Method for cultivating tobacco mutants on basis of EMS (ethyl methanesulfonate) mutagenesis
A technology of mutants and tobacco, applied in botany equipment and methods, seed and rhizome treatment, plant genetic improvement, etc., can solve gene function disturbance, target gene function loss, and there is no very detailed mutagenesis of the main tobacco variety K326 Technology optimization system and other issues
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Embodiment 1
[0035] Embodiment 1: Tobacco variety K326 seed pretreatment
[0036] At room temperature 25°C, take 61.5mL of 1 mol / L K 2 HPO 4 and 38.5mL of 1mol / L KH 2 PO 4 Mix well to prepare 100mL solution, then dilute 10 times to 1000mL to prepare 0.1mol / L potassium phosphate buffer. Potassium phosphate buffer solution is generally prepared and used immediately. About 40,000 seeds were collected in a 50 mL centrifuge tube, and 50 mL of 0.1 mol / L potassium phosphate buffer solution with pH 7.0 was added for pretreatment at 25°C for 6 hours. Put it into the mixer during pretreatment to ensure that the seeds are fully treated. After the pretreatment, the seeds were blotted dry with filter paper and then air-dried naturally. After weighing, they were divided into 19 parts on average (6 gradients × 3 times + 1 control), each with about 2000 grains, and set aside.
Embodiment 2
[0037] Embodiment 2: EMS concentration situation and seed treatment
[0038]The EMS buffer was prepared with 0.1mol / L phosphate buffer (pH 7.0), and the volume fractions of EMS were 0%, 0.30%, 0.50%, 0.70%, 0.70%, 1.10%, and 1.50%. Prepare 1% EMS mother solution first, and then prepare working solution according to Table 1. Among them, all pipette tips and centrifuge tubes that come into contact with the EMS solution should be placed in the sodium hypochlorite solution so that the toxicity of the EMS is oxidized and eliminated. Pour tobacco K326 seeds into a centrifuge tube filled with EMS solution, soak each concentration of EMS at 28°C for 8h, 12h and 16h respectively, and the treatment and time numbers of different volume fractions are A8-F16, in which potassium phosphate buffer The control solution was named CK (Table 2). All the centrifuge tubes were placed in a mixer at 28°C at 40 r / min, dipped and shaken in the dark.
[0039] Table 1 EMS solution preparation table
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Embodiment 3
[0043] Example 3: Cleaning of EMS treated tobacco seeds and EMS waste liquid treatment
[0044] Rinse the seeds in the centrifuge tube with phosphate buffer solution 50 times, add to 50mL of the centrifuge tube each time, tighten the tube cap (to prevent leakage of EMS residue), and then shake it up and down about 30 times. Finally, pour it into a small sieve, mark it and rinse it with running water for 0.5 h. The cleaned seeds were blotted dry with filter paper and then dried naturally for later use.
[0045] After the treatment was completed, the EMS waste liquid obtained from the first 5 washes was poured into 1mol / L NaOH solution for digestion.
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