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Method for preparing high-quality rehmannia protoplast

A protoplast, high-quality technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, plant cells, etc., can solve the problems of low yield and poor quality of protoplasts, and achieve high content, regular morphology, well-structured effect

Pending Publication Date: 2022-05-27
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The invention discloses a method for preparing high-quality rehmannia root protoplasts, which effectively solves the problems of poor quality and low yield of protoplasts

Method used

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  • Method for preparing high-quality rehmannia protoplast
  • Method for preparing high-quality rehmannia protoplast
  • Method for preparing high-quality rehmannia protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] A method for preparing high-quality rehynomold protoplasts, including the following steps:

[0074] (1) Rehmannia leaf pretreatment:

[0075] Take the seedlings of the rehmannia sterile seedlings that have been cultivated for about 15 days, take about 0.5g of the leaves that have been fully unfolded, cut the main leaf veins, cut into thin strips of about 0.1cm × 2.0cm, and pay attention to holding the leaves gently when cutting the leaves to prevent damage to the leaves.

[0076] The cut ground yellow leaf strips are soaked in buffer, placed in an incubator, 25 °C, protected from light and let stand for 2 h ( Figure 1 A)；

[0077] Buffers include: MES 0.02 M, Mannitol 0.5 M, KCl 0.009 M, CaCl 2 0.005 M, pH 5.7-5.8, solvent is deionized water.

[0078] (2) Enzymatic digestion:

[0079]Clip the pretreated rehmannia leaf strips with forceps into a centrifuge tube filled with 10 mL enzymatic lysate, wrap in tin foil, and enzymatically lyse at 25 °C, 55 r / min for 4 h ( Figure 1 ...

Embodiment 2

[0093] Prepare the diaphragm protoplasts according to the method of Example 1, take step (3) centrifuge after centrifugation of the protoplasts added to buffer to 1 mL resuspending, and take the finally obtained isolated purified protoplasts, respectively, added dropwise on the blood cell counting plate, under a light microscope to observe the counting. After DAPI staining of protoplasts ( Figure 2), perform in vivo protoplast counts, and the results are shown in Table 2.

[0094] Table 2

[0095]

[0096] The resulting yield of high-quality geoxanthrophytes was (1.42±0.11) ×10 7 Quantity / mL (equivalent to 2.82×10 7 g -1 FW)。

Embodiment 3

[0098] Prepare the rehmannia protoplasts according to the method of Example 1, and set up a control group composed of different enzymatic solutions:

[0099] Table 3

[0100]

[0101] The results are as follows Figure 3 As shown, the protoplasts prepared in groups 1-6 have cell aggregation, incomplete enzymatic lysis, irregular morphology, low yield, many dead cells, etc., in contrast, the protoplasts prepared in Example 1 are uniformly dispersed, morphologically regular, structurally intact, and the living protoplast content is high ( Figure 4 )。

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Abstract

The invention discloses a method for preparing high-quality rehmannia protoplast, which comprises the following steps: (1) rehmannia leaf pretreatment: taking rehmannia seedling leaves, cutting the rehmannia seedling leaves into thin strips, and soaking the thin strips in a buffer solution in a dark place; (2) enzymolysis: transferring the pretreated rehmannia leaves into an enzymatic hydrolysate, and oscillating in a dark place for enzymolysis; (3) protoplast separation; (4) purifying the protoplast: resuspending the separated protoplast by using a buffer solution; adding the resuspended liquid to the upper layer of the sucrose salt solution, and centrifuging; after centrifugation, the middle layer is a protoplast band. The method is simple, the prepared protoplast is regular in shape, complete in structure, high in living protoplast content and good in quality, and a foundation is laid for research in the fields of heredity and molecular biology, such as genetic transformation, rapid propagation, somatic cell hybridization and breeding of good varieties by using the rehmannia protoplast as an experimental material.

Description

Technical field [0001] The present invention belongs to the field of protoplast preparation technology, specifically relates to a method of preparing high-quality lycopene protoplasts. Background [0002] Rehmannia glutinosa Libosch is a perennial herb of the family Metaphyllaceae, whose roots contain catalol, dixanthine, cardiac glycosides, polysaccharides and other bioactive substances, with a variety of effects such as nourishing yin and tonifying blood, clearing heat and vitality, and filling the pulp, and is widely used in clinical practice. [0003] Since rehmannia seeds are highly heterozygous, their hybrid offspring are prone to serious separation, and the vegetative propagation method using multi-generational roots in production is easy to cause virus infection, so the use of ex vivo culture and rapid propagation technology is the main way to obtain excellent varieties by genetic improvement of rehmannia. However, at present, the relatively mature exoplant culture regene...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206
Inventor 杨艳会杨慕荣董柯炜崔柳青李瑞芳张高阳伊艳杰谭曦王驰
Owner HENAN UNIVERSITY OF TECHNOLOGY
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