A Rapid, Highly Efficient and Economical Method of <i>Agrobacterium</i>-Mediated <i>In planta</i> Transient Transformation in Living Onion Epidermis

<div><p>Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the <i>in vivo</i> behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and <i>Agrobacterium</i>-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient <i>in planta</i> transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and <i>Agrobacterium</i>-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.</p></div

Schematic diagram of forming agroinfiltration bubble by injecting agroinfiltration liquid into the interface between adaxial epidermis and mesophyll of onion bulb scale.

<p>(A) Syringe. (B) Agroinfiltration liquid. (C) Mesophyll of onion bulb scale. (D) Adaxial epidermis of onion bulb scale.</p

Effects of bacterial concentrations and durations of agroinfiltration.

<p>Note: three samples per combination of bacterial concentration and infiltration duration were investigated for ten 2 mm² epidermal areas. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples, and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis.</p

Comparison of agroinfiltration and particle bombardment methods on transformation in onion epidermis.

<p>Note: three samples per unit of infiltration components were investigated for ten 2 mm² epidermal areas. The efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area) in the part of transformation efficiency, and the standard errors were calculated by using Excel.</p

<i>Agrobacterium</i> mediated <i>in planta</i> transient transoformationin living onion epidermal cells.

<p>(A–F) Operational process of the modified agroinfiltration, (A) Onion bulb without outer scales, (B, C) The cut onion bulb prepared for subsequent injection, (D) The injection of <i>Agrobacteria</i>, (E) Bind injected cut scales together with elastic for further incubation, scale bar = 2.5 cm. (F) The magnification of injection location, scale bar = 4 mm. (G–V) Onion epidermal cells were transformed with constructs of pCM1205-RFP (G, H, I and J), pLPGM202 (K, L, M and N), pLPGM413 (O, P, Q and R) and pLPGM113 (S, T, U and V). Bright field images (G, K, O and S), UV excited fluorescence images (L, P, T and H), UV excited DAPI staining images (I, M, Q and U) and the merged images of fluorescence and DAPI (J, N, R and V), scale bar = 10 µm.</p

Effects of different components of agroinfiltration liquid (OD₆₀₀ = 0.10).

<p>Note: three samples per unit of infiltration components were investigated for ten 2 mm² epidermal areas. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis. The symbol “-”, represents that the agroinfiltration liquid included all the components except the referred component.</p

Comparison of transient transformation efficiencies in different plant materials by using agroinfiltration.

<p>Note: three samples per material were investigated for ten 2 mm² areas of agroinfiltrated epidermal cells. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis. For onion two-day pretreatment was conducted before agroinfiltration.</p

Effects of different pretreatment time of onion before <i>Agrobacterium</i> infection.

<p>Note: three repeat samples for each treatment were investigated by observing ten 2 mm² epidermal areas of each sample. Each efficiency value represents the mean of transformation efficiencies of thirty replicates of 2 mm² epidermal areas from three samples (the percentage of positive cells in total cells per unit area), and the standard errors were calculated by using Excel. Different capital and lowercase letters within the same column exhibit significant difference at the 1% and 5% probability level according to the Duncan test of SPSS 10.0 statistic analysis.</p