阅读说明：本技术 一种白花鬼针草染色体常规制片的制作方法 (Method for preparing bidens alba chromosome conventional slide ) 是由 沈伟 岑湘涛 于 2021-07-07 设计创作，主要内容包括：本发明涉及一种白花鬼针草染色体常规制片的制作方法,具体包括以下步骤：根尖预处理培养；染色体制片；预处理；解离：取上述经预处理的根尖,在常温下用乙醇：38％HCl＝1:2分别进行解离,解离时间都为15分钟；染色：选取上述解离的根尖用不同染色剂1％结晶紫染色5分钟；观察拍照：用普通光学显微镜进行观察,对观察到的染色进行拍照。本发明优点在于：可获得细胞分散程度好,染色体收缩程度良好,染色体形态易辨别,提供了一种操作步骤简单又效率高的制片方法,此方法可用于白花鬼针草染色体数目的统计和观察。(The invention relates to a method for preparing a bidens alba chromosome conventional slide, which specifically comprises the following steps: pretreating and culturing root tips; preparing a chromosome; pre-treating; dissociation: taking the pretreated root tip, and treating the root tip with ethanol at normal temperature: dissociation was carried out with 38% HCl at 1:2 for 15 min; dyeing: selecting the dissociated root tips, and dyeing the root tips for 5 minutes by using 1% crystal violet of different dyeing agents; and (4) observing and photographing: the observation was carried out with a general optical microscope, and the observed staining was photographed. The invention has the advantages that: the method has the advantages of good cell dispersion degree, good chromosome contraction degree and easy chromosome morphology discrimination, and provides a flaking method with simple operation steps and high efficiency.)

1. A manufacturing method of a bidens alba chromosome conventional slide, which is characterized by comprising the following steps:

1) pre-treating and culturing root tips: firstly soaking the bidens alba seeds in clean water for twenty minutes, cleaning yellow substances outside the seed coats in the soaking process, selecting the bidens alba seeds with full particles by using tweezers, putting the bidens alba seeds in culture dishes paved with qualitative filter paper, putting the seeds on the qualitative filter paper at intervals, putting about 25 full bidens alba seeds in each culture dish, putting a proper amount of clean water in the culture dishes, paving a layer of qualitative filter paper on the seeds, putting a small amount of clean water, finally putting the culture dishes in a 28 ℃ culture box for culturing for 2-3 days, and adopting a root tip in vitro method to carry out root tip pretreatment when the root tips grow to 0.25 cm;

2) chromosome flaking: washing the pretreated root tip with distilled water for 3 times, soaking in distilled water for ten minutes, transferring to dissociation solution for dissociation, washing with distilled water for 3 times, and soaking in distilled water for 10 minutes; dyeing by using a dyeing agent for 5 minutes, sucking the dyeing solution by using absorbent paper after dyeing, then carrying out color separation and tabletting by using 45% acetic acid, sucking the dyeing solution by using the absorbent paper after color separation is finished, then starting tabletting, cutting off the extension area part of the root tip by using an operating knife, leaving the meristem area part, and then dripping a drop of clear water on the glass slide; then covering a cover glass, placing a piece of filter paper on the glass slide, lightly beating the filter paper by using tweezers to uniformly disperse the chromosome, placing the pressed glass slide under a common optical microscope for microscopic examination and observing the chromosome, finding out clear metaphase mitotic phase cells and taking a picture;

3) pretreatment: uniformly taking materials of the bidens parviflora root tips at 8:30 in the morning, selecting the root tips which germinate to 0.25cm and are milky without root hairs, putting the root tips into a centrifugal tube containing a pretreatment reagent, and respectively soaking the root tips in 0.002 mol/L8-hydroxyquinoline for 4 hours for pretreatment under the dark condition at 25 ℃;

4) dissociation: taking the pretreated root tip, and treating the root tip with ethanol at normal temperature: dissociation was carried out with 38% HCl at 1:2 for 15 min;

5) dyeing: selecting the dissociated root tips, and dyeing the root tips for 5 minutes by using 1% crystal violet of different dyeing agents;

6) and (4) observing and photographing: the observation was carried out with a general optical microscope, and the observed staining was photographed.

Technical Field

The invention relates to a method for preparing a bidens alba chromosome conventional slide.

Background

Bidens alba, plant of Bidens of Compositae of Campanulaceae of Bidens, can be used as medicine. The dry medicinal materials are identified to be strip-shaped. The stem is blunt and quadrangular. The lower lobe 3 or not; the middle leaf has a handle, three leaves and 3 small leaves, the shape of an ellipse or an oval is oval, the tip is sharp, the base part is nearly round or is in a wide wedge shape, the shape is asymmetric, and the edge has sawteeth. How to provide a manufacturing method for the conventional preparation of the bidens parviflora chromosome, which has simple operation steps and high efficiency, becomes a research direction.

Disclosure of Invention

The invention aims to provide a method for preparing a conventional preparation of bidens alba chromosomes, which aims to solve the problems in the background technology.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a manufacturing method of a conventional slide preparation of bidens alba chromosomes specifically comprises the following steps:

1) pre-treating and culturing root tips: firstly soaking the bidens alba seeds in clean water for twenty minutes, cleaning yellow substances outside the seed coats in the soaking process, selecting the bidens alba seeds with full particles by using tweezers, putting the bidens alba seeds in culture dishes paved with qualitative filter paper, putting the seeds on the qualitative filter paper at intervals, putting about 25 full bidens alba seeds in each culture dish, putting a proper amount of clean water in the culture dishes, paving a layer of qualitative filter paper on the seeds, putting a small amount of clean water, finally putting the culture dishes in a 28 ℃ culture box for culturing for 2-3 days, and adopting a root tip in vitro method to carry out root tip pretreatment when the root tips grow to 0.25 cm;

2) chromosome flaking: washing the pretreated root tip with distilled water for 3 times, soaking in distilled water for ten minutes, transferring to dissociation solution for dissociation, washing with distilled water for 3 times, and soaking in distilled water for 10 minutes; dyeing by using a dyeing agent for 5 minutes, sucking the dyeing solution by using absorbent paper after dyeing, then carrying out color separation and tabletting by using 45% acetic acid, sucking the dyeing solution by using the absorbent paper after color separation is finished, then starting tabletting, cutting off the extension area part of the root tip by using an operating knife, leaving the meristem area part, and then dripping a drop of clear water on the glass slide; then covering a cover glass, placing a piece of filter paper on the glass slide, lightly beating the filter paper by using tweezers to uniformly disperse the chromosome, placing the pressed glass slide under a common optical microscope for microscopic examination and observing the chromosome, finding out clear metaphase mitotic phase cells and taking a picture;

3) pretreatment: uniformly taking materials of the bidens parviflora root tips at 8:30 in the morning, selecting the root tips which germinate to 0.25cm and are milky without root hairs, putting the root tips into a centrifugal tube containing a pretreatment reagent, and respectively soaking the root tips in 0.002 mol/L8-hydroxyquinoline for 4 hours for pretreatment under the dark condition at 25 ℃;

4) dissociation: taking the pretreated root tip, and treating the root tip with ethanol at normal temperature: dissociation was carried out with 38% HCl at 1:2 for 15 min;

5) dyeing: selecting the dissociated root tips, and dyeing the root tips for 5 minutes by using 1% crystal violet of different dyeing agents;

6) and (4) observing and photographing: the observation was carried out with a general optical microscope, and the observed staining was photographed.

The invention has the advantages that: the method has the advantages of good cell dispersion degree, good chromosome contraction degree and easy chromosome morphology discrimination, and provides a flaking method with simple operation steps and high efficiency.

Detailed Description

The invention is illustrated below by means of specific examples, without being restricted thereto.

Examples

A manufacturing method of a conventional slide preparation of bidens alba chromosomes specifically comprises the following steps:

1) pre-treating and culturing root tips: firstly soaking the bidens alba seeds in clean water for twenty minutes, cleaning yellow substances outside the seed coats in the soaking process, selecting the bidens alba seeds with full particles by using tweezers, putting the bidens alba seeds in culture dishes paved with qualitative filter paper, putting the seeds on the qualitative filter paper at intervals, putting about 25 full bidens alba seeds in each culture dish, putting a proper amount of clean water in the culture dishes, paving a layer of qualitative filter paper on the seeds, putting a small amount of clean water, finally putting the culture dishes in a 28 ℃ culture box for culturing for 2-3 days, and adopting a root tip in vitro method to carry out root tip pretreatment when the root tips grow to 0.25 cm;

2) chromosome flaking: washing the pretreated root tip with distilled water for 3 times, soaking in distilled water for ten minutes, transferring to dissociation solution for dissociation, washing with distilled water for 3 times, and soaking in distilled water for 10 minutes; dyeing by using a dyeing agent for 5 minutes, sucking the dyeing solution by using absorbent paper after dyeing, then carrying out color separation and tabletting by using 45% acetic acid, sucking the dyeing solution by using the absorbent paper after color separation is finished, then starting tabletting, cutting off the extension area part of the root tip by using an operating knife, leaving the meristem area part, and then dripping a drop of clear water on the glass slide; then covering a cover glass, placing a piece of filter paper on the glass slide, lightly beating the filter paper by using tweezers to uniformly disperse the chromosome, placing the pressed glass slide under a common optical microscope for microscopic examination and observing the chromosome, finding out clear metaphase mitotic phase cells and taking a picture;

3) pretreatment: uniformly taking materials of the bidens parviflora root tips at 8:30 in the morning, selecting the root tips which germinate to 0.25cm and are milky without root hairs, putting the root tips into a centrifugal tube containing a pretreatment reagent, and respectively soaking the root tips in 0.002 mol/L8-hydroxyquinoline for 4 hours for pretreatment under the dark condition at 25 ℃;

4) dissociation: taking the pretreated root tip, and treating the root tip with ethanol at normal temperature: dissociation was carried out with 38% HCl at 1:2 for 15 min;

5) dyeing: selecting the dissociated root tips, and dyeing the root tips for 5 minutes by using 1% crystal violet of different dyeing agents;

6) and (4) observing and photographing: the observation was carried out with a general optical microscope, and the observed staining was photographed.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.