Comparison of endoplasmic reticulum targeted and non-targeted cytoplasmic GFP as a selectable marker in citrus protoplast transformation

In order to improve transformation efficiency in citrus two green fluorescent protein (GFP) constructions, an endoplasmic reticulum targeted (GFP-ER) and cytoplasmic targeted (Cy-GFP), were used to study expression and stability of GFP in transgenic plants. Results from citrus protoplast-GFP transformation showed that GFP-ER had brighter fluorescence than the Cy-GFP. Although both of these constructs gave transient expression at the protoplast level, the non-targeted GFP gave less stable expression than targeted GFP at the micro-calli or plant level. Stable expression of GFP after 4 weeks of culture was observed in about 1.0% and 0.1% of the initial GFP positive cells in GFP-ER and Cy-GFP experiments, respectively. At the mature plant level, there were distinct fluorescent differences between shoots transformed with the two GFP constructs. Moreover, the fluorescence intensity varied among different parts of the plant, being higher in new expanding leaf flush than in old leaves, even when using the same construct. These results demonstrate that GFP-ER-targeted gene expression in citrus tissues is more stable than the Cy-GFP, thereby improving transformation efficiency and the subsequent recovery of transgenic plants. The protoplast-GFP transformation method in citrus could have an advantage over the routinely used Agrobacterium-mediated citrus transformation system that always requires the use of an antibiotic resistance gene.