CN114279780B - Chromosome and karyotype analysis method using dragon fruit stem tip as material - Google Patents
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Abstract
The invention relates to a chromosome and karyotype analysis method using a dragon fruit stem tip as a material. Specifically, young stem tips of dragon fruits are used as experimental materials, and chromosome morphology observation and research are performed by utilizing the characteristic that the young stem tips have meristematic cells with vigorous division. The method comprises the following steps: cutting off healthy and tender stem tips of dragon fruits, and then dissecting; placing the dissected stem tip into colchicine and 8-hydroxyquinoline mixed solution for chromosome concentration; soaking stem tip with KCl; refrigerating and fixing the stem tip by using a fixing liquid; cleaning the fixed root tip, and putting the root tip into HCl for dissociation; cleaning the dissociated stem tip, and placing the stem tip into a mixed solution of cellulase and pectase for enzymolysis; putting the stem tip after cleaning and enzymolysis into double distilled water for post hypotonic treatment; putting the post-hypotonic stem tip into a fixed liquid for cooling treatment; the stem tip was placed on a slide, cut, stained, and pelleted for microscopic examination. The invention solves the problems of difficult material taking of the aerial roots of the dragon fruits, troublesome material taking of the root tips of the dragon fruits, destructive effect on plants and unstable experimental effect. The invention takes the stem tip of the dragon fruit as a material for the first time, realizes the chromosome karyotype analysis of the dragon fruit, and provides more convenient and stable technical support for the related researches of the karyotype research of the chromosome of the dragon fruit, ploidy identification of germplasm resources and the like.
Description
Technical Field
The invention relates to a chromosome and karyotype analysis method using a stem tip of a dragon fruit as a material, belonging to the research field of plant cytology and cytogenetics.
Background
Plant chromosomes are polymers containing important genetic information in plant cells that are readily stained with basic dyes during cell division and can be observed under a microscope to appear rod-like or cylindrical. The chromosome karyotype analysis is a method of analyzing chromosome information by observing and researching chromosome characteristic information such as chromosome number, morphological characteristics, positions of a fiber attachment point, presence or absence of telomeres, and the like.
Pitaya is a novel tropical specialty fruit of the genus Pacific (Cactaceae) or Serpentis (Seleniereus) genus, originating in tropical rainforest areas of central america. The chromosome cardinal number of plants of the genus dipperstick is 11 and large logarithmic varieties all belong to diploids (2n=2x=22), while triploid, tetraploid, galloid, hexaploid dragon fruit plants have been obtained by studies such as germplasm collection, artificial cross breeding (Liu Shunzhi, liu Zhenghao, lin Runyi, etc. white pulp dragon fruit chromosome making technology and karyotype analysis [ J ]. Guangdong agricultural science, 2015,42 (03): 115-118+193). Chromosome related studies on dragon fruits are still in the development stage, and few reports on karyotype analysis studies are available. The method for analyzing and researching the core type of the dragon fruit basically adopts a conventional root tip tabletting method or a dragon fruit aerial root tabletting method, compared with the root tip tabletting method, the method for tabletting the dragon fruit aerial root is more convenient, but the method for accelerating the growth of the dragon fruit aerial root is unstable, materials are difficult to obtain, the root tip is relatively troublesome to obtain, and the success rate of experiments is low. Therefore, a more convenient and stable method for researching the chromosome karyotype of the dragon fruit is established, and important technical support is provided for chromosome observation and karyotype analysis of the dragon fruit, ploidy identification of germplasm resource classification and breeding work.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a chromosome karyotype analysis and research method using a stem tip of a dragon fruit as a material, so that various chromosome forms can be clearly observed under a microscope, and karyotype and ploidy researches can be carried out.
In order to achieve the above object, the present invention provides the technical method as follows:
step 1: material drawing and pretreatment
Healthy and tender stem tips of the dragon fruits are selected, and dissected to obtain central stem tips of dragon fruit plants;
step 2: concentrating
The stem tip of the dragon fruit obtained by dissection is placed into a mixed solution of 0.2 percent colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for at least 2 hours at normal temperature.
Step 3: anterior hypotonic
The concentrated stem tip is soaked with 0.075M KCl at normal temperature.
Step 4: front fixing
The stem tip is put into a Carnot's fixing solution (absolute ethyl alcohol: glacial acetic acid=3:1), and is placed at 4 ℃ for refrigeration and fixing for more than 4 hours.
Step 5: dissociation of
The stem tip from which the Carnot fixative was washed was placed in 1M HCl and dissociated.
Step 6: enzymolysis
Placing the dissociated stem tip into a mixed solution of 2.5% cellulase and 2.5% pectase, and standing at normal temperature for at least 1 hr.
Step 7: post hypotonic
Washing the stem tip subjected to enzymolysis, and putting into double distilled water for hypotonic treatment for 10-30min.
Step 8: post-fixing
The stem tip with low permeability is put into Carnot's fixing solution, refrigerated and fixed at 4deg.C for 20-30min, if it can not be made into tablet in time, the stem tip can be rinsed and put into 75% alcohol, and stored at 4deg.C.
Step 9: dyeing and tabletting
Placing stem tip on a glass slide, cutting 1-2mm, dripping 10% modified phenol fuchsin staining solution, covering a cover glass, staining for 20-40min, washing off excessive dye with distilled water after staining, and sucking off excessive liquid with filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass.
Step 10: microscopic examination
The observation is carried out by using an optical microscope, firstly, the observation is carried out by using a low power mirror, and after finding a proper visual field and splitting phase, the observation is carried out by turning to a high power mirror, and the photographing is carried out.
Further: in the step 1, healthy and tender stem tips of dragon fruit plants are selected as materials, and are dissected.
Further: the specific operation process of the step 2 is that the stem tip of the dragon fruit plant obtained by dissection is placed into a mixed solution of 0.2 percent colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for 2 hours at normal temperature.
Further: step 3 is specifically performed by immersing the stem tip in 0.075M KCl for 120min.
Further: the specific operation process of the step 4 is that the stem tip is put into a Carnot's fixing solution, and is refrigerated for more than 4 hours at the temperature of 4 ℃, wherein the Carnot's fixing solution is absolute ethyl alcohol: glacial acetic acid=3:1.
Further: the specific operation process of the step 5 is that the stem tip after washing off the Carnot's fixative is put into 1M HCl and left for 90min at normal temperature for dissociation.
Further: the specific operation process of the step 6 is that the dissociated stem tip is put into the mixed solution of 2.5 percent of cellulase and 2.5 percent of pectase, and is placed for 1h at normal temperature.
Further: step 7, the specific operation process is that the stem tip subjected to enzymolysis is cleaned and put into double distilled water to be hypotonic for 30min.
Further: the specific operation process of the step 8 is that the stem tip with low permeability is put into a Carnot's fixing solution, refrigerated and fixed for 30min at 4 ℃, if the stem tip cannot be made into tablets in time, the stem tip can be put into 75% alcohol after being rinsed, and the stem tip is preserved at 4 ℃.
Further: step 9, the specific operation process is that the stem tip is placed on a glass slide, 2mm is gently cut, 10% of modified phenol fuchsin staining solution is dripped, a cover glass is covered, the stem tip is stained for 40min, after the staining is finished, the excess dye is washed by distilled water, and the excess liquid is sucked by filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass.
Drawings
FIG. 1 is a photograph of an undiscovered stem tip of a dragon fruit of example 1 of the present invention.
FIG. 2 is a photograph of the shoot tip of an anatomic pitaya of example 1 of the present invention.
FIG. 3 is a 100-fold microscopic photograph of the chromosome of the stem tip of the dragon fruit in example 1 of the present invention under extracellular vision.
FIG. 4 is a graph showing the comparison of the root tip, aerial root and dissected stem tip of a dragon fruit of the present invention.
FIG. 5 is a photograph of the root tip of the dragon fruit of comparative example 1 of the present invention under a microscope.
FIG. 6 is a photograph of a 100-fold microscopic examination of the extracellular field of view of a chromosome of a aerial root of a dragon fruit of comparative example 1 of the present invention.
FIG. 7 is a 100-fold photomicrograph of the stem tip chromosome of the 'white meat' dragon fruit of example 2 of the present invention in extracellular view.
FIG. 8 is a 100-fold microscopic photograph of the stem tip chromosome of the 'Jindu Yi' Dragon fruit in example 2 of the present invention under extracellular vision.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific examples, it being understood that these examples are provided only for illustrating the invention and are not intended to limit the scope of the invention.
Example 1 dragon fruit stem tip was selected as the experimental material, and the specific operation steps were as follows:
step 1: material drawing and pretreatment
Healthy and tender stem tips of the dragon fruits are selected and dissected to obtain the central stem tip of the dragon fruit plant.
Step 2: concentrating
The stem tip of the dragon fruit obtained by dissection is placed into a mixed solution of 0.2% colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for 2 hours at normal temperature.
Step 3: anterior hypotonic
The concentrated stem tip is soaked with 0.075M KCl at normal temperature.
Step 4: front fixing
The stem tip is put into a Carnot's fixing solution (absolute ethyl alcohol: glacial acetic acid=3:1), and is placed at 4 ℃ for refrigeration and fixing for more than 4 hours.
Step 5: dissociation of
The stem tip from which the Carnot fixative was washed was placed in 1M HCl and dissociated.
Step 6: enzymolysis
Placing the dissociated stem tip into a mixed solution of 2.5% cellulase and 2.5% pectase, and standing at normal temperature for 1 hr.
Step 7: post hypotonic
Washing the stem tip subjected to enzymolysis, and putting into double distilled water for hypotonic treatment for 10-30min.
Step 8: post-fixing
The stem tip with low permeability is put into Carnot's fixing solution, refrigerated and fixed at 4deg.C for 30min, and if it can not be made into tablet in time, the stem tip can be rinsed and put into 75% alcohol, and stored at 4deg.C.
Step 9: dyeing and tabletting
Placing the stem tip on a glass slide, slightly cutting 1-2mm, dripping 10% modified phenol fuchsin staining solution, covering a cover glass, staining for 20-40min, washing the excess dye with distilled water after the staining is finished, and sucking the excess liquid with filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass.
Step 10: microscopic examination
The chromosome is counted according to the picture, which is obtained by observing with an optical microscope, observing with a low power mirror, finding a proper visual field and splitting phase, and then transferring to a high power mirror for photographing, as shown in fig. 3, taking under a 100-time oil mirror.
Comparative example 1 dragon fruit root tip and dragon fruit aerial root were selected as experimental materials, and the specific operation is as follows;
step 1: drawing materials
The root tip of the dragon fruit plant is selected for cleaning, and the aerial root is reserved.
Step 2: concentrating
The root tip and aerial root of the dragon fruit are put into a mixed solution of 0.2% colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for 2 hours at normal temperature.
Step 3: anterior hypotonic
The concentrated root tip and aerial root are soaked in 0.075M KCl for 30min at normal temperature.
Step 4: front fixing
The root tip and the aerial root are placed into a Carnot's fixing solution (absolute ethyl alcohol: glacial acetic acid=3:1), and are placed at 4 ℃ for refrigeration and fixing for more than 4 hours.
Step 5: dissociation of
Placing the root tip and the aerial root after washing off the Carnot's fixative in 1M HCl, and dissociating for 50min at normal temperature.
Step 6: enzymolysis
The dissociated root tip and the aerial root are put into a mixed solution of 2.5 percent of cellulase and 2.5 percent of pectase and treated for 1 hour at normal temperature.
Step 7: post hypotonic
Cleaning the root tip and the aerial root after enzymolysis, and hypotonic for 10min in double distilled water.
Step 8: post-fixing
The root tip and the aerial root which are hypotonic are put into a Carnot fixing solution, refrigerated and fixed for 30min at 4 ℃, and if the root tip and the aerial root cannot be made into tablets in time, the root tip and the aerial root can be put into 75 percent alcohol after being rinsed, and the preserved at 4 ℃.
Step 9: dyeing and tabletting
Placing root tip and aerial root on a glass slide, slightly cutting 1-2mm, dripping 10% modified phenol fuchsin staining solution, covering a cover glass, staining for 40min, washing off excessive dye with distilled water after the staining is finished, and sucking off excessive liquid with filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that root tips, aerial root tissues and cells can be uniformly dispersed on the glass.
Step 10: microscopic examination
The observation is carried out by using an optical microscope, firstly, the observation is carried out by using a low power mirror, the proper visual field and the split phase are found, then the image is transferred to a high power mirror for photographing, and as shown in fig. 5, the image is obtained by photographing under a 100-time oil mirror.
Results
The roots of the dragon fruit plants are relatively tiny, and the main roots are fewer and have more fibrous roots; the dragon fruit grows slowly, long time is needed from planting to stably collecting root tips, and the root tips are collected to easily damage dragon fruit plants; because the root tips of the dragon fruits are smaller, the meristematic regions of the root systems cannot be accurately cut, and more dispersed split phases cannot be easily obtained, so that 1 slide specimen can be prepared only by using a plurality of root tips. The dragon fruit seeds are cultivated through sand culture and water culture, and the phenomenon that root systems are weak due to the fact that the dragon fruit seeds are small is found, and the dragon fruit seeds are easy to break when taken out of the sand culture; although the root system of the seed under water planting cannot be broken, the seed is too small and inconvenient to operate. The integral effect of the aerial root is better than that of the root tip of the dragon fruit, but the means of aerial root culture of the dragon fruit is not mature, so that the aerial root cannot be stably obtained in a large scale. The observation and karyotype analysis of plant chromosome need more than 20 cells, and the method of root tip and aerial root all results in long time for obtaining high-quality glass slide, and the root tip and aerial root are selected as materials, so that the workload is greatly increased.
Conclusion(s)
Compared with the flaking technology of the root tip and the aerial root of the dragon fruit, the chromosome flaking and nuclear analysis method using the young stem tip of the dragon fruit as the material has the characteristics of convenient sampling, easy obtaining of more divisions and the like, and can not influence the growth and development conditions of the dragon fruit plant. Meanwhile, higher working efficiency can be obtained, and the workload of workers is reduced.
Example 2 'white meat' and 'Jindu one' were chosen as experimental materials for this experimental example, and the specific procedure is as follows: step 1: material drawing and pretreatment
Healthy and tender stem tips of the dragon fruits are selected and dissected to obtain the central stem tip of the dragon fruit plant.
Step 2: concentrating
The stem tip of the dragon fruit obtained by dissection is placed into a mixed solution of 0.2% colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for 2 hours at normal temperature.
Step 3: anterior hypotonic
The concentrated stem tip is soaked with 0.075M KCl at room temperature for 120min.
Step 4: front fixing
The stem tip is put into a Carnot's fixing solution (absolute ethyl alcohol: glacial acetic acid=3:1), and is placed at 4 ℃ for refrigeration and fixing for more than 4 hours.
Step 5: dissociation of
Putting the stem tip after washing off the Carnot's fixative into 1M HCl, and dissociating for 90min at normal temperature.
Step 6: enzymolysis
The dissociated stem tip is put into a mixed solution of 2.5 percent of cellulase and 2.5 percent of pectase and treated for 1 hour at normal temperature.
Step 7: post hypotonic
And cleaning the stem tip subjected to enzymolysis, and putting the stem tip into double distilled water for hypotonic for 30min.
Step 8: post-fixing
The stem tip with low permeability is put into Carnot's fixing solution, refrigerated and fixed at 4deg.C for 30min, and if it can not be made into tablet in time, the stem tip can be rinsed and put into 75% alcohol, and stored at 4deg.C.
Step 9: dyeing and tabletting
Placing the stem tip on a glass slide, slightly cutting 1-2mm, dripping 10% modified phenol fuchsin staining solution, covering a cover glass, staining for 40min, washing off excessive dye with distilled water after the staining is finished, and sucking off excessive liquid with filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass.
Step 10: microscopic examination
The chromosome is counted according to the picture, as shown in fig. 7 and 8, which is obtained by photographing under a 100-fold oil microscope.
In summary, the technical scheme of the invention can fully and effectively realize the aim of the invention, and the sequence, the steps and the functional principle related by the invention are fully verified in the embodiment, so that the expected efficacy and the aim can be achieved.
The invention has the following beneficial effects:
the materials are tender dragon fruit stem tips, can be directly obtained, are easy to obtain, are not limited by growth period, are not destructive to plants compared with root tip materials, and are convenient and quick without cultivating seeds for germination.
More split phases can be obtained by concentrating the mixture of colchicine and 8-hydroxyquinoline.
The dissociation uses acidolysis method, and the dissociation time is short, and is effectual.
In the experiment, the stem tip is treated by the mixed solution of the cellulase and the pectase, so that excessive pectin in the stem tip of the dragon fruit can be effectively removed, and the microscopic examination is clearer.
Post-hypotonic helps to disperse chromosomes, but too long results in easy cell disruption when tabletted.
Claims (6)
1. The chromosome and karyotype analysis method using the stem tip of the dragon fruit as a material is characterized by comprising the following steps of:
step 1: material drawing and pretreatment
Selecting healthy and tender stem tips of dragon fruit plants as materials, and dissecting the materials to obtain the central stem tips of the dragon fruit plants;
step 2: concentrating
Placing the stem tip of the dragon fruit obtained by dissection into a mixed solution of 0.2% colchicine and 0.002M 8-hydroxyquinoline, and standing at normal temperature for at least 2 hours;
step 3: anterior hypotonic
Soaking the concentrated stem tip in 0.075M KCl at normal temperature for 120min;
step 4: front fixing
Putting the stem tip into a Carnot's fixing solution, and standing at 4 ℃ for refrigeration and fixing for more than 4 hours, wherein the Carnot's fixing solution is absolute ethyl alcohol: glacial acetic acid = 3:1;
step 5: dissociation of
Putting the stem tip washed with the Carnot's fixative into 1M HCl for dissociation;
step 6: enzymolysis
Placing the dissociated stem tip into a mixed solution of 2.5% cellulase and 2.5% pectase, and standing for 1h at normal temperature;
step 7: post hypotonic
Washing the stem tip subjected to enzymolysis, and putting the stem tip into double distilled water for hypotonic treatment for 10-30min;
step 8: post-fixing
Placing the post-hypotonic stem tip into Carnot's fixing solution, refrigerating at 4deg.C for fixing for 20-30min, if it is unable to make tablet in time, rinsing the stem tip, placing into 75% alcohol, and preserving at 4deg.C;
step 9: dyeing and tabletting
Placing stem tip on a glass slide, cutting 1-2mm, dripping 10% modified phenol fuchsin staining solution, covering a cover glass, staining for 20-40min, washing off excessive dye with distilled water after staining, and sucking off excessive liquid with filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass slide;
step 10: microscopic examination
The observation is carried out by using an optical microscope, firstly, the observation is carried out by using a low power mirror, and after finding a proper visual field and splitting phase, the observation is carried out by turning to a high power mirror, and the photographing is carried out.
2. The method for analyzing the chromosome and the karyotype by using the stem tip of the dragon fruit as a material according to claim 1, which is characterized in that: in the step 2, the stem tip of the dragon fruit plant obtained by dissection is placed into a mixed solution of 0.2% colchicine and 0.002M 8-hydroxyquinoline, and the mixture is placed for 2 hours at normal temperature.
3. The method for analyzing the chromosome and the karyotype by using the stem tip of the dragon fruit as a material according to claim 1, which is characterized in that: in step 5, the stem tip from which the Carnot's fixative is washed is placed in 1M HCl and left at normal temperature for 90min for dissociation.
4. The method for analyzing the chromosome and the karyotype by using the stem tip of the dragon fruit as a material according to claim 1, which is characterized in that: in the step 7, the stem tip subjected to enzymolysis is cleaned and put into double distilled water for hypotonic for 30min.
5. The method for analyzing the chromosome and the karyotype by using the stem tip of the dragon fruit as a material according to claim 1, which is characterized in that: in step 8, the stem tip with low permeability is put into Carnot's fixing solution, refrigerated and fixed at 4 ℃ for 30min, if the stem tip cannot be made into tablets in time, the stem tip can be rinsed and then put into 75% alcohol, and the stem tip is preserved at 4 ℃.
6. The method for analyzing the chromosome and the karyotype by using the stem tip of the dragon fruit as a material according to claim 1, which is characterized in that: in the step 9, the stem tip is placed on a glass slide, 2mm is cut, 10% of modified phenol fuchsin staining solution is dripped, a cover glass is covered, the glass slide is stained for 40min, after the staining is finished, the excess dye is washed by distilled water, and the excess liquid is sucked by filter paper; in the tabletting process, a pencil with a rubber is used for lightly knocking the cover glass, so that stem tip tissues and cells can be uniformly dispersed on the glass.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645360A (en) * | 2012-04-16 | 2012-08-22 | 北京林业大学 | Lagerstroemia plant stem tip chromosome tablet preparation method |
| CN103931497A (en) * | 2014-04-05 | 2014-07-23 | 云南省农业科学院花卉研究所 | Method for improving seedling rate of tissue culture seedlings of hylocereus undulatus britt |
| CN104871755A (en) * | 2015-04-17 | 2015-09-02 | 广德县菁菁果业专业合作社 | Greenhouse cultivation method of dragon fruit |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645360A (en) * | 2012-04-16 | 2012-08-22 | 北京林业大学 | Lagerstroemia plant stem tip chromosome tablet preparation method |
| CN103931497A (en) * | 2014-04-05 | 2014-07-23 | 云南省农业科学院花卉研究所 | Method for improving seedling rate of tissue culture seedlings of hylocereus undulatus britt |
| CN104871755A (en) * | 2015-04-17 | 2015-09-02 | 广德县菁菁果业专业合作社 | Greenhouse cultivation method of dragon fruit |
Non-Patent Citations (1)
| Title |
|---|
| 白肉火龙果染色体制片技术及核型分析;刘顺枝 等;广东农业科学(第03期);第115-118页 * |
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