Preparation method and application of sugarcane stem tissue protoplast
A technology for protoplasts and sugarcane stems, which is applied in the field of preparation of sugarcane stem tissue protoplasts, can solve problems such as inability to carry out genetic transformation work, difficulty in preparing protoplasts from sugarcane stems, etc., achieves highlighting the value of popularization and application and commercial development potential, and optimizes supporting facilities. The effect of conversion system, widely applicable value
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Embodiment 1
[0049] The preparation of sugarcane stem tissue protoplast, described preparation method comprises the steps:
[0050] (1) Material cultivation: such as figure 1 As shown, the sugarcane stem tissue of the age of 2 months is collected, and the part of the shell at the base of the material is peeled off, leaving only the soft white tender stem part, and the tender stem part obtained is cut into 0.5mm tender stem segments with a scalpel;
[0051] (2) Enzymatic hydrolysis: put the cut tender stem segments into the lysate buffer at a ratio of 20mL lysate / g tender stem segments, place on a shaker at 25rpm at room temperature, and enzymolyze in the dark for 4.5h until the protoplasts are complete Dissociate from the leaves of the small section of the tender stem, and microscopically examine the enzymatic hydrolysis results;
[0052] (3) Termination of enzymatic hydrolysis: add an equal volume of pre-cooled W5 solution to the solution after enzymatic hydrolysis in step (2), pass thro...
Embodiment 2
[0057] The transient expression application of sugarcane stem tissue protoplast, described application comprises the steps:
[0058] (1) Add 10μL to a 2mL centrifuge tube at a time 35S : GFP Plasmid (10~20μg, within 5kb in size), 100μL protoplasts, mix thoroughly, add 110μL PEG4000 solution, mix gently immediately, and let stand in the dark at room temperature for 15min;
[0059] (2) Terminate the transformation reaction: add 1mL W5 solution, mix by inverting, and centrifuge horizontally (4°C, 100 × g) for 5min, discard the supernatant to collect protoplasts;
[0060] (3) Add 1mL W5 solution to resuspend the protoplasts, gently blow open the protoplasts that are still agglomerated, and place them on a shaker at 25rpm in the dark for 18-24h at slow speed;
[0061] (4) Take the protoplasts cultured in the dark and observe under the microscope (such as image 3 shown).
[0062] according to image 3 The protoplast prepared from the middle sugarcane stem tissue adopts the tr...
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