Efficient Rutin and Quercetin Biosynthesis through Flavonoids-Related Gene Expression in Fagopyrum tataricum Gaertn. Hairy Root Cultures with UV-B Irradiation

Abstract

Transformed hairy roots had been efficiently induced from the seedlings of Fagopyrum tataricum Gaertn. due to the infection of Agrobacterium rhizogenes. Hairy roots were able to display active elongation with high root branching in 1/2 MS medium without growth regulators. The stable introduction of rolB and aux1 genes of A. rhizogenes WT strain 15834 into F. tataricum plants was confirmed by PCR analysis. Besides, the absence of virD gene confirmed hairy root was bacteria-free. After six different media and different sources of concentration were tested, the culturing of TB7 hairy root line in 1/2 MS liquid medium supplemented with 30 g l(-1) sucrose for 20 days resulted in a maximal biomass accumulation (13.5 g l(-1) fresh weight, 1.78 g l(-1) dry weight) and rutin content (0.85 mg g(-1)). The suspension culture of hairy roots led to a 45-fold biomass increase and a 4.11-fold rutin content increase in comparison with the suspension culture of non-transformed roots. The transformation frequency was enhanced through preculturing for 2 days followed by infection for 20 min. The UV-B stress treatment of hairy roots resulted in a striking increase of rutin and quercetin production. Furthermore, the hairy root lines of TB3, TB7, and TB28 were chosen to study the specific effects of UV-B on flavonoid accumulation and flavonoid biosynthetic gene expression by qRT-PCR. This study has demonstrated that the UV-B radiation was an effective elicitor that dramatically changed in the transcript abundance of ftpAL, FtCHI, FtCHS, FtF3H, and FtFLS-1 in F. tataricum hairy roots.

Keywords: UV-B; flavonoid biosynthetic genes; genetic transformation; hairy root; rutin; tartary buckwheat.

FIGURE 1

Effects of preculture time (A) and Agrobacterium rhizogenes infection time. (B) Development of hairy roots from a petiole segment within 10 days. (C) Development of hairy roots from cotyledon within 5–7 days. (D) On transformation frequency of Fagopyrum tataricum.

FIGURE 2

Polymerase chain reaction (PCR) analysis for the presence of rolB, aux1, and virD genes in hairy root lines of F. tataricum transformed by A. rhizogenes strain 15834. Lane M, marker. Lane 1, non-transformed roots [negative control (NC)]; Lane 2, plasmid DNA [positive control (PC)]; Lanes 4–13, transformed hairy root clones.

FIGURE 3

Rapidly growing hairy root line TB7 culture in liquid MS medium without growth regulators after 3 weeks.

FIGURE 4

Hairy root line TB7 growth as a function of time, and kinetics of rutin production. Points represent mean ± SE of three replicates. Each experiment was performed in triplicate. Means with common letters are not significantly different at P < 0.05 according to Duncan’s multiple range test.

FIGURE 5

The effects of UV radiation on transcript abundance of flavonoid biosynthetic genes. Data shown are the average mean ± SE of three replicates (n = 3). Different letters indicate statistical significane (P < 0.05) according to one-way ANOVA and Duncan’s multiple range test. (A) The expression of flavonoid biosynthetic genes in hairy root lines TB3, TB7, and TB28 of F. tataricum; (B) the expression of flavonoid biosynthesis genes in hairy root lines TB3, TB7, and TB28 of F. tataricum. after UV-B treatment.