A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata L. Walp.)

摘要

Development of the cowpea leaf transient expression system using a fluorescent reporter (At-UBQ3pro:ZsGreen). a A 3–4-week old cowpea plant showing the first five trifoliate leaves numbered from apex to base. The central terminal leaflets from trifoliate leaves at positions 3 and 4 were used in experiments. Direct injection of Agrobacterium suspension into cowpea leaves using a syringe b without or c with a needle. d Detached, leaflet pieces, infiltrated with Agrobacterium containing the constitutive AtUBQ3pro:ZsGreen reporter, resting on filter paper on top of solid “Medium 4”, infiltrated side in contact with filter paper. eAtUBQ3pro:ZsGreen reporter fluorescence detected in infiltrated leaf cells 2 days after incubation. Inset shows an individual fluorescing leaf cell. f Removal of the lower leaf epidermis using cello tape. g Sonication of leaf pieces with epidermis removed in a flask containing Agrobacterium in a water bath at room temperature, followed by h shaking at 100 rpm for 30 min and, i incubation on filter paper on top of solid “Medium 4” (lower leaf side in contact with the filter paper). j Higher numerical frequencies of AtUBQ3pro:ZsGreen leaf cell fluorescence were obtained when the epidermis was removed, but this was not statistically significant. Transient transformation frequency was calculated as % of cells with fluorescence in the field of total cells (minimum of 200 counted cells) ± SD. Scale bars: a = 5 cm; b, c = 0.5 cm; d, f and i = 1 cm; e = 20 µm