Author
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IVIC-HAYMES, SNEZANA - UNIV OF BELGRADE, YO |
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Smigocki, Anna |
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Submitted to: Mid Atlantic Plant and Molecular Biology Proceedings
Publication Type: Abstract Only Publication Acceptance Date: 8/12/2002 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Nonproprietary sugar beet transformation methods are plagued by low transformation frequencies and lack of reproducibility. In efforts to optimize the methods, we established highly embryogenic sugar beet cell suspension cultures for transformation by the particle bombardment method. Callus obtained from leaf discs of greenhouse-grown FC607 plants was propagated in liquid medium for 2 weeks, sieved, and then plated for 1 day on agar medium prior to bombardment. After 2 to 5 days callus was passed to selection medium containing either kanamycin or paromomycin or no antibiotics. Transformation vectors carried the reporter gene uidA (GUS) fused to either the osmotin (Osm) or proteinase inhibitor II (Pin2) gene promoter, or the EGFP gene under control of the double 35S promoter. Transient GUS expression monitored 2 days after bombardment showed 900 to 3000 blue units per bombarded plate of 0.2 g of suspension cells. Transient EGFP expression visualized with epifluorescence microscope showed similar number of fluorescent cells per bombarded plate. Both GUS and EGFP expression decreased significantly during the initial 14 days of culture. Stably transformed GUS (+) calli were obtained as early as 3 weeks following bombardment at a frequency of 0.25 - 9 calli per bombarded plate but no GUS (+) shoots regenerated even though plated control cell suspensions were highly embryogenic. Further modulation of the plant growth regulator composition in the media may promote the regeneration of transgenic shoots. |
