Poster: Late and Moved Abstracts

Abs # 1375: Enhancing b-glucan content in wheat by transformation

Presenter:	Yong, Weidong , yongwd@purdue.edu
Authors	Yong, Weidong  (A)   Carpita, Nicholas C (A)
Affiliations:	(A): Purdue University

The pollen-tube pathway method is a simple and useful way to genetically transform wheat and other species. From previous studies we determined that the potential transformation frequency based on Southern blot and PCR is around 1% in the first generation. However, in next generation, the foreign DNA was rarely detected and expression was lost. We are exploring possible explanations for this effect. In the meantime, we are using this transformation system in an attempt to enhance the content of a mixed-linkage (1,3),(1,4)-b-D-glucan by antisense inhibition of the enzymes that degrade it during development. 5’ primers for RT-PCR were designed by comparing EST sequences of wheat with known b-D-glucan exohydrolase and endo-1,4-b-glucanase genes of barley. The cDNAs of two genes were isolated from young leaves of wheat (cv. Chinese Spring) by RT-PCR. Antisense oriented cDNAs of exo- and endoglucanases driven by glutelin endosperm-specific promoter were constructed in the binary vector, pCAMBAI1301.1, and used for pollen-tube transformation. Increased efficiency was attempted by applying a vacuum to the stem just below the head during application plasmid DNA solutions or Agrobacterium harboring the plasmids. The transgenic seedlings were detected using both GUS and PCR analysis. From T0 plant, no PCR or GUS positive transgenic line was found with Agrobacterium. However, plasmid DNA treatment of 263 flowers resulted in 167 seeds. Five of the seeds produced plants that tested positive through PCR analysis, and one plant that also exhibited GUS positive in the root. The influence of the antisense genes in seed will be analyzed upon seed set. This work has been supported by The Indiana 21st Century Research & Technology Fund