Background: Trigonella foenum-graecum L. commonly known as fenugreek is a rich source of important medicinal metabolite, i.e. trigonelline.

Objective: In this study, hairy roots culture as a novel method for trigonelline production was evaluated.

Methods: For optimizing the hairy roots culture of Trigonella foenum-graecum, three strains of Agrobacterium rhyzogenes (ATCC15834, MSU440 and K599) via two inoculations methods including scotch and vacuum pump were used to agro-infiltration. Two elicitors including methyl jasmonate (0, 25, 50, 100 and 200 µM) and chitosan (0, 50, 100, 150 and 200mglˉ') were added to liquid medium as abiotic and biotic elicitors in various concentrations, respectively.

Results: The trigonelline content was increased via elicitation by methyl jasmonate and chitosan against control condition. The maximum trigonelline (36.7 and 37.3 mM/g D.W) were observed in 100 µM of methyl jasmonate and 150 mg/l of chitosan, respectively.

Conclusion: All parts of the seedling (crown, stem and leaf) were able to produce the hairy roots. Also, the highest dry weight of hairy root was obtained by A. rhizogenes strain 15834. The transformation of fenugreek using Agrobacterium rhizogenes to form hairy root cultures has the potential benefits of fast growth and rates of secondary metabolite production equal to or greater than that found for the intact plant.