Method for preparing masson pine protoplasts
A protoplast and masson pine technology, applied to plant cells, etc., can solve the problems of lack of masson pine preparation methods, etc., and achieve significant economic and social benefits, low price, and strong vitality
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Embodiment 1
[0023] A method for preparing masson pine protoplasts, the operation steps are as follows:
[0024] (1) Isolation: The embryonic suspension cells of masson pine subcultured for 4 months were cultured at a centrifugal force of 20× g Centrifuge for 6 min under the conditions of, discard the supernatant, and take out the centrifuged cells for use;
[0025] (2) Enzymatic hydrolysis: place the centrifugal pelleted cells obtained in step (1) in an Erlenmeyer flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a speed of 55 rpm, at a temperature of 21.5-22.5°C, dark Enzymatic hydrolysis under light conditions for 8 h, you can get a mixed solution containing protoplasts; the enzymatic hydrolysis solution consists of the following components in mass and volume concentration: cellulase RS 25 g / L, cellulase R-10 8 g / L, pectinase Y-23 8 g / L, isolating enzyme R-10 2 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L, magnesium sulfate ...
Embodiment 2
[0029] A method for preparing masson pine protoplasts, the operation steps are as follows:
[0030] (1) Isolation: The embryonic suspension cells of masson pine subcultured for 4 months were cultured at a centrifugal force of 20× g Centrifuge for 8 min under the conditions of, discard the supernatant, and take out the centrifuged cells for later use;
[0031] (2) Enzymatic hydrolysis: place the centrifugal pelleted cells obtained in step (1) in an Erlenmeyer flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a speed of 55 rpm, at a temperature of 21.5-22.5°C, dark Enzymatic hydrolysis under light conditions for 5 hours, you can get a mixed solution containing protoplasts; the enzymatic hydrolysis solution is composed of the following components in mass and volume concentration: cellulase RS 35 g / L, cellulase R-10 4 g / L, pectinase Y-23 6 g / L, isolating enzyme R-10 2 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L, magne...
Embodiment 3
[0035] A method for preparing masson pine protoplasts, the operation steps are as follows:
[0036] (1) Isolation: The embryonic suspension cells of masson pine subcultured for 4 months were cultured at a centrifugal force of 20× g Centrifuge for 7 min under the conditions of, discard the supernatant, and take out the centrifuged cells for later use;
[0037] (2) Enzymatic hydrolysis: place the centrifugal pelleted cells obtained in step (1) in an Erlenmeyer flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a rotation speed of 60 rpm, at a temperature of 21.5-22.5°C, dark Enzymatic hydrolysis under light conditions for 6 hours can obtain a mixed solution containing protoplasts; the enzymatic hydrolysis solution consists of the following components with mass and volume concentration: cellulase RS 30 g / L, cellulase R-10 6 g / L, pectinase Y-23 6 g / L, isolating enzyme R-10 4 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L,...
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