Identification and Characterization of Two Putative Citrus Phosphomannose Isomerase (CsPMI) Genes as Selectable Markers for Mature Citrus Transformation

Abstract

Two Citrus sinensis (L.) phosphomannose isomerase (PMI) genes, CsPMI1 and CsPMI2, were evaluated as novel selectable markers in mature citrus transformation. Transgenic shoots produced after transformation of Kuharske rootstock with each PMI construct were selected on six treatments of mannose and sucrose. For CsPMI1, there were no significant differences among the various mannose and sucrose treatments for the mean number of positive shoots (PS), the mean transformation efficiency based on the number of shoots (TES), or the mean transformation efficiency based on the number of explants (TEE). However, for the CsPMI2 gene, the number of transgenics produced in two treatments (7.5 g L−1 mannose + 22.5 g L−1 sucrose and 15 g L−1 mannose + 15 g L−1 sucrose) was significantly greater than the sucrose control for TES at 4.2% and 3.7%, respectively. Moreover, TEE at 4.2% in the 15 g L−1 mannose + 15 g L−1 sucrose treatment, supported the TES value. Most of the transgenic lines demonstrated higher in vivo and in vitro enzyme assays compared with the wild-type control. CsPMI2 provided acceptable selection in mature citrus, and it will be applied in future intragenic research.