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Method for storing potato viruses and viroid in host for long time at ultralow temperature

A potato virus, long-term preservation technology, used in microorganism-based methods, viruses, horticultural methods, etc.

Pending Publication Date: 2022-01-14
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the preservation of potato virus and endovirus by cryopreservation technology

Method used

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  • Method for storing potato viruses and viroid in host for long time at ultralow temperature
  • Method for storing potato viruses and viroid in host for long time at ultralow temperature
  • Method for storing potato viruses and viroid in host for long time at ultralow temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The cryopreservation of embodiment 1 with poisonous shoot tip

[0034] The experimental operation method is as follows:

[0035] (1) The tube plantlets of potato "Zihuabai" which are highly sensitive to PVS and PSTVd ​​were used as the experimental material, and the shoot tip was cut and transferred laterally into a disposable plastic petri dish containing MS, pH 5.8 medium.

[0036] (2) After sealing the culture dish, transfer it to the tissue culture room for culture for 1 week until the axillary buds germinate into 1 cm stem segments.

[0037] (3) Transfer the shoot tip in step (2) to a petri dish to harden the seedlings in a dark environment at 4°C for 3 weeks.

[0038] (4) Take 0.5-2.0 mm (carrying 5-6 leaf primordia) long shoot tips from the virus-carrying plants that have been hardened and pre-cultured.

[0039] (5) Pre-cultivation: This step is carried out on MS medium containing 0.3M sucrose and 8g / L agar, and treated at 4°C in the dark for at least 16h after...

Embodiment 2

[0045] The influence of embodiment 2 shoot tip size on pathogen storage rate

[0046] Operation steps are the same as embodiment 1, only change the shoot tip length used in the step (4). Take 0.5mm (2-3 leaf primordia) and 1.5mm (carry 5-6 leaf primordia) shoot tips respectively on hardened and pre-cultivated virus-carrying plants, by changing the size of the test shoot tips, study the effect of this variable on Effects on pathogen preservation and removal efficiency. Other parameters and experimental procedures were consistent with Example 1.

[0047] The measurement results are shown in Table 1:

[0048] Table 1 The effect of shoot tip size on the virus-free rate and virus preservation rate of the shoot tip cryopreservation of "Zihuabai" test-tube plantlets with PVS and PSTVd

[0049]

Embodiment 3

[0050] RT-PCR detection of plant pathogen carrying situation before and after cryopreservation in embodiment 3

[0051] A total of five RT-PCR tests were performed in this experiment. The first time is the detection of the poison source potato to the "Zihuabai" greenhouse virus transmission; the second time is to check the test tube seedlings with pathogens; the third time is to detect the third (9th week) plants of the regenerated subculture , as the result of the initial inspection, it is convenient for real time RT-qPCR virus quantitative detection; the fourth time is to detect the plants of the 6th (18th week) subculture after regeneration to obtain the preservation efficiency of the virus by ultra-low temperature freezing of the infected shoot tip; The fifth time is to detect the results of grafting and friction transmission to identify whether the regenerated pathogen has normal infection ability. The specific implementation method is as follows:

[0052] (1) RNA extra...

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Abstract

The invention discloses a method for storing potato viruses and viroid in a host for a long time at an ultralow temperature. According to the method, Zihuabai potato test-tube plantlets infected with potato virus S (PVS) and potato spindle viroid (PSTVd) as test materials, and virus stem tips are stored for a long time by using a droplet vitrification ultralow-temperature storage method. The method comprises the following steps: cutting the stem tips, culturing, hardening seedlings, taking the stem tips, pre-culturing, loading, carrying out vitrification treatment, preparing droplets, carrying out liquid nitrogen cryopreservation, unfreezing and unloading. The regeneration rate of the stem tips stored at the ultralow temperature is 52-60%, and the storage rates of the PVS and the PSTVd are 100%.

Description

technical field [0001] The invention relates to the technical field of low-temperature preservation, in particular to a method for long-term ultra-low temperature preservation of potato virus and viroid hosts. Background technique [0002] Plant viruses and viroids are cell obligate parasites, which can only replicate autonomously in host cells with vitality and use the raw materials in the host cells, and do not have the ability to survive without the host. [0003] The long-term preservation of viruses and viroids is of great significance and is the prerequisite for disease-resistant breeding of animals and plants. In the past few decades, the production of plant-based vaccines has provided new strategies and avenues for the prevention and treatment of human diseases. It has been reported that viruses have the potential to be applied in the pharmaceutical industry through nanotechnology, and the prerequisite for the wide application of viruses is the long-term preservatio...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01H4/00C12R1/94
CPCC12N7/00A01H4/008C12N2770/34051
Inventor 李经纬王乔春王敏锐张万萍徐秀红黄廷敏
Owner GUIZHOU UNIV
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