CN104450859A - Morphological analysis method of crop seed endosperm cells - Google Patents
Morphological analysis method of crop seed endosperm cells Download PDFInfo
- Publication number
- CN104450859A CN104450859A CN201410811254.1A CN201410811254A CN104450859A CN 104450859 A CN104450859 A CN 104450859A CN 201410811254 A CN201410811254 A CN 201410811254A CN 104450859 A CN104450859 A CN 104450859A
- Authority
- CN
- China
- Prior art keywords
- cell
- click
- morphological analysis
- image
- caryopsis
- 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
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a morphological analysis method of crop seed endosperm cells. The morphological analysis method comprises the following steps: fabricating resin slices; dyeing the slices; collecting images; pretreating the images; carrying out morphological analysis of cells; and the like. The images are pretreated by accurately drawing and stroking the outlines of seed cells by virtue of a pen tool of Photoshop software. The morphological analysis method can be used for analyzing the morphological features of the endosperm cells, and can also be used for further analyzing the features of endosperm cell contents (starch and protein). The endosperm cells are subjected to accurate treatment and analysis in situ through the Photoshop and Image-Pro Plus software, so that errors are greatly reduced, and massive and full-automatic analysis can be achieved. The defects of cell loss, inaccurate analysis result and the like in a conventional method are overcome; a theoretical basis is provided for breeding, processing and utilizing crops; and the morphological analysis method has important significance in improvement of the quality and yield of the seeds.
Description
Technical field
The present invention relates to plant seed detection technique field, particularly relate to a kind of morphological analysis method of cereal seed albuminous cell.
Background technology
The seed of the cereal crop such as paddy rice, wheat, corn is the main source of mankind's grain, animal feed and industrial raw material.Albuminous cell is the important component part of cereal seed, and its growth and substantial situation decide weight (grain weight) and the quality of seed.The grain of cereal seed is heavy has significant dependency with the number of albuminous cell, and the increase of endosperm appearance object causes the increase that grain is heavy.The particle shape (grain length, grain are wide, grain is thick) of cereal seed is by the impact of the morphological feature such as number, length, width of albuminous cell.Therefore, the morphological feature of albuminous cell is analyzed, from the growth of cytology angle research cereal seed and can enrich, and then illustrate the cytology reason causing seed quality generation difference, for the breeding of cereal, processing and utilization provide theoretical foundation, the quality and yield improving seed is had great importance.
At present, following 2 kinds of methods are mainly adopted to carry out to the research of cereal seed albuminous cell: 1. to utilize cellulase to decompose Formation of Endosperm Cell Walls, separated by albuminous cell and analyze; 2. utilize microtomy to be cut by albuminous cell, then demonstrate profile and the content post analysis of albuminous cell by staining technique.Wherein, 1. there is larger drawback in method: a) operation of cellulase decomposition Formation of Endosperm Cell Walls is more complicated, very strict to the requirement of enzyme concn and enzymolysis time, excessive concentration, overlong time will cause cell walls to be destroyed, and concentration is too low, the time too short will cause albuminous cell be separated insufficient, finally cause the analysis of albuminous cell inaccurate; B) process of cellulase decomposition albuminous cell easily causes the loss of cell, finally causes endosperm appearance object really not statistical uncertainty; C) albuminous cell be separated is mixture, accurately cannot locate the morphological feature of endosperm different sites albuminous cell; D) characteristic of albuminous cell content (starch and albumen) cannot be analyzed.And 2. method solves method many drawbacks 1. well, but still existing defects: a) profile of stained cells is clear not, causes the difficulty of analysis; B) means analyzed are single, process is complicated, the at substantial time, and tolerance range is lower; C) cannot analyze in enormous quantities, and the morphological feature analyzed very little.
At present, the scholar of existing many life sciences utilizes the morphological feature of various professional software to micro-object to analyze, as: 1. with Smileview, manual measurement is carried out to amyloplast and proteoplast; 2. by geometrical property, the measurement liver volume of Image J software analysis rice fecula grain; 3. the axial length of PractiStat software analysis wheat Large and small starch grains is used; 4. the geometrical property etc. of Photoshop and Image-Pro Plus software analysis wheat endosperm starch small grain is used.Wherein first three methods complicated operation, randomness is comparatively large, causes error very large; And the 4. kind method first have employed " the evil spirit rod " of Photoshop software and " eraser " instrument and carry out scratching to starch small grain and to scheme and painted, " count/size " instrument of recycling Image-Pro Plus software is analyzed accurately to all colouring starch small graiies, compare first three methods, greatly simplifie operation steps, and be able to batch the morphological feature of evaluating objects, easy and simple to handle, result is accurate.But the 4. kind method still there is following defect: a) " evil spirit rod " instrument of Photoshop software is only applicable to target that is as single in starch small grain, proteoplast etc., homogeneous, be not suitable for the target including many heterogeneous contents, such as include the cereal albuminous cell of a large amount of proteoplast, starch small grain; B) " evil spirit rod " instrument of Photoshop software is only applicable to comparatively small amt and the target of comparatively disperseing with " eraser " instrument, because when destination number is a lot of and distribution is very compact, " evil spirit rod " instrument just cannot fast by each target area separately, need to distinguish target by other instruments (as " lasso trick " instrument), the region that this instrument is chosen simultaneously is constituency, cannot preserve, disposablely must process all targets; C) coloring process is too complicated, accurately must process by the edge of " eraser " instrument to each target, and workload is still very large.Existing technological method can not meet the morphological feature analyzing cereal seed albuminous cell, because: 1. the albuminous cell of cereal seed is close to each other, and includes a large amount of proteoplast and starch small grain, and " evil spirit rod " instrument accurately cannot sketch the contours the profile of cell; 2. sketch the contours according to " lasso trick " instrument, then need to retouch limit and painted to each cell, the albuminous cell quantity of cereal seed is numerous, and workload is huge; 3. shading operations needs accurately to process by the edge of " eraser " instrument to each target, operates more loaded down with trivial details.Therefore, need to need badly improve existing albuminous cell morphological analysis method and optimize, invent a kind of morphological analysis method of cereal seed cell.
Through groping for a long time, we utilize " pen tool " of Photoshop software to carry out easy, quick, accurate pre-treatment to the displaing micro picture of albuminous cell, the obvious picture of founder cell clear-cut, contrast, Image-Pro Plus software is used to analyze accurately cell again, define a set of effective analytical procedure, well compensate for now methodical defect.Summary of the invention
In order to overcome the deficiency of existing albuminous cell morphological analysis method, the invention provides a kind of morphological analysis method of cereal seed albuminous cell.
The technical solution adopted in the present invention is as follows:
A morphological analysis method for cereal seed albuminous cell, comprises the following steps:
(1) making of resin slicer:
1. draw materials and fix: the seed gathering cereal, the complete caryopsis of careful taking-up, is immersed in 4% paraformaldehyde--24h in 2.5% glutaraldehyde stationary liquid;
2. rinsing and dehydration: the caryopsis after fixing with the abundant rinsing of 0.1M phosphoric acid buffer, then with 30%-50%-70%-90%-100%-100% ethanol, caryopsis is dewatered step by step, every grade of 15min;
3. resin penetration, embedding and polymerization: with 25%-50%-75%-100%-100% London white glue resin, caryopsis is permeated step by step, every grade of 12h, then the caryopsis permeated is put in imbedded mold, inject virgin resin, be placed on by imbedded mold in baking oven, 60 DEG C of polymerization 1-2 days, make hardening of resin;
4. block and semithin section is repaiied: craft is carried out to embedded block and repaiies block, to prune Excess resin around caryopsis, cut with ultramicrotome the section that thickness is 2 microns, section is transferred on the water droplet on slide glass, slide glass is placed on 45 DEG C of hot-plates dry, section is flattened;
(2) dyeing of cutting into slices: with iodine liquid, sarranine-methyl violet dye liquor or white dyes dye liquor to the section statining flattened, by setting off by contrast cell outline to the dyeing of entocyte or directly carrying out dyeing showed cell profile to cell walls;
(3) collection of image: the morphological structure of the upper albuminous cell of shooting section under an optical microscope, preserve picture:
1., when using iodine liquid or sarranine-methyl violet dye liquor to dye, normal light pattern is adopted to take;
2., when using white dyes dye liquor to dye, fluorescence mode is adopted to take;
(4) pre-treatment of image: the picture opening shooting with Photoshop software, utilizes the profile of pen tool to seed cell to sketch the contours accurately, then retouches limit with paintbrush tool to path, preserves picture;
(5) morphological analysis of cell: the picture opening pre-treatment with Image-Pro Plus software, " Count/Size " instrument of utilization carries out morphological analysis to albuminous cell, derives the morphological feature analyzed.
In step of the present invention (4), the pre-treatment concrete operations of described image are as follows:
1. the picture of shooting is opened with Photoshop software, region to be analyzed is selected with " rectangular marquee ", click " editor "-" copy " of menu bar, click again " file "-" newly-built ", in the dialog box ejected, resolving power is changed into " 300 ", background content changes into " background colour ", clicks the painting canvas (background is black) that " determination " newly-built pixel is identical with region to be analyzed;
2. " editor "-" stickup " of menu bar is clicked, by region duplication to be analyzed on new painting canvas;
3. select " pen tool ", one by one cell walls is accurately sketched the contours, press " ESC " key and complete and sketch the contours;
4. open " the Paths panel ", double-click " path icon ", input path title, click " determination " storing path;
5. select " paintbrush tool ", in " paintbrush tool hurdle ", " size " is changed into " 4 " pixel, " hardness " changes into " 100% ";
6. select " pen tool ", " path " that right click is finished, in popup menu, select " stroke path ", select " paintbrush tool " in dialog box, click " determination ", region to be analyzed clearly demonstrates the profile of albuminous cell;
7. right click " Background From Layer " in " Layer panel ", " duplicate layer " is selected in popup menu, click the layer that " determination " generation name is called " background copy ", drag this layer to top, right click " path ", in popup menu, select " stroke path ", " paintbrush tool " is selected in dialog box, click " determination ", demonstrating background is black, and cell outline is the image of white;
8. click " file "-" being stored as " of menu bar, save as the harmless picture of TIF form.
Compared with existing albuminous cell morphological analysis method, advantage of the present invention is:
(1) the morphological analysis method being applicable to various types of grain seed endosperm cell is established.
(2) conjunctive use resin slicer, dyeing, photomicrograph, software processes and analytical technology, cereal seed albuminous cell is carried out to the morphological analysis of original position, intuitively easy, accurate positioning, reliable results, not only can compare the Morphological Differences of different varieties cereal seed albuminous cell, can also the morphological feature of more same kind cereal seed different sites albuminous cell.
(3) not only can be used for analyzing the morphological feature of albuminous cell, the characteristic of albuminous cell content (starch, albumen) can also be analyzed further.
(4) in position cell outline accurately sketched the contours by the pen tool of Photoshop software and retouch limit, overcome the defect of " evil spirit rod ", " lasso trick ", " eraser " instrument, considerably reduce error, simplify operation, save a large amount of analysis time.
(5) can the morphological feature of a large amount of albuminous cell of in enormous quantities, full automatic analysis by " count/size " instrument of Image-Pro Plus software, easy and simple to handle, result is accurate, with a high credibility, and application prospect is broad.
Accompanying drawing explanation
Fig. 1 is the Photomicrograph of iodine staining rice paddy seed section;
Fig. 2 is the picture with generating after Photoshop software processes Fig. 1, shows cell walls profile clearly;
Fig. 3 is the picture with generating after Photoshop software processes Fig. 2, and background is black, and cell walls profile is white;
Fig. 4 is the picture with generating after Image-Pro Plus software analysis Fig. 3, shows the cell numbering participating in calculating;
Fig. 5 is the morphological feature result figure obtained after Image-Pro Plus software analysis Fig. 3.
Fig. 6 is the Photomicrograph of white dyes dyeing wheat seed section;
Fig. 7 is the picture with generating after Photoshop software processes Fig. 6, shows cell walls profile clearly;
Fig. 8 is the picture with generating after Photoshop software processes Fig. 7, and background is black, and cell walls profile is white;
Fig. 9 is the picture with generating after Image-Pro Plus software analysis Fig. 8, shows the cell numbering participating in calculating;
Figure 10 is the morphological feature result figure obtained after Image-Pro Plus software analysis Fig. 8.
Figure 11 is the Photomicrograph of sarranine-methyl violet staining corn seed section;
Figure 12 is the picture with generating after Photoshop software processes Figure 11, shows cell walls profile clearly;
Figure 13 is the picture with generating after Photoshop software processes Figure 12, and background is black, and cell walls profile is white;
Figure 14 is the picture with generating after Image-Pro Plus software analysis Figure 13, shows the cell numbering participating in calculating;
Figure 15 is the morphological feature result figure obtained after Image-Pro Plus software analysis Figure 13.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
Embodiment one: the morphological analysis of rice paddy seed albuminous cell
1. the making of resin slicer:
(1) draw materials and fix:
1. 0.2mol/L phosphoric acid buffer (pH 7.4) is prepared:
A) mother liquor A (the 0.2mol/L Sodium phosphate dibasic aqueous solution): take 71.64g Na
2hPO
412 H
2o, is settled to 1000mL with distilled water;
B) mother liquor B (0.2mol/L biphosphate sodium water solution): take 31.21g NaH
2pO
42 H
2o, is settled to 1000mL with distilled water;
C) according to A liquid: the ratio mixing of B liquid=81:19, regulates pH to 7.4 with A liquid or B liquid.
2. the 10% paraformaldehyde aqueous solution is prepared:
A) take 20g paraformaldehyde in 250mL triangular pyramidal bottle, add 200mL distilled water, put into the magnetic stir bar of 25*9mm specification;
C) 0.1mol/L aqueous sodium hydroxide solution is prepared: take 40mg sodium hydroxide in 10mL centrifuge tube, add 10mL distilled water, mix; Preparation 0.1mol/L aqueous hydrochloric acid: measure the concentrated hydrochloric acid that 4.2mL massfraction is 36.5%, be settled to 500mL with distilled water;
D) in Erlenmeyer flask, dropwise drip 0.1mol/L aqueous sodium hydroxide solution just to clarify to the paraformaldehyde aqueous solution;
E) be cooled to room temperature, regulate pH to 7.4.
3. 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid is prepared: 100mL 0.2mol/L phosphoric acid buffer (the pH 7.4)+80mL 10% paraformaldehyde aqueous solution+20mL 25% glutaraldehyde water solution (traditional Chinese medicines group, FEM import packing) fully mix, regulate pH to 7.4 with sodium hydroxide or aqueous hydrochloric acid.
4. rice paddy seed is taken off from spike of rice, clever shell is carefully peelled off with dissecting needle, take out complete caryopsis, selecting the normal caryopsis of particle shape is put in penicillin bottle, 10mL 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid is added rapidly in penicillin bottle, make caryopsis be immersed in stationary liquid, be put in 4 DEG C of refrigerators and fix 12h, make caryopsis softening and preliminary fixing;
5. take out caryopsis with tweezers, open cross-section from middle part for softening caryopsis with sharp blade, cut the tissue block that 1mm in the middle part of caryopsis is thick, be again soaked in the stationary liquid in penicillin bottle, be put in 4 DEG C of refrigerators and fix 48h, make tissue and cell fully fixing.
(2) rinsing and dehydration:
1. 0.1mol/L phosphoric acid buffer (pH 7.4) is prepared: 100mL 0.2mol/L phosphoric acid buffer+100mL distilled water, fully mixes.
2. carefully suck stationary liquid with clean suction pipe, do not encounter caryopsis section, then the caryopsis after fixing with the rinsing of 0.1mol/L phosphoric acid buffer, altogether rinsing 5 times, each 30min, fully washes away stationary liquid.
3. compound concentration is the aqueous ethanolic solution of 30%, 50%, 70% and 90%, dewaters step by step to caryopsis according to the gradient of 30%-50%-70%-90%-100%-100%: every grade of 10mL, dehydration 15min.
(3) resin penetration, embedding and polymerization:
1. London white glue (LR White) resin is prepared: measure 40mL resin in 50mL centrifuge tube, taking 0.8g catalyzer (Benzoyl Peroxide) pours in centrifuge tube, magnetic agitation 20min makes abundant dissolving, uses after being put in 4 DEG C of refrigerator 24h;
2. compound concentration is the resin ethanolic soln of 25%, 50% and 75%: resin and dehydrated alcohol form according to the ratio mixture of 1:3,1:1 and 3:1;
3. according to the gradient of 25%-50%-75%-100%-100%, caryopsis is permeated step by step: every grade of 5mL, be put in 4 DEG C of refrigerators, infiltration 12h.
4. clamp caryopsis side gently with tweezers, transverse section is put in imbedded mold down, slowly inject virgin resin submergence caryopsis with 200mL liquid-transfering gun, avoid occurring bubble.
5. be placed in baking oven by imbedded mold, 60 DEG C of polymerizations 1-2 days (time must control within 2 days, prevents from causing resin shrinkage, causes section difficulty), make hardening of resin.
(4) block and semithin section is repaiied:
1. first with file, sample surfaces embedding medium is ground off, expose caryopsis;
2. then embedded block is clipped on the specimen holder of slicing machine, 2mm is exposed on top, to prune embedding medium with the angle at 45 ° with horizontal plane by the surrounding of sharp blade at caryopsis under stereoscopic microscope, cone-shaped body is accomplished in its side, and upper hexagon parallel is below accomplished in the front of sample;
3. mounting glass cutter, is placed in cutter holder and clamps;
4. adjust the position of embedded block and glass cutter, tool setting under stereoscope, make the knife edge and sample in the face of neat:
Whether a) open the backlight of bottom, close other head lights, feed makes sample face and the knife edge close, can see an appearance bright wisp on sample face, utilize this bright wisp judgement sample to align with cutter;
B) first adjust upper and lower two limits, sample face parallel with the knife edge: whether two limits up and down observing bright wisp are parallel, if not parallel, then adjust specimen holder and rotate knob, make it parallel;
C) left and right adjusting sample face is again parallel with the knife edge: whether the limit, two, left and right observing bright wisp is isometric, if Length discrepancy, then adjusts the knife edge and rotates knob, make it equal;
D) finally adjust the parallel with the knife edge up and down of sample face: move up and down sample face, about observation bright wisp, whether the length on two limits changes, if change, then adjusts sample face and rotates knob, make its length constant;
5. after tool setting completes, feed make the knife edge and sample close, sample face lower edge and the knife edge contour, rotate sample arm lifting button sample arm is moved up and down, till making the knife edge just switch to tissue;
6. slice thickness is set to 2 microns, carry out hand section: the right hand slowly rotates sample arm lifting button and starts section, in the whole process of section, the front of embedded block at the uniform velocity, lentamente moves down, the glass knife edge cuts section gradually, and left hand takes eyelash pencil to be placed on gently directly over section front edge, prevents it curling, this process will remain stable, prevents eyelashes from encountering the knife edge or poking section;
7. get clean slide glass, drip distilled water, carefully clamp slicing edge with tweezers, by section to be placed on gently on water droplet with horizontal plane angle at 45 °, dry on 45 DEG C of hot-plates, section is flattened.
8. be put in baking oven by section, 60 DEG C are dried 12h, prevent flake in dyeing course.
2. the dyeing of section:
(1) iodine liquid preparation: 5g iodine+10g potassiumiodide+85mL distilled water, dilutes 80 times as dye liquor with 50% glycerine after dissolving completely.
(2) iodine staining: after dripping iodine liquid, lucifuge leaves standstill 10min, with basis of microscopic observation after cover glass mounting;
3. the collection of image: the microstructure of observing albuminous cell by the microscopical normal light pattern of Olympus BX53, takes pictures with subsidiary CCD, obtains displaing micro picture 1.
4. the pre-treatment of image:
(1) picture 1 of shooting is opened with Photoshop software, region to be analyzed is selected with " rectangular marquee ", click " editor "-" copy " of menu bar, click again " file "-" newly-built ", in the dialog box ejected, resolving power is changed into " 300 ", background content changes into " background colour ", clicks the painting canvas (background is black) that " determination " newly-built pixel is identical with region to be analyzed;
(2) " editor "-" stickup " of menu bar is clicked, by region duplication to be analyzed on new painting canvas;
(3) select " pen tool ", one by one cell walls is accurately sketched the contours, press " ESC " key and complete and sketch the contours;
(4) open " the Paths panel ", double-click " path icon ", input path title, click " determination " storing path;
(5) select " paintbrush tool ", in " paintbrush tool hurdle ", " size " is changed into " 4 " pixel, " hardness " changes into " 100% ";
(6) select " pen tool ", " path " that right click is finished, in popup menu, select " stroke path ", select " paintbrush tool " in dialog box, click " determination ", region to be analyzed clearly demonstrates the profile of albuminous cell, as Fig. 2;
(7) right click " Background From Layer " in " Layer panel ", " duplicate layer " is selected in popup menu, click the layer that " determination " generation name is called " background copy ", drag this layer to top, right click " path ", " stroke path " is selected in popup menu, select " paintbrush tool " in dialog box, click " determination ", demonstrating background is black, cell outline is the image of white, as Fig. 3;
(8) click " file "-" being stored as " of menu bar, save as the harmless picture of TIF form.
5. the morphological analysis of cell:
(1) setting of scale:
1. C1 type micrometer picture is opened with Image-Pro Plus software, click " Measure "-" Calibration "-" Spatial " of menu bar, " New " newly-built scale is clicked in the window ejected, " Name " up one " spatial cal 0 " place under hurdle taps, input scale name " 10X ";
2. " Unit " one hurdle select " μm " as unit, choose " convert when change unit ";
3. " Image " of " Units/pixel " in hurdle is clicked, eject " scaling " dialog box, show a scale in the scale picture upper left corner simultaneously, mouse is moved on on the whippletree of this scale, tap left button, cursor just becomes little hand immediately, just drag with this Bar end scale, again little hand is moved on to successively the two ends, left and right of whippletree, " magnifying glass " instrument of utilization, drag these two end points to this line place of scale on picture both sides with left button, in " scaling " dialog box, fill out scale length, click " OK " close box.
(2) define scale: " Measure "-" Calibration "-" the Select Spatial " clicking menu bar, in the window ejected, click newly-built " 10X " scale.
(3) morphological analysis:
1. click " Measure "-" count/size " of menu bar, eject " count/size " dialog box;
2. " Measure "-" the Select Measurements " on the dialog box menu hurdle is clicked, eject " SelectMeasurements " dialog box, click the required parameter measured, here we select " Area (area) ", " Roundness (circularity) " " Size (length) (long axis length) " and " Size (width) (minor axis length) ", and click " OK " returns " count/size " dialog box;
3. click " Automatic Dark Objects ", " Measure Objects ", " Apply FilterRanges " and " Display Objects " check box on hook, click the calculating that " Count " button completes parameter, display is participated in by picture each cell label calculated, as Fig. 4;
4. click " View "-" the Measurement Data " on the dialog box menu hurdle, obtain the statistic data of albuminous cell morphological feature, as Fig. 5.Wherein square measure is μm
2, circularity unit is μm
2/ μm
2, unit of length is μm.
5. the implication of each parameter:
A) " Area ": the area representing measuring object;
B) " Roundness ": the circularity of indicated object, calculation formula=(girth
2)/(4* π * area); The circularity of circle object is 1, and other shape circularity is greater than 1;
C) " Size (length) ": indicated object along main shaft (major axis) direction Object Projection profile two border parallel lines between distance diameter;
D) " Size (width) ": indicated object along countershaft (minor axis) direction Object Projection profile two border parallel lines between distance diameter.
Embodiment two: the morphological analysis of wheat seed albuminous cell
1. the making of resin slicer:
(1) draw materials and fix:
1. 0.2mol/L phosphoric acid buffer (pH 7.4) is prepared: method is with embodiment one.
2. the 10% paraformaldehyde aqueous solution is prepared: method is with embodiment one.
3. 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid is prepared: method is with embodiment one.
4. wheat seed is taken off from the wheat head, clever shell is carefully peelled off with dissecting needle, take out complete caryopsis, selecting the normal caryopsis of particle shape is put in penicillin bottle, 10mL 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid is added rapidly in penicillin bottle, make caryopsis be immersed in stationary liquid, be put in 4 DEG C of refrigerators and fix 12h, make caryopsis softening and preliminary fixing;
5. take out caryopsis with tweezers, open cross-section from middle part for softening caryopsis with sharp blade, cut the tissue block that 1mm in the middle part of caryopsis is thick, be again soaked in the stationary liquid in penicillin bottle, be put in 4 DEG C of refrigerators and fix 48h, make tissue and cell fully fixing.
(2) rinsing and dehydration: method is with embodiment one.
(3) resin penetration, embedding and polymerization: method is with embodiment one.
(4) block and semithin section is repaiied: method is with embodiment one.
2. the dyeing of section:
(1) white dyes dye liquor preparation: 10mg white dyes+1mL water, fully mixing dissolves rear dilution 10 times as dye liquor.
(2) white dyes dyeing:
1. section is put on hot-plate and is heated to 55 DEG C;
2. white dyes dye liquor dyeing 10min is dripped;
3. 10min is washed, basis of microscopic observation after hot-plate is dried.
3. the collection of image: the microstructure of observing albuminous cell by the microscopical fluorescence mode of Olympus BX51, takes pictures with subsidiary CCD, obtains the displaing micro picture 6 of cell.
4. the pre-treatment of image: method, with embodiment one, obtains picture 7 and 8.
5. the morphological analysis of cell: method, with embodiment one, obtains picture 9 and 10.
Embodiment three: the morphological analysis of corn seed albuminous cell
1. the making of resin slicer:
(1) draw materials and fix:
1. 0.2mol/L phosphoric acid buffer (pH 7.4) is prepared: method is with embodiment one.
2. the 10% paraformaldehyde aqueous solution is prepared: method is with embodiment one.
3. 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid is prepared: method is with embodiment one.
4. corn seed is taken off from corn ear, selecting the normal caryopsis of particle shape is put in penicillin bottle, adds 10mL 4% paraformaldehyde-2.5% glutaraldehyde stationary liquid rapidly, make caryopsis be immersed in stationary liquid in penicillin bottle, be put in 4 DEG C of refrigerators and fix 12h, make caryopsis softening and preliminary fixing;
5. take out caryopsis with tweezers, open cross-section from middle part for softening caryopsis with sharp blade, cut the tissue block that 1mm in the middle part of caryopsis is thick, be again soaked in the stationary liquid in penicillin bottle, be put in 4 DEG C of refrigerators and fix 48h, make tissue and cell fully fixing.
(2) rinsing and dehydration: method is with embodiment one.
(3) resin penetration, embedding and polymerization: method is with embodiment one.
(4) block and semithin section is repaiied: method is with embodiment one.
2. the dyeing of section:
(1) sarranine-methyl violet dye liquor preparation:
1. sarranine dye liquor: 1g sarranine+10mL 95% dissolve with ethanol, then be settled to 100mL;
2. methyl violet dye liquor: 0.5g methyl violet+100mL distilled water.
(2) sarranine-methyl violet staining:
1. section is put on hot-plate and is heated to 55 DEG C;
2. sarranine dye liquor dyeing 12min is dripped;
3. wash 5min, hot-plate is dried;
4. methyl violet dye liquor dyeing 5min is dripped;
5. 5min is washed, basis of microscopic observation after hot-plate is dried.
3. the collection of image: method, with embodiment one, obtains the displaing micro picture 11 of cell.
4. the pre-treatment of image: method, with embodiment one, obtains picture 12 and 13.
5. the morphological analysis of cell: method, with embodiment one, obtains picture 14 and 15.
Claims (4)
1. a morphological analysis method for cereal seed albuminous cell, comprises the following steps: the making of (1) resin slicer; (2) dyeing of cutting into slices; (3) collection of image; (4) pre-treatment of image; (5) morphological analysis of cell, is characterized in that, the pre-treatment of step (4) image utilizes the pen tool of Photoshop software sketch the contours accurately the profile of seed cell and retouch limit.
2. morphological analysis method according to claim 1, it is characterized in that, concrete steps are as follows:
(1) making of resin slicer
1. draw materials and fix: the seed gathering cereal, the complete caryopsis of careful taking-up, is immersed in 4% paraformaldehyde--24h in 2.5% glutaraldehyde stationary liquid;
2. rinsing and dehydration: the caryopsis after fixing with the abundant rinsing of 0.1M phosphoric acid buffer, then with 30%-50%-70%-90%-100%-100% ethanol, caryopsis is dewatered step by step, every grade of 15min;
3. resin penetration, embedding and polymerization: with 25%-50%-75%-100%-100% London white glue resin, caryopsis is permeated step by step, every grade of 12h, then the caryopsis permeated is put in imbedded mold, inject virgin resin, be placed on by imbedded mold in baking oven, 60 DEG C of polymerization 1-2 days, make hardening of resin;
4. block and semithin section is repaiied: craft is carried out to embedded block and repaiies block, to prune Excess resin around caryopsis, cut with ultramicrotome the section that thickness is 2 microns, section is transferred on the water droplet on slide glass, slide glass is placed on 45 DEG C of hot-plates dry, section is flattened;
(2) dyeing of cutting into slices: with iodine liquid, sarranine-methyl violet dye liquor or white dyes dye liquor to the section statining flattened, by setting off by contrast cell outline to the dyeing of entocyte or directly carrying out dyeing showed cell profile to cell walls;
(3) collection of image: the morphological structure of the upper albuminous cell of shooting section under an optical microscope, preserves picture;
(4) pre-treatment of image: the picture opening shooting with Photoshop software, utilizes the profile of pen tool to seed cell to sketch the contours accurately, then retouches limit with paintbrush tool to path, preserves picture;
(5) morphological analysis of cell: the picture opening pre-treatment with Image-Pro Plus software, " Count/Size " instrument of utilization carries out morphological analysis to albuminous cell.
3. morphological analysis method according to claim 1 and 2, is characterized in that, in step (3), the gatherer process of image carries out under an optical microscope:
1., when using iodine liquid or sarranine-methyl violet dye liquor to dye, normal light pattern is adopted to take;
2., when using white dyes dye liquor to dye, fluorescence mode is adopted to take.
4. morphological analysis method according to claim 1 and 2, is characterized in that, in step (4), the pre-treatment concrete operations of described image are as follows:
1. the picture of shooting is opened with Photoshop software, region to be analyzed is selected with " rectangular marquee ", click " editor "-" copy " of menu bar, click again " file "-" newly-built ", in the dialog box ejected, resolving power is changed into " 300 ", background content changes into " background colour ", clicks the painting canvas (background is black) that " determination " newly-built pixel is identical with region to be analyzed;
2. " editor "-" stickup " of menu bar is clicked, by region duplication to be analyzed on new painting canvas;
3. select " pen tool ", one by one cell walls is accurately sketched the contours, press " ESC " key and complete and sketch the contours;
4. open " the Paths panel ", double-click " path icon ", input path title, click " determination " storing path;
5. select " paintbrush tool ", in " paintbrush tool hurdle ", " size " is changed into " 4 " pixel, " hardness " changes into " 100% ";
6. select " pen tool ", " path " that right click is finished, in popup menu, select " stroke path ", select " paintbrush tool " in dialog box, click " determination ", region to be analyzed clearly demonstrates the profile of albuminous cell;
7. right click " Background From Layer " in " Layer panel ", " duplicate layer " is selected in popup menu, click the layer that " determination " generation name is called " background copy ", drag this layer to top, right click " path ", in popup menu, select " stroke path ", " paintbrush tool " is selected in dialog box, click " determination ", demonstrating background is black, and cell outline is the image of white;
8. click " file "-" being stored as " of menu bar, save as the harmless picture of TIF form.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410811254.1A CN104450859A (en) | 2014-12-23 | 2014-12-23 | Morphological analysis method of crop seed endosperm cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410811254.1A CN104450859A (en) | 2014-12-23 | 2014-12-23 | Morphological analysis method of crop seed endosperm cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104450859A true CN104450859A (en) | 2015-03-25 |
Family
ID=52897605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410811254.1A Pending CN104450859A (en) | 2014-12-23 | 2014-12-23 | Morphological analysis method of crop seed endosperm cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450859A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849125A (en) * | 2015-06-11 | 2015-08-19 | 扬州大学 | Method for separating different space parts of fresh sample caryopses of wheat |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
| CN106568758A (en) * | 2016-11-11 | 2017-04-19 | 西北师范大学 | Method of using three dimensional reconstruction imaging technology to analyze structure of root tip cells of arabidopis thaliana |
| 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 |
| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
| CN110763165A (en) * | 2019-10-26 | 2020-02-07 | 福建农林大学 | Method for measuring area of single acinus of pharyngeal gland of bee |
| CN117517029A (en) * | 2024-01-05 | 2024-02-06 | 南京农业大学三亚研究院 | Method for making resin slice of wheat mature period grain |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062212A (en) * | 2014-07-17 | 2014-09-24 | 扬州大学 | Method for analyzing geometric characteristics of plant cell amyloid and proteosome |
-
2014
- 2014-12-23 CN CN201410811254.1A patent/CN104450859A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062212A (en) * | 2014-07-17 | 2014-09-24 | 扬州大学 | Method for analyzing geometric characteristics of plant cell amyloid and proteosome |
Non-Patent Citations (3)
| Title |
|---|
| 余徐润等: "Image-Pro Plus软件在小麦淀粉粒显微图像分析中的应用", 《电子显微学报》 * |
| 俞轶平: "Photoshop抠图法之钢笔工具的使用", 《科技资讯》 * |
| 李栋梁等: ""高粱胚乳细胞与母体细胞发育关系的研究"", 《中国农业科学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849125A (en) * | 2015-06-11 | 2015-08-19 | 扬州大学 | Method for separating different space parts of fresh sample caryopses of wheat |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
| CN106568758A (en) * | 2016-11-11 | 2017-04-19 | 西北师范大学 | Method of using three dimensional reconstruction imaging technology to analyze structure of root tip cells of arabidopis thaliana |
| CN106568758B (en) * | 2016-11-11 | 2019-01-22 | 西北师范大学 | The method as technology analysis arabidopsis root-tip cells structure is reassembled into using three-dimensional |
| 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 |
| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
| CN110763165A (en) * | 2019-10-26 | 2020-02-07 | 福建农林大学 | Method for measuring area of single acinus of pharyngeal gland of bee |
| CN117517029A (en) * | 2024-01-05 | 2024-02-06 | 南京农业大学三亚研究院 | Method for making resin slice of wheat mature period grain |
| CN117517029B (en) * | 2024-01-05 | 2024-04-16 | 南京农业大学三亚研究院 | Method for making resin slice of wheat mature period grain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104450859A (en) | Morphological analysis method of crop seed endosperm cells | |
| Smith et al. | Serial block face scanning electron microscopy in cell biology: Applications and technology | |
| Paul et al. | Characterisation of mycelial morphology using image analysis | |
| Hollandi et al. | Nucleus segmentation: towards automated solutions | |
| CN103398999B (en) | Based on the detection method of carrotene distribution in the Isochrysis galbana of Raman spectroscopy | |
| CN100567943C (en) | A kind of section statining method of skeletal muscle band | |
| DE112014002043T5 (en) | A charged particle beam device and sample observation method | |
| CN104374601B (en) | A kind of resin slicer preparation method of kernel maturity seed | |
| CN104406834A (en) | Marine fish sperm staining method | |
| CN109142398A (en) | A method of improving arabidopsis seed image quality | |
| Sivaguru et al. | Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy | |
| De Smet et al. | An easy and versatile embedding method for transverse sections | |
| Höög et al. | Whole‐cell investigation of microtubule cytoskeleton architecture by electron tomography | |
| Czymmek et al. | Imaging plant cells by high-pressure freezing and serial block-face scanning electron microscopy | |
| CN106568758A (en) | Method of using three dimensional reconstruction imaging technology to analyze structure of root tip cells of arabidopis thaliana | |
| Khoshravesh et al. | Leaf microscopy applications in photosynthesis research: identifying the gaps | |
| Webb et al. | Volume scanning electron microscopy: serial block-face scanning electron microscopy focussed ion beam scanning electron microscopy | |
| CN111829859B (en) | Efficient poplar seed transparent dyeing and three-dimensional imaging method thereof | |
| CN105699312A (en) | Method for identifying material of leather product | |
| Noyce et al. | A practical method for staging grapevine inflorescence primordia in season 1, with improved description of stages | |
| Roselli et al. | New data‐driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume | |
| CN104390834A (en) | Sarranine and methyl violet mixed staining method for resin slices and staining solution thereof | |
| Reagan et al. | Focused ion beam-scanning electron microscopy for investigating plasmodesmal densities | |
| CN108872166B (en) | Method for realizing three-dimensional visualization of rape seed single cells and oil drops by double fluorescence labeling | |
| Kolotuev | Work smart, not hard: How array tomography can help increase the ultrastructure data output |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150325 |