Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection.
This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.
Zernova
Olga
O
Department of Crop Sciences, Edward R Madigan Laboratory, University of Illinois, Urbana, IL 61801, USA.
Zhong
Wei
W
Zhang
Xing-Hai
XH
Widholm
Jack
J
D001483:Base Sequence; D019470:Biolistics; D015152:Blotting, Northern; D015139:Blotting, Southern; D017931:DNA Primers; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013025:Glycine max / Q000235:genetics*; D046509:Tissue Culture Techniques; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
D017931:DNA Primers; D010940:Plant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase