Embryogenesis in culture of isolated microspore of broccoli

The process of embryogenesis and technological experimental protocol has been studied and applied to produce doubled haploid plants from microspore cultured in vitro in broccoli B. oleracea L. convar. botrytis (L.) Alef. var. italica Plenck. It was shown that successful embryoid development occurred from microspore isolated from buds 4-5 mm long, containing microspores at late vacuolated stage and pollen grain at twocell stage. The optimal temperature of treatment was 32 C° within 2 days after culture was launched. The embryoids were produced from the following broccoli accessions: Arcadia F1, Everest, Green Valiant, Marathon F1, and Furio. The highest embryoid yield was obtained from accession Green Valiant, and consisted of 140 embryoids per Petri dish, whereas the lowest yield was in Furio, up to 3 embryoids per Petri dish. The first microspore division was observed in all accessions in 2-3 days of cultivation. Further development of embryoids went either directly into usual embryoid or into suspensor-like structures. The embryoids with suspensor developed more slowly than embryoids without one. We observed the embryoid formation not only at distal end towards microspore originated the suspensor-like structure but also the formation of chain of embryoids, and all variation of twin embryoid combinations. The study of process of embryogenesis in isolated microspores in vitro showed that this method can be used both to produce doubled haploid plants and study the developmental stages of zygotic embryos and suspensors.