A method for isolating protoplasts of Prunus lucidum and its application
A technology of protoplasts and green calyx plums, applied in biochemical equipment and methods, enzymes, plant cells, etc., can solve the problems of low conversion rate, high heterozygosity, and difficulties in breeding new varieties of plum blossoms, and achieve easy access , the effect of great application value
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Embodiment 1
[0045] Example 1 Preparation method of plum blossom protoplasts
[0046] The present invention relates to a kind of preparation method of plum blossom protoplast, described method comprises the following steps:
[0047] (1) Select the 3-month-old plum blossom branches, cut the stem segments into 8-10 cm long stem segments, peel off the bark and completely immerse them into a 50 mL centrifuge tube containing 25-30 mL of enzymatic hydrolyzate. 6 to 8 rhizome segments can be placed, and the enzymatic hydrolysis solution is: 2.0% (m / v) cellulase R-10, 0.75% (m / v) isolating enzyme R-10, 0.5M mannitol, 20mM KCl, 10mM CaCl 2 , 20mM MES, 0.1% BSA and 0.3% PVP, pH 5.7-5.8;
[0048] (2) Gently screw on the cap of the tube, enzymolysis in a water bath at 27-28°C for 40-50 minutes, avoid shaking during the period;
[0049] (3) Gently transfer the stems to a 50mL centrifuge tube containing 30mL of washing solution with tweezers, shake vigorously up and down for 25-30s, and discard the ste...
Embodiment 2
[0055] Example 2 Effects of different plum varieties on protoplast yield and vigor
[0056] During protoplast isolation, different plum varieties had significant effects on protoplast yield and vigor. The present invention is provided with green calyx plum ('small green calyx', 'flying green calyx'), Gongfenmei ('Jiangnan Gongfen', 'Fenpi Gongfen'), cinnabar plum ('pink cinnabar', 'Wuyu' Yu') were treated with a total of 6 varieties. The stem segments of different varieties were peeled and immersed in the enzymatic hydrolysis solution (the enzymatic hydrolysis solution was: 2.0% (m / v) cellulase R-10, 1.0% (m / v) isolating enzyme R-10, 0.5M mannitol, 20mM KCl, 10mM CaCl 2 , 20 mM MES, 0.1% BSA, pH 5.7-5.8), enzymatic hydrolysis at room temperature (25 °C) for 2 h. By measuring the yield and vigor of the isolated protoplasts, the suitable plum blossom varieties were determined. The results are shown in Table 1. The protoplast yield and activity were compared comprehensively. ...
Embodiment 3
[0059] Example 3 Influence of exogenous additives on inhibition of protoplast browning
[0060] In the above protoplast isolation process, the protoplasts obtained from different plum varieties after soaking and enzymatic hydrolysis process all had the phenomenon of browning, and the protoplast obtained from the green calyx plum variety had the lightest color. In order to explore the method of inhibiting the browning of protoplasts and improve the activity of protoplasts, the present invention set a total of green calyx plum ('small green calyx', 'flying green calyx') at 0.1% (m / v) antioxidant vitamin C (Vc) , 0.3% (m / v) adsorbent polyvinylpyrrolidone (PVP) and 0.3% (m / v) activated carbon (AC) and other browning inhibitors in a total of 6 samples. After peeling the plum stem section, immerse it in the enzymatic hydrolysis solution containing the above inhibitor (the enzymatic hydrolysis solution is: 2.0% (m / v) cellulase R-10, 1.0% (m / v) isolated enzyme R-10, 0.5M Mannitol, 20...
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