Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn)

Abstract

The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum 'Hokkai T10' cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3(,) H1, FtF3'H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat.

Keywords: Agrobacterium rhizogenes; HPLC; anthocyanin; polypropanoid biosynthetic genes; rutin; tartary buckwheat.

FIGURE 1

Flavonoid pathway scheme. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavones 3-hydroxylase; FLS, flavonol synthase; F3’H, flavonoid 3′-hydroxylase; ANS, anthocyanidin synthase; DFR, dihydroflavonol-4 reductase.

FIGURE 2

Sequential stages of hairy root induction in Fagopyrum tataricum ‘Hokkai T10’. (a) Hokkai T10 seeds. (b) De-hulled seeds. (c) Germinated seeds on a ½ MS solid medium. (d) Three-day-old seedlings. (e) Five-day-old seedlings transferred to the Magenta box. (f) Explant preparation for Agrobacterium infection. (g) Explant infection in Agrobacterium inoculum. (h) Hairy root induction. (i) Profuse hairy roots 10 days after infection. (j) Hairy root growth 2 weeks after infection. (k) Rapid hairy root growth on a fresh medium. (l) Hairy root growth 14 days after culture in a ½ MS liquid medium.

FIGURE 3

Development of F. tataricum ‘Hokkai T10’ hairy roots as influenced by different A. rhizogenes strains. Data from three replicates were compared 14 days after culturing in a ½ MS liquid medium. Mean values indicated by the same letter in a column are not different at P < 0.05, according to Duncan’s multiple range test.

FIGURE 4

Expression levels of phenylpropanoid biosynthetic pathway genes observed using qRT-PCR at 14 days after culturing of hairy roots. Expression level of each gene is relative to that of H3 (Ar1 = R1000, Ar2 = R1200, Ar3 = 13333, Ar4 = 15834, Ar5 = R1601, Ar6 = LBA9402, Ar7 = A4).