738249A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257089A (en) * | 2020-02-18 | 2020-06-09 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN113039955A (en) * | 2021-01-22 | 2021-06-29 | 韶关学院 | Method for inhibiting loquat flowering |
| CN113702131A (en) * | 2021-09-06 | 2021-11-26 | 海南大学 | Paraffin section of camellia oleifera flower bud and preparation method and application thereof |
| WO2024013605A1 (en) * | 2022-07-12 | 2024-01-18 | Diapath S.P.A. | Improved composition for the treatment of biological, cytological, histological and autopsical samples |
| WO2024013606A1 (en) * | 2022-07-12 | 2024-01-18 | Diapath S.P.A. | Method for preparing biological, histological, autopsical, cytological samples and composition therefor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103503647A (en) * | 2012-06-18 | 2014-01-15 | 江苏省中国科学院植物研究所 | Method for regulating flowering time of Lycoris sprengeri Comes ex Baker |
| CN107655731A (en) * | 2017-02-08 | 2018-02-02 | 云南省农业科学院花卉研究所 | For the larger explant leaf bud of carnation and the paraffin section method of bud |
| CN107702955A (en) * | 2017-08-24 | 2018-02-16 | 胡雨逸 | A kind of preparation method of paraffin section |
| CN108827964A (en) * | 2018-07-05 | 2018-11-16 | 南京林业大学 | A kind of method of quick determining green bamboo Pistil And Stamen developmental process |
-
2018
- 2018-12-25 CN CN201811595700.4A patent/CN109738249A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103503647A (en) * | 2012-06-18 | 2014-01-15 | 江苏省中国科学院植物研究所 | Method for regulating flowering time of Lycoris sprengeri Comes ex Baker |
| CN107655731A (en) * | 2017-02-08 | 2018-02-02 | 云南省农业科学院花卉研究所 | For the larger explant leaf bud of carnation and the paraffin section method of bud |
| CN107702955A (en) * | 2017-08-24 | 2018-02-16 | 胡雨逸 | A kind of preparation method of paraffin section |
| CN108827964A (en) * | 2018-07-05 | 2018-11-16 | 南京林业大学 | A kind of method of quick determining green bamboo Pistil And Stamen developmental process |
Non-Patent Citations (3)
| Title |
|---|
| 仲文晶 等: ""‘金娃娃’萱草花芽分化前期内源激素动态变化的研究"", 《北方园艺》 * |
| 林秀琴 等: ""甘蔗花芽分化及花序发育过程的石蜡切片显微观察与分析"", 《热带作物学报》 * |
| 王艳芳 等: ""不同发育时期苹果果实组织石蜡切片制作探究"", 《果树学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257089A (en) * | 2020-02-18 | 2020-06-09 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN111257089B (en) * | 2020-02-18 | 2020-10-27 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN113039955A (en) * | 2021-01-22 | 2021-06-29 | 韶关学院 | Method for inhibiting loquat flowering |
| CN113702131A (en) * | 2021-09-06 | 2021-11-26 | 海南大学 | Paraffin section of camellia oleifera flower bud and preparation method and application thereof |
| WO2024013605A1 (en) * | 2022-07-12 | 2024-01-18 | Diapath S.P.A. | Improved composition for the treatment of biological, cytological, histological and autopsical samples |
| WO2024013606A1 (en) * | 2022-07-12 | 2024-01-18 | Diapath S.P.A. | Method for preparing biological, histological, autopsical, cytological samples and composition therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109738249A (en) | A kind of production method of paraffin section that observing Process of Flower Bud Differentiation | |
| CN105973673B (en) | A kind of paraffin section method of eucalyptus tissue | |
| CN102967493B (en) | Rapid paraffin sectioning method for plant tissue | |
| CN109855936B (en) | Preparation method of plant tissue section with complete tissue and favorable microscopic observation | |
| CN104849110B (en) | A kind of paraffin section method of plant tissue | |
| CN111257089B (en) | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid | |
| CN107843470B (en) | Method for making skin tissue slice | |
| CN103940648A (en) | Preparation method for gill tissue paraffin section | |
| CN107167350B (en) | Preparation method of paraffin section of eggplant rhizome tissue | |
| CN107702959A (en) | A kind of paraffin section method for vegetable material serial dehydration | |
| CN109374373A (en) | A kind of Apple paraffin section fast method for preparing | |
| CN106546473B (en) | One kind embedding flaking method for sugarcane Different node lateral bud | |
| CN109211606A (en) | A kind of fast method for preparing of pears tissue paraffin section de | |
| CN107702955B (en) | Preparation method of paraffin section | |
| CN108332989B (en) | Method for analyzing high-starch-content cereal grains and observing internal structure | |
| CN110132673B (en) | Method for preparing needle mushroom fruiting body tissue slices | |
| CN107576552A (en) | A kind of paraffin section colouring method for observing Chinese Rose infection processs | |
| CN110926907A (en) | Method suitable for making equine animal ovary paraffin section | |
| RU2530612C1 (en) | Method for nematode preparation for morphological and histological examination | |
| CN107202720B (en) | A kind of paraffin section method of pomegranate seed | |
| CN109632425A (en) | A kind of paraffin section production method of Chinese herbaceous peony underground rhizome | |
| CN108333013A (en) | A kind of preparation method of jujube flower paraffin section | |
| CN105806680A (en) | Quick paraffin sectioning method of plant tissues | |
| JP2008239487A (en) | Liquid for rapidly fixing hardly permeable tissue | |
| CN109374378A (en) | A kind of method of beet tops paraffin embedding film-making |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190510 |