Plant chromosome karyotyping

Summary

Various organisms in nature have relative stability in the form, structure and number of their chromosomes. The complete set of chromosomes contained in the gametes of diploid organisms is called a chromosome set. The number of chromosomes in this group, their morphological features, the length of the two arms of the chromosome, the location of the attachment point, and the presence or absence of the secondary constriction, follower, etc. constitute the chromosome group type, also called karyotype, of the species. Chromosome histotyping, or karyotyping, is the process of measuring, comparatively analyzing, pairing, grouping, and arranging chromosomes by measuring, comparatively analyzing, pairing, grouping, and arranging chromosome specimens and their photographs, as well as performing morphological analysis. Chromosome karyotyping can provide an important basis for cytogenetic categorization, inter-species relatedness, and the study of chromosome number and structural variation.

Operation method

Plant chromosome karyotyping

Materials and Instruments

Material:
Broad bean (Vicia faba, 2n = 12) seeds.
Apparatus:
Microscope (with camera unit), thermostatic incubator, water bath, alcohol lamp, scissors, shackles, dissecting needles, slides, coverslips, filter paper, film rolls, transparent rulers, glue, etc.
Reagents:
① colchicine
② 8-hydroxy chirp solution
③ hematoxylin
① Colchicine ② 8-hydroxy chirp solution ③ Hematoxylin ④ Trichloroacetaldehyde hydrate
⑤ Potassium iron iso
⑥ Anhydrous ethanol
⑦ Glacial acetic acid
⑧ 1 mol/L hydrochloric acid
⑨ 50% Mitochondria propionate
⑩ Anhydrous sodium sulfite
⑪ Hydroquinone
⑫ Anhydrous sodium carbonate
⑪ Potassium apothecary
⑭ Sodium thiosulfate
⑮ Boric acid
⑯ 28% acetic acid

Move

The basic process of plant chromosome karyotyping can be divided into the following steps:

(i) Material preparation

Root tip: Select healthy and dry broad bean seeds, put them in a beaker and soak them in water overnight at room temperature. After the seeds absorbed water and swelled, they were placed in a constant temperature incubator at 20 ℃ for moisturizing and cultivation. When the roots were 1~2 cm long, the root tips were cut off and set aside.

(ii) Pretreatment

In order to obtain more cells in the mid-cell division phase and at the same time shorten and disperse the chromosomes, the root tips can be pretreated as follows.

(1) 0.05%~0.2% aqueous solution of colchicine was treated for 2~4 h. The root tips were then treated with 0.002 mol of colchicine.

(2) 0.002 mol/L 8-hydroxy wow solution was treated for 3~4 h. The root tips were then treated with 0.002 mol/L 8-hydroxy wow solution.

(3) Root tips were immersed in distilled water and treated at low temperature for 24 h at 1-4 ℃.

(iii) Fixation

Root tips were washed with distilled water and fixed in Carnosol (glacial acetic acid: anhydrous ethanol = 1:3) fixative for 30~60 min at room temperature, then soaked in 90% ethanol→80% ethanol→70% ethanol (each for 30 min) for conversion, and then placed in 70% ethanol in a refrigerator at 4 ℃ for final storage.

(iv) Dissociation

The fixed materials were rinsed with distilled water for two times, and then put into a preheated 1 mol/L hydrochloric acid solution at 60 ℃ for about 5 min. Remove the hydrochloric acid, rinse with distilled water for 3 times, and soak the material in distilled water for 2 h. The material was then soaked in distilled water for 2 hours.

(v) Staining solution and preparation

1. Staining solution

The formula of propionic acid-ferric potassium isothymol iso iso iso iso iso iso iso iso iso iso iso as follows.

Storage group: Liquid A: 2 g of hematoxylin dissolved in 100 mL of 50% propionic acid; Liquid B: 0.5 g of ferricyanide dissolved in 100 mL of 50% propionic acid.

Staining solution: Liquid A and Liquid B are mixed in equal amounts, add 2 g of hydrated trichloroacetaldehyde to every 5 mL of Liquid A and Liquid B, dissolve thoroughly and shake well, store for use after Id.

2. Preparation

Select the dissociated root tip, put it on a clean slide, add 1 drop of the above staining solution and stain for about 20 min, cover the slide. Tap the coverslip with the eraser tip of a pencil to make the root tip cells evenly dispersed.

(VI) Microscopic examination

Place the prepared slides under a microscope and look for cells with dividing phases.

(vii) Measurement and comparison of chromosome photographs

1. Measurement

Select cells in the middle stage of mitosis, with well-dispersed chromosomes, no overlapping, complete number, clear morphology, and each chromosome in the same plane for measurement.

2. Micrographing, rinsing and magnification

Selected cells that meet the requirements under the microscope are micrographically photographed, then developed, and the negative with clear image is selected, enlarged, and washed to produce a clear photograph of the chromosome morphology. At the same time of microphotography, the microscope stage micrometer is photographed with the same magnification, enlarged, and then the magnification is calculated according to the actual length on the photograph.

3. Measurement and calculation

Accurately measure the total length of each chromosome and the length of the two arms of each chromosome on the enlarged photograph (to the middle of the node).

The following indicators are commonly used for chromosome morphology analysis.

Absolute chromosome length (μm) = [magnified chromosome length (mm) / magnification] X 1000

Relative chromosome length (%) = [Absolute chromosome length (μm) / Total chromosome length (μm)] X 100% Arm ratio = Long arm (μm) / Short arm (μm)

Stromal index = short arm (μm) / length of the chromosome (μm)

4. Cut and Paste and Pairing

Each chromosome on the enlarged photograph was cut out, and homologous chromosomes were paired based on visual inspection and the relative length of the chromosomes, arm ratio, position of the mitotic index, presence and position of the secondary constriction marks, and presence and morphological size of the follower.

5. Arrangement and Pasting

Arrange the paired chromosomes in order from the largest to the smallest. When arranging the chromosomes, make sure that the mitotic grains of each pair of chromosomes are arranged in a straight line, and that the short arm is on top and the long arm is on the bottom. For chromosomes of equal length, place the chromosome with the longer short arm in front. With the somatic chromosomes lined up last, the sex chromosomes and extra chromosomes are lined up separately.

The arranged homologous chromosomes are pasted on the drawing paper in the order of chromosome numbering, with care being taken that the mitoses are in the same straight line when pasting.

6. Classification

Determine the morphological type of chromosome based on the location of the mitotic site. The arm ratio reflects the position of the mitotic site on the chromosome (Table 29-1). The term sat is used to represent chromosomes with followers, which may or may not be included in the calculation of chromosome length, but need to be noted.

Table 29-1 Chromosome Morphology Types

Arm Ratio	Chromosome type	Symbol
1.0	Median mitotic chromosome	M
1.0~1.7	Middle mitotic chromosome	m
1.7~3.0	Near-middle mitotic chromosome	sm
3.0~7.0	Proximal filament chromosome	st
7.0 or more	Telomeric chromosome	t

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