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Title: DIRECT GENE TRANSFER TO SUGAR BEET LEAVES

Author
item IVIC-HAYMES, SNEZANA - UNIV OF BELGRADE, YO
item Smigocki, Anna

Submitted to: American Society of Sugarbeet Technologists
Publication Type: Abstract Only
Publication Acceptance Date: 10/10/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Sugar beet transformation methods in the public domain are not readily reproducible and yield low transformation frequencies. These methods utilize sugar beet cotyledons, shoot basal tissues, and hypocotyl callus generated in tissue culture for gene transfer experiments. We developed a particle bombardment transformation method that uses leaves from greenhouse grown plants. Leaf discs or squares were excised from surface sterilized FC607 leaves and placed on B1 medium (Doley and Saunders, 1989, Plant Cell Rep 8:222-25) with added mannitol and sorbitol. Leaf tissues were bombarded with the uidA (GUS) gene under control of the osmotin (OSM) gene promoter or with the enhanced green fluorescent protein (EGFP) gene fused to the double 35S CaMV promoter. After 2 days, leaf fragments were transferred to B1 medium (2 fragments per petri plate) and cultured at 31 degree C in the dark. Two days after bombardment, 1 to 30 GUS(+) units per leaf fragment were observed. With the EGFP gene, 20 to 150 fluorescent cells per leaf fragment were visualized with an epifluorescence microscope. Both GUS and EGFP expression decreased significantly during the first 2 weeks of culture. After 6 to 8 weeks, embryogenic callus was removed from the bombarded leaf discs and analyzed for GUS expression. Presence of the GUS and the selectable marker (NPTII) gene was confirmed by PCR analysis. GUS(+) shoots regenerated from several GUS(+) calli. The advantages over previously published transformation methods include an abundant source of leaf material from greenhouse grown plants, the ease of handling leaf material in tissue culture, and the overall rapid regeneration of transgenic shoots within three months.