Bud Length, Plating Density, and Incubation Time on Microspore Embryogenesis in Brassica napus

Abstract

Despite a large body of work on Brassica napus (L) microspore embryogenesis, the effects of bud length, plating density, and incubation time have not been extensively studied. Flower bud length (1–1.9, 2–2.9, 3–3.9, and 4–4.9 mm), plating density (1 × 104 and 2 × 104 microspores·mL−1 of medium), and temperature duration (2 and 6 days at 30°C) were tested to determine how they affected embryogenic processes in B. napus, cv. Regent. The highest embryo yield was obtained with a microspore density of 2 × 104 microspores·mL−1 from 2- to 2.9-mm buds incubated for 2 or 6 days at 30°C. There was a significant interaction between bud length and density with the duration of exposure to temperature. For buds 2–2.9 mm long, the highest level of embryogenesis (13.5 embryos·mL−1) was related to a culture density of 2 × 104 microspores·mL−1 and incubation for 6 days at 30°C, and for 3- to 3.9-mm-long buds, the highest level of embryogenesis (3 embryos·mL−1) was with 1 × 104 microspores·mL−1 and 6 days' incubation at 30°C. Higher normal regeneration (42%) was observed from embryos derived from 6 days of incubation rather than incubation for 2 days (24%); more callus (45%) and secondary embryos (31%) formed from embryos after incubating cultures for 2 days. Microspore embryogenesis in B. napus could be improved by choosing proper bud length, plating density, and duration of incubation at a high temperature.