CN107132097B - Slicing method suitable for observing anatomical structures of flower organs of rubber trees - Google Patents
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Abstract
A slicing method suitable for observing the anatomical structure of a flower organ of a rubber tree comprises the following steps: (1) placing 1 or more of the cut female flowers, male flowers and flower stalks of the rubber trees in a fixing solution, pumping out air, and fixing for a period of time; (2) soaking and washing the material obtained in the step (1) with a phosphate buffer solution, a mixed solution of the phosphate buffer solution and an OCT embedding agent and the OCT embedding agent in sequence, and then embedding the frozen section by using the OCT embedding agent; (3) and (3) slicing the embedded material obtained in the step (2), and then, sticking the slices and sealing the slices for observation. The method does not use volatile and toxic reagents, is simple and quick to operate, and has good repeatability and universality.
Description
Technical Field
The invention belongs to the technical field of plant tissue chemistry, relates to a slicing method of plant tissues, and particularly relates to a slicing method suitable for observing anatomical structures of organs of rubber tree flowers.
Background
The rubber tree is a conical inflorescence, grows from leaf axillary or scale axillary, is isogenic to male and female plants, and has isogenic flowers in the same sequence; female flowers are planted on the top of the flower branch, and each inflorescence usually has 3-20 female flowers, and the base of each female flower has a green disc; the ovary is superior, the style is short or none, the stigma grows on the ovary, the axis placenta is usually 3 chambers, and each chamber has one side of a raw ovule; the male flower is smaller than the female flower, the number is more, no green flower disc is provided, no petal is provided, the basal part of the calyx grows together, the calyx has 5 teeth cracks or 5 deep cracks, the color is beige, 5-10 stamens are arranged on the frontal filament column of the two rounds, and a large amount of pollen is provided in the medicine chamber. The rubber tree seedling tree flowers at 5-6 years, and the bud grafting tree flowers at 3-4 years; usually, blossoming is carried out twice a year, spring flowers are obtained in 3-4 months, summer flowers are obtained in 5-6 months, and spring flowers are used as the main flowering period. The flowering period of female flowers of rubber trees is 10-25 days, and the flowering period of male flowers is 12-27 days, which are different under the influence of factors such as climate and illumination.
At present, the organ structure of the flowers of the rubber tree is well understood, but the anatomical structure of the flowers of the rubber tree is rarely studied. The current method for observing the anatomical structure of the floral organ of the rubber tree is still the method for observing the mammary duct of the rubber tree, which is based on the methods reported by Shi ZQ, Hu ZH, A historical method for rubber-bearing tissues of plants, ActButt Sin, 1965,13,179-182, and the key link is to treat the tissues containing the rubber with a glacial acetic acid solution of iodine and bromine, and observe the tissues under an optical microscope after paraffin section. Several improvements were made to the method by Hao Biz, Wu J L, Biology of genetics in Hevea and technology, Chinese J trops, 2004,25(4):1-7) when applied to the visualization of ducts in the bark of rubber trees by Heyi, et al: 1) the fixing solution replaces FAA fixing solution with 80% ethanol, and aims to dissolve tannin substances and avoid mistaking stained reddish-brown tannin parenchyma cells as brown breast duct cells; 2) the collagen-containing tissue is placed in glacial acetic acid solution of iodine and bromine, and is treated for 36-48 hours at the temperature of 60-65 ℃, so that the color of the rubber can be darkened, and the mammary duct cells can be easily distinguished; the tissue can be softened, and smooth slicing is facilitated; 3) in the process of tissue transparency, n-butyl alcohol or tert-butyl alcohol with low toxicity is used for replacing dimethylbenzene with high toxicity.
However, the above-described paraffin sectioning method has three major drawbacks: 1) the steps are numerous, time-consuming, labels are easy to mistake, and slicing quality is not easy to repeat: the operation flow of the method has more than 40 steps, the observation from the material fixation to the slicing requires 7-10 days, and the label is easy to be mistaken when the material is more; in addition, because of many steps, if the operation is unskilled, the slicing quality is not easy to repeat; 2) for micro particles such as pollen grains, starch grains and the like exposed outside, the solution needs to be replaced or the container needs to be replaced, and the steps are various, so that the micro particles are easily washed away, and the micro particles cannot be observed on the slice; 3) because plant tissues are dyed by glacial acetic acid solution of iodine and bromine, the step has strong softening and corrosion on the plant tissues, flower organs are easily corroded and dissolved, and the complete structure is difficult to observe; 4) the used volatile toxic reagents are more: in addition to alcohol, corrosive bromine and glacial acetic acid, n-butanol and xylene, which are toxic, are also required. In addition, multiple steps operate at high temperatures, presenting a safety hazard.
Aiming at the defects, a rapid and safe slicing method suitable for observing the anatomical structure of the organ of the rubber tree flower is urgently needed to be established.
Disclosure of Invention
Therefore, the invention aims to provide a slicing method suitable for observing the anatomical structure of the organ of the flower of the hevea brasiliensis. The method does not use volatile toxic reagents, is simple and quick to operate, and has good repeatability and universality.
In order to achieve the purpose, the invention adopts the following technical scheme:
a slicing method suitable for observing the anatomical structure of a flower organ of a rubber tree comprises the following steps:
(1) placing 1 or more of the materials of female flower, male flower and flower stalk of the trimmed rubber tree in a fixing solution, pumping out air, and fixing for a period of time; the air can be pumped out by using a vacuum pump, so that the gas in the flower is exhausted, and the immersion and replacement speed of the solution is accelerated; trimming, for example, cutting the floral organ into a suitable small opening;
(2) soaking and washing the material obtained in the step (1) with a phosphate buffer solution, a mixed solution of the phosphate buffer solution and an OCT embedding medium and the OCT embedding medium in sequence to finish the transition of the material from the buffer solution to the OCT embedding medium, and then embedding the frozen section by using the OCT embedding medium;
(3) and (3) slicing the embedded material obtained in the step (2), and then, sticking the slices and sealing the slices for observation.
The organ of the rubber tree flower can not be sliced by paraffin by the conventional method of the rubber tree, and is easily softened and dissolved by iodine and bromine, so the prior literature for slicing the organ of the rubber tree flower is still unavailable. The traditional frozen section method for observing the organ of the rubber tree flower has poor frozen section effect mainly because unopened male flower buds and developed ovaries are closed, gaps exist in the buds, air exists in the buds, the buds are directly fixed without trimming, fixing liquid is difficult to immerse, tissues cannot be well fixed and solution cannot be well replaced, and the section effect is poor. In the invention, a small opening is trimmed on the unopened male flower bud and the developed ovary side surface, the upper end and the lower end of the flower stem are trimmed in parallel, and the internal gas can be exhausted by a vacuumizing method, so that the immersion and replacement speed of the solution is accelerated.
Preferably, for female flowers containing developed ovaries in step 1), the cutting is to cut 0.1-0.3cm × 0.1-0.3cm, preferably 0.2cm × 0.2cm of epidermis on the ovaries side to form an opening, which can accelerate gas discharge, stationary liquid permeation and solution replacement, and can make a plane to be kept horizontal during embedding;
for unopened male flower buds, the pruning is to prune a small opening of 0.1-0.3cm multiplied by 0.1-0.3cm, preferably 0.2cm multiplied by 0.2cm at the uplift part at one side of a calyx, the opening can accelerate gas discharge, stationary liquid permeation and solution replacement, and a plane can be manufactured to be beneficial to keeping the level when embedding;
for an open male flower, the trimming is to cut off an unfolded calyx, so that a plane can be manufactured to be beneficial to keeping the level during embedding;
for the flower stalk, the trimming is to cut a section of the flower stalk with the length of 0.3-0.8cm, preferably 0.5cm, below the ovary.
The above materials are carefully placed in the fixing solution respectively without violent shaking, so as to prevent the internal organs and pollen grains of the flower from being washed away.
Trimming can be performed using a single-sided blade.
Preferably, the fixing solution in step (1) is a phosphate buffer solution containing paraformaldehyde and glutaraldehyde.
Preferably, the mass concentration of the paraformaldehyde is 2-6%, preferably 4%, and the concentration of the glutaraldehyde is 1-5%, preferably 2.5%.
Preferably, the pH of the phosphoric acid buffer solution is 6 to 8, preferably 7.2.
In one embodiment, the fixative solution is a 0.01M pH 7.2 phosphate buffer of 4 wt% paraformaldehyde and 2.5 wt% glutaraldehyde.
The air discharge in the step (1) can be realized by the following processes: placing the container containing the fixing liquid into a container connected with a vacuum device, and evacuating for more than 0.5min, preferably 1-2 min. The fresh fixing liquid can be replaced after the air is exhausted and before the fixing is started.
Preferably, the fixing time in step (1) is 1 hour or more, preferably 2 to 4 hours. The fixing may preferably be carried out at room temperature.
Preferably, the concentration of the phosphate buffer in step (2) is 0.005-0.02M, preferably 0.01M, and the pH is 6-8, preferably 7.2. Soaking with phosphate buffer solution for removing paraformaldehyde and glutaraldehyde from the sample.
The OCT embedding medium is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, is widely used in immunohistochemical laboratories at present, and is used for supporting tissues during frozen section so as to increase continuity of the tissues and reduce wrinkles and fragmentation. Further, since the OCT mixture is water-soluble, it is soluble in water when the sheet is rinsed, and therefore, the background staining is not increased in the subsequent staining. The OCT mixture is used for infiltrating tissues in advance, and then the constant-cooling box slicing is carried out, so that the slicing quality can be improved. In order to improve the slicing quality, the OCT embedding medium used by the invention is a commercial product and is commercially available.
Preferably, the volume ratio of the phosphate buffer solution to the OCT embedding medium in the mixed solution of the phosphate buffer solution and the OCT embedding medium is 1:0.5-2, preferably 1: 1. The mixed solution was used for the transition between phosphate buffer and OCT embedding medium.
Preferably, the time of the soaking with the phosphate buffer is more than 10min, preferably 15-20min, to remove paraformaldehyde and glutaraldehyde from the sample, since the fixing solution is also the phosphate buffer, the washing with the phosphate buffer is more convenient here, the solution is not changed, and the phosphate buffer is close to the pH value and the internal salt concentration of the plant tissue itself, so that the plant tissue itself is not damaged too much, the time of the soaking with the mixed solution of the phosphate buffer and the OCT embedding medium is more than 20min, preferably 30min, for the transition between the phosphate buffer and the OCT embedding medium, and the time of the soaking with the OCT embedding medium is more than 40min, preferably 60min, for the OCT embedding medium to be soaked into the flower organ tissue. The immersion cleaning mode of firstly washing the buffer solution, then washing the phosphate buffer solution and the OCT embedding medium, and then immersing and washing the OCT embedding medium can prevent the replacement solution from changing too fast and prevent the tissues from shrinking or swelling.
In one embodiment, in step 3), 0.01M phosphate buffer solution with pH 7.2 is used for immersion washing for 15-20min to remove paraformaldehyde and glutaraldehyde from the sample; then, soaking and washing for 30min by using a mixed solution of one half of 0.01M phosphate buffer solution with the pH value of 7.2 and one half of OCT embedding medium for transition between the phosphate buffer solution and the OCT embedding medium; and finally, using an OCT embedding medium for immersion washing for 60min, wherein the OCT embedding medium is used for immersing the flower organ tissues.
Preferably, the frozen sections are embedded at a freezer stage temperature of-18 to-22 ℃; the embedding time is 15-30 min.
Preferably, 5 to 20 wt%, preferably 10 wt% glycerol in water is used for the sealing sheet in step (3).
Slicing can be carried out according to a conventional freezing slicing method; the sections can be taken on ordinary glass slides at room temperature.
Preferably, the slicing method of the present invention comprises the steps of:
1) placing 1 or 2 combinations of the female flowers, the male flowers and the flower stalks of the cut rubber trees into a centrifugal tube filled with stationary liquid;
2) opening the cover of the centrifugal tube, putting the centrifugal tube into a glass dryer connected with a vacuum pump, and exhausting for 1-2 min;
3) replacing fresh fixative, fixing at room temperature for 2-4 hr, and soaking with buffer solution for 3 times, 15min for the first time and 30min for the second time; the second time is 60 min;
4) transferring the material in the step 3) from the buffer solution to an OCT embedding medium;
5) supplementing the material in the OCT embedding medium in the step 4) by the OCT embedding medium, embedding the material in an embedding box, placing the embedding box on a freezing table at the temperature of minus 20 ℃, and condensing for 20 min;
6) taking out the material in the step 5), and slicing according to a conventional freezing slicing method;
7) sticking the slices with a common glass slide at room temperature;
8) the film was sealed with a sealing agent and observed with an optical microscope.
The centrifuge tube can be a common plastic or glass centrifuge tube with a cover; the centrifuge tube may be a 1.5-10mL tube. Advantageously, the volume of the fixative solution is one half to two thirds of the specification of the centrifuge tube. In one embodiment, the centrifuge tube format is a 5mL centrifuge tube; the volume of the fixative solution was 3 mL.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the operation is simple, and the time is saved. The experimental procedure is completed in two days at most, and the time is mainly spent in two links of fixing and slicing. Because the link is few, easy operation, the quality repeatability of section is good, and be difficult to mistake the label moreover.
(2) The operation steps are few, and the damage of internal organs of the flower and the washing-off of pollen grains during the replacement of the solution are avoided, so that the damage cannot be observed.
(3) Volatile toxic reagents are not used in the whole method, and all the steps are operated at room temperature and low temperature, so that potential safety hazards do not exist.
(4) Has the advantages of convenient material acquisition, labor and land saving, good repeatability and universality and batch screening.
Drawings
FIG. 1 is a schematic diagram of female flowers of a rubber tree before fixation;
FIG. 2 is a schematic view of an unopened male flower of a rubber tree treated before fixing;
FIG. 3 is a longitudinal sectional view of 9-year-old cut female flowers of rubber tree clone CATAS 7-33-97 under 5-fold objective bright field conditions, wherein 1 is calyx, 2 is stigma, 3 is ovule, 4 is ovary wall, 5 is placenta, 6 is calyx base, and 7 is flower stalk; asterisk indicates calyx abscission layer, white arrow indicates newborn milk duct, black arrow indicates epidermal hair, etc.; a scale: 200 μm, slice thickness: 14 μm, and the multiple of the objective lens is 5 times;
FIG. 4 is a longitudinal sectional view of a 9-year-old, bloomed male flower of rubber tree clone CATAS 7-33-97, under 5-fold objective brightfield conditions, wherein 1 is the calyx, 2 is the symbiotic filaments, 3 is the anther, 4 is the pollen grains, 5 is the shed anther, 6 is the basal portion of the calyx, and 7 is the flower stalk; white arrows indicate newborn milk ducts, black arrows indicate epidermal hairs, etc.; a scale: 100 μm, slice thickness: 14 μm, and the multiple of the objective lens is 5 times;
FIG. 5 is a longitudinal sectional view of a 9-year-old blooming male flower of rubber tree clone CATAS 7-33-97 under 5-fold objective brightfield conditions, wherein 1 is calyx, 2 is symbiotic filaments, 3 is anthers, 4 is pollen grains, 5 is shed anthers, 6 is calyx base, and 7 is flower stalk; white arrows indicate newborn milk ducts, black arrows indicate epidermal hairs, etc.; a scale: 200 μm, slice thickness: 12 μm, the objective multiple is 5 times;
FIG. 6 is a cross-sectional view of 9-year-old cut female flower stalks of rubber tree clone CATAS 7-33-97 under 5-fold objective bright field conditions, wherein 1 is epidermis, 2 is cortex, 3 is fiber layer, 4 is phloem, 5 is cambium, 6 is xylem, and 7 is marrow; white arrows indicate newborn milk ducts; a scale: 100 μm, slice thickness: 12 μm and a magnification of 5 times.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications can be made by those skilled in the art after reading the contents of the present invention, and those equivalents also fall within the scope of the invention defined by the appended claims.
Example 1 anatomical Structure Observation of female, male and petioles of rubber Tree
In this example, the anatomical structure of the flower organ was observed using the spring flower of a 9-year-old cut tree of rubber tree variety CATAS 7-33-97.
The experimental conditions are as follows: collecting inflorescences of 9-year-old cut tree branch ends of a rubber tree clone variety CATAS 7-33-97 in 4 months, and respectively collecting female flowers, male flowers, flower stalks and other materials. The trimming method comprises the following steps:
1) female flowers: picking the female flowers which are at the topmost end of the inflorescence and the ovaries begin to develop, cutting flower stalks by using a single-sided blade, cutting 0.2cm multiplied by 0.2cm of epidermis on one side of the expanded ovaries to form a small opening, and shown in figure 1, wherein the dotted lines show the parts of the female flower ovaries;
2) unopened male flower: picking up mature and unopened male flowers, cutting off flower stems by using a single-sided blade, and cutting small openings of 0.2cm multiplied by 0.2cm on the calyx on one side of the male flowers, as shown in figure 2, wherein the dotted line shows the positions of the trimmed male flower calyxes;
3) opened male flowers: picking up the open and non-withered male flowers, and removing flower stalks by using a single-sided blade without any treatment:
4) flower stem: picking the female flowers of the expanded ovaries, using a single-sided blade to prune a section of flower stalk with the length of 0.5cm below the symbiosis of the calyx at the upper and lower ends in parallel.
The above materials were placed in 3ml of a 0.01M phosphate buffer (pH 7.2) fixation solution containing 4 wt% paraformaldehyde and 2.5 wt% glutaraldehyde, respectively, evacuated for 2min, replaced with a new fixation solution, and fixed at room temperature for 3 h. Respectively soaking and washing once with three buffer solutions, wherein the first time is 0.01M phosphate buffer solution with pH of 7.2, and the soaking and washing time is 15 min; the second time is a mixed solution of one half of 0.01M phosphate buffer solution with pH 7.2 and one half of OCT embedding agent, and the immersion washing time is 30 min; the third time is OCT embedding medium, and the immersion time is 60 min. Immersing the washed floral organ material, and embedding the frozen section by using an OCT embedding medium, wherein the condensation time is 15-30 min; slicing by conventional freezing slicing technique at-18 to-22 deg.C; slicing the glass into slices with the thickness of 12-14 mu m, sticking the glass slides at normal temperature, and sealing the slices by 10 wt% of glycerol aqueous solution; the results were observed under an optical microscope.
The results show that: the results of observation under the bright field of the microscope were as follows:
1) the structure of the female flowers of the rubber trees is complete, and the female flowers comprise epidermal hair, stigma and short style, developed ovaries, pericarp, seeds and the structures of embryos, placenta, basal-part-synthesized calyx, flower stalks and the like inside the ovaries, the pericarp and the seeds, and also can see dark strip-shaped milk ducts and calyx abscission layers to be broken; the clarity and completeness of the slices was good, as shown in fig. 3;
2) the unopened male flower has a complete structure, is in an egg-shaped lanceolate shape, comprises epidermal hair, a calyx combined with the base part and a plurality of stamens which are arranged in two rounds and combined to generate a flower silk column, a large amount of pollen is arranged in a flower medicine chamber, and the clarity and the integrity of the slices are good, as shown in figure 4;
3) the structure of the opened male flower is complete, the flower comprises a calyx, anthers growing on a flower silk column and fallen anthers, a small amount of free pollen and structures such as a withered flower stem can be seen, and the clarity and the integrity of the section are good, as shown in fig. 5;
4) flower stem: the transverse cutting of the flower stalk of the female flower can show complete and clear tissue structures including epidermis, cortex, fibrous layer, phloem, newborn milk duct, cambium, xylem and marrow, and the clarity and integrity of the cut slice are good, as shown in fig. 6.
Example 2
Same as example 1, except for the following differences: the female flower cut is 0.1cm × 0.1cm, the unopened male flower cut is 0.1cm × 0.3cm, and the cut length of the flower stalk is 0.3cm and 0.8cm respectively.
Example 3
Same as example 1, except for the following differences: the fixative solution was 2 wt% paraformaldehyde and 5wt% glutaraldehyde in 0.01M pH 7.2 phosphate buffer; the soaking process is 0.005M pH 7.8 phosphoric acid buffer solution for the first time, and the soaking time is 10 min; the second time is a mixed solution of two-thirds of 0.005M phosphate buffer solution with pH value of 7.8 and one-third of OCT embedding agent, and the immersion washing time is 20 min; the third time is OCT embedding medium, and the immersion time is 40 min.
Example 4
Same as example 1, except for the following differences: the fixative solution was 6wt% paraformaldehyde and 1 wt% glutaraldehyde in 0.01M pH 7.2 phosphate buffer; the soaking process is 0.02M pH 6 phosphate buffer solution for the first time, and the soaking time is 15 min; one third of the mixed solution of 0.02M pH 6 phosphate buffer solution and two thirds of OCT embedding medium is used for immersion washing for 30 min; the third time is OCT embedding medium, and the immersion time is 60 min.
The tissue anatomical integrity and clarity observed for examples 2-4 were comparable to that observed for example 1.
The experimental results are combined, and the observation of various flower organs of the rubber tree proves that the tissue anatomical structure of the flower organs of the rubber tree can be completely and clearly displayed by using the slicing method. The slicing method provided by the invention can be used for rapidly and effectively displaying the anatomical structure of the organ of the rubber flower, and has a wide application prospect.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (7)
1. A slicing method suitable for observing the anatomical structure of a flower organ of a rubber tree comprises the following steps:
(1) placing 1 or more of the materials of female flower, male flower and flower stalk of the trimmed rubber tree in a fixing solution, pumping out air, and fixing for a period of time;
(2) soaking and washing the material obtained in the step (1) with a phosphate buffer solution, a mixed solution of the phosphate buffer solution and an OCT embedding agent and the OCT embedding agent in sequence, and then embedding the frozen section by using the OCT embedding agent;
(3) freezing and slicing the embedded material obtained in the step (2), and then sealing and observing the sliced material;
wherein, for female flowers containing developed ovaries in the step (1), the trimming is to cut off 0.1-0.3cm multiplied by 0.1-0.3cm of epidermis at the position of a bulge at one side of the ovaries;
for unopened male flower buds, a small opening of 0.1-0.3cm multiplied by 0.1-0.3cm is trimmed at the position of the swelling position on one side of a calyx;
for an open male flower, the trimming is to excise a deployed calyx;
for the flower stalks, the trimming is to cut a section of the flower stalks with the length of 0.3-0.8cm below the ovary;
a small opening is trimmed on the unopened male flower bud and the developed ovary side surface, the upper end and the lower end of the flower stem are trimmed in parallel, and the internal gas is exhausted by a vacuumizing method, so that the solution immersion and replacement speed is accelerated;
the concentration of the phosphate buffer solution in the step (2) is 0.005-0.02M, and the pH value is 6-8;
the volume ratio of the phosphate buffer solution to the OCT embedding medium in the mixed solution of the phosphate buffer solution and the OCT embedding medium is 1: 0.5-2;
in the step (2), the time for soaking and washing by using a phosphate buffer solution is 15-20min, the time for soaking and washing by using a mixed solution of the phosphate buffer solution and the OCT embedding medium is more than 20min, and the time for soaking and washing by using the OCT embedding medium is more than 40 min;
the temperature of a freezing table is-18 to-22 ℃ when the frozen section is embedded; the embedding time is 15-30 min.
2. The slicing method according to claim 1, wherein the fixing solution in step (1) is a phosphoric acid buffer solution containing paraformaldehyde and glutaraldehyde.
3. The slicing method according to claim 2, wherein the mass concentration of paraformaldehyde is 2-6wt%, and the concentration of glutaraldehyde is 1-5 wt%.
4. The slicing method as defined in any one of claims 2 to 3, wherein the pH of the phosphate buffer solution in the step (1) is 6 to 8.
5. The method for slicing in accordance with any one of claims 1 to 3, wherein the fixing solution in step (1) is 0.01M pH 7.2 phosphate buffer containing 4 wt% paraformaldehyde and 2.5 wt% glutaraldehyde.
6. The sectioning method according to any one of claims 1 to 3, wherein the time fixed in step (1) is 2 to 4 hours.
7. The slicing method as defined in any one of claims 1 to 3, wherein the sealing sheet in the step (3) uses a 10 wt% glycerin aqueous solution.
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| CN109060468A (en) * | 2018-06-11 | 2018-12-21 | 塔里木大学 | A kind of preparation method of the paraffin section of pear flower tissue |
| CN111982629B (en) * | 2020-08-25 | 2023-11-17 | 北京市农林科学院 | Ultrathin frozen slicing method for edible fungus tissue cells |
| CN113640080B (en) * | 2021-08-06 | 2023-05-23 | 中国农业科学院棉花研究所 | Method for preparing slice for cotton ovule space transcriptome analysis |
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| CN105092291A (en) * | 2015-09-09 | 2015-11-25 | 徐州工程学院 | Rapid frozen section method for camphor leaf |
| CN106501035A (en) * | 2016-10-17 | 2017-03-15 | 中国热带农业科学院橡胶研究所 | A kind of histochemical method of quick display rubber tree bark latex dust |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175501A (en) * | 2011-01-07 | 2011-09-07 | 山东省农业科学院作物研究所 | Method for preprocessing plant sample used for microscopic observation |
| CN104374601A (en) * | 2014-12-08 | 2015-02-25 | 扬州大学 | Mature grain seed resin section making method |
| CN105092291A (en) * | 2015-09-09 | 2015-11-25 | 徐州工程学院 | Rapid frozen section method for camphor leaf |
| CN106501035A (en) * | 2016-10-17 | 2017-03-15 | 中国热带农业科学院橡胶研究所 | A kind of histochemical method of quick display rubber tree bark latex dust |
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| 三叶橡胶花粉植株的获得;陈正华 等;《中国科学》;19790630(第6期);613-624页 * |
| 巴西橡胶树花药体细胞胚胎发生的研究;K.Das 等;《世界热带农业信》;20100930(第9期);1-3页 * |
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