Antibiotic resistance occurs when the use of antibiotics to treat bacterial infections or to select for transformed bacteria from in vitro cultures becomes ineffective. Although this phenomenon is primarily observed in medicine, it also affects the success of scientific research when bacterial-based genetic transformation experiments are performed. During the agrotransformation of plant cells, tissue, and organs, the acquisition of antibiotic resistance by Agrobacterium spp. has been widely observed, making it difficult to select for the transformed bacteria. The objective of this study was to develop a heat-shock protocol for the transformation of Agrobacterium rhizogenes without the spontaneous generation of antibiotic resistance, to increase its sensitivity and specificity to produce transgenic hairy roots. After the bacterium was transformed, it was cultured in liquid culture medium and plated on solid medium to isolate colonies. The genetic transformation of the bacteria and the plant tissue was verified by PCR and by β-glucuronidase assays. The reproducibility of the method was assessed among the A. rhizogenes strains LBA 9402, A4 and 15834 using streptomycin, kanamycin and ampicillin for selection.