This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

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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.