A simple and efficient agroinfiltration method in coffee leaves ( L.): assessment of factors affecting transgene expression.

The establishment of a simple, rapid and efficient transient expression system is a necessary tool for the functional validation of candidate genes in coffee biotechnology. The effects of strain, age of the donor plant, infiltration method, and infiltration medium on transgene expression in detached coffee leaves were evaluated. Regarding the effect of strain, the expression of was higher in GV3101-treated coffee disks than in LBA4404 and ATHV-treated samples. On the other hand, transient expression of was significantly higher in leaf disks from young plants (6-weeks-old) (13.1 ± 1.4%) than in mature tissue (12-weeks-old) (1.6 ± 1.2%). Transient expression was higher in detached coffee leaf disks from young plants infiltrated with one injection of 15 µL of strain GV3101::1303 suspended in MS salts supplemented with 30 g/L sucrose, 1.9 g/L MES and 200 uM AS with subsequent sanding of the abaxial epidermis. Using the optimized protocol, expression of the gene was observed 6, 24 and 48 h and 5 weeks after bacterial injection. DNA was extracted from coffee disks with positive GUS expression and specific and fragments were amplified 5 weeks post-agroinfiltration. On the other hand, using the optimized protocol, a specific (500 bp) fragment was amplified in the agro-infiltrated coffee leaf disks 5 weeks post-agroinfiltration with the plasmid pB427-35S-cry10Aa. Moreover, the expression of the gene in two infiltrated coffee leaf disks was verified by RT-PCR and an expected 500 bp fragment was amplified.