Method for conducting high-throughput and directed mutagenesis for sugarcane resistance to glyphosate by plasma

A method for conducting high-throughput and directed mutagenesis for sugarcane resistance to glyphosate by plasma, comprising the following steps:

• (a) irradiating sugarcane embryonic calli by a plasma instrument under a sterile condition for mutagenesis, wherein the mutagenesis power is 140-200 W, the discharging distance is 35-45 mm, the mutagenesis time is 110-140 s and the protective gas is nitrogen;
• (b) inoculating the sugarcane embryonic calli after mutagenesis in the above step in a solid medium CM2 and sealed by a film for buffering culture for 8-10 days;
• wherein the medium CM2 contains:
• a MS medium, 1.2-1.7 μmol·L−1 of 2,4-D, 28-32 g·L−1 of sucrose and 7.5-8.5 g·L−1 of agar, and the pH of the medium is 6.0-6.4;
• (c) inoculating the sugarcane calli after buffering culture in a first solid medium CM3 containing 1.2-1.7 μmol·L−1 of glyphosate, inoculating in a second solid medium CM3 containing 2.2-2.7 μmol·L−1 of glyphosate, then sealing each of the first and second solid medium CM3 with the calli by a film for screening culture for 8-10 days in each of the first and second solid medium CM3;
• wherein the medium CM3 contains:
• a MS medium, 28-32 g·L−1 of sucrose and 7.5-8.5 g·L−1 of agar, and the pH of the medium is 6.0-6.4;
• (d) transferring the embryonic calli survived in the above step to a solid differentiation medium CM4 for culture and conducting subculture after seedlings emerge;
• wherein the medium CM4 contains:
• a MS medium, 0.4-0.6 μmol·L−1 of NAA, 0.8-1.2 μmol·L−1 of Kt, 48-52 g·L−1 of sucrose and 7.5-8.5 g·L−1 of agar, and the pH of the medium is 6.0-6.4;
• (e) after growing to 20 mm-30 mm, transferring the seedlings in the above step to a medium CM5 for screening culture;
• wherein the medium CM5 contains:
• a MS medium, 4.8-5.2 μmol·L−1 of glyphosate, 28-32 g·L−1 of sucrose, 8 g·L−1 of agar, and the pH of the medium is 6.0-6.4;
• and
• (f) inoculating the seedlings survived in the above step in a medium CM4 for proliferation culture, transferring the seedlings to a medium CM6 for root-growth culture and seedling hardening, then transplanting the seedlings to a seedbed;
• after growing to 100 mm-150 mm, screening the seedlings by spraying 4.8-5.2 μmol·L−1 of glyphosate;
• wherein the survived seedlings are glyphosate-resistant strains;
• and
• wherein the medium CM6 contains:
• ½MS medium, 0.4-0.6 μmol·L−1 of NAA, 1.8-2.2 μmol·L−1 of 6-BA, 28-32 g·L−1 of sucrose, and the pH of the medium is 6.0-6.4.