Microspore derived embryo formation and doubled haploid plant production in broccoli (Brassica oleracea L. var italica) according to nutritional and environmental conditions

In cell culture, the maintenance of proper growing conditions is a key approach for improving the formation of embryos, and is useful in the production of doubled haploid (DH) plants. Optimal nutritional and environmental conditions for the microspore culture of Brassica oleracea L. var italica were determined in order to reduce time and effort in breeding. The optimal conditions for microspore embryo formation differed depending on genotype. Microspore-derived embryos (MDE) formation was influenced by the strength of the NLN medium, the microelement and sugar concentration, and the heat shock temperature and period. The 0.5XNLN liquid medium was the most favorable for MDE formation. The most efficient formation of MDE was observed in the 0.5X NLN liquid medium, without the addition of microelements. When 13 or 15% sucrose was added to the 0.5X NLN liquid medium, the amount of normal MDE formation increased. The optimum heat shock temperature and period for MDE formation was 32.5°C and 24 h, respectively. A polyploidy test indicated that 30% of the microspore derived plants were diploid throughout the embryogenesis process.