Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as a model for examining a range of processes, including hormonal regulation of embryo development. We examined the hormonal physiology of MDEs with the primary objective of testing the validity of using the MDE system as a model for hormonally-regulated development in zygotic embryos, through late stages. To do this we identified and quantified endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA) and a number of gibberellins (GAs), including GA19, GA20, GA1 and GA8 in both MDEs and zygotic embryos. The presence of GA19, together with its C19 metabolites indicates that the early-13 hydroxylation pathway is operative in both embryo systems. Gibberellins A4 and GA9 were also identified, thereby confirming the presence of the early non-hydroxylation pathway in B. napus MDEs and zygotic embryos. In general, the pattern of change of hormone (ABA, IAA, GA1 and GA20) content per embryo through embryogenesis was similar for MDEs and zygotic embryos. Indole-3-acetic acid and GA1 increased to a maximum at day 30 after culture (DAC) before decreasing. Abscisic acid levels increased to a maximum at approximately day 35, and declined in zygotic embryos but not in MDEs. GA20 increased to the final harvest at maturity, or day 40. The absolute content (g/embryo) of each hormone, however was appreciably lower (5- to 15- fold) in the MDEs. This was not the result of dilution into surrounding medium for ABA or IAA; GA1, however, did accumulate in the medium. Although there were absolute quantitative differences in the levels of IAA and ABA found in the two embryo systems, the similarities in the pattern of hormone changes suggests that the MDE system can serve as a useful model for examining the physiological roles of hormones during embryogenesis.