An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20

Abstract

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 μM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 μM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

“…In the present study, the application of Tween-20 did not improve the expression efficiency. Our result contrasts those reported by Zhao et al [63], who found that Tween-20 increased GUS expression by influencing the agroinfiltration efficiency. Co-agroinfiltration with Tween-20 caused fast tissue necrosis and cell death, potentially due to the induction of damage by Tween 20 to plant cells in the agroinfiltration system.…”

Within‐plant heterogeneity in fecundity and herbivory induced by localized DNA hypomethylation in the perennial herb Helleborus foetidus

“…In this respect, syringe infiltration of leaves with a zebularine solution was a more reliable application method relative to spraying (Puy et al., ) because it allowed closer control of the spatial distribution of the treatment and the actual volume of solution delivered to plants. Syringe infiltration is widely used in transient gene expression assays and other molecular transformation studies (Jelly et al., ; Zhao et al., ), but we are not aware of any prior application in ecological epigenetic experiments with wild plants. The results obtained here with H. foetidus illustrate the potential of the syringe infiltration technique in epigenetic experiments that require spatially well‐defined delivery of demethylating agents to specific parts of adult plants.…”

“…For each experimental session, syringe infiltration was used to deliver 0.5 mL of a 0.1 mM sterile solution of the DNA methylation inhibitor zebularine (Sigma‐Aldrich, St. Louis, MO, USA) in Milli‐Q water (Millipore Corp., Darmstadt, Germany) to the intercellular spaces of the parenchyma in leaves near the inflorescence borne by each treated ramet. The infiltration method consisted of pressing a needleless 1‐mL syringe against the underside of leaves and gently forcing the solution through the stomata into the leaf parenchyma (e.g., Jelly et al., ; Zhao et al., ) as illustrated in Fig. .…”

The Role of Plant Genotype, Culture Medium andAgrobacteriumon Soybean Plantlets Regeneration during Genetic Transformation

“…This method induces transient expression of genes in a plant by forcing Agrobacterium suspension into the internal leaf tissues using a syringe [19]. Zhao et al [20] reported transformation of Nicotiana benthamiana using this method with Agrobacterium strain harbouring pCAMBIA1301. The β-glucuronidase (GUS) expression and GUS activity showed increased transgene expression more than 6-fold for agroinfiltration suspension containing 20 μM 5-azacytidine, 0.56 mM ascorbic acid and 0.03% Tween-20.…”

“…This microprojectile or biolistic bombardment employs particle acceleration coated DNA into the target plant tissues. Once in the cells, the DNA becomes permanently integrated into the chromosomes of the host plant genome [20]. Although, there are numerous techniques used for transformation, all methods face the same challenges of inefficiency, lack of a routinely used protocol and the genotype specificity problem.…”