Effects of ascorbic acid, alpha-tocopherol, and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l−1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l−1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l−1) and ascorbic acid (5 and 10 mg l−1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l−1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l−1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l−1 α-tocopherol. When 50 mg l−1 α-tocopherol was combined with 5 or 10 mg l−1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l−1 of reduced glutathione and 5 mg l−l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.