Enhancing secondary embryogenesis in Brassica napus by selecting hypocotyl-derived embryos and using plant-derived smoke extract in culture medium

Induction of secondary embryogenesis on transformed androgenic microspore-derived embryos (MDEs) is a convenient approach to avoid chimerism and hemizygosis for the introduced transgene. In this work, we improved two aspects related to secondary embryogenesis in rapeseed (Brassica napus L. cv. Topas) MDEs: the identification of the best source of secondary embryos in the germinated MDEs and the increase in the production of secondary embryos (SEs). We performed a ploidy analysis of the different organs of MDEs-derived plantlets by flow cytometry. Our results showed that 60 % of the MDEs-derived plantlets were mixoploid, with 60 % of them having different ploidies for different organs. We concluded that hypocotyl-derived SEs present in general higher levels of genome duplication, which makes them a source of SEs better than cotyledons in terms of genetic stability and avoidance of hemizygosis. In order to increase production of SEs, we used plant-derived aqueous smoke extracts. The aim was to verify whether these extracts have a positive effect on secondary embryogenesis and if so, to identify the most efficient conditions of use. We tested smoke extracts at different incubation times, concentrations and methods of application to MDEs. The use of smoke extract, either prior to or during germination of MDEs, markedly enhances secondary embryogenesis. The best results were obtained with the use of smoke extract as a pretreatment, incubating MDEs for no longer than 15 min with a 1: 250 extract concentration.