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

Abstract

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 in vivo 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 Agrobacterium-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 in planta 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 Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Development of Agrobacterium-mediated transient transformation in persimmon (Diospyros kaki Thunb.)

“…Vacuum infiltration and syringe infiltration are the most common methods for Agrobacterium-mediated transient transformation (Takata et al, 2012;Xu et al, 2014). Compare with the vacuum infiltration, the syringe infiltration method has its unique advantages such as simplicity, easy performance, less requirement for laboratory apparatus and more suitable in the field experiments.…”

Isolation and functional analysis of MxNAS3 involved in enhanced iron stress tolerance and abnormal flower in transgenic Arabidopsis

“…This transient expression vector contains a green fluorescent protein (GFP) at SacI-SmaI sites. According to Xu et al (2014), the plasmids of MxNAS3-GFP were introduced into Allium cepa epidermal cells by injection. The fusion proteins of MxNAS3-GFP were observed and photographed by confocal microscopy.…”

Isolation and functional analysis of MdNAS1, with functions in improved iron stress tolerance and abnormal flower in transgenic tobacco

“…This transient expression vector contains a GFP at HindIII and SalI sites. According to Xu et al (2014), the plasmids of MdNAS1-GFP were introduced into Allium cepa epidermal cells by injection. The fusion proteins of MdNAS1-GFP were observed and photographed by confocal microscopy.…”