Hairy root culture of Strobilanthes cusia for the production and enhancement of indigo biosynthesis

Strobilanthes cusia is an indigo-yielding plant and scarcity of both wild and cultivated plants has increased the supply pressure of the natural dye. Since hairy root cultures are suitable for the in vitro production of secondary metabolites, the hairy root culture of Strobilanthes cusia was established using two Rhizobium rhizogenes strains (ATCC 15834 and MTCC 532). Polymerase chain reaction (PCR) using rol B primer confirmed the molecular evidence of hairy root transformation. The differential transformation frequency was studied based on factors like explants types (leaves, shoot tips, and stem); infection techniques, infection period, co-cultivation period, bacterial strain types, acetosyringone concentration, and antibiotic treatment conditions. Leaf explants, infected with ATCC 15834 exhibited the highest induction rate (58.67%) among the three explants. The most efficient hairy root transformation was achieved with the immersion technique, involving 10 min of infection and 5 days of co-cultivation using 100 µM acetosyringone during infection. When the indigo content of the hairy roots and different parts of in vitro plantlets were compared, the hairy roots produced 0.314 mg/g fresh weight (FW) of indigo, slightly more than the roots (0.21 mg/g FW), but less than the leaves (1.08 mg/g FW) of in vitro plantlets. Further, the indigo content of the hairy root cultures treated with 200 µM indican and 200 µM salicylic acid were enhanced up to 0.78 mg/g FW and 0.71 mg/g FW, respectively. Thus, this study reveals the potential of hairy roots of S. cusia for indigo biosynthesis, which may serve as a potential alternative source for natural indigo.