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

Shangqian Qin,Shiwen Lin,Bingwu Wang,Wanjun Zhang,Yanrong Liu,Jianping Yan

doi:10.3390/agronomy12020348

Shangqian Qin, Shiwen Lin + Show 4 more

Open Access

https://doi.org/10.3390/agronomy12020348

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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.

Highlights

Rhizobium rhizogenes, previously referred to as Agrobaterium rhizogenes, is a soil-borneGram-negative microbe that can infect dicots and induce hairy roots at the infected site [1]
We noticed the explants from the 2 week old leaves became necrotic after cocultivation, and the hairy root induction rate was the lowest compared to the other transformations (Figure 2)
We studied the effects of organic ingredients on the efficiency of hairy root induction for 4 week old leaf discs by R. rhizogenes strain LBA9402 infection

Summary

Introduction

Previously referred to as Agrobaterium rhizogenes, is a soil-borneGram-negative microbe that can infect dicots and induce hairy roots at the infected site [1]. R. rhizogenes-mediated plant transformation has been widely used in the production of secondary metabolites and is increasingly applied to studying plant secondary metabolism and/or gene expression regulation pathways due to the simple transformation procedure and the rapid growth of the hairy root [4,5]. Hairy root induced by R. rhizogenes infection has been used in tobacco for functional gene investigation [3]. Co-expression of basic helix–loop–helix protein (bHLH) and transcriptional activator Myb genes reportedly induced anthocyanin production in tobacco hairy roots [6]. Considering the culturing time needed, induction of hairy root by R. rhizogenes has many advantages over traditional transgenic technology in the study of gene function and regulation of secondary metabolism [9]

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