Non-destructive estimation of the amount of green fluorescent protein transiently expressing in Nicotiana benthamiana leaves with a viral vector

Abstract

A custom-made fluorescence measurement system was constructed for non-destructive and quantitative estimation of green fluorescent protein (GFP) transiently expressing in a Nicotiana benthamiana leaf with a viral vector. The system comprised a digital camera, a blue LED lamp, and a long wavelength-pass filter to acquire the brightness intensity of the green channel (G value) of a leaf surface image as a measure of fluorescence intensity. The genes encoding GFP and tobamoviral vector were transferred to N. benthamiana leaves 42 days post seeding by syringe infiltration with Agrobacterium for transient expression. The destructively measured GFP content in leaves as the ground-truth data started to increase at 3-4 days post infiltration (DPI), once showed a plateau at 6-8 DPI, and then increased again until 11 DPI. Regression analysis demonstrated that the r2 of the linear regression between the ground-truth GFP content and the G value was 0.86-0.87 when the G value at a given DPI was calibrated with the initial leaf color at 0 DPI for the same leaf. These results indicate that the constructed system and method could potentially be used to quantitatively and non-destructively estimate the amount of GFP transiently expressing in intact N. benthamiana leaves.