A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides)

Abstract

Background: The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.

Results: We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.

Conclusions: The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.

Figure 1

Expression of GFP in hybrid aspen using Agrobacterium -mediated vacuum infiltration.(A-F) Transient expression of GFP in aspen leaves. CaMV35S::EmGFP construct was transformed into hybrid aspen cuttings. Bright field image (A and D), GFP fluorescence (B and E), and GFP fluorescence and chloroplast autofluorescence (C and F) were captured using a fluorescence stereomicroscope. Scale bar = 5 mm (A-C). Areas with a white box in A, B, and C are magnified in D, E, and F, respectively. Scale bar = 1 mm (D-F). (G-O) Transient expression of GFP in various cell types in aspen leaves. GFP signals were observed in epidermal cells (G-I), guard cells (J-L), and mesophyll cells (M-O). GFP fluorescence (G, J, and M) and autofluorescence of chloroplasts (H, K, and N) were captured using a CLSM. GFP signal and autofluorescence of chloroplasts were merged (I, L, and O). Scale bar = 10 μm.

Figure 2

Transient transformation efficiency in hybrid aspen leaves.(A) Effect of infiltration medium on transient transformation. Overnight culture of Agrobacterium was re-suspended in individual infiltration medium containing 200 μM Acetosyringon and 0.0075% Silwet L-77 (final OD₆₀₀ = 0.5). The GFP signals were observed after three days of the transformation. Transformation efficiency was calculated by counting GFP positive cells in a 1.5-mm² leaf area. Values are means ± SE (n = 330). Asterisk indicates a statistically significant difference based on a Student’s t-test (P < 0.01). (B) Effect of Silwet L-77 concentration on transient transformation. Overnight culture of Agrobacterium was re-suspended in the medium [0.5 × MS, 5 mM MES-KOH (pH 5.6) and 200 μM Acetosyringon] (final OD₆₀₀ = 0.5). Appropriate Silwet L-77 was added to the solution. Values are means ± SE (n = 350). Means with different letters are different according to Tukey’s HSD test (P < 0.05). (C) Effect of bacteria density on transient transformation. Overnight culture of Agrobacterium was re-suspended in the medium [0.5 × MS, 5 mM MES-KOH (pH 5.6), 200 μM Acetosyringon and 0.015% Silwet L-77]. The final OD₆₀₀ was adjusted to 0.5, 1.0, and 2.0. Values are means ± SE (n = 480). Means with different letters are different according to Tukey’s HSD test (P < 0.05). The experiments shown are from two or three experiments each of which included three biological repeats. For each biological repeat, 40 to 60 sectors were measured.

Figure 3

Subcellular localization of Populus proteins and protein-protein interaction in hybrid aspen cells.(A-D) Transient GFP fusion protein expression assay in hybrid aspen leaves. (A) PttMTP1-GFP labels the tonoplast, (B) PttPrxQ-GFP labels the plastids, (C) PttC4H-GFP labels the ER and (D) PttGT47C-GFP labels the Golgi. After three days of the transformation, GFP signals, chlorophyll autofluorescence and fluorescence of organelle markers were captured using a CLSM. Columns show GFP fluorescence (left), fluorescence of organelle markers and chloroplast autofluorescence (middle, left), the superimposed images (Merged; middle, right), and bright field images (BF, right). Scale bar = 10 μm. (E) BiFC assay in aspen leaves using transient co-transformation. The plasmid constructs containing CaMV35S::nEYFP-AtRBR1 and CaMV35S::cEYFP-AtCYCD3;1 were used for BiFC assay. YFP signals and DAPI fluorescence were captured using a CLSM. Columns show YFP fluorescence (left), DAPI (middle, left), the superimposed images (Merged; middle, right), and bright field images (BF, right). Scale bar = 10 μm.

Figure 4

LUC reporter assay in hybrid aspen apices. The plasmid construct containing PttLHY2promoter::luc⁺ was transformed into aspen cuttings using the Agrobacterium-mediated vacuum infiltration assay. The apices were supplemented with luciferin solution at one day after the transformation. The LUC activity was detected by a cooled CCD camera beginning about 5 h following addition of substrate to obtain dynamic, real-time expression. Gray and white boxes indicate subjective night and day, respectively. Luminescence values are means ± SE (biological replicates n = 5).