Kiwifruit plant somatic cell mitosis metaphase chromosome slice and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a somatic cell mitosis metaphase chromosome slice of a kiwifruit plant and a preparation method of the somatic cell mitosis metaphase chromosome slice. The invention provides a preparation method of a somatic cell mitosis metaphase chromosome slice of a kiwifruit plant. The slice prepared by the method is relatively good in chromosome morphology and uniform in dispersion. The preparation method provided by the invention comprises the following steps: firstly, pretreating root tips of kiwi plants for 3 hours by adopting a 0.002 mol / L 8-hydroxyquinoline solution; then, a specific enzyme solution is adopted for enzymolysis for 100 min, so that the actinidia plant root tip cell wall removal effect is good, the background is clean, the number of material division phases is large, chromosomes are easy to observe, the obtained chromosomes are good in morphology and uniform in dispersion, and subsequent chromosome counting, karyotype analysis (such as chromosome fluorescence in situ hybridization) and other chromosome genetic researches of the actinidia plant are facilitated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a preparation method of a chromosome slice of mitosis metaphase of somatic cells of an Actinidia plant. Background Art

[0002] Kiwifruit belongs to the genus Actinidia of the family Actinidiaceae. It is a functional dioecious perennial deciduous vine berry. Kiwifruit has a unique flavor and is rich in vitamin C, phenolic substances, amino acids, minerals, dietary fiber and other nutrients that are beneficial to health. It is known as the "king of fruits" internationally. China is the origin center of Actinidia plants and a major producer of kiwifruit. Sichuan Province is a dominant kiwifruit producing area in China, and its cultivated area and output are among the highest in the country. According to the color of the flesh and the difference in the content of various substances, kiwifruit can be divided into red flesh, yellow flesh, green flesh and other types. Among them, yellow flesh kiwifruit contains a large amount of carotenoids, which makes its flesh golden yellow. It is rich in nutrients, containing vitamins, 20 kinds of amino acids and various mineral elements such as zinc, iron, calcium, phosphorus, selenium, etc., and has extremely high nutritional, medical, and health development and utilization value.

[0003] Plant chromosomes are carriers of genetic information and have extremely high stability. The number and morphological characteristics of chromosomes are among the most stable cytological characteristics. Chromosome preparation is the most basic and commonly used method in plant cytogenetics. The quality of chromosome preparation directly affects the subsequent chromosome observation and identification. Therefore, high-quality chromosome preparation is an important guarantee for chromosome identification, chromosome karyotype analysis, chromosome banding, chromosome structural variation, identification of chromosome special components and structures, fluorescence in situ hybridization and gene localization.

[0004] The chromosome ploidy variation of Actinidia plants is very rich. Cytological observations have found that Actinidia plants have a variety of ploidy types, including diploid (2n=2x=58), tetraploid (2n=4x=116), hexaploid (2n=6x=174) and octoploid (2n=8x=232). The chromosome base number of Actinidia has been determined to be 29. Due to the large chromosome base number (x=29), small morphology (0.6-1.5μm), complex ploidy and highly similar morphology of Actinidia plants, the karyotype analysis or banding analysis of Actinidia plants is relatively lagging, and the chromosome ploidy of many kiwifruit species or subspecies is not clear. Therefore, it is urgent to explore a simple and effective method for preparing metaphase chromosomes of somatic cell mitosis in Actinidia plants. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation of somatic cell mitosis metaphase chromosomes of Actinidia plants and a preparation method thereof, so as to solve the related problems existing in the above-mentioned prior art. By utilizing the preparation method provided by the present invention, a clearly visible preparation of somatic cell mitosis metaphase chromosomes of Actinidia plants can be prepared, and the method has the advantage of being simple and convenient.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a chromosome slice of a somatic cell of an Actinidia plant at mitosis metaphase, comprising the following steps:

[0008] The seeds of Actinidia genus are subjected to low temperature treatment and variable temperature culture in sequence to obtain Actinidia genus plant seedlings;

[0009] After taking the root tip of the Actinidia plant seedling, the root tip is sequentially subjected to pretreatment, fixation, enzymatic hydrolysis, post-infiltration, re-fixation, smearing and staining to obtain a preparation of the chromosomes of the somatic cell mitosis metaphase of the Actinidia plant;

[0010] The pretreatment liquid used in the pretreatment is 0.002 mol / L 8-hydroxyquinoline solution; the pretreatment time is 3 hours;

[0011] The enzyme preparation used in the enzymolysis is a mixed enzyme solution; the mixed enzyme solution includes cellulase and pectinase; the volume percentage of cellulase in the mixed enzyme solution is 3%, and the volume percentage of pectinase is 0.3%; the enzymolysis time is 100 minutes, and the temperature is 37°C.

[0012] The specific embodiments of the present invention optimize the pretreatment solution, pretreatment time and enzymatic hydrolysis time, and it is found that under the pretreatment solution, pretreatment time and enzymatic hydrolysis time provided by the present invention, a preparation with good chromosome morphology and uniform dispersion can be obtained.

[0013] Preferably, the condition parameters of the low temperature treatment are: 4°C sand storage for 60 days;

[0014] And / or, the variable temperature culture time is 14 days;

[0015] And / or, one cycle consists of one high temperature culture and one low temperature culture, the high temperature culture time is 8 hours; the low temperature culture time is 16 hours;

[0016] And / or, the temperature of the high temperature culture is 25°C;

[0017] And / or, the temperature of the low-temperature culture is 4°C.

[0018] Preferably, the length of the root tip is 0.5 cm.

[0019] Preferably, the fixative used for fixation is Carnoy's fixative;

[0020] and / or, the Carnoy's fixative comprises anhydrous ethanol and glacial acetic acid;

[0021] and / or, the volume ratio of anhydrous ethanol to glacial acetic acid in the Carnoy's fixative is 3:1;

[0022] And / or, the fixed temperature is 4°C and the time is >5h.

[0023] Preferably, the post-osmosis includes a step of adding clean water after the enzymatic hydrolysis is completed.

[0024] Preferably, the re-fixation includes the step of adding a fixative after the post-infiltration is completed;

[0025] And / or, the fixative is Carnoy's fixative; the Carnoy's fixative comprises anhydrous ethanol and glacial acetic acid;

[0026] and / or, the volume ratio of anhydrous ethanol to glacial acetic acid in the Carnoy's fixative is 3:1;

[0027] And / or, the re-fixation temperature is 25° C. and the time is 30 min.

[0028] Preferably, the staining includes pre-staining and counter-staining;

[0029] The pre-dyeing liquid used in the pre-dyeing is a 0.067 mol / L disodium hydrogen phosphate solution;

[0030] And / or, the pre-dyeing time is 30 min;

[0031] And / or, the mixed staining solution used for the counterstaining includes a sodium hydrogen phosphate-potassium dihydrogen phosphate mixed solution and a Giemsa staining solution;

[0032] and / or, the volume ratio of the sodium hydrogen phosphate-potassium dihydrogen phosphate mixed solution to the Giemsa staining solution is 20:1;

[0033] And / or, the re-staining time is 5-10 min.

[0034] Preferably, the Actinidia plant includes any one or more of Cuiyu, Donghong, Cuixiang, Jinshi, Hongyang, Jinyan, Maohua, Shaanxi Wanhong, Miliang No. 1 and Guichang.

[0035] The present invention provides a preparation of mitotic metaphase chromosomes of somatic cells of Actinidia plants prepared by the above-mentioned preparation method.

[0036] Further preferably, the Actinidia plant includes any one or more of Cuiyu, Donghong, Cuixiang, Jinshi, Hongyang, Jinyan, Maohua, Shaanxi Wanhong, Miliang No. 1 and Guichang.

[0037] The present invention provides the application of the above-mentioned metaphase chromosome preparation of somatic cell mitosis of Actinidia plants in the chromosome genetic research of Actinidia plants.

[0038] Further preferably, the Actinidia plant includes any one or more of Cuiyu, Donghong, Cuixiang, Jinshi, Hongyang, Jinyan, Maohua, Shaanxi Wanhong, Miliang No. 1 and Guichang.

[0039] The present invention discloses the following technical effects:

[0040] The invention provides a preparation method for a preparation of a chromosome slice of a somatic cell of an Actinidia plant in mitosis metaphase. The prepared chromosome slices prepared by the method have good morphology and are evenly dispersed. In the preparation method provided by the invention, a 0.002 mol / L 8-hydroxyquinoline solution is used to pretreat the root tip of the Actinidia plant for 3 hours, and a specific enzyme solution is used for enzymatic hydrolysis for 100 minutes, so that the cell wall removal effect of the root tip of the Actinidia plant is very good, the background is clean, the material has more division phases, and the chromosomes are easy to observe. At the same time, the chromosome morphology is good and evenly dispersed, which is convenient for subsequent chromosome counting and karyotype analysis (such as chromosome fluorescence in situ hybridization) of the Actinidia plant and other chromosome genetic research.

[0041] At the same time, the root tips of Actinidia plants are small, and the slice making method of the present invention can better control the position of root tip cells in slice making, reduce material loss, facilitate cell dispersion, and improve slice making efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0043] Figure 1 The microscopic effect diagrams of different pretreatment conditions on kiwifruit somatic cell chromosomes; a is the microscopic effect diagram of 0.002mol / L 8-hydroxyquinoline treatment for 2h; b is the microscopic effect diagram of 0.002mol / L 8-hydroxyquinoline treatment for 4h; c and d are the microscopic effect diagrams of 0.002mol / L 8-hydroxyquinoline treatment for 3h; eg are the microscopic effect diagrams of ice-water mixture treatment for 8h, 16h and 24h respectively; h is the blank control;

[0044] Figure 2The diagrams are the effect diagrams of different enzymatic hydrolysis times on the chromosomes of kiwifruit somatic cells; ag are the effect diagrams of enzymatic hydrolysis for 60min, 70min, 80min, 90min, 110min, 120min and 130min, respectively; hi is the effect diagram of enzymatic hydrolysis for 100min;

[0045] Figure 3 These are the chromosome number and morphology observation diagrams of ten different varieties of kiwifruit; among them, aj is Cuiyu, Donghong, Cuixiang, Jinshi, Hongyang, Jinyan, Maohua, Shaanxi Wanhong, Miliang No. 1 and Guichang respectively; the scale bar of a is 10μm, the scale bar of b is 20μm, the scale bar of c is 10μm, the scale bar of d is 10μm, the scale bar of e is 10μm, the scale bar of f is 10μm, the scale bar of g is 10μm, the scale bar of h is 10μm, the scale bar of i is 10μm, and the scale bar of j is 10μm. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1

[0052] A method for preparing chromosomes of somatic cell mitosis metaphase of Actinidia plant, comprising the following steps:

[0053] (1) Low-temperature treatment of seeds: Pick ripe kiwifruit and store them at room temperature. When the fruit is fully soft and ripe, peel it and put it into a fine gauze bag to crush it. Wash it in water to remove impurities and grains. Spread the seeds and place them in a dry and ventilated place to dry in the shade. After drying, place them in a dry and cool place for later use. Mix the seeds with wet sand in a mass ratio of 1: (10-20) and put them into a nylon bag. Put it in a 4℃ refrigerator and store it in the sand for about 60 days. Turn the seed layer every 15-20 days, and keep the humidity of the sand at the level of "kneading into a ball by hand, and loosening it when loosened".

[0054] (2) Root apex culture

[0055] After the seeds were treated with low temperature, they were placed in an incubator for 2 weeks (14 days) at 25°C (8 hours) and 4°C (16 hours) and treated with 0.06wt.% gibberellin solution. The germinated seeds were sown in seedling trays with an appropriate amount of seedling matrix (the mass ratio of peat soil, coconut shell and perlite in the seedling matrix was 3:1:1), with 100 seeds sown in each seedling tray and marked accordingly.

[0056] (3) Pretreatment and fixation

[0057] At 9:00-11:00 in the morning on a sunny day, young root tips with a length of about 0.5 cm were taken and rinsed in distilled water. After rinsing, they were treated with a pretreatment solution (0.002 mol / L 8-hydroxyquinoline) for 3 hours at 25°C in the dark. After the treatment, the pretreatment solution was sucked out with a pipette, and a newly prepared Carnoy's fixative (the volume ratio of anhydrous ethanol and glacial acetic acid in Carnoy's fixative was 3:1) was added and fixed at 4°C for more than 5 hours to obtain a mixture.

[0058] (4) Enzymatic hydrolysis

[0059] Remove the Carnoy's fixative from the mixture obtained in step (3), add deionized water to wash 3 times, and then soak in deionized water for 30 minutes. After the material is soaked, the fixative is thoroughly cleaned. Remove the clean water, cut about 1mm of the active division part of the root tip with a blade, dry the water on filter paper, add about 20μL of enzyme solution (the volume percentage of cellulase in the enzyme solution is 3%, and the volume percentage of pectinase is 0.3%), ensure that the root tip material is immersed in the enzyme solution, and enzymolysis at 37℃ for 100min.

[0060] (5) Post-hypotonicity and re-fixation

[0061] After the enzymatic hydrolysis is completed, the enzyme solution is gently aspirated, and about 500 μL of clean water is added. After soaking for 10 min, the clean water is aspirated and 500 μL of Carnoy's fixative is added.

[0062] (6) Smear

[0063] After adding Carnoy's fixative, fix at 25℃ for another 30 minutes before smearing. When smearing, pour out the fixative, use the tip of tweezers to gently pick up the material and evenly apply it to the center of the slide, then quickly bake it over the flame of an alcohol lamp.

[0064] (7) Dyeing

[0065] Place the slide upside down on a white porcelain plate with a sleeper pad, and perform pre-staining (pre-staining) treatment before staining. The specific steps are: treat the material with pre-staining solution (0.067mol / L disodium hydrogen phosphate solution (pH 7.0)) for about 30 minutes. Pour out the pre-staining solution and then perform re-staining. Mix the mixed solution (0.067mol / L disodium hydrogen phosphate and 0.067mol / L potassium dihydrogen phosphate in a volume ratio of 2:1) with Giemsa staining solution in a volume ratio of 20:1 (pre-prepared and used), and the staining time is about 5-10 minutes.

[0066] (8) Microscopic examination

[0067] After the slides are dried, they are observed under an Olympus microscope. Find the cell division items under a low-power microscope, then switch to a high-power microscope for observation, make records, and store at -20°C for future use.

[0068] Example 2 Effect of different pretreatment conditions on microscopic examination of kiwifruit somatic cell chromosome preparation

[0069] A method for preparing chromosomes of somatic cell mitosis metaphase of Actinidia plant, comprising the following steps:

[0070] (1) Low-temperature treatment of seeds: Pick ripe kiwifruit and store them at room temperature. When the fruit is fully soft and ripe, peel it and put it into a fine yarn mesh bag to crush it. Wash it in water to remove impurities and grains. Spread the seeds and place them in a dry and ventilated place to dry in the shade. After drying, place them in a dry and cool place for later use. Mix the seeds and wet sand in a mass ratio of 1:15 and put them into a nylon bag. Store them in a 4℃ refrigerator for about 60 days. Turn the seed layer every 18 days, and keep the humidity of the sand at a level that "it can be squeezed into a ball by hand, but it will fall apart when you loosen it."

[0071] (2) Root apex culture

[0072] After the seeds were treated with low temperature, they were placed in an incubator for 2 weeks at 25°C (8h) and 4°C (16h) and treated with 0.06wt.% gibberellin solution. The germinated seeds were sown in seedling trays with an appropriate amount of seedling matrix (the mass ratio of peat soil, coconut shell and perlite in the seedling matrix was 3:1:1), 100 seeds were sown in each seedling tray, and corresponding marks were made.

[0073] (3) Pretreatment and fixation

[0074] At 9:00-11:00 in the morning on a sunny day, take the young and tender root tips of about 0.5 cm in length, rinse them in distilled water, and then treat them with pretreatment solution (0.002 mol / L 8-hydroxyquinoline or ice-water mixture) at 25°C in the dark, where the time of 8-hydroxyquinoline is 2h, 3h or 4h, and the time of ice-water mixture treatment is 8h, 16h or 24h. At the same time, a blank control is designed, that is, fixation is performed directly without pretreatment. After the treatment, the pretreatment solution is sucked out with a pipette, and the newly prepared Carnoy's fixative (the volume ratio of anhydrous ethanol and glacial acetic acid in Carnoy's fixative is 3:1) is added and fixed at 4°C for more than 5h to obtain a mixture.

[0075] (4) Enzymatic hydrolysis

[0076] Remove the Carnoy's fixative from the mixture obtained in step (3), add deionized water to wash 3 times, and then soak in deionized water for 30 minutes. After the material is soaked, the fixative is thoroughly cleaned. Remove the clean water, cut about 1mm of the active division part of the root tip with a blade, dry the water on filter paper, add about 20μL of enzyme solution (the volume percentage of cellulase in the enzyme solution is 3%, and the volume percentage of pectinase is 0.3%), ensure that the root tip material is immersed in the enzyme solution, and enzymolysis at 37℃ for 100min.

[0077] (5) Post-hypotonicity and re-fixation

[0078] After the enzymatic hydrolysis is completed, the enzyme solution is gently aspirated, and about 500 μL of clean water is added. After soaking for 10 min, the clean water is aspirated and 500 μL of Carnoy's fixative is added.

[0079] (6) Smear

[0080] After adding Carnoy's fixative, fix at 25℃ for another 30 minutes before smearing. When smearing, pour out the fixative, use the tip of tweezers to gently pick up the material and evenly apply it to the center of the slide, then quickly bake it over the flame of an alcohol lamp.

[0081] (7) Dyeing

[0082] Place the slide upside down on a white porcelain plate with a sleeper pad and perform pre-staining (pre-staining) treatment before staining. The specific steps are: treat the material with pre-staining solution (0.067 mol / L disodium hydrogen phosphate solution (pH 7.0)) for about 30 minutes. Pour out the pre-staining solution and then perform re-staining. Mix the mixed solution (0.067 mol / L disodium hydrogen phosphate and 0.067 mol / L potassium dihydrogen phosphate in a volume ratio of 2:1) with Giemsa staining solution in a volume ratio of 20:1 (prepared and used immediately), and the staining time is about 8 minutes.

[0083] (8) Microscopic examination

[0084] After the slides are dried, they are observed under an Olympus microscope. Find the cell division items under a low-power microscope, then switch to a high-power microscope for observation, make records, and store at -20°C for future use. Figure 1 As shown. The pretreatment link is an important link in the preparation of kiwifruit chromosomes. Therefore, in this example, 0.002mol / L 8-hydroxyquinoline (2h, 3h or 4h) and ice water mixture (8h, 16h or 24h) were used for pretreatment under dark conditions at 25°C, and a blank control was set up, that is, fixation was performed directly without pretreatment, and the effects of different pretreatment conditions on kiwifruit chromosome preparation were compared. The results showed that: after 0.002mol / L 8-hydroxyquinoline treatment for 2h, the overall mitotic phase was too small, and the chromosomes were easy to stick together ( Figure 1 a); 0.002mol / L 8-hydroxyquinoline treatment for 4h, fewer division phases, poor chromosome morphology, easy to overlap, difficult to count ( Figure 1 b); the kiwifruit chromosomes were very rarely divided after being treated with ice-water mixture for 8h, 16h and 24h ( Figure 1 eg), especially in the metaphase, it is difficult to observe chromosomes; the blank control (no pretreatment) is difficult to observe chromosomes ( Figure 1 h in the figure). A comprehensive analysis of the preparation effects under different pretreatment conditions revealed that the 0.5 cm slice cut when the kiwifruit root tip grew to 1.0 cm was treated with 0.002 mol / L 8-hydroxyquinoline for 3 h. The material had more mitotic phases and chromosomes were easy to observe. The chromosomes had better morphology and were evenly dispersed, which was convenient for subsequent chromosome counting and related research ( Figure 1 c and d).

[0085] Example 3 Effect of different enzymolysis times on the preparation of kiwifruit somatic cell chromosomes

[0086] A method for preparing chromosomes of somatic cell mitosis metaphase of Actinidia plant, comprising the following steps:

[0087] (1) Low-temperature treatment of seeds: Pick ripe kiwifruit and store them at room temperature. When the fruit is fully soft and ripe, peel it and put it into a fine yarn mesh bag to crush it. Wash it in water to remove impurities and grains. Spread the seeds and place them in a dry and ventilated place to dry in the shade. After drying, place them in a dry and cool place for later use. Mix the seeds and wet sand in a mass ratio of 1:10 and put them into a nylon bag. Store them in a 4℃ refrigerator for about 60 days. Turn the seed layer every 15 days, and keep the humidity of the sand at a level that "it can be squeezed into a ball by hand, but it will fall apart when you loosen it."

[0088] (2) Root apex culture

[0089] After the seeds were treated with low temperature, they were placed in an incubator for 2 weeks at 25°C (8h) and 4°C (16h) and treated with 0.06wt.% gibberellin solution. The germinated seeds were sown in seedling trays with an appropriate amount of seedling matrix (the mass ratio of peat soil, coconut shell and perlite in the seedling matrix was 3:1:1), 100 seeds were sown in each seedling tray, and corresponding marks were made.

[0090] (3) Pretreatment and fixation

[0091] At 9:00-11:00 in the morning on a sunny day, young root tips with a length of about 0.5 cm were taken and rinsed in distilled water. After rinsing, they were treated with a pretreatment solution (0.002 mol / L 8-hydroxyquinoline) for 3 hours at 25°C in the dark. After the treatment, the pretreatment solution was sucked out with a pipette, and a newly prepared Carnoy's fixative (the volume ratio of anhydrous ethanol and glacial acetic acid in Carnoy's fixative was 3:1) was added and fixed at 4°C for more than 5 hours to obtain a mixture.

[0092] (4) Enzymatic hydrolysis

[0093] Remove the Carnoy's fixative from the mixture obtained in step (3), add deionized water to wash 3 times, and then add deionized water to soak for 30 minutes. After the material is soaked, the fixative is thoroughly cleaned. Remove the clean water, cut about 1mm of the active root tip with a blade, absorb the water on the filter paper, and add about 20μL of enzyme solution (the volume percentage of cellulase in the enzyme solution is 3%, and the volume percentage of pectinase is 0.3%) to ensure that the root tip material is immersed in the enzyme solution, and enzymolysis is performed at 37°C. The enzymolysis time is set to 60min, 70min, 80min, 90min, 100min, 110min, 120min or 130min.

[0094] (5) Post-hypotonicity and re-fixation

[0095] After the enzymatic hydrolysis is completed, the enzyme solution is gently aspirated, and about 500 μL of clean water is added. After soaking for 10 min, the clean water is aspirated and 500 μL of Carnoy's fixative is added.

[0096] (6) Smear

[0097] After adding Carnoy's fixative, fix at 25℃ for another 30 minutes before smearing. When smearing, pour out the fixative, use the tip of tweezers to gently pick up the material and evenly apply it to the center of the slide, then quickly bake it over the flame of an alcohol lamp.

[0098] (7) Dyeing

[0099] Place the slide upside down on a white porcelain plate with a sleeper pad, and perform pre-staining (pre-staining) treatment before staining. The specific steps are: treat the material with pre-staining solution (0.067mol / L disodium hydrogen phosphate solution (pH 7.0)) for about 30 minutes. Pour out the pre-staining solution and then perform re-staining. Mix the mixed solution (0.067mol / L disodium hydrogen phosphate and 0.067mol / L potassium dihydrogen phosphate in a volume ratio of 2:1) with Giemsa staining solution in a volume ratio of 20:1 (prepared and used immediately), and the staining time is about 5 minutes.

[0100] (8) Microscopic examination

[0101] After the slides are dried, they are observed under an Olympus microscope. Find the cell division items under a low-power microscope, then switch to a high-power microscope for observation, make records, and store at -20°C for future use. Figure 2 As shown. Enzymatic hydrolysis is an important step in the wall removal hypotonic flame drying method. Therefore, this example explores the effect of different enzymatic hydrolysis times (60min, 70min, 80min, 90min, 100min, 110min, 120min and 130min) in step (4) on kiwifruit chromosome preparation. The results show that when the kiwifruit root tip is enzymatically hydrolyzed for 60min and 70min, the background is not clean due to insufficient enzymatic hydrolysis, and there are more impurities around ( Figure 2 a and b in the figure); at 80 and 90 min of enzymatic hydrolysis, some impurities were present due to insufficient enzymatic hydrolysis, and the chromosome morphology was poor ( Figure 2 c and d); at 110min and 120min of enzymatic hydrolysis, the chromosomes were dispersed due to excessive enzymatic hydrolysis and were difficult to count ( Figure 2 e and f); when the enzymatic hydrolysis lasted for 130 min, the root tip cells were easily broken and the chromosomes were easily lost due to excessive enzymatic hydrolysis ( Figure 2 g in the figure). Comprehensive analysis of the preparation effects under different enzymatic hydrolysis conditions showed that the root tip cell wall removal effect was the best when the enzymatic hydrolysis lasted for 100 min, with dispersed chromosomes, good morphology, and a clean background ( Figure 2 h and i).

[0102] Example 4 Application of kiwifruit somatic cell chromosome preparation

[0103] A method for preparing chromosomes of somatic cell mitosis metaphase of Actinidia plant, comprising the following steps:

[0104] (1) Low-temperature treatment of seeds: Pick ripe kiwifruit (varieties are shown in Table 1) and store them at room temperature. When the fruit is fully soft and ripe, peel it and put it into a fine yarn mesh bag to crush it. Wash it in water to remove impurities and grains. Spread the seeds and place them in a dry and ventilated place to dry in the shade. After drying, place them in a dry and cool place for later use. Mix the seeds and wet sand in a mass ratio of 1:20 and put them into a nylon bag. Put it in a 4℃ refrigerator and store it in the sand for about 60 days. Turn the seed layer every 20 days, and keep the humidity of the sand at the level of "kneading into a ball by hand, and loosening it when loosened".

[0105] Table 1 Test materials

[0106]

[0107] (2) Root apex culture

[0108] After the seeds were treated with low temperature, they were placed in an incubator for 2 weeks at 25°C (8h) and 4°C (16h) and treated with 0.06wt.% gibberellin solution. The germinated seeds were sown in seedling trays with an appropriate amount of seedling matrix (the mass ratio of peat soil, coconut shell and perlite in the seedling matrix was 3:1:1), 100 seeds were sown in each seedling tray, and corresponding marks were made.

[0109] (3) Pretreatment and fixation

[0110] At 9:00-11:00 in the morning on a sunny day, young root tips with a length of about 0.5 cm were taken and rinsed in distilled water. After rinsing, they were treated with a pretreatment solution (0.002 mol / L 8-hydroxyquinoline) for 3 hours at 25°C in the dark. After the treatment, the pretreatment solution was sucked out with a pipette, and a newly prepared Carnoy's fixative (the volume ratio of anhydrous ethanol and glacial acetic acid in Carnoy's fixative was 3:1) was added and fixed at 4°C for more than 5 hours to obtain a mixture.

[0111] (4) Enzymatic hydrolysis

[0112] Remove the Carnoy's fixative from the mixture obtained in step (3), add deionized water to wash 3 times, and then soak in deionized water for 30 minutes. After the material is soaked, the fixative is thoroughly cleaned. Remove the clean water, cut about 1mm of the active division part of the root tip with a blade, dry the water on filter paper, add about 20μL of enzyme solution (the volume percentage of cellulase in the enzyme solution is 3%, and the volume percentage of pectinase is 0.3%), ensure that the root tip material is immersed in the enzyme solution, and enzymolysis at 37℃ for 100min.

[0113] (5) Post-hypotonicity and re-fixation

[0114] After the enzymatic hydrolysis is completed, the enzyme solution is gently aspirated, and about 500 μL of clean water is added. After soaking for 10 min, the clean water is aspirated and 500 μL of Carnoy's fixative is added.

[0115] (6) Smear

[0116] After adding Carnoy's fixative, fix at 25℃ for another 30 minutes before smearing. When smearing, pour out the fixative, use the tip of tweezers to gently pick up the material and evenly apply it to the center of the slide, then quickly bake it over the flame of an alcohol lamp.

[0117] (7) Dyeing

[0118] Place the slide upside down on a white porcelain plate with a sleeper pad, and perform pre-staining (pre-staining) treatment before staining. The specific steps are: treat the material with pre-staining solution (0.067 mol / L disodium hydrogen phosphate solution (pH 7.0)) for about 30 minutes. Pour out the pre-staining solution and then perform re-staining. Mix the mixed solution (0.067 mol / L disodium hydrogen phosphate and 0.067 mol / L potassium dihydrogen phosphate in a volume ratio of 2:1) with Giemsa staining solution in a volume ratio of 20:1 (prepared and used immediately), and the staining time is about 10 minutes.

[0119] (8) Microscopic examination

[0120] After the slides were dried, they were observed under an Olympus microscope. The cell division items were found under a low-power microscope, and then switched to a high-power microscope for observation. Records were made and stored at -20°C for later use. The chromosome numbers and ploidy levels of different kiwifruits were determined at the same time. The results are shown in Table 2. The chromosome division items of the ten kiwifruit varieties during metaphase are as follows: Figure 3 shown.

[0121] Table 2 Chromosome ploidy of ten kiwifruit varieties

[0122] Serial number Material Name Ploidy a Jade 2n=4x=116 b Donghong 2n=2x=58 c Cuixiang 2n=4x=116 d Jinsh 2n=4x=116 e Hongyang 2n=2x=58 f Jin Yan 2n=4x=116 g Hairy flower 2n=2x=58 h Shaanxi Late Red 2n=2x=58 i Miliang No.1 2n=6x=174 g Noble 2n=6x=174

[0123] From Table 2 and Figure 3 It can be seen that the chromosome ploidy of Hongyang, Donghong, Shaanxi Wanhong and Hairy Flower kiwifruit are all diploid (2n=2x=58), the chromosome ploidy of Jinyan, Jinshi, Cuiyu and Cuixiang kiwifruit are all tetraploid (2n=4x=116), and the chromosome ploidy of Guichang and Miliang No. 1 kiwifruit are all hexaploid (2n=6x=174).

[0124] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a chromosome slice of somatic cell mitosis metaphase of an Actinidia plant, characterized in that: The following steps are involved: The seeds of Actinidia genus are subjected to low temperature treatment and variable temperature culture in sequence to obtain Actinidia genus plant seedlings; After taking the root tip of the Actinidia plant seedling, the root tip is sequentially subjected to pretreatment, fixation, enzymatic hydrolysis, post-infiltration, re-fixation, smearing and staining to obtain a preparation of the chromosomes of the somatic cell mitosis metaphase of the Actinidia plant; The pretreatment liquid used in the pretreatment is 0.002 mol / L 8-hydroxyquinoline solution; the pretreatment time is 3 hours; The enzyme preparation used in the enzymolysis is a mixed enzyme solution; the mixed enzyme solution includes cellulase and pectinase; the volume percentage of cellulase in the mixed enzyme solution is 3%, and the volume percentage of pectinase is 0.3%; The enzymatic hydrolysis time is 100 min and the temperature is 37°C.

2. The preparation method according to claim 1, characterized in that: The condition parameters of the low temperature treatment are: 4°C sand storage for 60 days; And / or, the variable temperature culture includes high temperature culture and low temperature culture; And / or, the variable temperature culture time is 14 days; And / or, one cycle consists of one high temperature culture and one low temperature culture, the high temperature culture time is 8 hours; the low temperature culture time is 16 hours; And / or, the temperature of the high temperature culture is 25°C; And / or, the temperature of the low-temperature culture is 4°C.

3. The preparation method according to claim 1, characterized in that: The length of the root tip is 0.5 cm.

4. The preparation method according to claim 1, characterized in that: The fixative used in the fixation is Carnoy's fixative; and / or, the Carnoy's fixative comprises anhydrous ethanol and glacial acetic acid; and / or, the volume ratio of anhydrous ethanol to glacial acetic acid in the Carnoy's fixative is 3:1; And / or, the fixed temperature is 4°C and the time is >5h.

5. The preparation method according to claim 1, characterized in that: The post-osmosis includes the step of adding clean water after the enzymatic hydrolysis is completed.

6. The preparation method according to claim 1, characterized in that: The re-fixation comprises the step of adding a fixative after the post-infiltration is completed; And / or, the fixative is Carnoy's fixative; the Carnoy's fixative comprises anhydrous ethanol and glacial acetic acid; and / or, the volume ratio of anhydrous ethanol to glacial acetic acid in the Carnoy's fixative is 3:1; And / or, the re-fixation temperature is 25° C. and the time is 30 min.

7. The preparation method according to claim 1, characterized in that: The staining includes pre-staining and counter-staining; The pre-dyeing liquid used in the pre-dyeing is a 0.067 mol / L disodium hydrogen phosphate solution; And / or, the pre-dyeing time is 30 min; And / or, the mixed staining solution used for the counterstaining includes a sodium hydrogen phosphate-potassium dihydrogen phosphate mixed solution and a Giemsa staining solution; and / or, the volume ratio of the sodium hydrogen phosphate-potassium dihydrogen phosphate mixed solution to the Giemsa staining solution is 20:1; And / or, the re-staining time is 5-10 min.

8. The preparation method according to claim 1, characterized in that: The Actinidia plants include any one or more of Cuiyu, Donghong, Cuixiang, Jinshi, Hongyang, Jinyan, Maohua, Shaanxi Wanhong, Miliang No. 1 and Guichang.

9. A preparation of mitotic metaphase chromosomes of somatic cells of Actinidia plants prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the preparation of somatic mitotic metaphase chromosomes of Actinidia plants according to claim 9 in chromosome genetic research of Actinidia plants.

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