Plant Regeneration from Haploid Suspension and Protoplast Cultures from Isolated Microspores of Maize

Abstract

Summary Isolated maize microspores were used to initiate haploid suspension and protoplast cultures capable of plant regeneration. Exposure of 10-day induced microspores to a medium containing 9.0 µM 2,4-D resulted in the proliferation of androgenic proembryos into mucilaginous cultures that, after several cycles of selective sub-culture, were enriched for densely cytoplasmic cell clusters. Suspension cultures ranging from yellowish brown, coarse textured, thick, and slow growing to bright yellow, finer textured, and rapidly growing were obtained from the same population of microspores. Re-plating onto callus induction medium resulted in the formation of nodular, embryogenic callus from which haploid plants could be recovered upon transfer to regeneration medium. Enzymatic digestion of suspension cultures followed by density gradient centrifugation yielded totipotent protoplasts. The results from this study expand the list of available maize germplasm amenable to protoplast culture to include a Lancaster-related genotype and add the microspore to the limited list of explants capable of being used directly as a source of embryogenic suspension cultures and totipotent protoplasts.