CN107462454B - Dyeing method of amygdalus mongholicus anther intracellular polysaccharide granules - Google Patents
Dyeing method of amygdalus mongholicus anther intracellular polysaccharide granules Download PDFInfo
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- CN107462454B CN107462454B CN201610382818.3A CN201610382818A CN107462454B CN 107462454 B CN107462454 B CN 107462454B CN 201610382818 A CN201610382818 A CN 201610382818A CN 107462454 B CN107462454 B CN 107462454B
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Classifications
-
- 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
-
- 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
- G01N2001/305—Fixative compositions
Abstract
The invention discloses a dyeing method of polysaccharide particles in almond anther cells, which is characterized in that a peach anther cell slice is dyed for 0.5h by using a dye solution prepared in advance under the dark condition, redundant dye solution is firstly absorbed by using filter paper, then 0.5% sodium metabisulfite solution is respectively used for color separation and distilled water is used for color washing, the slice is placed under a microscope for observing the polysaccharide particles after being dried and the slice can be made into a permanent slice after being fixed by neutral gum. Under microscope observation, the polysaccharide particles in the tonsil cells were stained to a vivid red or deep red color, and the more the polysaccharide particle content, the more the staining. The method can observe the distribution conditions of the amygdaloid epidermal cells, the cells of the inner wall of the medicine chamber, the cells of the middle layer, the tapetum cells, the pollen grains and the polysaccharide grains at the connecting tissues in different periods, and is used for researching the functions of the polysaccharide grains in pollen development, anther cracking and anther sugar metabolism.
Description
Technical Field
The invention discloses a dyeing method of polysaccharide particles in almond anther cells, which is characterized in that a peach anther cell slice is dyed for 0.5h by using a dye solution prepared in advance under the dark condition, redundant dye solution is firstly absorbed by using filter paper, then 0.5% sodium metabisulfite solution is respectively used for color separation and distilled water is used for color washing, the slice is placed under a microscope for observing the polysaccharide particles after being dried and the slice can be made into a permanent slice after being fixed by neutral gum. Under microscope observation, the polysaccharide particles in the tonsil cells were stained to a vivid red or deep red color, and the more the polysaccharide particle content, the more the staining. The method can observe the distribution conditions of the amygdaloid epidermal cells, the cells of the inner wall of the medicine chamber, the cells of the middle layer, the tapetum cells, the pollen grains and the polysaccharide grains at the connecting tissues in different periods, and is used for researching the functions of the polysaccharide grains in pollen development, anther cracking and anther sugar metabolism.
Background
Almond (Amygdalus communis L.) The almond apricot, named as almond, belongs to the genus amygdalus of the genus peaches of the family rosaceae, is a perennial deciduous fruit tree and one of the world famous nut tree species, and the annual yield and trade volume of the almond apricot belong to the first of four nuts (almond, walnut, hazelnut and pistachio). The almond kernel has rich nutrition and good taste, is deeply loved by consumers, can be eaten raw,or processing into parched kernel and glutinous kernel with special taste of Mel, and salt taste of Capsici fructus; takes almond as one of raw materials, and can be processed into chocolate candy, high-grade cake, dry fruit can and the like. The almond volatile oil can be used for preparing high quality oil, cosmetic balm, vanishing cream, almond cheese, almond water, almond emulsion, almond bran, etc., and can be used for eliminating freckle and sweating sole and axilla. The mandelic emulsion oil can also be used for treating skin pruritus. The Chinese external trades also make almond into various tonics, such as almond milk and almond wine, and use bitter almond to make sedative and analgesic, and make amygdalin hydroxide new drug in the United states for adjuvant treatment of cancer; or used as breakfast to treat diabetes, hypercholesterolemia, obesity, coronary heart disease, children epilepsy, and gastropathy. In addition, cherry gum secreted by almond kernels is a good industrial raw material. The almond flowers are beautiful and aromatic, are important woody flowers and honey-source plants in early spring, and are also greening tree species and mountain land soil-fixing tree species. The resin secreted by the tonsil trunk can be processed into gum arabic and cotton fabric coloring agent. The almond wood is hard, has good glossiness and light red and beautiful color, and can be used for manufacturing high-grade furniture and exquisite manual craft woodware. The method can also be used for extracting the anti-phenolic oxidant from the almond shell by a new process and processing the almond shell into the activated carbon.
The almond is mainly distributed in Kaishi areas of the Uygur autonomous region in Xinjiang, and is also cultivated sporadically in areas such as Kurler, Aksu, Artush, Hotan and the like, the local varieties such as olecranon, leatheroid, hemp shell, Zhanfeng, Shuangguo, Hanfeng and the like are taken as main parts in the areas, and the cultivation areas of foreign introduced varieties such as Nonpareil, Mission and the like are increased rapidly. The 2006-year data of the department of forestry, Uyghur autonomous region, Sinkiang, show that, Xinjiang almonds are usually cultivated in an agricultural and forestry intercropping mode, the area is about 9647 hm2, the total yield is about 721 t, and the yield of many big trees grown in two thirty years is lower, which is only equivalent to 1/10-1/5 of the yield of almonds cultivated in developed countries such as California and Iran. Along with the popularization of the cultivation technology, the yield of the almond in China is improved, the cultivation area is rapidly increased, and the main production area of the almond cultivated in Xinjiang is mainly distributed in the Kwangshi areaThe current cultivation area of the vehicle, the Yingjisha county and the surrounding areas is about 7.3 kilohm2With a resulting area of about 4 kilohm2The fruit bearing area is about 1.3 kilohm2The total yield in 2014 is about 4 ten thousand t. Although the almond variety in China has abundant resources, the yield is low, and the yield of only a few excellent single plants reaches 20 kg. The main reason is that the economic benefit is influenced by early spring, low temperature and late frost. In addition, in the sexual reproduction stage of the amygdala, the low-temperature stress can cause structural and functional abnormalities of pollen, fertilization failure and other serious consequences. The freezing injury can also cause the spread of diseases such as the death of branches and flower buds due to freezing, the weak tree vigor, the rot disease and the like, and seriously influences the production development of fruit trees. In the earlier research, the subject group finds that the sugar metabolism plays an important role in the development and cold resistance of the almond anther. In order to research the change of saccharides in cells of different stages and different parts of the almond anther, the subject group invents a dyeing method of polysaccharide granules in cells of the almond anther, which can better observe the distribution condition of the polysaccharide granules at epidermal cells, cells of the inner wall of an anther chamber, tapetum cells, pollen grains and connective tissues of the almond anther and is used for researching the action and the change of the polysaccharide in pollen development, anther cracking and carbohydrate metabolism. In previous experiments, conventional I was used2The KI staining method stains polysaccharide particles, but since light is emitted from bottom to top under the microscope, conventional I2the-KI dyeing method cannot effectively distinguish polysaccharide particles from shadows, and the method can dye the polysaccharide particles into bright red, so that the granular polysaccharide particles are clearly visible, and the method can be used for researching the functions of the polysaccharide in pollen development, anther dehiscence and anther sugar metabolism.
Disclosure of Invention
For the conventional I2the-KI staining method stains the polysaccharide particles blue, but since light is emitted from the bottom up when the polysaccharide particles are observed with a microscope, the imaging system cannot distinguish the polysaccharide particles well. The invention discloses a dyeing method of polysaccharide granules in almond anther cells, which is characterized in that a prepared dye solution is used for dyeing almond anther slices processed by a traditional paraffin slicing method under the condition of keeping out of the sun, and after the almond anther slices are dyed for 0.5h, filtering is firstly usedThe paper absorbs the excess dye solution, then the color separation is carried out with 0.5% sodium metabisulfite solution, the color washing is carried out with distilled water, the section is taken out and dried, and the section is placed under a microscope to observe the polysaccharide particles. Or soaking the slices cleaned with distilled water in 90% ethanol for 3min, soaking in anhydrous ethanol for 3min, soaking in 1/2 anhydrous ethanol +1/2 xylene mixed solution for 3min, soaking in xylene for 5min, and adding neutral gum to make into permanent slices. The patented method has the following advantages: 1. the polysaccharide particles can be dyed to be bright red, and the polysaccharide particles of cells of all parts of the anther are clearly visible. 2. The experiment is easy to operate, and the dyeing time is easy to control. 3. The observation can be carried out without loading the slide, and the cost is lower. 4. Can also be made into permanent slices for study and teaching experiment of almond anther.
Detailed description of the invention
(1) Material fixation, FAA fixative solution (glacial acetic acid: 38% formaldehyde: 70 ethanol =5:5: 90) was prepared, anthers at different periods were fixed in the FAA fixative solution, the fixative solution was changed once after 7 days, and the fixative solution was changed again after 14 days, and then the fixative solution and the samples were stored together in a4 ℃ refrigerator. (2) Solution preparation, 0.5% periodic acid solution, 1N hydrochloric acid, colorless fuchsin solution (Schiff's solution) and 0.5% sodium metabisulfite solution are prepared respectively. A0.8% periodic acid solution was prepared by adding 0.8g periodic acid to 100ml distilled water; the preparation method of the 1N hydrochloric acid is that 8.5mL of concentrated hydrochloric acid (with the content of 22 percent) is added into 91.5mL of distilled water; the colorless fuchsin solution (Schiff's solution) is prepared by adding basic fuchsin 0.8g into boiling distilled water 100mL, dissolving for 10min, cooling to 45-50 deg.C, filtering, adding hydrochloric acid 1N 10mL into the filtrate, cooling to 25 deg.C, adding sodium metabisulfite 0.5g, shaking in a triangular flask, mixing, and storing in brown bottle (or wrapping the bottle in black cloth) in dark place. A0.5% sodium metabisulfite solution was prepared by adding 0.5g of sodium metabisulfite solution to 100mL of distilled water. (3) Slicing paraffin, taking out flos Almond flower in FAA stationary liquid, soaking flos Almond flower in 50% ethanol for 1 hr, 70% ethanol for 1 hr, 85% ethanol for 1 hr, 95% ethanol for 1 hr, 100% ethanol for 0.5 hr, and bottling 100% ethanolSoaking in ethanol for 0.5h, soaking in 1/2 ethanol +1/2 xylene mixed solution for 0.5h, soaking in 1/3 ethanol +2/3 xylene mixed solution for 0.5h, soaking in xylene for 1h, soaking fresh xylene for 0.5h, soaking 5mL xylene +4g paraffin in a 40 ℃ oven for 6h, soaking 3mL xylene +5g paraffin in a 50 ℃ oven for 6h, soaking pure paraffin in a 60-62 ℃ oven for 6h, replacing with fresh paraffin for 2 times, soaking for 6h each time, pouring flos Pruni Armeniacae dulcis flower and paraffin into a paper box, adjusting the position of flos Pruni dulcis flower with a dissecting needle, soaking the paper box in cold water for 12h after the paraffin in the paper box is solidified, taking out the paper box, air drying, peeling off the paper box, cutting the paraffin coated with flos Pruni dulcis flower into small blocks with a dissecting knife, fixing on small wooden stakes, cutting anther into 12 μm slices with a semi-automatic slicer, spreading on a 36-40 deg.C spreading machine for 2h, drying on a 36-38 deg.C drying machine for 12h, soaking the slices in xylene liquid for 5min, soaking in new xylene liquid for 5min, soaking in 1/2 xylene +1/2 ethanol for 5min, soaking in 1/3 xylene +2/3 ethanol for 5min, soaking in absolute ethanol for 3min, soaking in 95% ethanol for 3min, soaking in 85% ethanol for 3min, soaking in 70% ethanol for 3min, soaking in 50% ethanol for 3min, soaking in 30% ethanol for 3min, and soaking in clear water for 3min to obtain almond anther slices. (4) Dyeing and color separation treatment, namely dripping 0.5% periodic acid solution on almond anther slice tissues, soaking for 10min, then soaking the slices in a basin filled with distilled water for 3min, then soaking the slices in new distilled water for 3min, fishing out the slices, drying the slices, dripping colorless fuchsin solution (Schiff's solution) on the almond anther slice tissues, dyeing for 30min in the dark, then sucking off the redundant dye solution by using filter paper, soaking for 2 times by using 0.5% sodium metabisulfite solution, soaking for 3min in the distilled water for 2-3min each time, fishing out the slices, and drying. (5) And (4) photographing and observing, namely placing the section under a microscope for photographing and observing. (6) Permanent section preparation and observation, soaking the section soaked in distilled water for 3min in 90% alcohol for 3min, soaking in absolute ethyl alcohol for 3min, soaking in 1/2 mixed solution of absolute ethyl alcohol and 1/2 dimethylbenzene for 3min, soaking in 1/3 mixed solution of absolute ethyl alcohol and 2/3 dimethylbenzene for 3min, soaking in dimethylbenzene for 5min, taking out, wiping off residual dimethylbenzene liquid at the bottom of the glass slide, placing in clean and dry A4On the paper, when the xylene liquid on the upper part of the glass slide is about to volatilize, 1-2 drops of neutral gum are dropped on the almond anther tissue for sealing, then the sealed slices are baked in an oven at 38-42 ℃ for 12h to obtain permanent slices, and the permanent slices are placed under a microscope for observation.
Claims (2)
1. A dyeing method of polysaccharide particles in almond anther cells is characterized in that almond anther is fixed in FAA fixing solution for preservation, then the almond anther is cut into 12 mu m slices by a paraffin slicing method, the almond anther slices are dyed by dye solution prepared in advance under the dark condition, the slices are taken out and dried after color separation, the slices are placed under a microscope for observing the polysaccharide particles, or are made into permanent slices after being fixed by neutral gum, and then photographing observation is carried out;
the almond anther is cut into 12 mu m slices by a paraffin slicing method, which is characterized in that: taking out almond anther in FAA stationary liquid, soaking the almond anther in 50% ethanol for 1h, soaking in 70% ethanol for 1h, soaking in 85% ethanol for 1h, soaking in 95% ethanol for 1h, soaking in 100% ethanol for 0.5h, soaking another bottle in 100% ethanol for 0.5h, soaking in 1/2 ethanol +1/2 xylene mixed solution for 0.5h, soaking in 1/3 ethanol +2/3 xylene mixed solution for 0.5h, soaking in xylene for 1h, soaking in new xylene for 0.5h, soaking 5mL of xylene +4g of solid paraffin in a 40 ℃ oven for 6h, soaking 3mL of xylene +5g of solid paraffin in a 50 ℃ oven for 6h, soaking pure paraffin in a 60-62 ℃ oven for 6h, replacing new paraffin solution for 2 times, soaking for 6h each time, pouring the almond anther and the paraffin solution into a paper box, adjusting the position of the almond anther by using a dissecting needle, after wax liquid in the paper box is solidified, soaking the paper box in cold water for 12 hours, taking out the paper box, airing, peeling off the paper box, cutting the paraffin coated with the almond anther into small blocks by using a scalpel, fixing the small blocks on small wooden stakes, cutting the anther into 12-micron slices by using a semi-automatic slicer, spreading the slices on a 36-40 ℃ spreading machine for 2 hours, drying the slices on a 36-38 ℃ drying machine for 12 hours, soaking the slices in dimethylbenzene liquid for 5 minutes, soaking new dimethylbenzene liquid for 5 minutes, soaking l/2 dimethylbenzene +1/2 ethanol for 5 minutes, soaking l/3 dimethylbenzene +2/3 ethanol for 5 minutes, soaking in absolute ethanol for 3 minutes, soaking in 95% ethanol for 3 minutes, soaking in 85% ethanol for 3 minutes, soaking in 70% ethanol for 3 minutes, soaking in 50% ethanol for 3 minutes, soaking in 30% ethanol for 3 minutes, and soaking in clear water for 3 minutes to obtain almond anther slices;
the dye liquor prepared in advance is characterized in that: a0.8% periodic acid solution was prepared by adding 0.8g periodic acid to 100ml distilled water; the preparation method of the 1N hydrochloric acid is that 8.5mL of 22 percent concentrated hydrochloric acid is added into 91.5mL of distilled water; the colorless fuchsin solution is prepared by adding 0.8g basic fuchsin into 100mL boiled distilled water, dissolving for 10min, cooling to 50 deg.C, filtering, adding 10mL 1N hydrochloric acid into the filtrate, cooling to 25 deg.C, adding 0.5g sodium metabisulfite, shaking in a triangular flask, mixing, and storing in brown flask in dark place;
the dyeing and color separation treatment is characterized in that: dripping 0.8% periodic acid solution on almond anther slice tissue, soaking for 10min, soaking the slice in a basin filled with distilled water for 3min, soaking the slice in new distilled water for 3min, taking out the slice, air drying, dripping colorless magenta solution on the almond anther slice tissue, dyeing for 30min in the dark, sucking off the redundant dye solution with filter paper, soaking for 2 times with 0.5% sodium metabisulfite solution for 2-3min each time, soaking in distilled water for 3min, taking out the slice, and air drying;
the permanent slice is characterized in that: soaking the slices soaked in the distilled water for 3min in 90% alcohol for 3min, soaking in absolute ethyl alcohol for 3min, soaking in 1/2 mixed solution of absolute ethyl alcohol and 1/2 xylene for 3min, soaking in l/3 mixed solution of absolute ethyl alcohol and 2/3 xylene for 3min, soaking in xylene for 5min, taking out, wiping off residual xylene liquid at the bottom of the glass slide, placing on clean and dry A4 paper, dripping 1-2 drops of neutral gum on the tonsil medicinal tissue for sealing when the xylene liquid on the upper part of the glass slide is about to volatilize, and baking the sealed slices in an oven at 38-42 ℃ for 12h to obtain permanent slices.
2. Dyeing process according to claim 1, characterized in that: the method for fixing the almond anther in the FAA stationary liquid for storage is characterized in that: anthers at different periods were fixed in FAA fixative, the fixative was changed once after 7 days, and the fixative was changed again after 14 days, and then the fixative was stored in a4 ℃ refrigerator together with the samples.
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| CN105136547A (en) * | 2015-08-21 | 2015-12-09 | 新疆农业科学院园艺作物研究所 | Korla fragrant pear filament cell stereo dyeing method |
| CN105571920A (en) * | 2014-10-13 | 2016-05-11 | 新疆农业大学 | Stereoscopic dyeing method for cell slide of Korla pear |
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| CN105571920A (en) * | 2014-10-13 | 2016-05-11 | 新疆农业大学 | Stereoscopic dyeing method for cell slide of Korla pear |
| CN105136547A (en) * | 2015-08-21 | 2015-12-09 | 新疆农业科学院园艺作物研究所 | Korla fragrant pear filament cell stereo dyeing method |
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