Pro-embryos of Lupinus spp. produced from isolated microspore culture

Several species of lupin (Lupinus spp.) are grown in Australia as crop and pasture plants. Lupin breeding, and legume breeding in general, is constrained by the inability to produce doubled haploid (DH) plants, which would accelerate the selection and release of new varieties. This technology is still in the developmental phase for legumes, although other major grain crops such as wheat, barley, and canola successfully use DHs on a commercial scale. A new, reproducible method of microspore culture that leads to cell division and pro-embryos in lupin is reported here. Microspores at the late uninucleate stage of development are mechanically isolated from lupin buds and embryogenesis induced by a combined heat shock and sucrose starvation stress treatment. Addition of further components to the growth medium promotes division of up to 50% of microspores to greater than or equal to 16 cells within 24 h. Further development of these multicellular structures or pro-embryos appears to be limited by the rigid outer exine layer, which needs to rupture for continued cell division to the globular embryo stage. Further research is required to break this barrier to development of haploid lupin embryos.