Large-scale microspore culture technique for mutation–selection studies in Brassica napus

Rapid isolation of oilseed rape (Brassica napus L.) microspores was accomplished by mechanical homogenization of whole racemes with buds no longer than 4.5 mm. After filtration and multiple washes, spores were incubated overnight in liquid medium at 30 °C in the dark, before plating in fresh medium at a density of 100 000 spores/mL. After 14 days in culture, gentle agitation on a shaker improved the rapid development of normal embryos and later, when transferred to light and liquid B5G medium (B5 + 0.1 mg/L gibberellic acid), further agitation enhanced the rate of maturation and germination. With this method 200–400 million spores can be processed in one batch, resulting in a final yield of several hundred thousand embryos within 3 weeks. Applications of the system for mutation–selection in B. napus are outlined.