CN105547793B - A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method - Google Patents
A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method Download PDFInfo
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- CN105547793B CN105547793B CN201610021972.8A CN201610021972A CN105547793B CN 105547793 B CN105547793 B CN 105547793B CN 201610021972 A CN201610021972 A CN 201610021972A CN 105547793 B CN105547793 B CN 105547793B
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- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 27
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 26
- 235000005822 corn Nutrition 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 240000008042 Zea mays Species 0.000 title abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 18
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 13
- 239000011630 iodine Substances 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000008439 repair process Effects 0.000 claims abstract description 9
- 229920001651 Cyanoacrylate Polymers 0.000 claims abstract description 8
- 239000004830 Super Glue Substances 0.000 claims abstract description 8
- 239000011521 glass Substances 0.000 claims description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000004043 dyeing Methods 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 claims description 2
- 241000209149 Zea Species 0.000 claims 2
- 229920002472 Starch Polymers 0.000 abstract description 13
- 235000019890 Amylum Nutrition 0.000 abstract description 10
- 229920005989 resin Polymers 0.000 abstract description 6
- 239000011347 resin Substances 0.000 abstract description 6
- 238000010186 staining Methods 0.000 abstract description 6
- 238000009826 distribution Methods 0.000 abstract description 4
- 238000005520 cutting process Methods 0.000 abstract description 3
- 239000012188 paraffin wax Substances 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- 230000008595 infiltration Effects 0.000 description 6
- 238000001764 infiltration Methods 0.000 description 6
- 230000035515 penetration Effects 0.000 description 5
- 238000007711 solidification Methods 0.000 description 5
- 230000008023 solidification Effects 0.000 description 5
- 206010044565 Tremor Diseases 0.000 description 4
- 210000000720 eyelash Anatomy 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000004568 cement Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000010422 painting Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920000832 Cutin Polymers 0.000 description 1
- 240000007643 Phytolacca americana Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of corn mature seed farinaceous albumen nail polish aid in whole slices preparation method, including seed stickup fixation, repair block, nail polish embedding, repair the steps such as block, semithin section, section statining and observation again, be characterized in：It is fixed on 1. being pasted mature seed using super glue on resin-base；2. water white transparency nail polish is used as embedding medium, Rapid embedding endosperm tissue；3. establishing a kind of fast and convenient dry incision technology suitable for corn mature seed farinaceous albumen, the whole slices of seed are obtained；4. section has multiple use, the form and size and the spatial distribution in endosperm tissue of iodine staining or polarisation observation amylum body can be carried out.The present invention solves the problems, such as that paraffin section and resin slicer can not prepare corn mature seed whole slices, the defects of dry cutting method can not prepare farinaceous albumen section is overcome simultaneously, so as to provide technical support to study the morphosis of corn mature seed farinaceous albumen.
Description
Technical field
The present invention relates to plant microscopic tissue sections technical field, more particularly to a kind of corn mature seed farinaceous albumen
Whole slices preparation method.
Background technology
Corn is one of main source crop of mankind's grain, animal feed and the raw material of industry.Corn seed contains larger
Endosperm, be the important vegetative storage tissue of corn, its development and substantial situation decide the weight and quality of seed.Corn
Endosperm is divided into cutin and the class of silty two, and the former enriches albuminous cell, and the latter's albuminous cell does not enrich.Starch is that endosperm is main
Reserve substance, its form, size and the distribution in endosperm tissue are always the problem of corn breeding scholar pays special attention to.
Observation On The Morphology is carried out to corn embryosperm, first has to cut into slices to seed.Corn embryosperm morphosis at present
Research be concentrated mainly on development seed, and the research to mature seed is seldom, and the research of especially farinaceous albumen is rarely reported,
Main cause shows the following aspects：1. the classical paraffin section technology of conventional plant tissue slice, but this method
It is unsuitable for rich amyloid mature seed.2. resin embedding corn mature seed is utilized, although section, maize seed can be made
Son is excessive, it is difficult to complete endosperm section is obtained, and resin embedding process is comparatively laborious time-consuming, and embedding medium is expensive.③
External someone is cut into slices using dry cutting method to corn seed, but is only applicable to horny endosperm, and farinaceous albumen can not be completed, and
And it can not also obtain complete seed section using dry cutting method.Therefore need badly to existing microsection manufacture method carry out improvement and it is excellent
Change, invent a kind of simple and rapid whole slices preparation method for corn mature seed farinaceous albumen.
The content of the invention
In order to overcome the shortcomings of above-mentioned existing microsection manufacture method, the invention provides a kind of corn mature seed silty embryo
Newborn nail polish aids in whole slices preparation method.
The technical solution adopted in the present invention is as follows：
A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method, including the stickup of seed to consolidate
Determine, repair block, embedding, repair the steps such as block, semithin section, section statining and observation again, it is characterised in that：Will be into using super glue
Ripe seed, which is pasted, to be fixed on resin-base；Using water white transparency nail polish as embedding medium, Rapid embedding endosperm tissue.
Comprise the following steps that：
(1) seed, which is pasted, fixes：Corn mature seed is crosscutting from seed middle part, it is pasted and fixed on and has been gathered with super glue
On the resin-base of conjunction；The area of resin-base is more than sample area, and descending in shape for resin-base is uniform；The horizontal stroke of resin-base
Sectional area is greater than sample, and sample trembles influence section during preventing section, while resin-base is also less than the big of specimen holder
It is small, so as to which base is clamped completely；Super glue pastes sample and base and needs overnight, to cement completely, to prevent the hair that trembles
It is raw；
(2) slightly repair：Block slightly is repaiied to sample progress using double-edged razor blade and glass cutter on ultramicrotome, sample surface is repaiied
Pat whole, it is ensured that sample surface is the cross section of seed；
(3) refine：Use sharp glass cutter instead and refine block is carried out to flattened sample surfaces, it is in mirror to make sample surfaces
Face；
(4) nail polish embeds：The hairbrush worn with nail polish picks a small amount of nail polish, the sample table to having accomplished minute surface
Smeared in right amount in face, it is ensured that be completely covered, action is soft；Drying at room temperature 15 minutes；
(5) block is repaiied again：Nail polish unnecessary on sample surfaces is cut off using glass cutter, sample is switched to and stops immediately.By
It is limited in penetration depth, therefore can not cuts more, otherwise can wastes the sample being saturated；
(6) semithin section：The section that thickness is 2 microns is cut to sample, will be cut into slices on the water droplet being placed on slide, 45
Dried on degree Celsius roasting piece platform, flatten section；
(7) dyeing observation：Lucifuge stands 10 minutes after iodine solution is added dropwise, with micro- Microscopic observation after cover glass mounting；
Section after iodine staining, or do not dye carry out 50% glycerol concentrations mounting after, respectively in the polarisation with CCD
Observe and take pictures under microscopical visible mode and polarisation pattern, you can obtain the microstructure (Fig. 1) of whole seed, amylum body
Form and size in endosperm tissue and the distribution in endosperm (Fig. 2), the polarized light image (Fig. 3) of amylum body.Polarisation is observed
50% glycerol concentrations mounting must be used, it is easily dry during using water seal piece, there is bubble, influence observing effect.
Step (2) is specifically：Resin-base is clipped on the specimen holder of slicer, in the stereomicroscope observation of slicer
It is lower to be tried one's best equating with the sharp blade position that cut into slices；Install glass cutter afterwards, by it is appropriate to knife after, use and return knife
Mode, which rotates sample arm lifting button, makes sample arm move up and down, while control operation panel, makes the base with glass cutter slow
Advance, until the knife edge just switches to the whole section of material, untill abundant equating.
Slice thickness is 500 nanometers during sample refine block described in step (3).
Step (4) is specifically：1. nail polish is smeared：After minute surface is accomplished in section, the hairbrush carried using nail polish is picked
Enough nail polish, uniformly slowly it is applied on whole sample surface, action is soft, avoids hairbrush from drawing and arrives sample, has on section
Cut can influence the visual effect of gained section；Also to smear uniformly, avoid producing aeration observation；2. nail polish permeates：
After painting wipes nail polish, 15 minutes or so are stood, allows nail polish to carry out fully infiltration and appropriate solidification；Time of penetration mistake
Short, nail polish infiltration is insufficient, and good fixation can not be carried out to amylum body, and the long position that can cause infiltration of time of repose is coagulated
Gu really up to the mark, section is frangible；After nail polish solidification, embedding is completed at the position permeated；The present invention is using nail polish as embedding
Agent, and the position for further solving sample is fixed, establishes the technical problems such as embedding method, smearing method and time of penetration.
Compared with existing microsection manufacture method, advantage of the invention is that：
(1) the quick whole slices preparation method suitable for corn mature seed farinaceous albumen is established；
(2) use water white transparency nail polish that it is more economical to be compared to resin etc. as Rapid embedding agent；
(3) operating process is easy, easy to learn, easy to spread；
(4) the sample pre-treatments steps such as fixed, embedding are saved, it is more convenient；
(5) nail polish is compared to resin and various fixers, and toxicity is lower, uses safer；
(6) can be more quick and the whole slices of seed be intactly cut；
(7) this method, which obtains section, multiple use：Specific stain or polarisation can be carried out to amylum body with iodine solution
Observation." the cross garland " of amylum body is clear in structure under iodine staining amylum body or polarisation, and contrast is obvious, is easy to observe.Quick letter
Just, and section can reuse after soaking and washing.
Brief description of the drawings
Fig. 1 is that first beautiful 335 mature seeds (being rich in farinaceous albumen) nail polish aids in whole slices to corn variety, is cut into slices through iodine
Dyeing, shows distribution of the starch in endosperm tissue under visible light；
Fig. 2 amplifies for Fig. 1 regional areas, shows the form and size of amylum body in farinaceous albumen；
Fig. 3 is picture of the endosperm section under petrographic microscope, is shown " the cross garland " of amylum body.
Embodiment
With reference to example figure, the invention will be further described：
1st, the preparation of section sample：
Seed, which is pasted, to be fixed：Corn ripe seed is polished to target slice position along transverse direction with file first, then
Sample blocks are obtained using sharp cutter or scroll saw are crosscutting in the middle part of corn seed；By size is suitable and the uniform resin bottom of shape
Seat top is polished and clean with alcohol wipe with file, and appropriate super glue is added dropwise after alcohol is dry, sample blocks are disposed across into tree
In glue on fat base, flicking 10 seconds, stand overnight and can ensure that firm pasting.The cross-sectional area of resin-base is greater than
Sample, sample trembles influence section during preventing section, while resin-base is also less than the size of specimen holder, so as to by base
Clamp completely；Super glue pastes sample and base and needs overnight, to cement completely, to prevent generation of trembling；
2nd, block is repaiied：
(1) slightly repair：
1. resin-base is clipped on the specimen holder of slicer, it is ensured that clip；
2. tried one's best equating with the sharp blade position that will cut into slices under the stereomicroscope of slicer；
3. installing glass cutter, it is placed on tool rest and clamps；
4. adjusting the position of sample blocks and glass cutter, to knife under stereoscope, the knife edge and sample surface is set to align as far as possible：
A) backlight of bottom is opened, closes other illuminating lamps, feed makes sample surface close with the knife edge, it can be seen that sample
Occur a bright wisp on face, whether alignd with knife using this bright wisp judgement sample；
B) it is parallel with the knife edge that two sides above and below sample surface are first adjusted：Whether parallel two sides up and down of bright wisp are observed, if not
It is parallel, then specimen holder turn knob is adjusted, makes its parallel；
C) left and right for adjusting sample surface again is parallel with the knife edge：Whether isometric observe the side of left and right two of bright wisp, if Length discrepancy,
Knife edge turn knob is then adjusted, makes its equal；
D) the upper and lower parallel with the knife edge of sample surface is finally adjusted：Move up and down two sides of sample surface, observation bright wisp or so
Whether length changes, if changing, adjusts sample surface turn knob, makes its length constant；
5. after the completion of knife, feed makes the knife edge be approached with sample, and sample surface lower edge and the knife edge are contour, rotates sample arm lifting
Button makes sample arm move up and down, and makes untill the knife edge just switches to material；
6. using returning the mode of knife quickly come sample arm of picking up and down, while control operation panel, make the bottom with glass cutter
Seat is advanced slowly, and makes the abundant equating of sample surface；
(2) refine：Change sharp glass cutter, it is ensured that the edge of a knife flushes.Re-start it is above-mentioned to knife after, rotate clockwise sample
Arm lifts button, and refine is carried out to sample, and slice thickness is 500 nanometers, and sample surface is accomplished into minute surface；
(4) nail polish embeds：
1. nail polish is smeared：After minute surface is accomplished in section, the hairbrush carried using nail polish picks enough nail polish, uniformly
Slowly it is applied on whole sample surface, action is soft, avoids hairbrush from drawing and arrives sample, has cut to influence gained on section and cuts
The visual effect of piece；Also to smear uniformly, avoid producing aeration infiltration；
2. nail polish permeates：After painting wipes nail polish, 15 minutes or so are stood, allows nail polish fully permeate and fit
When solidification；Time of penetration is too short, and nail polish infiltration is insufficient, and good fixation, time of repose mistake can not be carried out to amylum body
Length can cause the position solidification of infiltration really up to the mark, cut into slices frangible；After nail polish solidification, embedding is completed at the position permeated；
4th, cut into slices：
(1) block is repaiied again：After the completion of embedding, by the somewhat withdrawing, then backward of glass cutter before under stereoscopic sem observation slowly
Feed forward, sample arm is rotated clockwise, nail polish unnecessary on sample surface is all cut away, to switching to intact sample face just
Untill；It because penetration depth is limited, therefore can not cut more, otherwise can waste the sample being saturated；
(2) semithin section：
1. slice thickness is arranged to 2 microns, hand section is carried out：The right hand slowly rotates sample arm lifting button and starts to cut into slices,
During the entire process of section, left hand takes eyelash pencil to be gently placed on the surface of section front edge, prevents its curling；Work as section
When being cut out more, eyelash pencil to be placed on section it is following, prevent it to be pasted onto on glass cutter；This process is the most key, left hand
Remain stable, prevent eyelashes from encountering the knife edge or poke section；
2. taking clean slide, appropriate distilled water is added dropwise, slicing edge is carefully clamped with tweezers, will section with and it is horizontal
Face is gently placed on water droplet into 45 degree of angles, is dried on 45 degrees Celsius of roasting piece platforms, is flattened section；
3. section is put in baking oven, 60 degrees Celsius 12 hours, prevent flake in dyeing course.
5th, dye and take pictures：
(1) iodine staining：
1. iodine solution is prepared：5 grams of iodine add 10 grams of KIs to add 85 milliliters of distilled water, dilute 80 with 50% glycerine after being completely dissolved
It is used as dye liquor again.
2. iodine staining (dye starch, in bluish violet)：Lucifuge stands 10 minutes after iodine solution is added dropwise, with micro- after cover glass mounting
Microscopic observation；
(2) take pictures：
Section is inclined with Olympus BX53 respectively after 50% glycerol concentrations mounting of above-mentioned iodine staining mounting or use
The microstructure of the normal optical mode and polarisation pattern observation albuminous cell of light microscope, is taken pictures with subsidiary CCD, is obtained
The moderate optical microphotograph picture of high resolution, contrast (Fig. 1 and Fig. 2) and polarisation picture (Fig. 3).
Claims (4)
1. a kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method, including seed to paste and fix, be thick
Repair, refine, nail polish embedding, repair again block, semithin section and dyeing observation the step of, it is characterised in that：Will using super glue
Mature seed, which is pasted, to be fixed on resin-base；Using water white transparency nail polish as embedding medium, Rapid embedding endosperm tissue；Tool
It is as follows that body includes step：
(1) seed, which is pasted, fixes：Corn mature seed is crosscutting from seed middle part, it is pasted and fixed on what is polymerize with super glue
On resin-base；The area of resin-base is more than sample area, and descending in shape for resin-base is uniform；
(2) slightly repair：Block slightly is repaiied to sample progress using double-edged razor blade and glass cutter on ultramicrotome, sample surface repaired flat
It is whole, it is ensured that sample surface is the cross section of seed；
(3) refine：Use sharp glass cutter instead and refine block is carried out to flattened sample surfaces, it is in minute surface to make sample surfaces；
(4) nail polish embeds：The hairbrush worn with nail polish picks a small amount of nail polish, and the sample surfaces for having accomplished minute surface are entered
Row is appropriate to be smeared, it is ensured that is completely covered, action is soft；Drying at room temperature 15 minutes；
(5) block is repaiied again：Nail polish unnecessary on sample surfaces is cut off using glass cutter, sample is switched to and stops immediately；
(6) semithin section：The section that thickness is 2 microns is cut to sample, will be cut into slices on the water droplet being placed on slide, 45 is Celsius
Dried on the roasting piece platform of degree, flatten section；
(7) dyeing observation.
2. microsection manufacture method according to claim 1, it is characterised in that in step (3), slice thickness is during refine block
500 nanometers.
3. microsection manufacture method according to claim 1, it is characterised in that appropriate to smear nail polish process in step (4)
In, to smear uniformly and prevent bubble.
4. microsection manufacture method according to claim 1, it is characterised in that step (7) is：The section iodine solution of flattening in
Dark place is dyed 5 minutes, is observed under an optical microscope；Or 50% glycerine mounting of undyed section, in petrographic microscope
Lower observation.
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| CN108332989B (en) * | 2018-02-27 | 2020-12-29 | 江南大学 | Method for analyzing high-starch-content cereal grains and observing internal structure |
| CN108802080A (en) * | 2018-08-16 | 2018-11-13 | 扬州大学 | A kind of home position observation method of endosperm starch growth ring in corn mature seed |
| CN110132963A (en) * | 2019-04-26 | 2019-08-16 | 安徽省农业科学院烟草研究所 | A kind of method of Rapid identification waxy corn germplasm |
| CN110646257B (en) * | 2019-08-27 | 2022-05-24 | 中国科学院东北地理与农业生态研究所 | Flaking method for observing aerial root microstructure of corn and application thereof |
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| 外源激素作用下小麦胚乳细胞大小的动态分析;李睿等;《湖北农业科学》;20050131(第01期);35-36页 * |
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