An Optimized Tobacco Hairy Root Induction System for Functional Analysis of Nicotine Biosynthesis-Related Genes

Abstract

Rhizobium rhizogenes-mediated plant hairy root induction is a convenient method for functional study of root-specific genes. To develop an optimized tobacco hairy root induction system and study gene function in nicotine biosynthesis, we investigated hairy root induction by three R. rhizogenes strains, R1601, K599, and LBA9402, on different media with leaf discs from plants of different ages, and we observed that the strain LBA9402 used for explant infection exhibited the highest hairy root induction rate with 4 and 8 week old leaf discs of the tobacco ‘Coker176’ on 2/3MS medium, and it could also be used as a cargo delivering foreign genes to hairy roots. Overexpression of MsSPL12 gene, an alfalfa (Medicago sativa) SQUAMOSA promoter binding protein-like (SPL) transcription factor, significantly improved nicotine production in transgenic hairy roots, reaching 1.38–1.85 mg/g compared to 0.5 mg/g of the controls. Expression analysis of the nicotine biosynthesis and transport-related genes responding to methyl-jasmonate (MeJA) treatment revealed a significant upregulation of NtMPO2 responsible for increased nicotine biosynthesis in MsSPL12 transgenic hairy roots. Our results establish a high-throughput approach for gene functional characterization in the hairy roots of a tobacco elite cultivar, ‘Coker176’, as well as suggest a system for efficiently manipulating tobacco nicotine biosynthesis.