A kind of extraction method of Lycoris plant protoplast

A technology of Lycoris genus and protoplast, which is applied in the direction of plant cells and the like, and achieves the effect of simple and easy operation and high repeatability of the extraction method.

Active Publication Date: 2021-02-26
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Separation and purification of Lycoris protoplasts, and their development and utilization will lay the foundation for the subsequent use and improvement of germplasm through bioengineering. It has broad application prospects. Domestic and foreign research has not seen the separation and purification of protoplasts from different species of Lycoris genus. Reports on methods and applications

Method used

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  • A kind of extraction method of Lycoris plant protoplast
  • A kind of extraction method of Lycoris plant protoplast
  • A kind of extraction method of Lycoris plant protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Lycoris bulb protoplast isolation and application

[0041] (1) Select Lycoris seedlings with a diameter of 0.5 cm (1-year-old seedlings produced by Lycoris cuttings and propagation), darken for 10 hours, cut off the bulbs of the seedlings, weigh 5 g of bulbs, and cut the bulbs into thin strips with a width of about 1 mm;

[0042] (2) Inject 5ml of Reagent A to treat the fragmented material obtained in step (1), carry out enzymatic hydrolysis in the dark, at 22°C, shake slowly on the shaker for 8 hours, the shaker speed is 50rpm, and remove the tip after the enzymolysis is completed. Gently pipette the mixed solution with the pipette tip to fully release the protoplasts;

[0043] The reagent A composition is shown in Table 4

[0044] Table 4 Example 1 Reagent A component

[0045]

[0046] (3) Filter the protoplast solution obtained in step (2) with a 50 μm filter, collect the filtrate, and centrifuge at 50 g for 5 min at 4° C. The precipitate was repea...

example 1

[0047] The composition of reagent B described in example 1 is shown in Table 5, and the composition of reagent C is shown in Table 6

[0048] Table 5 Reagent B components

[0049]

[0050] Table 6 Reagent C components

[0051]

[0052] Gently pipette the protoplasts obtained in step (3) with a pipette tip with the tip removed to make them evenly distributed. Take a clean hemocytometer, use a pipette to absorb 10 μl of protoplast body fluid to fill the counting area, repeat counting 5 times, and take the average value. According to the weight 5g that adds in the step (1), calculate the protoplast cell number that every gram of bulb obtains 2.88 * 10 7 pcs / g.

[0053] Analysis of galantamine content in Lycoris bulbs:

[0054] Take 1ml of Lycoris bulb protoplasts prepared above as the sample to be tested, add 2mL of 70% ethanol, ultrasonically extract twice, each time for 28min, and then centrifuge at 12000rpm for 10min, take the supernatant, blow it with nitrogen and c...

Embodiment 2

[0057] Example 2 Isolation and Application of Lycoris Leaf Protoplasts

[0058] (1) Select Lycoris seedlings with a diameter of 1.0 cm (1-year-old seedlings produced by cuttings and propagation of Lycoris lycoris), treat them in the dark for 12 hours, cut off the leaves of the seedlings, weigh 10 g of leaves, and cut the leaves into pieces about 1 mm wide;

[0059] (2) Inject 10ml of Reagent A to treat the fragments obtained in step (1), carry out enzymatic hydrolysis in the dark, at a temperature of 28°C, shake slowly on a shaker for 5 hours, the speed of the shaker is 50rpm, and remove the tip after the enzymolysis is completed. Gently pipette the mixed solution with the pipette tip to fully release the protoplasts;

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Abstract

The invention provides a method for extracting protoplasts of Lycoris plants, the method comprising the following steps: (1) treating Lycoris plant seedlings in the dark for 10-12 hours, taking young leaves or bulbs and chopping them; (2) using a reagent A process the fragment material obtained in step (1), and slowly shake it on a shaking table to release protoplasts; (3) filter the protoplast solution obtained in step (2) with a 40-50 μm filter screen, and centrifuge the filtrate for 8-10min , wash the precipitate with Reagent B, centrifuge repeatedly, and finally use Reagent C to suspend the protoplasts. The extraction method of Lycoris plant protoplasts of the present invention has a large number of protoplasts and good integrity, can be directly used for the detection of Lycoris alkaloids galantamine, and can be used for protoplast fusion and protoplast transformation Provide important materials.

Description

technical field [0001] The invention relates to the technical field of plant protoplasts, in particular to a method for extracting plant protoplasts of the genus Lycoris. Background technique [0002] Lycoris spp. is a bulbous herb of the family Amaryllidaceae (Amaryllidaceae). Many species in the genus Lycoris are unique ornamental and medicinal flowers in China. High medicinal value. Narciscin has anti-tumor activity, and lycorine can block the cell cycle and induce apoptosis in the cancer cell line HL-60. Galantamine and Ricolamine, as acetylcholinesterase inhibitors, can affect the brain's nicotinic receptors and inhibit acetylcholinesterase activity, and are the first-choice drugs for the clinical treatment of Alzheimer's disease (Alzheimer's disease, early symptoms of Alzheimer's disease) one. So far, wild Amaryllis resources are still the most important source of medicinal galantamine, but the slow growth of the plant itself and the plundering of resources have red...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 李青竹蔡友铭张永春杨柳燕许俊旭王桢周琳
Owner SHANGHAI ACAD OF AGRI SCI
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