Microspore embryogenesis and plant regeneration in Brussels sprouts (Brassica oleracea L. var. gemmifera)

• Two modes of Brussels sprouts microspore embryogenesis were described.
• Cold pretreatment enhanced embryogenesis via increasing the viability of microspores.
• AVG improved multinucleate structures to further develop in microspore culture.

As a tool in genetic engineering, isolated microspore cultures have the remarkable quality to produce doubled haploids for plant breeding and gene mapping. Protocols were developed for the induction of microspore-derived embryos and generation of doubled haploid plants from isolated microspores of Brussels sprouts. A cytological analysis showed that two modes of Brussels sprouts microspore embryogenesis exist: a direct route via embryos which was the major developmental pathway, and an indirect route via calli. Cold pretreatment (4 °C) and aminoethoxyvinylglycine addition were tested to determine whether and how they could improve embryogenesis. Cold pretreatment improved the viability of microspores, and stimulated to produce embryos directly. Similarly, aminoethoxyvinylglycine had a positive effect on the number of embryos produced via improving multinucleate structures to further develop. The combination of cold pretreatment for 24 h and 1 μM aminoethoxyvinylglycine addition significantly enhanced microspore embryogenesis efficiency, especially with low responsive genotype ‘1076’ for which it was increased by about 82-fold. Subsequently, the use of solid B5 medium with 1% agarose and 2% sucrose (w/v) achieved an efficient rate of plant regeneration, and more than 67% of regenerated plants were spontaneous diploids.