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Effective selection and regeneration of transgenic sugarcane plants using positive selection system

Zhang, Muqing, Zhuo, Xiaolei, Wang, Jihua, Wu, Yang, Yao, Wei, Chen, Rukai
In vitro cellular & developmental biology 2015 v.51 no.1 pp. 52-61
2,4-D, biolistics, callus, color, enzyme activity, gene expression, genes, genetic markers, genetically modified organisms, geneticin, kanamycin kinase, mannose, naphthaleneacetic acid, phosphinothricin acetyltransferase, polymerase chain reaction, sugarcane
We compared the transformation frequency of a positive selection system, which employs an Escherichia coli-derived pmi (phosphomannose isomerase) gene as the selectable marker and mannose as the selective agent, to commonly used negative selection systems of bar (phosphinothricin acetyltransferase) /phosphinothricin (PPT), npt II (neomycin phosphotransferase) /G418 (geneticin), and hpt (hygromycin phosphotransferase) /Hygromycin. Transgenic sugarcane plants were obtained following biolistic transformation of callus with four different selectable marker genes. Under different selective agents, transformed callus was selectively proliferated on MS medium containing 13.57 μM of 2,4-D, regenerated on MS medium with 4.44 μM of BA, 9.29 μM of KT, and 0.89 μM of NAA, and rooted on half of MS medium with 13.38 μM of NAA and 0.44 μM of 6-BA. The results demonstrated that the transformation frequency of 4.2% scored in the pmi/mannose positive system was significantly higher than those in the negative selection systems (0.47% for bar/PPT, 1.38% for npt II/G418, and 0.63% for hpt/Hygromycin, respectively). Transgenic sugarcane plants using the pmi/mannose system were confirmed by polymerase chain reaction (PCR) analysis and chlorophenol red (CPR) assay. The CPR assay was found to efficiently and qualitatively detect the transgenic sugarcane from the nontransformed plants because medium color changes in the CPR assay were highly responsive to pmi gene expression and enzyme activity. Thus, a pmi/mannose positive selection system for sugarcane transformation was developed to obviate the use of negative selection systems based on herbicides and antibiotics.