High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Introduction

Plant transformation integrates foreign genes into the plant nuclear genome. The development of different transformation protocols in various plants has enabled advances in plant molecular biology and crop improvements (Saifi et al., 2020). Agrobacterium is routinely used as a plant gene transformation vehicle as it naturally possesses the ability to transfer a segment of its plasmid DNA (T-DNA) into its host nucleus, which ultimately leads to integration of the T-DNA into the nuclear genome (Tzfira et al., 2004). During 1980s and early 1990s, generating transgenic plants by leaf disc-based Agrobacterium-mediated transformation required laborious plant tissue culture and regeneration steps. In 1993, a simple floral vacuum infiltration method was developed in Arabidopsis for stable transformation, overcoming the tedious tissue culture requirements (Bechtold et al., 1993). Later, the vacuum infiltration step was replaced by floral dipping where the developing floral tissues are dipped into a solution containing Agrobacterium, sucrose and the surfactant Silwet L-77 (Clough & Bent, 1998). Because of the simplicity and reliability of this floral dip method, it is now the commonly used transformation method in Arabidopsis. This protocol has also been shown to work in certain Brassicaceae plants (Bent, 2006). Floral dip transformation may be feasible in plants such as wheat, maize, tomato, flax, Medicago truncatula and Setaria viridis (Agarwal et al., 2009; Bastaki & Cullis, 2014; Martins et al., 2015; Mu et al., 2012; Trieu et al., 2000; Yasmeen et al., 2009).

In Agrobacterium-mediated transformation protocols, the concentration of bacterial inoculum has been considered crucial to the success of plant transformation. In the commonly used floral dip protocol, bacterial cells are grown to stationary phase (OD₆₀₀=2.0), pelleted and resuspended in inoculation medium to OD₆₀₀≥0.8 (Clough & Bent, 1998; Zhang et al., 2006). Here, we tested whether a low concentration of Agrobacterium inoculum affects the plant transformation rate. Our data showed that, contrary to our expectation, using an extremely low density of Agrobacterium inoculum (OD₆₀₀=0.002) in the floral dip method still warrants relatively high transformation rate in Arabidopsis.

Method

Results and discussion

Four Agrobacterium inocula from high to low concentrations (OD₆₀₀=1, 0.1, 0.01, 0.002) were used in floral dip transformation to test the effect of bacterial concentration on the transformation rate. As shown in Figure 1 (Underlying data (Wang, 2020)), similar transformation rate (approximately 0.60%) was observed under all tested bacterial concentration. Notably, the transformation efficiency remains unchanged even though the Agrobacterium inoculum was diluted 500 times form OD₆₀₀ = 1 to OD₆₀₀ = 0.002. Therefore, it is feasible to dramatically reduce the Agrobacterium inoculum concentration in the floral dip method. Regardless of the inoculum concentration, transforming eight Arabidopsis plants grown in a single pot produced about 36 T1 transgenic lines on average, which is sufficient for most studies.

Figure 1. The effect of Agrobacterium concentration on transformation rate in floral dip method.

Transformation rates were calculated as [(# of hygromycin-resistant seedlings) / (total # seedlings tested)] × 100%. The data are shown as mean ± SE from six independent repeats in one representative experiment. The same letters denote no statistically significant difference according to one-way ANOVA (p<0.05).

Standard floral dip protocols use high concentrations of Agrobacterium inoculum, which requires growing large bacterial cultures (Clough & Bent, 1998; Zhang et al., 2006). Our study showed that Agrobacterium inoculum can be diluted to as low as OD₆₀₀ = 0.002 without sacrificing the transformation efficiency. Thus, the volume of bacterial culture used in each transformation experiment could be greatly reduced. For example, diluting 0.1 ml of overnight culture (OD₆₀₀ = 2) to OD₆₀₀ = 0.002 gives ~100 ml bacterial inoculum, which is sufficient in most transformation experiments. Such improvement allows researchers to culture small volume of a large number of Agrobacterium strains in parallel and use the diluted cultures to carry out high-throughput transformation of a large number of different constructs into Arabidopsis plants.

Data availability

Open Science Framework: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum project.

https://doi.org/10.17605/OSF.IO/YF6AE (Wang, 2020)

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).