INCORPORATION OF THE GUS GENE INTO ORCHIDS VIA EMBRYO ELECTROPHORESIS

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  • 1 Florist & Nursery Crops, USDA, ARS, Beltsville, MD 20705-2350

The apical meristems of Calanthe orchid embryos were exposed to 1 mg/ml pBI-121 DNA in an electric field. pBI-121 contains the GUS marker gene glucoronidase under the control of the 35 S cauliflower mosaic virus promoter. A pipette containing 0.3% agarose and acetate buffer containing the DNA was placed on one end of the embryo; while the opposite end was in contact with a pipette containing only buffer and agarose. Uptake of the DNA into the meristem was monitored by 4′6-diamidino-2-phenylindole (DAPI) fluorescence. Optimal uptake occurred after 10 min of electrophoresis at 10 volts and 0.5 milliamps. Under these conditions, 55% of the embryos survived the treatment and 57% of those which survived were transformed as measured by GUS-positive staining. Leaves from 6 month old plants which developed from the transformed embryos expressed specific patterns of GUS staining.

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