Induction of secondary embryogenesis in microspore-derived embryos of Brassica napus L.

Abstract

High frequencies of secondary embryos can be induced when microspore-derived embryos (MDEs) of Brassica napus cv. ‘Topas’ were cultured at low density in the dark or in indirect, low intensity light (15–20 μmol m −2 s −1), as well as via mechanical wounding, or by addition of auxins to the culture medium. Best results in terms of the number of secondary embryos per explant and reduced manipulative steps were obtained in low density cultures of intact MDEs in darkness, without the addition of growth regulators. Intact and wounded MDEs cultured at low densities responded differently to light. In darkness, 50–60% of the intact MDEs developed 20–80 secondary embryos per primary embryo, but when cultured in continuous light (150–200 μmol m −2 s −1) secondary embryogenesis was suppressed, only 19–23% of the MDEs developed pro-embryogenic structures. In contrast, wounded MDEs responded equally well in dark and light culture conditions. Around 60–100% of the transversely wounded MDEs, independently of the embryo stage, developed 10–50 secondary embryos in the wounded and surrounding areas of the explant, while culture of cotyledons alone, chopped, or transversely wounded, developed no secondary embryos at all. Embryogenic capacity was highest when the hypocotyl part was present in the explant. Secondary embryos that developed from intact as well as wounded MDEs could be dissected from the parental tissue and could develop further into plantlets.