Slide preparation method for rapidly obtaining root tip chromosome of sweet potato and application of slide preparation method

Abstract

The invention discloses a slide preparation method for rapidly obtaining root tip chromosomes of Shenghua and application, and belongs to the technical field of chromosome slide preparation. The flaking method comprises the following steps: carrying out cuttage water culture on node vines at the overground part of the ginseng and the sweet potatoes, and inducing to generate adventitious roots; the root tips of the adventitious roots are taken and placed in a pretreatment solution to be pretreated; the pretreated root tips are placed in a fixing solution to be fixed; sequentially carrying out acidolysis and enzymolysis on the fixed root tips; taking root tip meristematic zone tissues after acidolysis and enzymolysis, placing the root tip meristematic zone tissues on a glass slide, dyeing, tabletting, and placing on a heating plate for heating; according to the flaking method disclosed by the invention, the division phase metaphase cells with clean flaking background, clear chromosome morphological structure, obvious chromosome overflow mark and good dispersion can be rapidly obtained, the chromosome number statistics and karyotype analysis are convenient, and the flaking method has important significance on ploidy identification, variety identification, genetic relationship identification and the like of the sweet potatoes.

Description

Technical Field

[0001] The invention belongs to the technical field of chromosome preparation, and in particular relates to a preparation method and application of chromosomes of ginseng potato root tips for rapid acquisition. Background Art

[0002] Ginseng potato Dioscorea alata L. ), belonging to the Dioscorea genus of the Dioscorea family, is a widely cultivated yam variety. The tubers of yam are rich in nutrients such as starch, polysaccharides and mucin. They also accumulate a variety of physiologically active ingredients such as anthocyanins, flavonoids, dioscin, and allantoin, and have high nutritional and health value.

[0003] As the carrier of the main genetic material, changes in the number and structure of chromosomes usually cause corresponding genetic effects. Chromosome karyotype analysis is an effective method to detect genetic differences in variety resources at the chromosome level, taking metaphase chromosomes as the research object. Genetic differences are evaluated through characteristics such as chromosome length, long-short arm ratio, presence of satellites, and centromere position, thereby providing an important basis for genetic differences between variety resources. This analysis method has been widely used in many plants, but it is rarely used in ginseng potatoes. Therefore, it is necessary to carry out karyotype analysis research on ginseng potatoes. However, due to the complex genetic background of ginseng potatoes, large differences in chromosome number and insignificant morphological characteristics, clarifying the chromosome karyotype of ginseng potatoes is of great significance for promoting the utilization, analysis and genetic breeding research of ginseng potatoes.

[0004] Plant root tip chromosome pressing usually directly obtains root tip meristem cells from the root system of the underground part of the plant for preparation. This process is limited by the growth state of the plant root system and the time of sampling, and it is easy to damage the root system, resulting in the plant's inability to survive. The root tip of ginseng potato is easy to lignify. The existing method usually directly obtains root tip meristem cells from the root system in the soil, and the resulting chromosome constriction is not obvious; in addition, the ploidy of ginseng potato is complex, and the chromosomes are small, making it difficult to clearly distinguish the chromosome morphology and perform karyotype analysis. Obtaining ginseng potato root tips using existing methods is likely to damage its root system, and even cause some rare ginseng potato germplasm resources to be lost due to root damage. At present, there have been reports of using matrix to cultivate yam spores or tissue culture seedlings to obtain root tips, but only a few yam varieties can produce spores, and the spore-producing period is long; and tissue culture has high technical requirements, requiring a large amount of professional equipment and operating skills. Therefore, developing a method that can quickly, conveniently and with high quality obtain root tip meristem cells of ginseng potato is of great significance for chromosome preparation and karyotype analysis of ginseng potato. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for quickly obtaining root tip chromosomes of ginseng potato, which can quickly obtain metaphase cells with clean preparation background, clear chromosome morphology and structure, obvious chromosome spill marks, and good dispersion, which is convenient for chromosome number statistics and karyotype analysis.

[0006] Another object of the present invention is to provide a ginseng potato root tip chromosome slide prepared by the preparation method.

[0007] Another object of the present invention is to provide an application of the slice making method or the ginseng potato root tip chromosome slide in ploidy identification, variety identification or kinship identification of ginseng potatoes.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for rapidly obtaining chromosomes of root tips of ginseng potatoes, the method comprising the following steps: The above-ground noded vines of ginseng potatoes are taken for cutting and hydroponics to induce adventitious roots; the root tips of the adventitious roots are taken and placed in a pretreatment solution for pretreatment; the pretreated root tips are placed in a fixing solution for fixation; the fixed root tips are subjected to acid hydrolysis and enzymolysis in sequence; the root tip meristem tissue after acid hydrolysis and enzymolysis is taken and placed on a slide, stained, pressed, and placed on a heating plate for heating.

[0009] Preferably, the noded vines are obtained by cutting the aerial vines of ginseng potato into noded stem segments with 2 leaves and 1 node.

[0010] Preferably, the hydroponic cutting method comprises fixing the noded vines on a planting plate, and floating the planting plate in a hydroponic tank, into which pure water is added for root induction.

[0011] Preferably, the culture temperature of the cutting hydroponic culture is 20-25°C; the light intensity of the cutting hydroponic culture is 100-200 μmol / m 2 / s; the culture time of the cutting hydroponic culture is 7 to 10 days.

[0012] Preferably, the root tip of the adventitious root refers to the root tip portion of the front end of the root system that is 0.8 to 1.2 cm long; the root tip of the adventitious root is collected between 8:00 and 9:00 in the morning.

[0013] Preferably, the pretreatment solution comprises 8-hydroxyquinoline, colchicine, saturated p-dichlorobenzene or bromonaphthalene; and the fixative comprises Carnoy's fixative.

[0014] Preferably, the acid solution used in the acid hydrolysis is HCl solution; the temperature of the acid hydrolysis is 55-65° C.; and the time of the acid hydrolysis is 5-15 min.

[0015] Preferably, the enzymatic hydrolysis solution used in the enzymatic hydrolysis contains cellulase and pectinase; the enzymatic hydrolysis temperature is 30-40° C.; and the enzymatic hydrolysis time is 5-15 min.

[0016] The present invention also provides a ginseng potato root tip chromosome slide prepared by the slide making method.

[0017] The present invention also provides an application of the slide making method or the ginseng potato root tip chromosome slide in ploidy identification, variety identification or kinship identification of ginseng potatoes.

[0018] Beneficial effects of the present invention: The present invention provides a preparation method for rapidly obtaining root tip chromosomes of ginseng potato and its application. The method induces aerial node-bearing vines of ginseng potato to generate adventitious roots by cutting and hydroponics. Compared with the existing method of obtaining root tip meristem cells from plant roots or obtaining root tip meristem cells by tissue culture, the root tip meristem cells of ginseng potato can be rapidly and conveniently obtained without destroying the root system of ginseng potato. The cells can be used for chromosome preparation, and metaphase cells with clean background, clear chromosome morphology and structure, obvious chromosome spill marks and good dispersion can be obtained, which is convenient for chromosome number statistics and karyotype analysis, and has important significance for ploidy identification, variety identification, kinship identification and the like of ginseng potato. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The 2-leaf 1-node stem segment of Gan Ziyun No. 1 ginseng potato in Example 1; Figure 2 The hydroponic rooting induction of Gan Ziyun No. 1 ginseng potato vines in Example 1; wherein A is the picture above the foam board after planting, and B is the picture below the foam board after planting; Figure 3 The root tips induced by hydroponics in Example 1; A is a 2-leaf 1-node stem segment and the induced root tip, and B is an enlarged view of the root tip; Figure 4 The root tip induced by hydroponic culture of Gan Ziyun No. 1 ginseng potato and the slice preparation effect of the root tip in Example 1; wherein A is the root tip induced by hydroponic culture of Gan Ziyun No. 1 ginseng potato, and B is the slice preparation effect of the root tip (scale is 10 μm); Figure 5 The chromosome number and karyotype of Gan Ziyun No. 1 ginseng potato in Example 1; wherein A is a chromosome metaphase map, B is a chromosome morphology map, and C is a karyotype pattern map; Figure 6 The root tips induced by hydroponics of Lijiang purple ginseng sweet potato and the slice preparation effect of the root tips in Example 2; wherein A is the root tips induced by hydroponics of Lijiang purple ginseng sweet potato, and B is the slice preparation effect of the root tips (scale is 10 μm); Figure 7 The chromosome number and karyotype of the Lijiang purple ginseng potato in Example 2; wherein A is a chromosome metaphase map, B is a chromosome morphology map, and C is a karyotype pattern map; Figure 8 This is the slice preparation effect of the root tip of Gan Ziyun No. 1 ginseng potato group in comparative example 1 (scale bar is 10 μm); Fig. 9 This is the preparation effect of the root tip directly obtained from the root of Gan Ziyun No. 1 ginseng potato in Comparative Example 2 (scale is 10 μm); Fig.10 This is the slice preparation effect of the bacon tip of Lijiang purple ginseng potato group in comparative example 3 (scale bar is 10μm); Fig.11 This is the preparation effect of the root tip directly obtained from the root of Lijiang purple ginseng potato in comparative example 4 (scale is 10μm); Fig.12 The root system induced by hydroponic cuttings of single-leaf and single-node stem segments of Gan Ziyun No. 1 ginseng potato in Comparative Example 5; Fig.13 This is the slice preparation effect of the root tip of Gan Ziyun No. 1 ginseng potato in Comparative Example 5 (scale bar is 10 μm); Fig.14 The root system induced by hydroponic cuttings of single-leaf and single-node stem segments of Lijiang purple ginseng potato in Comparative Example 6; Fig.15 This is the slice preparation effect of the root tip of Lijiang purple ginseng potato in Comparative Example 6 (scale bar is 10 μm). DETAILED DESCRIPTION

[0020] The present invention provides a method for rapidly obtaining chromosomes of root tips of ginseng potatoes, the method comprising the following steps: The above-ground noded vines of ginseng potatoes are taken for cutting and hydroponics to induce adventitious roots; the root tips of the adventitious roots are taken and placed in a pretreatment solution for pretreatment; the pretreated root tips are placed in a fixing solution for fixation; the fixed root tips are subjected to acid hydrolysis and enzymolysis in sequence; the root tip meristem tissue after acid hydrolysis and enzymolysis is taken and placed on a slide, stained, pressed, and placed on a heating plate for heating.

[0021] In the present invention, there is no special limitation on the variety of the ginseng potato, which can be conventionally selected according to actual needs, for example, including but not limited to Gan Ziyun No. 1 ginseng potato or Lijiang purple ginseng potato. In the present invention, the above-ground node vines of the ginseng potato are taken for cutting and hydroponic cultivation to induce the formation of adventitious roots. The node vines are preferably obtained by cutting the above-ground vines of the ginseng potato into node stem segments with 2 leaves and 1 node. The cutting and hydroponic method preferably includes fixing the node vines on a planting plate, and floating the planting plate in a hydroponic tank, and adding pure water to the hydroponic tank for root induction. The fixing method of the node vines can be conventionally selected according to actual needs; as an optional embodiment, the node vines are fixed to the planting plate using a water-absorbing sponge. The aperture for planting on the planting plate can be conventionally selected according to actual needs. As an optional embodiment, the aperture is preferably 3~8cm, more preferably 5cm or 4~5cm or 5~7cm. The amount of pure water added can be conventionally selected according to actual needs. As an optional embodiment, the root induction process also includes replacing pure water; the time interval for the replacement can be conventionally selected according to actual needs, preferably 1 to 2 days. The culture temperature of the cutting hydroponic culture is preferably 20 to 25°C, more preferably 20°C or 21°C or 22°C or 23°C or 24°C or 25°C; the light intensity of the cutting hydroponic culture is preferably 100 to 200 μmol / m 2 / s, more preferably 150 μmol / m 2 / s or 125~175μmol / m 2 / s or 130~150μmol / m 2 / s or 150~170μmol / m 2 / s; the culture time of the cutting hydroponics can be conventionally selected according to the growth of adventitious roots, preferably, cultured until 2-3 cm adventitious roots grow out of the internodes of the ginseng potato vines; as an optional embodiment, the culture time of the cutting hydroponics is preferably 7-10 days, more preferably 7 days or 8 days or 9 days or 10 days.

[0022] In the present invention, the root tip induced to generate adventitious roots is placed in a pretreatment solution for pretreatment. The root tip is preferably the root tip part of 0.8~1.2cm at the front end of the root system, and more preferably the root tip part of 1cm at the front end of the root system. The collection time of the root tip can be conventionally selected according to actual needs, preferably 8:00~9:00 in the morning. The type of the pretreatment solution can be conventionally selected according to actual needs, preferably including 8-hydroxyquinoline, colchicine, saturated p-dichlorobenzene or bromonaphthalene, etc.; more preferably 8-hydroxyquinoline. The concentration of the pretreatment solution can be conventionally selected according to actual needs. For example, when the pretreatment solution is 8-hydroxyquinoline, the concentration is preferably 0.001~0.005mol / L, more preferably 0.002mol / L~0.003mol / L. The pretreatment temperature is preferably 2~6°C, more preferably 4°C; the pretreatment time is preferably 3~5h, more preferably 4h.

[0023] In the present invention, the pretreated root apex is placed in a fixative for fixation. The type of the fixative can be conventionally selected according to actual needs, for example, including Carnoy's fixative; the Carnoy's fixative is preferably a mixture of anhydrous ethanol and glacial acetic acid in a volume ratio of 3:1. The fixation time can be conventionally selected according to actual needs, preferably 18 to 30 hours, more preferably 24 hours or 22 to 24 hours or 24 to 26 hours. If it is necessary to retain the root apex, as an optional embodiment, the fixed root apex can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0024] In the present invention, the fixed root apex is subjected to acid hydrolysis and enzymolysis in sequence. In the present invention, before acid hydrolysis and enzymolysis, it is preferred that the root apex is subjected to hypotonic treatment. The method of the hypotonic treatment can be conventionally selected according to actual needs. As an optional embodiment, the hypotonic treatment includes: cleaning the fixed root apex, immersing it in a saline solution for hypotonic treatment; the cleaning method can be conventionally selected according to actual needs, preferably using distilled water for cleaning; the number of cleanings is preferably 1 to 3 times, more preferably 2 times; the type of saline solution can be conventionally selected according to actual needs, such as NaCl solution or KCl solution; the concentration of the saline solution can be conventionally selected according to actual needs, and the mass fraction is preferably 0.5% to 1%, more preferably 0.75%; the time of the hypotonic treatment is preferably 20 to 40 minutes, more preferably 30 minutes. In the present invention, after the hypotonic treatment, it is preferred that the root apex is cleaned. The cleaning method can be conventionally selected according to actual needs, preferably using distilled water for cleaning; the number of cleanings is preferably 1 to 3 times, more preferably 2 times. In the present invention, the acid solution used in the acid hydrolysis is preferably an HCl solution; the concentration of the acid solution is preferably 0.1~0.5mol / L, more preferably 0.25mol / L. The acid hydrolysis method is preferably to carry out acid hydrolysis in a metal bath. The temperature of the acid hydrolysis is preferably 55~65°C, more preferably 60°C. The time of the acid hydrolysis is preferably 5~15min, more preferably 10min. In the present invention, after the acid hydrolysis, it is preferred to also clean the root apex. The cleaning method can be conventionally selected according to actual needs, preferably using distilled water for cleaning; the number of cleanings is preferably 1~3 times, more preferably 2 times. In the present invention, the enzymatic solution used for the enzymatic hydrolysis preferably contains cellulase and pectinase; in the enzymatic hydrolysis solution, the mass percentage of the cellulase is preferably 1% to 4%, more preferably 2.5%; in the enzymatic hydrolysis solution, the mass percentage of the pectinase is preferably 1% to 4%, more preferably 2.5%; in the enzymatic hydrolysis solution, the enzymatic activity of the cellulase is preferably> 7IU / g, more preferably> 17.5IU / g or> 28IU / g; the enzymatic activity of the pectinase is preferably> 1.5IU / mg, more preferably> 3.75IU / mg or> 6IU / mg. The enzymatic hydrolysis method is preferably to perform enzymatic hydrolysis in a water bath. The enzymatic hydrolysis temperature is preferably 30 to 40°C, more preferably 37°C. The enzymatic hydrolysis time is preferably 5 to 15min, more preferably 10min. In the present invention, after the enzymatic hydrolysis, it is preferred to also clean the root tip. The cleaning method can be conventionally selected according to actual needs, preferably using distilled water for cleaning; the number of cleanings is preferably 1 to 3 times, more preferably 2 times. In the present invention, after cleaning, it is preferred to further include a hypotonic treatment.The method of the hypotonic treatment can be conventionally selected according to actual needs. Preferably, the root tip is placed in distilled water at room temperature for hypotonic treatment. The time of the hypotonic treatment is preferably 20 to 40 minutes, more preferably 30 minutes.

[0025] In the present invention, the root apical meristem tissue after acid hydrolysis and enzymolysis is placed on a slide, stained, pressed, and placed on a heating plate for heating. The method for obtaining the root apical meristem tissue can be conventionally selected according to actual needs, for example, cutting with a dissecting needle or a scalpel. The staining preferably includes: dripping a dye solution, covering with a cover glass after staining, and absorbing excess dye solution. The dye solution can be conventionally selected according to actual needs, preferably a carbofuchsin solution. The dyeing time is preferably 10 to 20 minutes, more preferably 15 minutes. The method of absorbing excess dye solution is preferably to absorb excess dye solution with filter paper. The method of pressing can be conventionally selected according to actual needs. As an optional embodiment, the soft eraser head of a pencil is used for knocking; the knocking time can be conventionally selected according to the degree of morphological dispersion of the root apical meristem tissue, preferably 3 to 8 minutes, more preferably 5 minutes. In the present invention, the glass slide after staining and pressing is placed on a heating plate for heating to make the slide preparation more stable. The heating temperature is preferably 60 to 70°C, more preferably 65°C. The heating time is preferably 0.5 to 1.5 min, more preferably 1 min.

[0026] In the present invention, as an optional embodiment, the steps of the method for quickly obtaining chromosomes of ginseng potato root tips are as follows: Inducing root tips: During the vigorous growth period of the aboveground vines, select healthy, disease-free and insect-free mature vines of ginseng potatoes, cut them into 2-leaf 1-node stem segments, fix the stem segments with absorbent sponges on planting boards with a pore size of 3-8 cm, and float the planting boards in a hydroponic tank. Add pure water to the hydroponic tank to induce rooting. Keep the room temperature at 20-25℃ and the light intensity at 100-200μmol / m 2 / s conditions to induce adventitious roots; replace pure water every 1 to 2 days; after 7 to 10 days of induction culture, 2 to 3 cm adventitious roots can be seen in the internodes of the ginseng potato vines.

[0027] Root tip pretreatment: Cut young root tips 0.8~1.2cm between 8:00~9:00 in the morning, place them in 0.001~0.005mol / L 8-hydroxyquinoline solution, and pretreat them at 2~6℃ for 3~5h.

[0028] Fixation: Wash the root apex with distilled water 1 to 3 times and fix it in Carnoy's fixative for 18 to 30 hours; the Carnoy's fixative is composed of anhydrous ethanol and glacial acetic acid, and the volume ratio of anhydrous ethanol to glacial acetic acid is 3:1. If the root apex needs to be preserved, it can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0029] Dissociation: At room temperature, wash the root tip with distilled water 1-3 times, and treat it in 0.5%-1% KCl solution for 20-40 minutes; continue to wash the root tip with distilled water 1-3 times, and then acid hydrolyze it in a 55-65℃ metal bath with 0.1-0.5mol / L HCl solution for 5-15 minutes; wash the root tip with distilled water again 1-3 times, and then use enzymatic solution to hydrolyze it in a 30-40℃ water bath for 5-15 minutes; the enzymatic solution consists of cellulase, pectinase and distilled water; in the enzymatic solution, the mass percentage of the cellulase is 1%-4%, and the mass percentage of the pectinase is 1%-4%; in the enzymatic solution, the enzyme activity of the cellulase is >7IU / g, and the enzyme activity of the pectinase is >1.5IU / mg. Wash the root tip with distilled water 1-3 times, and place it in distilled water for 20-40 minutes of hypotonic treatment at room temperature.

[0030] Staining and slide preparation: Use a blade to cut a 1-2 mm section of the root apical meristem, place it on a glass slide, add Carbol fuchsin solution, stain for 10-20 min, cover with a coverslip, absorb excess dye with filter paper, and tap with the eraser end of a pencil for 3-8 min. Wait until the root apex morphology is dispersed, place it on a 60-70°C heating plate and heat for 0.5-1.5 min to make the slide stable.

[0031] The present invention also provides a ginseng potato root tip chromosome slide prepared by the slide making method.

[0032] The present invention also provides an application of the slide making method or the ginseng potato root tip chromosome slide in ploidy identification, variety identification or kinship identification of ginseng potatoes.

[0033] The preparation method of the present invention can quickly and conveniently obtain ginseng potato root tip meristem cells without destroying the ginseng potato root system, and the prepared ginseng potato root tip chromosome slide has a clean background, well-dispersed cells in the mitotic metaphase, clear chromosome morphology and structure, and obvious chromosome overflow marks. The prepared ginseng potato root tip chromosome slide is observed under an Olympus microscope, and the chromosome metaphase diagram can be quickly found and photographed, so that the chromosome can be morphologically observed and counted according to the picture, which is convenient for chromosome number statistics and karyotype analysis, and can be used for ploidy identification, variety identification, kinship identification, etc. of ginseng potato.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1 A method for quickly obtaining chromosomes from the root tip of ginseng potato. Gan Ziyun No. 1 ginseng potato was selected as the experimental material of this example. The specific operation steps are as follows: (1) Inducing root tips: During the vigorous growth period of the aboveground vines, select the healthy, disease-free and insect-free mature vines of Gan Ziyun No. 1 ginseng potato and cut them into 2-leaf 1-node stem segments ( Figure 1 ), use absorbent sponge to fix the noded vines on the foam board, the hole diameter of the foam board for planting is 5cm, float the foam board in the hydroponic tank, add pure water to the hydroponic tank to induce rooting ( Figure 2 , where A is the picture above the foam board after planting, and B is the picture below the foam board after planting); at room temperature 22℃ and light intensity 150μmol / m 2 / s conditions to induce adventitious roots; pure water was replaced every other day, and on the 7th day, adventitious roots of 2-3 cm were observed at the internodes of the ginseng potato vines ( Figure 3 , where A is a 2-leaf 1-node stem segment and the induced root tip, and B is an enlarged view of the root tip).

[0038] (2) Root tip pretreatment: Cut about 1 cm of young root tips between 8:00 and 9:00 in the morning, place them in 0.002 mol / L 8-hydroxyquinoline solution, and pretreat them at 4°C for 4 hours.

[0039] (3) Fixation: Wash the root apex twice with distilled water and fix it in Carnoy's fixative (V anhydrous ethanol: V glacial acetic acid = 3:1) for 24 h. If the root apex needs to be preserved, it can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0040] (4) Dissociation: At room temperature, the root tip was washed twice with distilled water, and then subjected to hypotonic treatment in 0.75% KCl solution for 30 min; the root tip was washed twice with distilled water, and then acidified with 0.25 mol / L HCl solution in a 60°C metal bath for 10 min; the root tip was washed twice with distilled water again, and then hydrolyzed with enzymatic solution (2.5% cellulase + 2.5% pectinase + 95% distilled water) in a 37°C water bath for 10 min, where the cellulase activity in the enzymatic solution was >17.5 IU / g and the pectinase activity was >3.75 IU / mg; the root tip was washed twice with distilled water, and finally subjected to hypotonic treatment in distilled water at room temperature for 30 min.

[0041] (5) Staining and slide preparation: Use a blade to cut 1-2 mm of the root apical meristem, place it on a glass slide, add Carbol fuchsin solution, stain for 15 min, cover with a coverslip, absorb excess stain with filter paper and tap with the eraser of a pencil for 5 min. When the root apex morphology is dispersed, place it on a 65°C heating plate and heat for 1 min to make the slide stable.

[0042] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Figure 4 As shown, the chromosomes were then morphologically observed, counted, and karyotyped according to the pictures. The results are as follows Figure 5 shown.

[0043] Depend on Figure 4 It can be seen that the slide prepared by the method of the present invention has a clean background, clear chromosome morphology and structure, and obvious chromosome overflow marks. Figure 5 It can be seen that the number of chromosomes of Gan Ziyun No. 1 ginseng potato is 40, which is diploid. The chromosome karyotype formula is 2n=2x=40=14sm+26m. The relative length of chromosomes is 2n=40=2L+12M2+24M1+2S. The relative length is 1.51%~4.73%. The chromosome length ratio is 3.13, the arm ratio is 1.17~2.78, and the average arm ratio is 1.69. Chromosomes with arm ratios greater than 2 account for 20% of the total chromosomes. The nuclear asymmetry coefficient is 62.64%, and the karyotype is "2B".

[0044] Example 2 A method for quickly obtaining chromosomes from the root tip of ginseng potato, selecting Lijiang purple ginseng potato as the experimental material of this example, and the specific operating steps are as follows: (1) Inducing root tips: During the vigorous growth period of the aboveground vines, select healthy, disease-free and mature vines of Lijiang purple ginseng potato, cut them into stem segments with 2 leaves and 1 node, and use absorbent sponges to fix the vines with nodes on a foam board with a pore size of 5 cm for planting. Float the foam board in a hydroponic tank, and add pure water to the hydroponic tank to induce rooting. 2 / s conditions to induce adventitious roots; pure water was replaced every other day, and on the 10th day, adventitious roots of 2 to 3 cm could be seen in the internodes of the ginseng potato vines.

[0045] (2) Root tip pretreatment: Cut about 1 cm of young root tips between 8:00 and 9:00 in the morning, place them in 0.002 mol / L 8-hydroxyquinoline solution, and pretreat them at 4°C for 4 hours.

[0046] (3) Fixation: Wash the root apex twice with distilled water and fix it in Carnoy's fixative (V anhydrous ethanol: V glacial acetic acid = 3:1) for 24 h. If the root apex needs to be preserved, it can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0047] (4) Dissociation: At room temperature, the root tip was washed twice with distilled water, and then subjected to hypotonic treatment in 0.75% KCl solution for 30 min; the root tip was washed twice with distilled water, and then acidified with 0.25 mol / L HCl solution in a 60°C metal bath for 10 min; the root tip was washed twice with distilled water again, and then hydrolyzed with enzymatic solution (2.5% cellulase + 2.5% pectinase + 95% distilled water) in a 37°C water bath for 10 min, where the cellulase activity in the enzymatic solution was >17.5 IU / g and the pectinase activity was >3.75 IU / mg; the root tip was washed twice with distilled water, and finally subjected to hypotonic treatment in distilled water at room temperature for 30 min.

[0048] (5) Staining and slide preparation: Use a blade to cut 1-2 mm of the root apical meristem, place it on a glass slide, add Carbol fuchsin solution, stain for 15 min, cover with a coverslip, absorb excess stain with filter paper and tap with the eraser of a pencil for 5 min. When the root apex morphology is dispersed, place it on a 65°C heating plate and heat for 1 min to make the slide stable.

[0049] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Figure 6 As shown, the chromosomes were then morphologically observed, counted, and karyotyped according to the pictures. The results are as follows Figure 7 shown.

[0050] Depend on Figure 6 It can be seen that the slide prepared by the method of the present invention has a clean background, clear chromosome morphology and structure, and obvious chromosome spill marks. Figure 7 It can be seen that the number of chromosomes of Lijiang purple ginseng potato is 80, tetraploid, the chromosome karyotype formula is 2n=4x=80=60sm+20m, the relative length of chromosomes is 2n=80=4L+32M2+40M1+4S, the relative length is 1.43%~4.55%, the arm ratio is 1.53~2.73, the chromosome length ratio is 3.19, the average arm ratio is 1.95, and the chromosomes with arm ratio greater than 2 account for 40% of the total chromosomes. The nuclear asymmetry coefficient is 66.08%, and the karyotype is "2B".

[0051] Example 3 A method for quickly obtaining chromosomes from the root tip of ginseng potato. Gan Ziyun No. 1 ginseng potato was selected as the experimental material of this example. The specific operation steps are as follows: (1) Inducing root tips: During the vigorous growth period of the aboveground vines, select the healthy, disease-free and insect-free mature vines of Gan Ziyun No. 1 ginseng potato, cut them into stem segments with 2 leaves and 1 node, and use absorbent sponges to fix the noded vines on a foam board with a 3 cm hole diameter for planting. Float the foam board in a hydroponic tank, and add pure water to the hydroponic tank to induce rooting. Keep the room temperature at 20 °C and the light intensity at 200 μmol / m 2 / s conditions to induce adventitious roots; pure water was replaced every 2 days, and on the 8th day, adventitious roots of 2 to 3 cm were visible in the internodes of the ginseng potato vines.

[0052] (2) Root tip pretreatment: Cut the young root tip about 0.8 cm between 8:00 and 9:00 in the morning, place it in 0.001 mol / L 8-hydroxyquinoline solution, and pretreat it at 2°C for 6 hours.

[0053] (3) Fixation: Wash the root apex once with distilled water and fix it in Carnoy's fixative (V anhydrous ethanol: V glacial acetic acid = 3:1) for 18 h. If the root apex needs to be preserved, it can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0054] (4) Dissociation: At room temperature, the root tip was washed once with distilled water, and then subjected to hypotonic treatment in 0.5% KCl solution for 40 min; the root tip was washed once with distilled water, and then acid-hydrolyzed in 0.1 mol / L HCl solution in a 65°C metal bath for 15 min; the root tip was washed once with distilled water, and then hydrolyzed in a 40°C water bath with an enzymatic solution (1% cellulase + 1% pectinase + 98% distilled water) for 15 min, where the cellulase activity in the enzymatic solution was >7 IU / g and the pectinase activity was >1.5 IU / mg; the root tip was washed once with distilled water, and finally subjected to hypotonic treatment in distilled water for 20 min at room temperature.

[0055] (5) Staining and slide preparation: Use a blade to cut 1-2 mm of the root apical meristem, place it on a glass slide, add Carbol fuchsin solution, stain for 20 min, cover with a coverslip, absorb excess stain with filter paper and tap with the eraser of a pencil for 8 min. When the root apex morphology is dispersed, place it on a 60°C heating plate and heat for 1.5 min to make the slide stable.

[0056] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10x field of view, observe the chromosome metaphase diagram under a 10×100x oil objective and take a picture. You can see that the slide background is clean, the chromosome morphology and structure are clear, and the chromosome overflow marks are obvious.

[0057] Example 4 A method for quickly obtaining chromosomes from the root tip of ginseng potato, selecting Lijiang purple ginseng potato as the experimental material of this example, and the specific operating steps are as follows: (1) Inducing root tips: During the vigorous growth period of the aboveground vines, select healthy, disease-free and mature vines of Lijiang purple ginseng potato, cut them into stem segments with 2 leaves and 1 node, and use absorbent sponges to fix the vines with nodes on a foam board with a hole diameter of 8 cm for planting. Float the foam board in a hydroponic tank, add pure water to the hydroponic tank to induce rooting; keep the room temperature at 25°C and the light intensity at 100 μmol / m 2 / s conditions to induce adventitious roots; pure water was replaced every other day, and on the 9th day, adventitious roots of 2 to 3 cm were visible in the internodes of the ginseng potato vines.

[0058] (2) Root tip pretreatment: Cut the young root tip about 1.2 cm between 8:00 and 9:00 in the morning, place it in 0.005 mol / L 8-hydroxyquinoline solution, and pretreat it at 6°C for 3 hours.

[0059] (3) Fixation: Wash the root apex three times with distilled water and fix it in Carnoy's fixative (V anhydrous ethanol: V glacial acetic acid = 3:1) for 30 h. If the root apex needs to be preserved, it can be transferred to 70% anhydrous ethanol and stored in a refrigerator at 4°C.

[0060] (4) Dissociation: At room temperature, the root tip was washed with distilled water for 3 times, and then subjected to hypotonic treatment in 1% KCl solution for 20 min; the root tip was washed with distilled water for 3 times, and then acidified with 0.5 mol / L HCl solution in a 55°C metal bath for 5 min; the root tip was washed with distilled water for 3 times, and then acidified with 0.5 mol / L HCl solution in a 55°C metal bath for 5 min; the root tip was washed with distilled water again for 3 times, and then acidified with enzymatic solution (4% cellulase + 4% pectinase + 92% distilled water) in a 30°C water bath for 5 min, where the cellulase activity in the enzymatic solution was >28 IU / g, and the pectinase activity was >6 IU / mg; the root tip was washed with distilled water for 3 times, and finally subjected to hypotonic treatment in distilled water at room temperature for 40 min.

[0061] (5) Staining and slide preparation: Use a blade to cut 1-2 mm of the root apical meristem, place it on a glass slide, add Carbol Fuchsin solution, stain for 10 min, cover with a coverslip, absorb excess stain with filter paper and tap with the eraser of a pencil for 3 min. Wait until the root apex morphology is dispersed, place on a 70°C heating plate and heat for 0.5 min to make the slide stable.

[0062] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10x field of view, observe the chromosome metaphase diagram under a 10×100x oil objective and take a picture. You can see that the slide background is clean, the chromosome morphology and structure are clear, and the chromosome overflow marks are obvious.

[0063] Comparative Example 1 A method for preparing chromosomes of root tips of ginseng potatoes, wherein Gan Ziyun No. 1 ginseng potato is selected as the experimental material of this comparative example, and the difference from Example 1 is that the root tips are obtained by tissue culture, and the specific operation steps are as follows: Tissue culture to induce root tips: Take the above-ground vine stem segments of Gan Ziyun No. 1 ginseng potato, rinse with tap water for 30 minutes, rinse with sterile water 3 times; soak in 75% ethanol solution for 30 seconds, rinse with sterile water 3 times; disinfect with 0.1% HgCl2 solution for 8 minutes, rinse with sterile water 3 times; cut the stem segments into MS culture medium containing 2 mg / L 6-BA and 0.2 mg / L NAA, and 2-3 cm adventitious roots will grow in about 20 days.

[0064] The root apex pretreatment, fixation, dissociation, staining and slide preparation steps are the same as those in Example 1.

[0065] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that the background of the slide prepared from the root tip obtained by tissue culture method is blurred, and the chromosome morphology structure cannot be clearly observed.

[0066] Comparative Example 2 A method for preparing chromosomes of root tips of ginseng potatoes, wherein Gan Ziyun No. 1 ginseng potato is selected as the experimental material of this comparative example, and the difference from Example 1 is that the root tips are obtained directly from the root system of the underground part, and the specific operation steps are as follows: Obtaining root tips: In the Gan Ziyun No. 1 ginseng potato plantation, carefully dig up the root soil to expose the roots, cut 2-3 cm adventitious roots with scissors, rinse them in tap water, and rinse them 3 times with sterile water to obtain adventitious roots.

[0067] The root apex pretreatment, fixation, dissociation and staining and slide preparation steps are the same as those in Example 1.

[0068] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that the background of the slide prepared from the root tip directly obtained from the underground part of the root system of ginseng potato is blurred, and the chromosome morphological structure cannot be clearly observed.

[0069] Comparative Example 3 A method for preparing chromosomes of root tips of ginseng potatoes, selecting Lijiang purple ginseng potatoes as experimental materials for this comparative example, which is different from Example 2 in that the root tips are obtained by tissue culture, and the specific operation steps are as follows: Tissue culture to induce root tips: Take the above-ground vine stem segments of Lijiang purple ginseng potato, rinse with tap water for 30 minutes, rinse with sterile water 3 times; soak in 75% ethanol solution for 30 seconds, rinse with sterile water 3 times; disinfect with 0.1% HgCl2 solution for 8 minutes, rinse with sterile water 3 times; cut the stem segments into MS culture medium containing 2 mg / L 6-BA and 0.2 mg / L NAA, and 2-3 cm adventitious roots will grow in about 20 days.

[0070] The root apex pretreatment, fixation, dissociation, staining and slide preparation steps are the same as those in Example 2.

[0071] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Fig.10 As shown. Fig.10 It can be seen that the background of the slide prepared from the root tip obtained by tissue culture method is blurred, and the chromosome morphology structure cannot be clearly observed.

[0072] Comparative Example 4 A method for preparing chromosomes of root tips of ginseng potatoes, selecting Lijiang purple ginseng potatoes as experimental materials for this comparative example, which is different from Example 2 in that the root tips are obtained directly from the root system of the underground part, and the specific operation steps are as follows: Obtaining root tips: In the Lijiang purple ginseng potato plantation, carefully dig up the root soil to expose the roots, cut 2-3 cm adventitious roots with scissors, rinse them in tap water, and rinse them 3 times with sterile water to obtain adventitious roots.

[0073] The root apex pretreatment, fixation, dissociation, staining and slide preparation steps are the same as those in Example 2.

[0074] Chromosome observation and counting: Place the prepared slide on an Olympus microscope, find the observation target within the 10×10 magnification field of view, observe the chromosome metaphase diagram under a 10×100 magnification oil lens and take a photo. The results are as follows: Fig.11 As shown. Fig.11 It can be seen that the background of the slide prepared from the root tip directly obtained from the underground part of the root system of ginseng potato is blurred, and the chromosome morphological structure cannot be clearly observed.

[0075] It can be seen from the chromosome metaphase diagram observed on the slides prepared by the methods of Examples 1-2 and Comparative Examples 1-4 that the division period is not easy to control and the degree of differentiation is high, so the cell mass is not easy to disperse during the slice pressing, resulting in a blurred slice making effect. The slides prepared by the method of the present invention have a clean background, well-dispersed metaphase cells, clear chromosome morphology and structure, and obvious chromosome overflow marks, which are convenient for chromosome number statistics and karyotype analysis, and can be used for ploidy identification, variety identification, and kinship identification of ginseng potatoes.

[0076] Comparative Example 5 A method for preparing chromosomes of ginseng potato root tips, selecting Gan Ziyun No. 1 ginseng potato as the experimental material of this comparative example, which is different from Example 1 in that single-leaf single-node nodular stem segments are used for cutting hydroponics, and the remaining steps are the same as those of Example 1.

[0077] The root system induced by hydroponic cuttings of single-leaf single-node stem segments is as follows Fig.12 As shown in the figure, it can be seen that the root system induced by cuttings of single-leaf single-node stem segments is thick and strong, and the root tip meristem is not easy to spread out during the preparation process. The prepared slides were placed under an Olympus microscope for chromosome observation and counting. The metaphase diagram of chromosome division was observed and photographed under a 10×100 oil lens. The results are shown in the figure. Fig.13 As shown in the figure, it can be seen that the background of the slide made from the root tip obtained from the root system induced by cutting hydroponics of a single-leaf single-node stem segment is blurred, and the chromosome morphology structure cannot be clearly observed.

[0078] Comparative Example 6 A method for preparing chromosomes of root tips of ginseng potatoes, selecting Lijiang purple ginseng potatoes as experimental materials for this comparative example, which is different from Example 2 in that single-leaf single-node nodal stem segments are used for cutting hydroponics, and the remaining steps are the same as those of Example 2.

[0079] The root system induced by hydroponic cuttings of single-leaf single-node stem segments is as follows Fig.14 As shown in the figure, it can be seen that the root system induced by cuttings of single-leaf single-node stem segments is shortened and it is difficult to find the root tip meristem. The prepared slides were placed under an Olympus microscope for chromosome observation and counting. The metaphase diagram of chromosome division was observed and photographed under a 10×100 oil lens. The results are shown in the figure below. Fig.15 As shown in the figure, it can be seen that the background of the slide made from the root tip obtained from the root system induced by cutting hydroponics of a single-leaf single-node stem segment is blurred, and the chromosome morphology structure cannot be clearly observed.

[0080] From the chromosome metaphase images observed on the slides prepared by the methods of Examples 1-2 and Comparative Examples 5-6, it can be seen that the root system induced by the single-leaf single-node stem segment is short and thick, and it is difficult to find the root apical meristem. The root apical meristem is not easy to spread during the preparation process, resulting in a blurred preparation effect. The slide prepared by the method of the present invention has a clean background, well-dispersed metaphase cells, clear chromosome morphology and structure, and obvious chromosome overflow marks, which is convenient for chromosome number statistics and karyotype analysis, and can be used for ploidy identification, variety identification, and kinship identification of ginseng potatoes.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapidly obtaining chromosomes from root tips of ginseng potatoes, characterized in that: The film-making method comprises the following steps: Taking the above-ground noded vines of ginseng potatoes for cutting and hydroponics to induce adventitious roots; taking the root tips of the adventitious roots and placing them in a pretreatment solution for pretreatment; placing the pretreated root tips in a fixative for fixation; performing acid hydrolysis and enzymolysis on the fixed root tips in sequence; taking the root tip meristem tissue after acid hydrolysis and enzymolysis and placing it on a slide, staining, pressing, and heating on a heating plate; The node vines are obtained by cutting the aerial vines of ginseng potatoes into node stem segments with two leaves and one node; The cutting hydroponic method comprises fixing noded vines on a planting plate, floating the planting plate in a hydroponic tank, and adding pure water into the hydroponic tank for inducing rooting.

2. The tableting method according to claim 1, characterized in that: The culture temperature of the cutting hydroponic culture is 20-25°C; the light intensity of the cutting hydroponic culture is 100-200 μmol / m 2 / s; the culture time of the cutting hydroponic culture is 7 to 10 days.

3. The tableting method according to claim 1, characterized in that: The root tip of the adventitious root refers to the root tip part of the front end of the root system that is 0.8 to 1.2 cm long; the root tip of the adventitious root is collected at 8:00 to 9:00 in the morning.

4. The tableting method according to claim 1, characterized in that: The pretreatment solution includes 8-hydroxyquinoline, colchicine, saturated p-dichlorobenzene or bromonaphthalene; and the fixative includes Carnoy's fixative.

5. The tableting method according to claim 1, characterized in that: The acid solution used in the acid hydrolysis is HCl solution; the temperature of the acid hydrolysis is 55-65° C.; and the time of the acid hydrolysis is 5-15 minutes.

6. The tableting method according to claim 1, characterized in that: The enzymatic hydrolysis solution used in the enzymatic hydrolysis contains cellulase and pectinase; the enzymatic hydrolysis temperature is 30-40° C.; and the enzymatic hydrolysis time is 5-15 min.

7. A ginseng potato root tip chromosome slide prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the preparation method according to any one of claims 1 to 6 or the ginseng potato root tip chromosome slide according to claim 7 in ploidy identification, variety identification or kinship identification of ginseng potatoes.

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