title: 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 intoNicotiana benthamianaleaves 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.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China. huimin0230@163.com.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China. smilebaobao@outlook.com.
authors.forename: Zilong
authors.initials: Z
authors.lastname: Tan
authors.affiliation: Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China. wxj-329@163.com.
authors.forename: Xuejing
authors.initials: X
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China. wangyucheng@ms.xjb.ac.cn.|Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China. wangyucheng@ms.xjb.ac.cn.
authors.forename: Yucheng
authors.initials: Y
authors.lastname: Wang
keywords: 5-azacytidine; Tween-20; ascorbate acid; syringe agroinfiltration; transformation efficiency