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Method for determining preservation regeneration rate of plant cells

A plant cell and regeneration rate technology, which is applied to the preservation of human or animal bodies, measuring devices, instruments, etc., can solve the problems of complex operation process and inability to optimize ultra-low temperature solutions, and achieve the effect of extended functions

Inactive Publication Date: 2020-08-07
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation process of this method is complicated, and it takes more than 2 weeks for plants to resume cultivation after cryopreservation before judging whether the cryopreservation method is successful, so the cryopreservation program cannot be optimized in time

Method used

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  • Method for determining preservation regeneration rate of plant cells
  • Method for determining preservation regeneration rate of plant cells
  • Method for determining preservation regeneration rate of plant cells

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preparation example Construction

[0030] In the present invention, the method for preparing plant embryogenic callus described in step (1) preferably includes inducing and culturing the zygotic embryos of plant seeds in an induction medium for 2 weeks; the induction medium uses WPM as a basic medium , also includes the following components: 2,4D 1mg / L, polyvinylpyrrolidone (PVP) 1g / L, casein (ch) 1g / L, activated carbon (AC) 1g / L, sucrose ( SUC) and agar with a mass volume percentage of 0.3%. In the present invention, the source of the zygotic embryo of the plant seed is not particularly limited, and the callus is preferably induced from the zygotic embryo of the mature seed. The induction culture in the present invention is preferably dark culture, and the temperature of the dark culture is preferably 25°C. The preparation method of the induction medium is not particularly limited in the present invention. After mixing the components, sterilize at 121° C. and adjust the pH to 5.8.

[0031] When performing th...

Embodiment 1

[0041] 1. The cryopreservation process of Magnolia officinalis embryogenic cell line:

[0042] Magnolia officinalis embryogenic callus induced by mature seed zygotic embryo; in ultra-clean bench, in sterilized 1.5ml centrifuge tube, add 1ml WPM (containing 3% sucrose, pH 5.8) liquid culture At this time, the scale is level with the lowest point of the 1ml meniscus. Use tweezers to pick the magnolia magnolia embryogenic callus in good growth state, transfer it to a 1.5ml centrifuge tube until the lowest point of the meniscus is equal to the scale of 1.25ml, and measure the magnolia embryogenic callus with a volume of 0.25ml Tissue: After loading treatment, vitrification treatment, cryopreservation, thawing in 40°C water bath, unloading treatment, washing and recovery culture.

[0043] 2. The most accurate time point for the determination of the survival rate of magnolia bark embryogenic callus after cryopreservation was determined by using the laser confocal scanning microscop...

Embodiment 2

[0046] The optimal vitrification solution (PVS2) treatment time was determined by detecting the ratio of dead cells and living cells of Magnolia officinalis embryogenic cells by laser confocal scanning microscope.

[0047] FDA can be combined with living cells and is excited to emit green fluorescence at 488nm, and the stronger the fluorescence, the stronger the cell viability. Propidium iodide (PI) can bind to dead cells and is excited at 545nm to emit red fluorescence. Using the characteristics of these two fluorescent dyes, FDA (10 μg / mL) was used to label live cells, and PI (10 μg / mL) was used to label dead cells.

[0048] Measure 2 batches of Magnolia officinalis embryogenic callus that volume is 0.25ml simultaneously, carry out following operation simultaneously: through loading treatment, vitrification treatment time 0 minutes, 5 minutes, 10 minutes, 30 minutes, 50 minutes, 70 minutes, 90 minutes Minute time gradient, cryopreservation, thawing in 40°C water bath, unloa...

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Abstract

The invention provides a method for rapidly and efficiently determining the regeneration rate of plant cells after ultralow-temperature preservation, and relates to the technical field of cell survival rate detection, and the method can rapidly judge the advantages and disadvantages of an ultralow-temperature preservation scheme and determine the optimal treatment time and intensity; according tothe invention, fluorescence detection can be carried out through the laser confocal scanning microscope, so that the function of the laser confocal scanning microscope is effectively expanded, theoretical support is provided for designing a plant ultralow-temperature preservation scheme, and the method has important significance for developing a new generation of ultralow-temperature preservationtechnology. In the embodiment of the invention, the ratio of the dead cells to the living cells detected by the laser confocal scanning microscope is consistent with the ultralow-temperature regeneration rate of the embryonic cells, so that the ratio of the dead cells to the living cells can be detected by using the laser confocal scanning microscope, and an optimal ultralow-temperature preservation scheme is determined in advance.

Description

technical field [0001] The invention belongs to the technical field of cell viability detection, and in particular relates to a method for measuring the preservation regeneration rate of plant cells. Background technique [0002] Cryopreservation generally refers to the technology and method of using liquid nitrogen (-196°C) or liquid nitrogen vapor to preserve biological materials. At liquid nitrogen temperature, all biochemical reactions in cells are temporarily stopped, allowing cells to be theoretically preserved indefinitely. Cryopreservation technology is widely used in the long-term preservation of germplasm resources of microorganisms, animals and plants. Compared with other existing in-situ protected area preservation and ex-situ preservation, the ultra-low temperature preservation technology of plants has permanent preservation, is not affected by changes in the natural environment, has high preservation efficiency, and is convenient for management. Compared with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64A01N1/02
CPCA01N1/0221A01N1/0226G01N21/6402G01N21/6486G01N2021/6419
Inventor 贾艳霞林亮马俊超李唯奇
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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