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 (spc^R), 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).

由于农杆菌在共培养阶段后过度生长导致外植体污染是农杆菌介导的植物转化中的常见问题。针对这一问题，本研究采取了另一种方法，通过携带壮观霉素抗性基因（spc ^R）的微型Tn5转座子，将根癌农杆菌AGL1突变体转化为特定氨基酸，共构建了3315个AGL1突变体。进一步筛选确定了 20 个假定的营养缺陷型，随后产生了三个突变体，其中一种特定氨基酸具有营养缺陷型。这些突变体是 AP5-2-51 苏氨酸营养缺陷型、AP5-5-2 半胱氨酸营养缺陷型和 AP5-7-27 色氨酸营养缺陷型。迷你 Tn农杆菌基因组中的5插入位置显示AP5-2-51突变体的插入位置在thrB基因（AAK86584.1；基因座标签At1D132_04580），而其他两个突变体无法通过TAIL-PCR技术鉴定。在以 AGL1 作为对照的新鲜日本晴水稻愈伤组织外植体上检查了这三个突变体转移 T-DNA (pCAMBIA1300 -eGFP-hpt )的有效性。结果表明，三个突变体的转化效率与 AGL1 没有显着差异（Tukey HSD，α = 0.05）。农杆菌的百分比还测量了对照和样品（三个突变体）的过度生长。有趣的是，AP5-2-51 突变体通过将农杆菌生长降低至 1.11%显示出防止过度生长的最高能力，而其他两个突变体将过度生长抑制至 15.56% (AP5-5-2) 和 12.22% (AP5-7- 27)。