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Isolated wheat microspore culture and plant regeneration, and, Electroporation of wheat anther culture-derived embryoids
Abstract
Part I. The use of doubled haploid plants in a wheat breeding program requires an efficient regeneration system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. The objective of this research was to determine a simple, efficient system for regenerating plants from isolated microspore culture. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. The effects of pre-isolation mannitol conditioning, cell density, culture dilution and sucrose centrifugations on microspore viability also were evaluated. Isolation by blending gave the highest initial microspore viability, determined by fluorescein diacetate staining (75% viability). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. A initial microspore density of $2 \times 10\sp5$ microspores per ml was necessary for continued microspore viability. Using blender isolation without mannitol conditioning, 109 haploid or spontaneously doubled haploid plants were regenerated. On the basis of this research, blender isolation with an initial density of $2 x 10\sp5$ microspores per ml is recommended. While this system regenerates plants as well as recently reported methods, the isolated microspore system needs further improvements to be efficient enough to be used for plant transformation or in a breeding program. Part II. Plant protoplasts have been successfully transformed for many crop plants. However, in cereal crops, plant regeneration from protoplasts is difficult and inefficient. The objective of this research was to determine if plants could be regenerated from electroporated anther culture derived embryoids and if the regenerated plants would be transformed. Electroporation at 500 $\mu$F gave the highest number of regenerable embryoids. The plasmid used for transformation contained the gus A and bar genes encoding for $\beta$-glucuronidase and Basta resistance. Embryoids were screened for PPT (Basta) resistance and regenerated plantlets were assayed for GUS activity. Of the 66 plants regenerated so far, 16 have the potential to be transformed. One plant is GUS positive. No embryoids have survived PPT selection.
Subject Area
Plant propagation|Agronomy
Recommended Citation
Gustafson, Vicki Diane, "Isolated wheat microspore culture and plant regeneration, and, Electroporation of wheat anther culture-derived embryoids" (1994). ETD collection for University of Nebraska-Lincoln. AAI9425283.
https://digitalcommons.unl.edu/dissertations/AAI9425283