Regeneration of buckwheat plantlets from hypocotyl and the influence of exogenous hormones on rutin content and rutin biosynthetic gene expression in vitro

We developed an in vitro regeneration system using the buckwheat species, Fagopyrum esculentum (Wenshatianqiao, WS) and Fagopyrum tataricum (Jiujiangkuqiao, JJ) in order to test the relationship between biosynthesis and accumulation of rutin and expression of genes encoding rutin biosynthetic enzymes. Hypocotyl explants were incubated on MS solid medium supplemented with 2,4-D and different concentrations of BA to optimize callus production from ‘WS’ and ‘JJ’, respectively. We also compared the rutin content in F. esculentum and F. tataricum plantlets regenerating with and without BA, after treatment with salicylic acid (SA) and methyl jasmonate (MeJA) for 12, 24, 48 h. The highest rutin content (5.01 mg gFW⁻¹) in regenerating plantlets was found in F. tataricum treated with SA for 24 h. We found no significant accumulation of rutin in F. esculentum. Following qRT-PCR analysis, gene expression patterns of rutin-biosynthesis-related genes regulated by SA and MeJA were found to be significantly different between the two buckwheat species. Four genes (FtCHS, Ft4CL, FtF3H and FtFLS) were up-regulated at different time points in ‘JJ’ following application of BA, SA, and MeJA, especially after 24 h MeJA treatment, where the FtCHS expression level was 11.55 times that of control. In ‘WS’ following the same treatment regime only two genes, FeCHS (2.67-fold) and Fe4CL (2.42-fold), were significantly up-regulated by MeJA and only at the latest time point (48 h); results for other genes and treatments were found to be close to or even lower than the control. These results provide an improved method for F. esculentum regeneration, extend the process to F. tataricum, and suggest that, although the gene expression patterns were quite different in F. esculentum and. F. tataricum, rutin content could be enhanced to a great extent by exogenous hormones.