CN104614211A - Paraffin sectioning method of Magnolia sieboldii K.Koch seeds - Google Patents
Paraffin sectioning method of Magnolia sieboldii K.Koch seeds Download PDFInfo
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- CN104614211A CN104614211A CN201510022858.2A CN201510022858A CN104614211A CN 104614211 A CN104614211 A CN 104614211A CN 201510022858 A CN201510022858 A CN 201510022858A CN 104614211 A CN104614211 A CN 104614211A
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Abstract
A paraffin sectioning method of Magnolia sieboldii K.Koch seeds comprises steps as follows: materials are obtained; fixation is performed by the aid of an FAA stationary liquid; step-by-step dehydration is performed by the aid of ethanol solutions with concentrations ranging from 65% to 100%; step-by-step transparency is realized from absolute ethyl alcohol to pure xylene; low-melting-point paraffin crumbs are placed in a small bottle containing 1/2 of the xylene until the xylene is completely saturated, paraffin impregnation is performed at the temperature of 40 DEG C for 24 h, then heat-insulating paraffin impregnation is performed by the aid of mixed paraffin in an incubator at the temperature of 60 DEG C, a mixed paraffin liquid is replaced twice a day, and paraffin impregnation is performed for 72-120 h totally; embedment is performed; sectioning with the thickness ranging from 6 mu m to 8 mu m is performed; sections are bonded to a glass slide by the aid of 2% gelatin, flattened on a water bath at the temperature of 35 DEG C for 2 h, and then placed in a drying oven at the temperature of 38 DEG C for drying; heat-insulating paraffin removal is performed by the aid of the pure xylene at the temperature of 30 DEG C for 40 min, and sections are aired; sealing is performed with neutral Canada balsam. With the adoption of the method, cell tissue of the Magnolia sieboldii K.Koch seeds can be well fixed, and sections are complete, distinct, easy to observe and simple to operate.
Description
Technical field
The present invention relates to a kind of vegetable seeds paraffin section method, particularly a kind of Tiannu Magnolia seed embryo paraffin section method.
Background technology
Tiannu Magnolia (
magnolia sieboldii K.Koch.) be the defoliation small arbor of Magnoliaceae, being rare ornamental plant, is country's three grades of national key protected plant, the wild magnolia that the Northeast of Ye Shi China is unique.
It is lower that Tiannu Magnolia seed has viability, and rest period is long, and germination rate is extremely low, grows the characteristics such as irregular.Greatly limit the amphigenetic success ratio of Tiannu Magnolia, thus cause sylvan life natural regeneration seedling famine.Tiannu Magnolia seedling-raising technique is complicated, and breeding difficulty is large, particularly uses seminal propagation speed slowly.Therefore, carry out and both there is learning value to the research of Seed Dormancy of Magnolia Sieboldii K. Koch and sprouting, there is again certain practical significance.Embryo is the main body of seed germination, and planting embryo after-ripening is the irregular main cause of Tiannu Magnolia seed development, and to the rare report of the research of Tiannu Magnolia embryo germination.
Paraffin section is the important experimental technique of Histological research, basic step comprises the drawing materials of sample, fixes, dewaters, transparent, waxdip, embedding, section, bonding die, dewaxing, a series of special processing such as dyeing and mounting.Experimental procedure is not complicated, but will make high-quality section, and the operation of each step is very important, especially dewaters the transparent stage, and the compatibility of different plant different tissues to medicine has very big-difference, needs to carry out groping and improving.And the correlative study report of Tiannu Magnolia embryo paraffin section technical elements seldom.
In sum, if the preparation method of Tiannu Magnolia embryo paraffin section can be found, for research Seed Dormancy of Magnolia Sieboldii K. Koch and sprouting provide technical support accurately, significant to announcement Seed Dormancy of Magnolia Sieboldii K. Koch mechanism.
Summary of the invention
The object of the invention is to: little for Tiannu Magnolia seed embryo, endosperm is many and endosperm fat content is high, it is difficult that the reasons such as waxdip is difficult cause seed microsection manufacture to operate, the problems such as observing effect is not good, find a kind of preparation method of Tiannu Magnolia seed embryo paraffin section simple to operation, make the biopsy tissues structural integrity of making, clear, be easy to observe.
Technical solution of the present invention is as follows:
1. a Tiannu Magnolia seed paraffin section method, is characterized in that carrying out as follows:
(1) draw materials with fixing
Initial point selection: by removing the full Tiannu Magnolia seed concentration 0.2% disinfecting solution of potassium permanganate 30min of aril, clean with running water;
Sample is fixed: clean seed thieving paper is blotted the surface of the seed moisture, peels off seed coat, and seed is put into rapidly the bottle filling FAA immobile liquid and fix, 4 DEG C of refrigerators are placed, fixing 24-72h;
(2) dewater: fixing material dewaters step by step through the ethanolic solution of 65% → 75% → 85% → 95% → 100% → 100% series concentration successively, each concentration gradient dewatering time 2h → 1.5h successively → 1.5h → 1h → 30min → 15min;
(3) transparent: through 4/5 absolute ethyl alcohol+3/5 dimethylbenzene → 1/5, absolute ethyl alcohol+2/5 dimethylbenzene → 2/5, absolute ethyl alcohol+1/5 dimethylbenzene → 3/5 absolute ethyl alcohol+4/5 dimethylbenzene → pure dimethylbenzene → pure dimethylbenzene, the volume ratio of continuous increase dimethylbenzene, transparent step by step, every grade of clearing time is followed successively by 1.5h → 1.5h → 1.5h → 1.5h → 40min → 20min;
(4) waxdip: by material transparent thoroughly bottle pour out the dimethylbenzene of 1/2 or 2/3, then by fusing point be 52-54 DEG C paraffin wax bits put into bottle, be added to dimethylbenzene completely saturated always; Waxdip 24h at 40 DEG C, afterwards again with blended wax 60 DEG C of insulation waxdips in incubator, changes twice blended wax liquid every day, waxdip 72-120h; Blended wax liquid to be merged by weight the ratio of 1:1 by fusing point to be 58-60 DEG C and fusing point the be paraffin of 50-52 DEG C to form;
(5) embed: material waxdip terminates rear horse back tweezers and transfers in the small paper box that liquid blended wax is housed gently, adjust positions of materials, small paper box is placed in the basin filling frozen water again and cools rapidly, after wax liquid surface solidification, rapid back-off carton submerged, then allow it naturally float in water, prevent crystallization;
(6) cut into slices: according to the position of material in wax stone and direction, wax stone is accomplished little trapezoidal, the candlestick then adhering to microtome is cut into slices, slice thickness 6-8 μm;
(7) bonding die: being evenly coated with thin layer mass volume ratio on the microslide of cleaning is 2% gelatin, wax band is put on microslide carefully, moves wax band to adjust position with the writing brush being moistened with water, after wax band flattens, microslide is placed on 2h on 30-35 DEG C of water-bath, makes wax band light face be close to microslide; Again microslide is put into 38 DEG C of baking ovens afterwards to dry;
(8) dewax: with pure dimethylbenzene 30 DEG C insulation dewaxing 40min in staining jar, outwell dimethylbenzene afterwards, dry section;
(9) mounting: cut into slices and to take out from staining jar, before treating that dimethylbenzene does not volatilize completely, 1 neutral canada balsam is dripped in centre in the material, is placed on cover glass side on the histotomy that drips and have natural gum, slowly puts down cover glass and got rid of completely by air; 40 DEG C of constant temperature oven dried overnight are put in the section of sealing, and make permanent section.
2. Tiannu Magnolia seed paraffin section method according to claim 1, is characterized in that: FAA immobile liquid consists of: concentration 38% formaldehyde, glacial acetic acid and concentration 70% ethanol, three's volume ratio is 1:1:18.
Good effect of the present invention is: the preparation method that have found a kind of suitable Tiannu Magnolia seed paraffin section, make the biopsy tissues structural integrity of making, clear, be easy to observe, observe for the form of Tiannu Magnolia seed development, seed During Removal of Dormancy and cell biology and provide practical technical support and guarantee, for disclosing a class seed embryonic development rule with kind of embryo after-ripening characteristic further, the research effectively carrying out correlation molecule mechanism establishes scientific basic.
Accompanying drawing explanation
Fig. 1 is Tiannu Magnolia earthing of seeds 0d embryo growth conditions figure (× 100) under optical microscope;
Fig. 2 is Tiannu Magnolia earthing of seeds 35d embryo growth change figure (× 100) under optical microscope;
Vt in figure: vascular tissue.
Fig. 3 is Tiannu Magnolia earthing of seeds 65d embryo growth change figure (× 100) under optical microscope;
Fig. 4 is Tiannu Magnolia earthing of seeds 110d embryo growth change figure (× 100) under optical microscope;
Fig. 5 is Tiannu Magnolia earthing of seeds 150d embryo growth change figure (× 100) under optical microscope;
En in figure: endosperm pl: plumule cot: cotyledon hyp: plumular axis.
Fig. 6 is Tiannu Magnolia chitting piece growth change figure (× 100) under optical microscope;
Vb in figure: vascular bundle.
Embodiment
Successfully prepared Tiannu Magnolia seed alternating temperature stratification 0,35,65,110, the paraffin section of 150d and chitting piece.
1. draw materials: in late September, 2011 gathers in the crops Tiannu Magnolia seed.
2. the pre-service of seed
(1) seed screening: seed is soaked in 72h in clear water, removes the unsubstantial seed floated.
(2) go aril: the seed hand after screening is rubbed, remove seed aril.
(3) kind, husky sterilization: with the liquor potassic permanganate of concentration 0.2%, 30min is carried out to seed and disinfect, 1h is carried out to river sand and disinfects, cleaner with running water.
(4) wet sand bed amasss: puddled with part by weight 1:3 by the wet sand of seed and water cut 60% and carry out replacing alternating temperature stratification vernalization high temperature 12-14 DEG C, low temperature 0-5 DEG C, every 5d alternating temperature once, and 150d.
(5) regularly sample: lamination 0,20,35,50,65,80,95,110,130,150d regularly samples, and with the husky potpourri of running water kind, goes sand to reserve seed for planting.
3. sample is fixed: clean seed thieving paper is blotted the surface of the seed moisture, peels off seed coat, and seed is put into rapidly the penicillin bottle filling FAA immobile liquid and fix, 4 DEG C of refrigerators are placed, fixing 24-72h.FAA immobile liquid consists of: concentration 38% formaldehyde, glacial acetic acid and concentration 70% ethanol, three's volume ratio is 1:1:18.
4. dewater: fixing material dewaters step by step through the ethanolic solution of 65% → 75% → 85% → 95% → 100% → 100% series concentration successively, each concentration gradient dewatering time 2h → 1.5h successively → 1.5h → 1h, 100% ethanol carries out at twice, first time 30min, second time 15min.
5. transparent: through 4/5 absolute ethyl alcohol+3/5 dimethylbenzene → 1/5, absolute ethyl alcohol+2/5 dimethylbenzene → 2/5, absolute ethyl alcohol+1/5 dimethylbenzene → 3/5 absolute ethyl alcohol+4/5 dimethylbenzene → pure dimethylbenzene → pure dimethylbenzene, the volume ratio of continuous increase dimethylbenzene, transparent step by step, every grade of clearing time is 1.5h, pure dimethylbenzene clearing time first time 40min, second time 20min.
6. waxdip: by material transparent thoroughly bottle pour out the dimethylbenzene of 1/2 or 2/3, then the paraffin wax of low melting point is considered to be worth doing (fusing point is 52-54 DEG C) and puts into bottle, be added to dimethylbenzene completely saturated always, waxdip 24h in the constant temperature oven of 40 DEG C of conditions.Then use blended wax liquid (to be 58-60 DEG C and fusing point be that the paraffin 1:1 of 50-52 DEG C mix by fusing point) in constant temperature oven 60 DEG C be incubated waxdips, change twice blended wax liquid every day, waxdip 72-120h.
7. embed: transfer in the small paper box that liquid above-mentioned blended wax is housed gently with tweezers immediately after material waxdip terminates, adjust positions of materials, small paper box is placed in the basin filling frozen water again and cools rapidly, after wax liquid surface solidification, rapid back-off carton submerged, then allow it naturally float in water, prevent crystallization.
8. cut into slices: according to the position of material in wax stone and direction, wax stone is accomplished little trapezoidal, the candlestick then adhering to microtome is cut into slices, slice thickness 6-8 μm.
9. bonding die: being evenly coated with thin layer mass volume ratio on the microslide of cleaning is 2% gelatin, wax band is put on microslide carefully, wax band can be moved with the writing brush being moistened with water to adjust position, after wax band flattens, microslide is placed on 2h on 30-35 DEG C of water-bath, makes wax band light face be close to microslide.Again microslide is put into 38 DEG C of baking ovens afterwards to dry.
10. dewax: with pure dimethylbenzene 30 DEG C insulation dewaxing 40min in staining jar, outwell dimethylbenzene afterwards, dry section.
11. mountings: cut into slices and to take out from staining jar, before treating that dimethylbenzene does not volatilize completely, 1 neutral canada balsam is dripped in centre in the material, is placed on cover glass side on the histotomy that drips and have natural gum, slowly puts down cover glass and got rid of completely by air.40 DEG C of constant temperature oven dried overnight are put in the section of sealing, and make permanent section.
12. microscopies and photograph: section be placed in Motic basis of microscopic observation and take a picture, by qualified slice sticker label, again to observe and to take a picture.
Claims (2)
1. a Tiannu Magnolia seed paraffin section method, is characterized in that carrying out as follows:
(1) draw materials with fixing
Initial point selection: by removing the full Tiannu Magnolia seed concentration 0.2% disinfecting solution of potassium permanganate 30min of aril, clean with running water;
Sample is fixed: clean seed thieving paper is blotted the surface of the seed moisture, peels off seed coat, and seed is put into rapidly the bottle filling FAA immobile liquid and fix, 4 DEG C of refrigerators are placed, fixing 24-72h;
(2) dewater: fixing material dewaters step by step through the ethanolic solution of 65% → 75% → 85% → 95% → 100% → 100% series concentration successively, each concentration gradient dewatering time 2h → 1.5h successively → 1.5h → 1h → 30min → 15min;
(3) transparent: through 4/5 absolute ethyl alcohol+3/5 dimethylbenzene → 1/5, absolute ethyl alcohol+2/5 dimethylbenzene → 2/5, absolute ethyl alcohol+1/5 dimethylbenzene → 3/5 absolute ethyl alcohol+4/5 dimethylbenzene → pure dimethylbenzene → pure dimethylbenzene, the volume ratio of continuous increase dimethylbenzene, transparent step by step, every grade of clearing time is followed successively by 1.5h → 1.5h → 1.5h → 1.5h → 40min → 20min;
(4) waxdip: by material transparent thoroughly bottle pour out the dimethylbenzene of 1/2 or 2/3, then by fusing point be 52-54 DEG C paraffin wax bits put into bottle, be added to dimethylbenzene completely saturated always; Waxdip 24h at 40 DEG C, afterwards again with blended wax 60 DEG C of insulation waxdips in incubator, changes twice blended wax liquid every day, waxdip 72-120h; Blended wax liquid to be merged by weight the ratio of 1:1 by fusing point to be 58-60 DEG C and fusing point the be paraffin of 50-52 DEG C to form;
(5) embed: material waxdip terminates rear horse back tweezers and transfers in the small paper box that liquid blended wax is housed gently, adjust positions of materials, small paper box is placed in the basin filling frozen water again and cools rapidly, after wax liquid surface solidification, rapid back-off carton submerged, then allow it naturally float in water, prevent crystallization;
(6) cut into slices: according to the position of material in wax stone and direction, wax stone is accomplished little trapezoidal, the candlestick then adhering to microtome is cut into slices, slice thickness 6-8 μm;
(7) bonding die: being evenly coated with thin layer mass volume ratio on the microslide of cleaning is 2% gelatin, wax band is put on microslide carefully, moves wax band to adjust position with the writing brush being moistened with water, after wax band flattens, microslide is placed on 2h on 30-35 DEG C of water-bath, makes wax band light face be close to microslide; Again microslide is put into 38 DEG C of baking ovens afterwards to dry;
(8) dewax: with pure dimethylbenzene 30 DEG C insulation dewaxing 40min in staining jar, outwell dimethylbenzene afterwards, dry section;
(9) mounting: cut into slices and to take out from staining jar, before treating that dimethylbenzene does not volatilize completely, 1 neutral canada balsam is dripped in centre in the material, is placed on cover glass side on the histotomy that drips and have natural gum, slowly puts down cover glass and got rid of completely by air; 40 DEG C of constant temperature oven dried overnight are put in the section of sealing, and make permanent section.
2. Tiannu Magnolia seed paraffin section method according to claim 1, is characterized in that: FAA immobile liquid consists of: concentration 38% formaldehyde, glacial acetic acid and concentration 70% ethanol, three's volume ratio is 1:1:18.
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Cited By (5)
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| CN105136546A (en) * | 2015-10-22 | 2015-12-09 | 国家林业局泡桐研究开发中心 | Paraffine slicing method for preventing maple leaf plants from leaf anthocyanin loss |
| CN107202720A (en) * | 2017-05-16 | 2017-09-26 | 安徽省农业科学院园艺研究所 | A kind of paraffin section method of pomegranate seed |
| CN109211606A (en) * | 2018-09-06 | 2019-01-15 | 吉林省农业科学院 | A kind of fast method for preparing of pears tissue paraffin section de |
| CN109374376A (en) * | 2018-11-09 | 2019-02-22 | 上海市农业科学院 | A kind of slice preparation method suitable for mushroom lamella Basidium morphologic observation |
| CN111272511A (en) * | 2020-03-19 | 2020-06-12 | 上海农林职业技术学院 | Method for making cucumber fruit thorn tumor paraffin slice |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136546A (en) * | 2015-10-22 | 2015-12-09 | 国家林业局泡桐研究开发中心 | Paraffine slicing method for preventing maple leaf plants from leaf anthocyanin loss |
| CN105136546B (en) * | 2015-10-22 | 2018-03-20 | 国家林业局泡桐研究开发中心 | A kind of red-leaf plants prevent the paraffin section method that blade anthocyanin is lost in |
| CN107202720A (en) * | 2017-05-16 | 2017-09-26 | 安徽省农业科学院园艺研究所 | A kind of paraffin section method of pomegranate seed |
| CN107202720B (en) * | 2017-05-16 | 2019-10-11 | 安徽省农业科学院园艺研究所 | A kind of paraffin section method of pomegranate seed |
| CN109211606A (en) * | 2018-09-06 | 2019-01-15 | 吉林省农业科学院 | A kind of fast method for preparing of pears tissue paraffin section de |
| CN109374376A (en) * | 2018-11-09 | 2019-02-22 | 上海市农业科学院 | A kind of slice preparation method suitable for mushroom lamella Basidium morphologic observation |
| CN111272511A (en) * | 2020-03-19 | 2020-06-12 | 上海农林职业技术学院 | Method for making cucumber fruit thorn tumor paraffin slice |
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