A protoplast-based transient expression system for melon (Cucumis melo L.)

Melon (Cucumis melo L.), an important horticultural and economic crop, is widely cultivated all over the world. Functional analysis of genes related to important agronomic traits will greatly promote the development of basic research in melon. The transient transformation system is a convenient method to analyze gene function in vivo and has been developed and applied in many species. Our objective was to develop an effective transformation system in melon. By comparing cotyledons and first true leaves, and balancing protoplast yield and protoplast vitality, optimal sampling tissue and enzymolysis time were obtained. Through analyzing effects of main parameters including PEG concentration, plasmid DNA amount and transfection time, on transformation efficiency, the optimal procedure for PEG-mediated transformation was obtained. Moreover, the subcellular localization of a GLK2-like transcription factor CmAPRR2 was analyzed by using this system. Results showed that first true leaves could yield more isolated protoplast than did cotyledons. The optimal time of enzymatic digestion is 6 h. The PEG concentration and plasmid DNA amount have a greater effect than incubation time on transformation efficiency. The peak transformaconcentration is 40% and 20 ig plasmid DNA is injected. In addition, subcellular localization showed that CmAPRR2 is a nuclear-localized protein. This research shows that the developed transient expression system based on protoplast isolation from first true leaves and PEG-mediated transformation is efficient and can be applied for functional gene analysis research in melon.