Sample treatment method for oat karyotype detection

Abstract

The invention relates to the technical field of biology, and particularly discloses a sample treatment method for oat karyotype detection, which comprises the following steps: S1, root tip pretreatment: taking root tip tissues after germination of oat seeds, and treating the root tip tissues in an N2O gas environment containing 0.05-0.1% colchicine for 2-3 hours, then transferring into a glacial acetic acid-ethanol mixed solution which is precooled to 4 DEG C and fixing for 15-20 minutes; s2, performing enzymolysis treatment: cutting the fixed root tip tissue to be 1-2mm, soaking the cut root tip tissue into enzymatic hydrolysate, and performing enzymolysis at 35-38 DEG C for 30-40 minutes; according to the method, the treatment time and pressure of the root tip tissue in the N2O gas environment containing 0.05%-0.1% of colchicine are controlled to be 0.5-0.8 MPa, and the treatment temperature is controlled to be 20-25 DEG C. According to the method, stagnation in the middle stage of cell division is effectively promoted, so that the dispersion effect of chromosomes is remarkably improved; the improved stationary liquid not only can effectively fix the chromosome morphology and prevent the chromosome morphology from changing in the subsequent treatment process, but also can significantly inhibit microbial contamination and prolong the preservation time of the sample.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, it is a method for sample processing for oat chromosome karyotype detection. Background Art

[0002] In plant genetics research, chromosome karyotype analysis is a crucial technique that helps to reveal information such as the genetic characteristics, evolutionary history, and genetic variation of species. As an important crop, the chromosome karyotype detection of oats is of great significance for variety identification, genetic improvement, and breeding work.

[0003] The main purpose of oat chromosome karyotype detection is to reveal the characteristics of oat chromosomes, including the number, morphology, size, centromere position, and satellites, etc. These characteristics together constitute the karyotype of oats. Through karyotype analysis, in-depth understanding of the genetic composition, evolutionary history, and genetic variation of oats can be achieved, providing important basis for oat genetic breeding, variety identification, and genetics research.

[0004] In traditional oat chromosome karyotype detection, the arrest effect at the metaphase of cell division is often not ideal, resulting in poor chromosome dispersion and affecting subsequent observation and analysis. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for sample processing for oat chromosome karyotype detection, so as to solve the problem that in traditional oat chromosome karyotype detection, the arrest effect at the metaphase of cell division is often not ideal, resulting in poor chromosome dispersion and affecting subsequent observation and analysis.

[0006] A method for sample processing for oat chromosome karyotype detection includes the following steps:

[0007] S1. Root tip pretreatment:

[0008] Take the root tip tissue after germination of oat seeds, place it in an N2O gas environment containing 0.05%-0.1% colchicine and treat for 2-3 hours, and then transfer it to an ice acetic acid-ethanol mixture pre-cooled to 4°C for fixation for 15-20 minutes;

[0009] S2. Enzymatic digestion treatment:

[0010] Cut 1-2 mm of the fixed root tip tissue and immerse it in the enzymatic digestion solution, and perform enzymatic digestion at 35-38°C for 30-40 minutes;

[0011] S3. Gradient dehydration:

[0012] After enzymatic digestion, perform gradient dehydration with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in sequence, and each treatment lasts for 3-5 minutes;

[0013] S4, Slide Preparation and Drying:

[0014] Place the dehydrated root tip tissue on a glass slide, add the modified fixative, gently press the cover slip after slightly heating with a flame, and leave it to dry at room temperature for 12 - 24 hours;

[0015] S5, Staining and Observation:

[0016] Stain with Giemsa stain for 10 - 15 minutes and observe the chromosome dispersion effect under a microscope.

[0017] Preferably, in S1, the environmental pressure of N2O gas is 0.5 - 0.8 MPa, and the treatment temperature is 20 - 25 °C.

[0018] Preferably, in S1, the volume ratio of the glacial acetic acid - ethanol mixture is 1:3.

[0019] Preferably, in S2, the enzyme digestion solution consists of 1 mL of citrate buffer, 0.03 - 0.05 g / mL of cellulase, 0.01 - 0.03 g / mL of pectinase, and 0.1% Triton X - 100.

[0020] Preferably, in S2, the pH of the citrate buffer is 4.8 - 5.2;

[0021] The preparation method of the citrate buffer is: add 0.5 g of trisodium citrate and 0.4 g of citric acid to every 50 mL of double - distilled water.

[0022] Preferably, in S4, the modified fixative is a methanol - glacial acetic acid mixture, the volume ratio of the methanol - glacial acetic acid mixture is 3:1, and the methanol - glacial acetic acid mixture contains 0.1% DTT.

[0023] Preferably, in S4, the modified fixative also contains 0.05% NaN3 to inhibit microbial contamination.

[0024] Preferably, in S5, before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 5 - 8 minutes to remove impurities on the chromosome surface.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] By controlling the treatment time and pressure of 0.5 - 0.8 MPa, and treatment temperature of 20 - 25 °C of the root tip tissue in an N2O gas environment containing 0.05% - 0.1% colchicine, the present invention effectively promotes the arrest at the metaphase of cell division, thus significantly improving the chromosome dispersion effect;

[0027] The improved fixative can not only effectively fix the chromosome morphology and prevent its changes during subsequent processing, but also significantly inhibit microbial contamination and extend the preservation time of the sample;

[0028] The gradient dehydration step ensures the sufficient dehydration of the root tip tissue before slide preparation, avoiding deformation and bubble generation during slide preparation;

[0029] Before staining, the slide is treated with proteinase K, effectively removing the impurities on the chromosome surface and improving the uniformity and clarity of staining;

[0030] The staining time and concentration of Giemsa stain are precisely controlled, ensuring the clear visualization and easy identification of chromosomes. Description of the Drawings

[0031] Figure 1 It is a schematic flow chart of the present invention;

[0032] Figure 2 It is the morphology and number of chromosomes in Example II of the present invention;

[0033] Figure 3 It is the chromosome karyotype analysis chart of chromosomes in Example II of the present invention;

[0034] Figure 4 It is the chromosome karyotype parameter table of chromosomes in Example II of the present invention. Detailed Embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] As Figure 1 shown:

[0037] The species name of the oat samples used in the following embodiments is "Qinghai 444";

[0038] Example 1:

[0039] Root tip pretreatment (S1):

[0040] Take 50 root tip tissues after germination of oat seeds.

[0041] Place them in an N2O gas environment containing 0.05% colchicine for 2 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20°C.

[0042] Subsequently, it was transferred to an acetic acid - ethanol mixture pre - cooled to 4°C and fixed for 15 minutes. The volume ratio of the acetic acid - ethanol mixture was 1:3.

[0043] Enzymatic digestion treatment (S2):

[0044] Cut 1 mm of the fixed root tip tissue.

[0045] Immerse it in the enzymatic digestion solution, which consists of 1 mL of citrate buffer (pH 4.8, prepared by adding 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double - distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X - 100.

[0046] Digest at 35°C for 30 minutes.

[0047] Gradient dehydration (S3):

[0048] After enzymatic digestion, dehydrate successively with double - distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradients, with each treatment for 3 minutes.

[0049] Slide preparation and drying (S4):

[0050] Place the dehydrated root tip tissue on a glass slide.

[0051] Drop the improved fixative (methanol - acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0052] Gently press the cover glass after slightly heating with a flame.

[0053] Leave it to dry at room temperature for 12 hours.

[0054] Staining and observation (S5):

[0055] Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0056] Stain with Giemsa stain for 10 minutes.

[0057] Observe the chromosome dispersion effect under a microscope.

[0058] Example 2:

[0059] Root tip pretreatment (S1):

[0060] Take 50 root tip tissues of germinated oat seeds.

[0061] Place them in an N2O gas environment containing 0.075% colchicine and treat for 2.5 hours. The pressure of the N2O gas environment is 0.65 MPa, and the treatment temperature is 22.5°C.

[0062] Subsequently, it was transferred to an acetic acid - ethanol mixture pre - cooled to 4°C for fixation for 17.5 minutes. The volume ratio of the acetic acid - ethanol mixture was 1:3.

[0063] Enzymatic digestion treatment (S2):

[0064] Cut 1.5 mm of the fixed root tip tissue.

[0065] Immerse it in the enzymatic digestion solution, which consists of 1 mL of citrate buffer (pH 5.0, preparation method: add 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double - distilled water), 0.04 g / mL of cellulase, 0.02 g / mL of pectinase, and 0.1% Triton X - 100.

[0066] Perform enzymatic digestion at 36.5°C for 35 minutes.

[0067] Gradient dehydration (S3):

[0068] After enzymatic digestion, perform gradient dehydration with double - distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in sequence, with each treatment lasting for 4 minutes.

[0069] Mounting and drying (S4):

[0070] Place the dehydrated root tip tissue on a glass slide.

[0071] Drop the improved fixing solution (methanol - acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0072] Gently press the cover glass after slightly heating with a flame.

[0073] Let it stand at room temperature for drying for 18 hours.

[0074] Staining and observation (S5):

[0075] Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 6.5 minutes to remove impurities on the chromosome surface.

[0076] Stain with Giemsa stain for 12.5 minutes.

[0077] The effect diagram of chromosome dispersion observed under the microscope is as Figure 2 shown; the karyotype analysis diagram of the chromosome is as Figure 3 shown; the karyotype parameter table of the chromosome is as Figure 4 shown.

[0078] Example 3:

[0079] Root tip pretreatment (S1):

[0080] Take 50 root tip tissues after germination of oat seeds.

[0081] Place them in an N2O gas environment containing 0.1% colchicine and treat for 3 hours. The pressure of the N2O gas environment is 0.8 MPa, and the treatment temperature is 25 °C.

[0082] Subsequently, transfer them to an ice acetic acid-ethanol mixture pre-cooled to 4 °C and fix for 20 minutes. The volume ratio of the ice acetic acid-ethanol mixture is 1:3.

[0083] Enzymatic digestion treatment (S2):

[0084] Cut 2 mm of the fixed root tip tissue.

[0085] Immerse it in the enzymatic digestion solution. The enzymatic digestion solution consists of 1 mL of citrate buffer (pH 5.2, preparation method: add 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double-distilled water), 0.05 g / mL of cellulase, 0.03 g / mL of pectinase, and 0.1% Triton X-100.

[0086] Digest at 38 °C for 40 minutes.

[0087] Gradient dehydration (S3):

[0088] After enzymatic digestion, dehydrate successively with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradients, and treat each level for 5 minutes.

[0089] Slide preparation and drying (S4):

[0090] Place the dehydrated root tip tissue on a glass slide.

[0091] Drop the improved fixing solution (methanol-ice acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0092] Gently press the cover glass after slightly heating with a flame.

[0093] Let it stand at room temperature and dry for 24 hours.

[0094] Staining and observation (S5):

[0095] Before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 8 minutes to remove impurities on the chromosome surface.

[0096] Stain with Giemsa stain for 15 minutes.

[0097] Observe the chromosome dispersion effect under a microscope.

[0098] Example 4: Compare the cell wall decomposition efficiency with / without Triton X-100

[0099] This example aims to explore the role of Triton X-100 in the enzymatic hydrolysate. By comparing the decomposition efficiency of the enzymatic hydrolysate with and without Triton X-100 on the cell wall of oat root tip tissue, the necessity of Triton X-100 in this sample treatment method is further verified.

[0100] Experimental materials and methods:

[0101] Root tip pretreatment (S1):

[0102] Take the root tip tissue after germination of oat seeds, and the treatment conditions are the same as those in Example 2.

[0103] Enzymatic hydrolysis treatment (S2, two groups with and without Triton X-100):

[0104] Group containing Triton X-100: The composition of the enzymatic hydrolysate is the same as that in Example 2, that is, it contains 1 mL of citrate buffer (pH 5.0), 0.04 g / mL of cellulase, 0.02 g / mL of pectinase and 0.1% Triton X-100.

[0105] Group without Triton X-100: Remove Triton X-100 from the enzymatic hydrolysate, and the other components are the same as those in Example 2.

[0106] Cut 1.5 mm of the fixed root tip tissue from both groups and immerse them in the corresponding enzymatic hydrolysate respectively, and perform enzymatic hydrolysis at 36.5 °C for 35 minutes.

[0107] Gradient dehydration (S3):

[0108] After enzymatic hydrolysis, the root tip tissues of both groups are sequentially dehydrated with double-distilled water, 70% ethanol, 95% ethanol and absolute ethanol in gradients, and each treatment lasts for 4 minutes.

[0109] Section preparation and drying (S4):

[0110] Place the dehydrated root tip tissues of both groups on glass slides respectively, and drop the same improved fixing solution (a mixed solution of methanol and glacial acetic acid with a volume ratio of 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0111] Gently press the cover glass after slightly heating with a flame, and leave it to dry at room temperature for 18 hours.

[0112] Observation and comparison:

[0113] Observe the decomposition of the cell wall of the root tip tissues of both groups under the microscope, and record and compare indicators such as the integrity of the cell wall, the degree of chromosome dispersion and the background clarity.

[0114] The experimental table is as follows:

[0115]

[0116] Note:

[0117] The column of "Cell wall decomposition" is used to record the degree of cell wall decomposition. For example, "Completely decomposed" means the cell wall is completely decomposed, while "Incompletely decomposed" means there is still some cell wall residue.

[0118] The column of "Chromosome dispersion degree" is used to describe the dispersion state of chromosomes. For example, "Good" means the chromosomes are evenly dispersed and easy to observe, while "Uneven" means the chromosomes are unevenly distributed and there is an aggregation phenomenon.

[0119] The column of "Background clarity" is used to evaluate the clarity of the background during observation. For example, "Clear" means the background is clean and there is no interference, while "Blurry" means there are impurities or a blurry phenomenon in the background.

[0120] Experimental results and analysis:

[0121] The group containing TritonX-100: The cell wall is decomposed more thoroughly, the chromosomes are dispersed well, the background is clear, and it is easy to observe.

[0122] The group without TritonX-100: The cell wall is incompletely decomposed, and there is still cell wall residue in some areas, resulting in uneven chromosome dispersion, a relatively blurry background, and greater difficulty in observation.

[0123] Example 5: Orthogonal experiment of N2O pressure and treatment time

[0124] Experimental purpose

[0125] Through orthogonal experimental design, explore the optimal parameter combination for the chromosome dispersion effect of oat root tips within the range of 0.5 - 0.8 MPa N2O gas pressure and 2 - 3 hours of treatment time, and verify the optimization effect of the pretreatment steps.

[0126] Experimental design

[0127] Experimental factors and levels

[0128] Factor 1: N2O pressure levels: 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa

[0129] Factor 2: Treatment time levels: 2 hours, 2.5 hours, 3 hours

[0130] Orthogonal table selection: L 12 (4×3 full-factor design), with a total of 12 groups of experiments, and each group is repeated 3 times.

[0131] Experimental grouping

[0132]

[0133]

[0134] Experimental procedures

[0135] Sample preparation (S1)

[0136] Select oat seeds of the same batch and germinate them until the root tip length reaches 1.5 - 2.0 cm.

[0137] Take 30 root tips for each group and evenly divide them into 3 replicate groups (10 root tips for each group).

[0138] N2O pretreatment (S2)

[0139] Place the root tips in a closed pressure vessel, introduce N2O gas to the target pressure (0.5 - 0.8 MPa), and keep the temperature constant at 25°C.

[0140] Treat according to the set time (2 / 2.5 / 3 hours).

[0141] Fixation and subsequent processing (S3 - S5)

[0142] After pretreatment, immediately transfer to precooled (4°C) glacial acetic acid - ethanol fixative (1:3) and fix for 20 minutes.

[0143] Subsequent steps are carried out normally:

[0144] Enzymolysis treatment: Cut 1 - 2 mm of the root tip meristem and immerse it in the enzymolysis solution (1 mL of citrate buffer, 0.03 - 0.05 g / mL cellulase, 0.01 - 0.03 g / mL pectinase, 0.1% Triton X - 100), and enzymolyze at 35 - 38°C for 30 - 40 minutes.

[0145] Gradient dehydration: Dehydrate with double - distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradient, 3 - 5 minutes for each step.

[0146] Section preparation and drying: Drop the modified fixative (methanol: glacial acetic acid = 3:1, containing 0.1% DTT, 0.05% NaN3), slightly heat with a flame and dry at room temperature for 12 - 24 hours.

[0147] Staining and observation: Pretreat with proteinase K (10 μg / mL) for 5 - 8 minutes, stain with Giemsa stain for 10 - 15 minutes, and observe under a microscope.

[0148] Evaluation indicators

[0149] Chromosome dispersion index (DI)

[0150] Randomly select 50 metaphase cells under the microscope and count the proportion of chromosomes with good dispersion (no overlap, clear morphology).

[0151] Formula:

[0152] Chromosome damage rate

[0153] Statistically analyze the proportion of chromosomes with breaks, adhesions or deformations.

[0154] Proportion of analyzable cells

[0155] Proportion of cells available for effective karyotype analysis (≥80% chromosome dispersion).

[0156] Data analysis and results

[0157] Main effect analysis

[0158] N2O pressure:

[0159] The DI of the 0.7 - 0.8 MPa group was significantly higher than that of the 0.5 - 0.6 MPa group, but the chromosome damage rate increased (>8%) at 0.8 MPa.

[0160] Treatment time:

[0161] The DI was highest after 3 hours of treatment (average 85%), but there was no significant difference between 2.5 hours and 3 hours.

[0162] Interaction analysis

[0163] 0.7 MPa × 2.5 hours: DI = 88.5%, damage rate = 4.2%, which is the optimal balance point.

[0164] 0.8 MPa × 3 hours: DI = 89.1%, but the damage rate reached 9.7%, which is not recommended.

[0165] Experimental conclusion

[0166] Verified by orthogonal experiments, the reasonable range of N2O pretreatment parameters (0.5 - 0.8 MPa, 2 - 3 hours) can be further optimized to a pressure of 0.6 - 0.7 MPa and a treatment time of 2.5 - 3 hours. This combination can significantly reduce the damage rate (≤5%) while ensuring the chromosome dispersion effect (DI≥85%), and is suitable for efficient karyotype analysis of hexaploid oats.

[0167] Example 6:

[0168] Experimental design: Verification of the protective effect of DTT on the structural integrity of chromosomes

[0169] Experimental purpose

[0170] Verify the protective effect of DTT (dithiothreitol) on the morphological integrity of chromosomes by observing the chromosomal structural differences of oats treated with modified fixative containing / without DTT through scanning electron microscopy (SEM).

[0171] Experimental grouping

[0172]

[0173] Note: For both groups of experiments, the pretreatment, enzymatic hydrolysis and dehydration parameters of Example 2 were used (N2O pressure 0.65 MPa, treatment time 2.5 hours), and only the fixative composition was different.

[0174] Experimental procedures

[0175] Sample preparation

[0176] Treat the oat root tips according to the method of Example 2 until the dehydration step (S3).

[0177] Divide the dehydrated root tips into two groups, and prepare slides using fixatives containing DTT (experimental group) and without DTT (control group) respectively.

[0178] Scanning electron microscope sample treatment

[0179] Critical point drying: Place the glass slide in a critical point dryer, replace ethanol with liquid CO2, and dry to a constant humidity.

[0180] Gold sputtering treatment: Use an ion sputtering instrument (model: JEOL JFC-1600) to sputter a 5 nm thick gold film to enhance conductivity.

[0181] SEM observation and data collection

[0182] Use a scanning electron microscope (model: Hitachi SU8010) to observe the surface morphology of chromosomes at an acceleration voltage of 10 kV.

[0183] Randomly select 20 well-dispersed chromosomes from each group and measure the following parameters:

[0184] Chromosome length (μm)

[0185] Chromosome width (μm)

[0186] Surface smoothness (scored on a scale of 0-5, 0 = severely wrinkled, 5 = smooth and flat)

[0187] Breakage rate (number of broken chromosomes / total number of observations × 100%)

[0188] Experimental results (table)

[0189]

[0190] Note: Data are expressed as mean ± standard deviation, n = 60 (20 chromosomes per group × 3 replicates).

[0191] Analysis of key results

[0192] Chromosome morphological integrity

[0193] Experimental group: The chromosome length and width were significantly greater than those of the control group, indicating that DTT maintains the chromosome in a stretched state by reducing disulfide bonds and reduces the contraction effect caused by glacial acetic acid.

[0194] Control group: The chromosome surface was significantly shrunken (smoothness score 2.1), and the length was shortened by approximately 18.5%.

[0195] Difference in breakage rate

[0196] The breakage rate in the DTT group was only 6.7%, significantly lower than 23.5% in the control group, proving that DTT can reduce mechanical damage to chromosomes during fixation.

[0197] Comparison of surface structure

[0198] SEM images showed that the edges of the chromosomes in the experimental group were clear and the chromatin fibers were arranged tightly; obvious cracks and fragments were visible on the surface of the control group.

[0199] Experimental conclusion

[0200] The improved fixing solution with 0.1% DTT can significantly improve the structural integrity of chromosomes, specifically manifested as: the chromosome length and width increase, being closer to the true physiological morphology;

[0201] The surface smoothness increased by 60% and the breakage rate decreased by 71.5%. This result verifies the technical necessity of DTT, which provides a high-precision sample basis for karyotype analysis by maintaining the stability of the chromosome ultrastructure.

[0202] Example 7: Root tip pretreatment (S1): Take 50 root tip tissues after germination of oat seeds. Place them in an N2O gas environment containing 0.065% colchicine and treat for 2.25 hours. The pressure of the N2O gas environment is 0.58 MPa and the treatment temperature is 21.5°C. Then transfer them to a pre-cooled glacial acetic acid-ethanol mixture at 4°C and fix for 16 minutes. The volume ratio of the glacial acetic acid-ethanol mixture is 1:3.

[0203] Enzymatic digestion treatment (S2): Cut 1.25 mm of the fixed root tip tissue. Immerse it in the enzymatic digestion solution, which consists of 1 mL of citrate buffer (pH 4.9, preparation method same as before), 0.035 g / mL cellulase, 0.015 g / mL pectinase, and 0.1% Triton X-100. Digest at 35.5°C for 32.5 minutes.

[0204] Gradient dehydration (S3): After enzymatic digestion, dehydrate successively with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradients, with each treatment for 3.5 minutes.

[0205] Slide preparation and drying (S4): Place the dehydrated root tip tissues on a glass slide. Dropwise add the modified fixative (a methanol - glacial acetic acid mixture with a volume ratio of 3:1, containing 0.1% DTT and also 0.05% NaN3). Gently press the cover slip after slightly heating with a flame. Leave it to dry at room temperature for 14 hours.

[0206] Staining and observation (S5): Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 5.8 minutes. Stain with Giemsa stain for 11 minutes. Observe the chromosome dispersion effect under a microscope.

[0207] Example 8: Root tip pretreatment (S1): Take 50 root tip tissues after germination of oat seeds. Place them in an N2O gas environment containing 0.085% colchicine for 2.8 hours. The pressure of the N2O gas environment is 0.72 MPa, and the treatment temperature is 23°C. Subsequently, transfer them to an ice acetic acid - ethanol mixture pre - cooled to 4°C for fixation for 18 minutes. The volume ratio of the ice acetic acid - ethanol mixture is 1:3.

[0208] Enzymatic digestion treatment (S2): Cut 1.75 mm of the fixed root tip tissues. Immerse them in the enzymatic digestion solution, which consists of 1 mL of citrate buffer (pH 5.1, prepared as described above), 0.045 g / mL cellulase, 0.025 g / mL pectinase, and 0.1% Triton X - 100. Digest at 37°C for 37.5 minutes.

[0209] Gradient dehydration (S3): After enzymatic digestion, dehydrate successively with double - distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in a gradient, with each treatment lasting 4.5 minutes.

[0210] Slide preparation and drying (S4): Place the dehydrated root tip tissues on a glass slide. Dropwise add the modified fixative (a methanol - glacial acetic acid mixture with a volume ratio of 3:1, containing 0.1% DTT and also 0.05% NaN3). Gently press the cover slip after slightly heating with a flame. Leave it to dry at room temperature for 20 hours.

[0211] Staining and observation (S5): Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 7.2 minutes. Stain with Giemsa stain for 13.5 minutes. Observe the chromosome dispersion effect under a microscope.

[0212] Example 9:

[0213] Root tip pretreatment (S1):

[0214] Take 50 root tip tissues after germination of oat seeds.

[0215] Place them in an N2O gas environment containing 0.06% colchicine for 2.2 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20°C.

[0216] Subsequently, it was transferred to an acetic acid - ethanol mixture pre - cooled to 4°C for fixation for 15 minutes. The volume ratio of the acetic acid - ethanol mixture was 1:3.

[0217] Enzymatic digestion treatment (S2):

[0218] Cut 1 mm of the fixed root tip tissue.

[0219] Immerse it in the enzymatic digestion solution, which consists of 1 mL of citrate buffer (pH 4.8, preparation method: add 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double - distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X - 100.

[0220] Digest at 35°C for 30 minutes.

[0221] Gradient dehydration (S3):

[0222] After enzymatic digestion, dehydrate successively with double - distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradients, with each treatment for 3 minutes.

[0223] Slide preparation and drying (S4):

[0224] Place the dehydrated root tip tissue on a glass slide.

[0225] Drop the improved fixing solution (methanol - acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0226] Gently press the cover slip after slightly heating with a flame.

[0227] Let it stand at room temperature for drying for 12 hours.

[0228] Staining and observation (S5):

[0229] Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0230] Stain with Giemsa stain for 10 minutes.

[0231] Observe the chromosome dispersion effect under a microscope.

[0232] Example ten:

[0233] Root tip pretreatment (S1):

[0234] Take 50 root tip tissues of germinated oat seeds.

[0235] Treat in an N2O gas environment containing 0.065% colchicine for 2.2 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20 °C.

[0236] Subsequently, transfer to a pre-cooled glacial acetic acid-ethanol mixture at 4 °C for fixation for 15 minutes. The volume ratio of the glacial acetic acid-ethanol mixture is 1:3.

[0237] Enzymatic digestion treatment (S2):

[0238] Cut 1 mm of the fixed root tip tissue.

[0239] Immerse in the enzymatic digestion solution. The enzymatic digestion solution consists of 1 mL of citric acid buffer (pH 4.8, preparation method: add 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double-distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X-100.

[0240] Perform enzymatic digestion at 35 °C for 30 minutes.

[0241] Gradient dehydration (S3):

[0242] After enzymatic digestion, perform gradient dehydration with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in sequence, with each treatment for 3 minutes.

[0243] Slide preparation and drying (S4):

[0244] Place the dehydrated root tip tissue on a glass slide.

[0245] Drop the improved fixing solution (methanol-glacial acetic acid mixture, volume ratio 3:1, containing 0.1% DTT, and also containing 0.05% NaN3).

[0246] Gently press the cover glass after slightly heating with a flame.

[0247] Let it stand at room temperature for drying for 12 hours.

[0248] Staining and observation (S5):

[0249] Before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0250] Stain with Giemsa stain solution for 10 minutes.

[0251] Observe the chromosome dispersion effect under a microscope.

[0252] Example XI:

[0253] Root tip pretreatment (S1):

[0254] Take 50 root tip tissues of germinated oat seeds.

[0255] Treat in an N2O gas environment containing 0.07% colchicine for 2.3 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20 °C.

[0256] Subsequently, transfer to a pre-cooled glacial acetic acid-ethanol mixture at 4 °C for fixation for 15 minutes. The volume ratio of the glacial acetic acid-ethanol mixture is 1:3.

[0257] Enzymatic digestion treatment (S2):

[0258] Cut 1 mm of the fixed root tip tissue.

[0259] Immerse it in the enzymatic digestion solution. The enzymatic digestion solution consists of 1 mL of citrate buffer (pH 4.8, prepared by adding 0.5 g of trisodium citrate and 0.4 g of citric acid to every 50 mL of double-distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X-100.

[0260] Digest enzymatically at 35 °C for 30 minutes.

[0261] Gradient dehydration (S3):

[0262] After enzymatic digestion, perform gradient dehydration with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in sequence, with each treatment for 3 minutes.

[0263] Slide preparation and drying (S4):

[0264] Place the dehydrated root tip tissue on a glass slide.

[0265] Dropwise add the modified fixative (methanol-glacial acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0266] Gently press the cover glass after slightly heating with a flame.

[0267] Let it stand at room temperature to dry for 12 hours.

[0268] Staining and observation (S5):

[0269] Before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0270] Stain with Giemsa stain solution for 10 minutes.

[0271] Observe the chromosome dispersion effect under a microscope.

[0272] Example XII:

[0273] Root tip pretreatment (S1):

[0274] Take 50 root tip tissues after germination of oat seeds.

[0275] Place them in an N2O gas environment containing 0.07% colchicine and treat for 2.5 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20 °C.

[0276] Subsequently, transfer them to an ice acetic acid - ethanol mixture pre-cooled to 4 °C and fix for 15 minutes. The volume ratio of the ice acetic acid - ethanol mixture is 1:3.

[0277] Enzymatic digestion treatment (S2):

[0278] Cut 1 mm of the fixed root tip tissue.

[0279] Immerse it in the enzymatic digestion solution. The enzymatic digestion solution consists of 1 mL of citrate buffer (pH 4.8, prepared by adding 0.5 g of trisodium citrate and 0.4 g of citric acid to 50 mL of double-distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X-100.

[0280] Digest at 35 °C for 30 minutes.

[0281] Gradient dehydration (S3):

[0282] After enzymatic digestion, dehydrate successively with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradients, with each treatment for 3 minutes.

[0283] Slide preparation and drying (S4):

[0284] Place the dehydrated root tip tissue on a glass slide.

[0285] Drop the modified fixative (methanol - ice acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0286] Gently press the cover slip after slightly heating with a flame.

[0287] Let it stand at room temperature and dry for 12 hours.

[0288] Staining and observation (S5):

[0289] Before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0290] Stain with Giemsa stain solution for 10 minutes.

[0291] Observe the chromosome dispersion effect under a microscope.

[0292] Example XII:

[0293] Root tip pretreatment (S1):

[0294] Take 50 root tip tissues of germinated oat seeds.

[0295] Place them in an N2O gas environment containing 0.08% colchicine for 3 hours. The pressure of the N2O gas environment is 0.5 MPa, and the treatment temperature is 20 °C.

[0296] Subsequently, transfer them to a pre-cooled acetic acid-ethanol mixture at 4 °C for fixation for 15 minutes. The volume ratio of the acetic acid-ethanol mixture is 1:3.

[0297] Enzymatic digestion treatment (S2):

[0298] Cut 1 mm of the fixed root tip tissue.

[0299] Immerse it in the enzymatic digestion solution. The enzymatic digestion solution consists of 1 mL of citrate buffer (pH 4.8, prepared by adding 0.5 g of trisodium citrate and 0.4 g of citric acid to every 50 mL of double-distilled water), 0.03 g / mL of cellulase, 0.01 g / mL of pectinase, and 0.1% Triton X-100.

[0300] Digest at 35 °C for 30 minutes.

[0301] Gradient dehydration (S3):

[0302] After enzymatic digestion, dehydrate successively with double-distilled water, 70% ethanol, 95% ethanol, and absolute ethanol in gradient, with each treatment for 3 minutes.

[0303] Slide preparation and drying (S4):

[0304] Place the dehydrated root tip tissue on a glass slide.

[0305] Drop the improved fixing solution (methanol-acetic acid mixture, volume ratio 3:1, containing 0.1% DTT and also containing 0.05% NaN3).

[0306] Gently press the cover glass after slightly heating with a flame.

[0307] Let it stand at room temperature for drying for 12 hours.

[0308] Staining and observation (S5):

[0309] Before staining, treat the slide with proteinase K at a concentration of 10 μg / mL for 5 minutes to remove impurities on the chromosome surface.

[0310] Stain with Giemsa stain for 10 minutes.

[0311] Observe the chromosome dispersion effect under a microscope.

[0312] All the standard parts used in the present invention can be purchased from the market. The special-shaped parts can be customized according to the descriptions in the specification and the attached drawings. The specific connection methods of each part all adopt the conventional means such as bolts, rivets, and welding that are mature in the prior art. The machinery, parts, and equipment all adopt the conventional models in the prior art. In addition, the circuit connection adopts the conventional connection method in the prior art, which will not be elaborated here. The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0313] In the description of the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. The meaning of "a plurality" is two or more, unless otherwise specifically defined.

[0314] In the present invention, unless otherwise clearly defined and limited, the terms such as "mounted", "connected", "connected to", and "fixed" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal connection of two components or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0315] In the present invention, unless otherwise clearly defined and limited, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over", and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "below", and "beneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0316] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0317] In the drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved, and other structures can refer to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0318] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A sample processing method for detecting the karyotype of oat chromosomes, characterized in that, It includes the following steps: S1. Root tip pretreatment: Take the root tip tissue after the germination of oat seeds, place it in an N2O gas environment containing 0.05%-0.1% colchicine and treat for 2-3 hours, then transfer it to an ice acetic acid-ethanol mixture pre-cooled to 4°C and fix for 15-20 minutes; S2. Enzymolysis treatment: Cut 1-2 mm of the fixed root tip tissue, immerse it in the enzymolysis solution, and enzymolyze at 35-38°C for 30-40 minutes; S3. Gradient dehydration: After enzymolysis, dehydrate successively with double-distilled water, 70% ethanol, 95% ethanol and absolute ethanol in gradients, and treat each level for 3-5 minutes; S4. Slide preparation and drying: Place the dehydrated root tip tissue on a glass slide, drop the modified fixing solution, slightly heat it with a flame and gently press the cover glass, and leave it to dry at room temperature for 12-24 hours; S5. Staining and observation: Stain with Giemsa stain for 10-15 minutes and observe the chromosome dispersion effect under a microscope.

2. The sample processing method for oat chromosome karyotype detection according to claim 1, wherein, In S1, the pressure of the N2O gas environment is 0.5-0.8 MPa and the treatment temperature is 20-25°C.

3. The sample processing method for oat chromosome karyotype detection according to claim 2, wherein, In S1, the volume ratio of the ice acetic acid-ethanol mixture is 1:

3.

4. The sample processing method for oat chromosome karyotype detection according to claim 1, wherein In S2, the enzymolysis solution consists of 1 mL of citrate buffer, 0.03-0.05 g / mL of cellulase, 0.01-0.03 g / mL of pectinase and 0.1% Triton X-100.

5. The sample processing method for oat chromosome karyotype detection according to claim 4, wherein In S2, the pH of the citrate buffer is 4.8-5.2; The preparation method of the citrate buffer is: add 0.5 g of trisodium citrate and 0.4 g of citric acid to every 50 mL of double-distilled water.

6. The sample processing method for oat chromosome karyotype detection according to claim 1, characterized in that, In S4, the modified fixing solution is a methanol-ice acetic acid mixture, the volume ratio of the methanol-ice acetic acid mixture is 3:1, and the methanol-ice acetic acid mixture contains 0.1% DTT.

7. The sample processing method for oat chromosome karyotype detection according to claim 6, wherein In S4, the modified fixing solution also contains 0.05% NaN3 for inhibiting microbial contamination.

8. The sample processing method for oat chromosome karyotype detection according to claim 1, wherein, In S5, before staining, treat the glass slide with proteinase K at a concentration of 10 μg / mL for 5-8 minutes to remove impurities on the chromosome surface.

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