The
Rhizobium rhizogene
-transformed root culture from
Physalis peruviana
L. (
P. peruviana
) may be a promising and novel source of valuable phenolics, including caffeoylputrescine (CP), which is known for antioxidant, antidiabetic, insect-resistant, disease-resistant, and neuroprotective properties. In this study, to improve the production efficiency of phytochemical components in
P. peruviana
hairy root cultures, we optimized various culture conditions, including the inoculum size, liquid volume, culture media type, carbon source, sucrose concentration, initial pH, and application of elicitors, to enhance the total phenolic content and CP yield in these hairy root cultures. The findings indicate that the use of sucrose as carbon source resulted in the highest biomass (13.28 g DW/L), total phenolic content (6.26 mg/g), and CP yield (2.40 mg/L). The White medium excelled in enhancing the total phenolic content (9.35 mg/g), whereas the B5 medium was most effective for the biomass (13.38 g DW/L) and CP yield (6.30 mg/L). A sucrose concentration of 5% was best for the biomass (18.40 g DW/L), whereas a sucrose concentration of 4% was ideal for the CP yield. Optimal culture conditions were as follows: an inoculum size of 0.5 g/100 mL, a liquid volume of 100 mL in a 250-mL flask, B5 medium, 4% sucrose, and a pH of 5.5. Among the tested elicitors, methyl jasmonate (MeJA) at 100 µM significantly increased the biomass (21.3 g/L), total phenolic content (23.34 mg/g), and CP yield (141.10 mg/L), which represent 0.96-, 2.12-, and 13.04-fold increases, respectively, over the control after 8 days. The optimized HR culture of
P. peruviana
provides a promising system to enhance the production of CP for pharmaceutical applications.