Preparation and conversion method of soybean protoplasts

A protoplast, soybean technology, applied in the field of plant genetic engineering, to achieve accurate transformation, improve the transformation rate, and solve the effects of difficult transformation

Pending Publication Date: 2019-09-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing method for preparing soybean protoplasts is to prepare the protoplasts of soybean young cotyledons with the enzymolysis solution of 1% cellulase and 0.1% pectinase, and the yield is 1×10 6 individual / g, while the transformation efficiency of soybean protoplasts using PEG is only 0.27%-0.4%

Method used

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  • Preparation and conversion method of soybean protoplasts
  • Preparation and conversion method of soybean protoplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Induction of soybean embryogenic callus and preparation of protoplasts:

[0042] 1) Pick the plump young pods of soybeans 14 days after flowering, rinse them with sterile water 3-4 times, and remove the impurities on the surface;

[0043] 2) The young pods are completely soaked in 20% sodium hypochlorite solution, sterilized by shaking on a shaker (180r / min, 25)°C for 20 minutes, and rinsed with sterile water 2-3 times;

[0044] 3) peel off the immature embryos of the immature pods with sterilized tweezers and scalpel, and remove the seed coat;

[0045] 4) Place the adaxial end of the immature embryo in D40 medium at constant temperature (25-26°C, 16h / 8h, 300μmol·m -2 ·s -1 ) induced for 30 days;

[0046] 5) selecting somatic embryos with a diameter of about 3 mm and transferring them to D20 medium for 20 days;

[0047] 6) After the somatic embryo grows to a size of 0.8-1 mm, it is transferred to a freshly prepared D20 medium for subculture for 5 times to obtain an ...

Embodiment 2

[0053] Preparation of soybean leaf protoplasts:

[0054] 1) Select the first three compound leaves after 14 days of soybean germination, and wash the surface of the leaves with sterile water to remove impurities;

[0055] 2) Cut the blade into thin strips of 0.5 mm with a scalpel and tweezers so as to fully contact with the enzyme solution, and take about 0.2 g for each sample;

[0056] 3) Add 0.2g strip leaves to 5mL mixed enzymolysis solution, seal and enzymolyze for 5h under the conditions of dark, temperature 25-26, ℃ 50r·min-1;

[0057] 4) Filter the enzymatic solution with a nylon mesh cell sieve (200 mesh), transfer the filtrate to a centrifuge tube at 800r min -1 Centrifuge for 5 minutes, draw the supernatant as carefully as possible, add 10mL W5 solution to wash twice, and obtain the crude and pure protoplasts;

[0058] 5) Add 5mL of W5-23% solution to the crude protoplasts, 800r·min -1 Centrifuge for 5 minutes, carefully suck out the protoplast band as much as pos...

Embodiment 3

[0062] Transformation of soybean leaf protoplasts:

[0063] 1) Adjust the density of leaf protoplast suspension to 5×10 with W5 solution 6 cells / mL, placed on ice for 1 h, after the protoplasts settled at the bottom of the test tube, carefully suck up the supernatant as much as possible with a pipette gun, resuspend the protoplasts with MMG suspension, and adjust the density to 2.5×106 / mL, stand at room temperature;

[0064] 2) Prepare 6-10 sterile centrifuge tubes, add 0.1mL of the protoplast resuspension, 0.01mL of the plasmid, and 0.11mL of a 30% PEG4000 solution to each test tube, wherein the concentration of the plasmid is 1.5 μg / μL, immediately invert and mix, and stand at room temperature for 10 minutes;

[0065] 3) Then add 0.4mL W5 solution to each centrifuge tube, gently invert the centrifuge tube to mix to terminate the transformation reaction, centrifuge at 200g / min for 2min at room temperature, and remove the supernatant;

[0066] 4) The protoplasts were resu...

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Abstract

The invention discloses a preparation and conversion method of soybean protoplasts, and relates to the technical field of plant gene engineering. The method comprises the following steps of (1) performing induction to obtain soybean embryogenic calluses; (2) adding an enzyme solution, performing enzymolysis on the embryogenic calluses, performing filtration and centrifuging, adding a W5-23% solution, and performing centrifuging so as to obtain purified protoplasts; and (3) putting the purified protoplasts on ice, performing treatment, performing re-suspending with MMG suspension liquid, then sequentially adding plasmids and a PEG4000 solution, performing uniform mixing, performing standing, adding a W5 solution, ending a conversion reaction, performing re-suspending on protoplasts obtained through centrifuging with a WI solution, performing shifting into a culture dish, and performing light-away room-temperature putting. According to the preparation and conversion method disclosed by the invention, the compounding ratio of the enzyme solution and PEG instantaneous conversion reaction conditions are optimized, the yield and the conversion rate of the soybean protoplasts are significantly increased, identification of soybean gene functions is facilitated, and a reliable basis is further provided for exploration of the preparation and conversion conditions of the protoplasts of other plants and other positions.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for preparing and transforming soybean protoplasts. Background technique [0002] Protoplasts have many advantages in genetic manipulation, germplasm breeding, etc., but most of the application of plant protoplast technology is based on efficient protoplast isolation system, that is, how to obtain protoplasts with high vitality and high yield. At present, PEG-mediated transient transformation is a general tool for detecting gene functions. It has the advantages of low cost, high transformation rate, short time, and no genotype restrictions. Establishing an efficient, fast, and accurate PEG transient transformation system will help Solving the problem that soybean is difficult to transform is of strategic significance to the research of high-throughput analysis of soybean genome. [0003] The existing method for preparing soybean protoplasts is to prepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206C12N2509/00
Inventor 苏彤姚陆铭张鑫王彪马晓红
Owner SHANGHAI JIAO TONG UNIV
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