Biotechnology and Bioprocess Engineering 2021; 26(4): 641-649  
Development of Auxotrophic Agrobacterium tumefaciens AGL1 by Tn5 Transposon for Rice (Oryza sativa L.) Transformation
Mohamed Sahrul Tamzil, Yuzer Alfiko, Andhika Faisal Mubarok, Sigit Purwantomo, Antonius Suwanto, and Sri Budiarti
Mohamed Sahrul Tamzil, Antonius Suwanto*, Sri Budiarti
Graduate School of Biotechnology, IPB University, Bogor 16680, Indonesia
Tel: +62-812-9973-680
Yuzer Alfiko, Andhika Faisal Mubarok, Sigit Purwantomo
Biotech Laboratory, Wilmar Benih Indonesia, Bekasi 17530, Indonesia
Contributed equally to this study
Received: July 30, 2020; Revised: November 23, 2020; Accepted: November 23, 2020; Published online: August 30, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

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Explant contamination due to Agrobacterium overgrowth after the co-cultivation stage is a common problem in Agrobacterium-mediated plant transformation. In order to overcome this issue, this research undertook another approach by generating auxotrophic Agrobacterium tumefaciens AGL1 mutants to a specific amino acid by mini Tn5 transposon carrying spectinomycin resistance gene (spcR), and a total of 3315 AGL1 mutants were successfully constructed. Further screening identified 20 putative auxotrophs, and subsequently produced three mutants carried auxotroph properties to one specific amino acid. These mutants were AP5-2-51 threonine auxotroph, AP5-5-2 cysteine auxotroph, and AP5-7-27 tryptophan auxotroph. The mini Tn5 insertion position in the Agrobacterium genome showed that the insertion position of AP5-2-51 mutants was in the thrB gene (AAK86584.1; locus tag At1D132_04580), while the other two mutants were unable to be identified by TAIL-PCR technique. The effectiveness of these three mutants to transfer T-DNA (pCAMBIA1300-eGFP-hpt) was examined on fresh Nipponbare rice callus explants with AGL1 as control. Results showed that transformation efficiency of the three mutants was not significantly different from AGL1 (Tukey HSD, α = 0.05). The percentages of Agrobacterium overgrowth in control and samples (three mutants) were also measured. Interestingly, the AP5-2-51 mutant indicated the highest ability to prevent overgrowth by reducing Agrobacterium growth to 1.11%, while the other two mutants suppressed the overgrowth to 15.56% (AP5-5-2) and 12.22% (AP5-7-27).
Keywords: Agrobacterium tumefaciens AGL1, overgrowth, auxotroph, mutagenesis, Tn5 transposon

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