Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/34614
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Type: Journal article
Title: The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum
Author: Able, J.
Rathus, C.
Godwin, I.
Citation: In Vitro Cellular & Developmental Biology-plant, 2001; 37(3):341-348
Publisher: C A B I Publishing
Issue Date: 2001
ISSN: 1054-5476
1475-2689
Statement of
Responsibility: 
Jason A. Able, Carl Rathus and Ian D. Godwin
Abstract: This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r s=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1−1 bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.
Keywords: Transformation; GUS; green fluorescent protein; particle inflow gun (PIG
Description: © 2001 Society for In Vitro Biology The original publication can be found at www.springerlink.com
RMID: RQF0000037
DOI: 10.1007/s11627-001-0061-7
Appears in Collections:Agriculture, Food and Wine publications

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