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Fluorescent screening of transgenic Arabidopsis seeds without germination

文献类型： 外文期刊

作者： Wei, S 1 ; Bravdo, BA 2 ; Shoseyov, O 3 ;

作者机构： 1.Hebrew Univ Jerusalem, Robert H Smith Inst Plant Sci & Genet Agr, IL-76100 Rehovot, Israel

2.Hebrew Univ Jerusalem, Otto Warburg Minerva Ctr Agr Biotechnol, Fac Agr Food & Environm Qual Sci, IL-76100 Rehovot, Israel

3.Jiangsu Acad Agr Sci, Inst Hort, Nanjing 210014, Peoples R China

关键词： Agrobacterium-mediated transformation;Endoplasmic-reticulum;Beta-glucosidase;Plant-cells;Marker genes;Thaliana;Expression;Retention;Proteins;Compartment

期刊名称：PLANT PHYSIOLOGY （ 影响因子：8.34； 五年影响因子：8.972 ）

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收录情况： SCI

摘要： 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 mum 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% +/- 0.349% 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.

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