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Determination method of activity of stem tip cells of plant

A determination method and a plant shoot tip technology are applied in the field of determination of plant shoot tip cell viability, can solve problems such as shoot tip damage, survival rate error, difficulty in obtaining, etc., and achieve the effects of simple procedure, high efficiency and easy operation.

Inactive Publication Date: 2015-07-22
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems when applied to plant shoot tips. On the one hand, there are many factors that affect TTC viability dyeing, such as TTC concentration, pH value, temperature, time, experimental materials, treatment methods, etc., and it is difficult to obtain standard TTC dyeing conditions; on the other hand The shoot tip, especially after cryopreservation treatment, was damaged to varying degrees, and some cells survived. When staining for TTC activity, different researchers have different understandings of the range and degree of red staining of TTC, which may lead to the staining of TTC activity in the shoot tip. There is a large error between the results and the actual survival rate of the shoot tip after recovery culture. In the actual staining and detection of viability in the past, the TTC viability staining result can only roughly represent the shoot tip viability value, which leads to the accuracy of TTC detection of the shoot tip after cryopreservation. poor

Method used

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  • Determination method of activity of stem tip cells of plant
  • Determination method of activity of stem tip cells of plant
  • Determination method of activity of stem tip cells of plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 The relationship between different dyeing temperatures and times and the vitality dyeing rate of fresh shoot tips

[0020] 1. Experimental materials

[0021] The variety used in the experiment was a sterile test-tube seedling of the potato variety "Huzi 83-213" preserved in the test-tube seedling bank of the National Germplasm Bank of the Chinese Academy of Agricultural Sciences.

[0022] 2. Experimental method and specific steps

[0023] — Obtaining potato shoot tips: select potato tube plantlets of subculture 30 days, and use a binocular dissecting microscope to peel off shoot tips with a size of about 1-2 mm under sterile conditions. The test-tube plantlets were subcultured on MS medium (pH=5.8) supplemented with 30g / L sucrose and 8g / L agar. The culture temperature is 18±1°C, and the light is 16h / d.

[0024] —TTC vitality staining: fresh healthy shoot tips were placed in 0.5% TTC-0.1M phosphate buffer (pH=7.0) for staining at 30°C, 40°C, and 50°C for 1, ...

Embodiment 2

[0030] Example 2 Rapid Determination of TTC Viability of Potato Stem Tips in Ultra-low Temperature Storage

[0031] 1. Experimental materials

[0032] The variety used in the experiment was a sterile test-tube seedling of the potato variety "Huzi 83-213" preserved in the test-tube seedling bank of the National Germplasm Bank of the Chinese Academy of Agricultural Sciences.

[0033] 2. Experimental method and specific steps

[0034] — Obtaining potato shoot tips: select potato tube seedlings that have been subcultured for about 30 days, and use a binocular dissecting microscope to peel off shoot tips with a size of about 1-2 mm under sterile conditions. The test-tube plantlets were subcultured on MS medium (pH=5.8) supplemented with 30g / L sucrose and 8g / L agar. The culture temperature is 18±1°C, and the light is 16h / d.

[0035] —Small drop vitrification cryopreservation: the shoot tips were pre-cultured in MS liquid medium containing 0.3mol / L and 0.5mol / L sucrose successivel...

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Abstract

The invention relates to a determination method of activity of stem tip cells of a plant. The method comprises the step of contacting a stem tip of the plant with a TTC (triphenyltetrazolium chloride) solution with a concentration of 0.45-0.55wt%. The activity of the stem tip cells of the plant can be quickly judged by using the method. The method is simple in procedure, easy to operate and high in efficiency and is an efficient path for quick determination of germplasm activity, especially the activity after ultralow temperature storage.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for measuring the vitality of plant shoot tip cells. Background technique [0002] Cryopreservation, that is, storing materials such as plant cells, tissues, or organs in steam or liquid nitrogen below -150°C, is considered to be a safe, economical, and effective method suitable for long-term preservation of vegetatively propagated crops. The detection of survival and regeneration after cryopreservation mainly adopts recovery culture and viability staining. It usually takes a long time to restore the culture, and it takes about half a month, one month or more under the conditions of suitable medium, light and temperature. Vitality staining, such as triphenyltetrazolium chloride (TTC) viability staining method, generally only takes 1-5 hours, or 10-30 hours. The principle of TTC method to measure cell viability is: triphenyltetrazolium chloride (TTC) is a redox pigment with...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 张金梅陈晓玲张海晶卢新雄辛霞尹广鹍何娟娟
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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