A kind of preparation method of masson pine protoplast
A protoplast, masson pine technology, applied in plant cells and other directions, 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 protoplasts of Pine massoniana, the operation steps of which are as follows:
[0024] (1) Separation: The embryogenic suspension cells of Pinus massoniana subcultured for 4 months were subjected to a centrifugal force of 20× g Centrifuge for 6 min under the conditions, discard the supernatant, and take out the centrifuged pelleted cells for later use;
[0025] (2) Enzymolysis: Place the centrifuged pelleted cells obtained in step (1) in a conical flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a rotation speed of 55 rpm. Enzymolysis under the condition of light for 8 h, the mixed solution containing protoplasts can be obtained; the enzymolysis solution is composed of the following components in mass volume concentration: cellulase RS 25g / L, cellulase R-10 8 g / L, pectinase Y-23 8 g / L, isolated enzyme R-10 2 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L, magnesium sulfate 0.25 g / L...
Embodiment 2
[0029] A method for preparing protoplasts of Pine massoniana, the operation steps of which are as follows:
[0030] (1) Separation: The embryogenic suspension cells of Pinus massoniana subcultured for 4 months were subjected to a centrifugal force of 20× g Centrifuge for 8 min under the conditions, discard the supernatant, and take out the centrifuged pelleted cells for later use;
[0031] (2) Enzymolysis: Place the centrifuged pelleted cells obtained in step (1) in a conical flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a rotation speed of 55 rpm. Enzymolysis for 5 h under the condition of light can obtain a mixed solution containing protoplasts; the enzymolysis solution is composed of the following components in mass volume concentration: cellulase RS 35g / L, cellulase R-10 4 g / L, pectinase Y-23 6 g / L, isolated enzyme R-10 2 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L, magnesium sulfate 0.25 g / L, chlori...
Embodiment 3
[0035] A method for preparing protoplasts of Pine massoniana, the operation steps of which are as follows:
[0036] (1) Separation: The embryogenic suspension cells of Pinus massoniana subcultured for 4 months were subjected to a centrifugal force of 20× g Centrifuge under the condition of 7 min, discard the supernatant, and take out the centrifuged pelleted cells for later use;
[0037](2) Enzymolysis: Place the centrifuged pelleted cells obtained in step (1) in a conical flask, add an equal volume of enzymatic hydrolysis solution, and then place it on a shaker with a rotation speed of 60 rpm. Enzymolysis under the condition of light for 6 h, the mixed solution containing protoplasts can be obtained; the enzymolysis solution is composed of the following components in mass volume concentration: cellulase RS 30g / L, cellulase R-10 6 g / L, pectinase Y-23 6 g / L, isolated enzyme R-10 4 g / L, potassium nitrate 1 g / L, sodium dihydrogen phosphate 0.35 g / L, magnesium sulfate 0.25 g / L, c...
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