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 producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 x 10(5) microspores per mi was necessary for continued microspore viability. One hundred and nine haploid or spontaneously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 x 10(5) microspores per ml is recommended for isolated microspore culture.