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:

• (1) sugarcane embryonic calli are irradiated 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;
• (2) the sugarcane embryonic calli after mutagenesis in the above step are inoculated to a solid medium CM2 and sealed by a film for buffering culture for 8˜10 days;
• 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;
• (3) the sugarcane calli after buffering culture are in turn inoculated to a solid medium CM3 containing 1.2˜-1.7 μmol·L−1 of glyphosate and to a solid medium CM3 containing 2.2˜2.7 μmol·L−1 of glyphosate, then sealed by a film for screening culture for 8˜10 days respectively;
• 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;
• (4) the embryonic calli survived in the above step are transferred to a solid differentiation medium CM4 for culture and subculture is conducted once after seedlings emerge;
• 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;
• (5) after growing to 20 mm-30 mm, the seedlings in the above step are transferred to a CM5 for screening culture;
• 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;
• (6) the seedlings survived in the above step are inoculated to a CM4 for proliferation culture, and then transferred to a CM6 for root-growth culture and seedling hardening, then transplanted to a seedbed;
• after growing to 100 mm˜150 mm, the seedlings are screened by spraying 4.8˜5.2 μmol·L−1 of glyphosate;
• the survived seedlings are glyphosate-resistant strains;
• 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.