ISOLATED WHEAT MICROSPORE CULTURE

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for prod ucing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four method s of isolating microspores from anthers (blending, stirring, maceratin g, and floating) were compared. Isolated microspores were washed and c ultured in liquid medium. The effects of pre-isolation mannitol condit ioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the hi ghest initial microspore viability (75%). Mannitol conditioning and pu rification by sucrose centrifugation had a detrimental effect on initi al viability. An initial microspore density of 2 x 10(5) microspores p er mi was necessary for continued microspore viability. One hundred an d nine haploid or spontaneously doubled haploid plants were regenerate d from microspores isolated without mannitol conditioning using the bl ending method. Based on this research, blender isolation with an initi al density of 2 x 10(5) microspores per ml is recommended for isolated microspore culture.