Abstract
Agrobacterium tumefaciens-mediated gene transfer is the most commonly used method for plant genetic engineering. However, during the period of A. tumefaciens culture, the effects of Agrobacterium culture before inoculation on genetic transformation are poorly understood. In the present study, we investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes, including virA, virB1, virC1, virD2, virD4 virE2, and virG. Spe treatment significantly induced the expression of all the studied genes, including virA, virB1, virC1, virD1, virD2, virD4, virE2, and virG. DTT treatment decreased reactive oxygen species accumulation. AS treatment activated the expression of the genes virA, virB1, virC1, virD1, virD2, virD4 and virG. All these effects resulted in increased T-DNA transfer. Additionally, combined Spe, 5-AzaC, DTT, and AS treatment improve Agrobacterium infection to a greater extent compared with their use alone, increasing T-DNA transfer by more than 8-fold relative to no treatment. Therefore, to improve genetic transformation, pretreatment of Agrobacterium during the culture period is important for improving genetic transformation efficiency.
Highlights
Genetic transformation is a method that transfers DNA of interest into the cell of an organism resulting in genetic alteration, and has been used in many areas of biotechnology, such as studies of gene function, genetic improvement, and molecular breeding
The results showed that 20 and 30 mM 5-AzaC treatment significantly decreased DNA methylation; 20 mM 5-AzaC reduced DNA methylation to a greater extent than 30 mM AzaC (Figure 1C), which is consistent with the trend of transfer DNA (T-DNA) transfer efficiency (Figure 1A)
DNA methylation usually correlates with gene expression; we further studied the expression of different vir genes in response to 5-AzaC treatment
Summary
Genetic transformation is a method that transfers DNA of interest into the cell of an organism resulting in genetic alteration, and has been used in many areas of biotechnology, such as studies of gene function, genetic improvement, and molecular breeding. To deliver foreign genes into host plants, three methods are mainly used: Agrobacterium tumefaciens- or Agrobacterium rhizogenesmediated plant transformation (Hinchee et al, 1988), protoplast transformation (Fischer and Hain, 1995), and particle bombardment (McCabe et al, 1988) Among these methods, Agrobacteriummediated plant genetic transformation has low cost, is the best choice for plant transformation, and is Agrobactium Treatments Affecting T-DNA Transfer the predominant method used to generate genetically modified plants. During the Agrobacterium infection period, the infected plants will induce a pathogen defense response to generate reactive oxygen species (ROS), and supplementation with of ROS scavenging factors in the co-cultivation medium, such as dithiothreitol (DTT), sodium thiosulfate (STS), and L-cysteine (L-Cys), can reduce ROS accumulation and increase the efficiency of transgene delivery. To improve T-DNA transfer, vacuum infiltration can be used, which could infiltrate plants in a short period of time and is more robust (Chen et al, 2014)
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