Fluorescent screening of transgenic Arabidopsis seeds without germination.
In this paper, we describe a reliable method for the screening and selection of Arabidopsis transgenic seeds within minutes without germination. Expression of the Aspergillus niger beta-glucosidase gene BGL1 in the plant's endoplasmic reticulum was used as a visual marker, together with 4-methylumbelliferyl-beta-D-glucopyranoside (MUGluc) as a substrate. Subsequent to incubation in a solution of MUGluc at room temperature for 2 to 15 min, transgenic seeds expressing BGL1 demonstrated a distinct fluorescent signal under UV light. Optimal screening conditions at room temperature were achieved between 75 and 450 microm MUGluc, at a pH of 2.5 to 5.0 and 2 to 5 min of incubation. No significant loss of viability was detected in transgenic seeds that were redried and stored for 45 d after incubation in MUGluc solution for 2 to 150 min. Transgenic plants expressing BGL1 displayed normal phenotypes relative to the wild type. Selection frequency was 3.1% +/- 0.34% for the fluorescence selection method, while kanamycin resistant selection resulted in only 0.56% +/- 0.13% using the same seed batch. This novel selection method is nondestructive, practical, and efficient, and eliminates the use of antibiotic genes. In addition, the procedure shortens the selection time from weeks to minutes.
Wei
Shu
S
The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Bravdo
Ben-Ami
BA
Shoseyov
Oded
O
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000235:genetics / Q000502:physiology*; D043326:Glucan 1,3-beta-Glucosidase / Q000235:genetics* / Q000378:metabolism; D007613:Kanamycin Resistance; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*; D012639:Seeds / Q000201:enzymology / Q000235:genetics*; D013050:Spectrometry, Fluorescence
D029681:Arabidopsis Proteins; D043326:Glucan 1,3-beta-Glucosidase