Abstract
Agrobacterium tumefaciens has been utilized for both transient and stable transformations of plants. These transformation methods have been used in fields such as breeding GM crops, protein production in plant cells, and the functional analysis of genes. However, some plants have significantly lower transient gene transfer and stable transformation rates, creating a technical barrier that needs to be resolved. In this study, Super-Agrobacterium was updated to ver. 4 by introducing both the ACC deaminase (acdS) and GABA transaminase (gabT) genes, whose resultant enzymes degrade ACC, the ethylene precursor, and GABA, respectively. A. tumefaciens strain GV2260, which is similar to other major strains (EHA105, GV3101, LBA4404, and MP90), was used in this study. The abilities of the Super-Agrobacterium ver. 4 were evaluated in Erianthus ravennae, Solanum lycopersicum “Micro-Tom,” Nicotiana benthamiana, and S. torvum. Super-Agrobacterium ver. 4 showed the highest T-DNA transfer (transient transformation) frequencies in E. ravennae and S. lycopersicum, but not in N. benthamiana and S. torvum. In tomato, Super-Agrobacterium ver. 4 increased the stable transformation rate by 3.6-fold compared to the original GV2260 strain. Super-Agrobacterium ver. 4 enables reduction of the amount of time and labor required for transformations by approximately 72%, and is therefore a more effective and powerful tool for plant genetic engineering and functional analysis, than the previously developed strains. As our system has a plasmid containing the acdS and gabT genes, it could be used in combination with other major strains such as EHA105, EHA101, LBA4404, MP90, and AGL1. Super-Agrobacterium ver. 4, could thus possibly be a breakthrough application for improving basic plant science research methods.
Highlights
Agrobacterium tumefaciens is an α-proteobacteria that causes crown gall disease in many agriculturally and economically important species, such as those from the families Rosaceae, Vitaceae, and the genus Juglans (Kado, 2014)
Since ethylene and Gamma-aminobutyric acid (GABA) suppress the transfer of T-DNA in different ways, we predicted that the introduction of AcdS and GabT activity into A. tumefaciens would be effective at increasing the T-DNA transfer
The accelerated growth period began 10 h after culturing, and after 18 to 26 h, the logarithmic growth phases were observed (Figure 1B). These results indicate that introducing the ACC deaminase (acdS) and GABA transaminase (gabT) at the same time in A. tumefaciens did not affect its bacterial growth
Summary
Agrobacterium tumefaciens is an α-proteobacteria that causes crown gall disease in many agriculturally and economically important species, such as those from the families Rosaceae (rose, apple, cherry, and pear), Vitaceae (grape), and the genus Juglans (walnut) (Kado, 2014). Transferred T-DNA is integrated into the plant genome via complicated plant cell systems (Guo et al, 2019), and results in crown gall disease. To utilize this unique ability of A. tumefaciens for research purposes, there has been a great deal of effort to remove its oncogenesis characteristics, and to develop a binary vector system (Zambryski et al, 1983; Hoekema et al, 1983; Bevan, 1984; Komari et al, 2006). There has been further effort to increase the T-DNA transfer frequency of A. tumefaciens; one effective strategy was to upregulate its vir gene expression levels. The application of vir gene inducers (Stachel et al, 1985; Stachel et al, 1986; Cangelosi et al, 1990; He et al, 2009; Hu et al, 2013), using ternary system (van der Fits et al, 2000), utilization of Super-binary vectors (Komari, 1990; Hiei et al, 1994; Ishida et al, 1996), and a modification of the Ori of the binary vector (Ye et al, 2011; Vaghchhipawala et al, 2018), have subsequently improved its transformation frequencies
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