The objective of this study was to determine a possible application of hairy roots as biotechnological producers of furanocoumarins, by establishing root clones of Glehnia littoralis after infection from Agrobacterium rhizogenes. None of the root clones grew under hormone-free conditions, but when cultured in the presence of indole-3-butyric acid, growth varied substantially across clones. Analysis of the insertion and expression of pathogenic rol (A, B TL , B TR , and C ) genes for 10 clones showed that the stable expression of rolC and irregular expression of rolB were detected in 2 clones, which exhibited active growth and higher furanocoumarin induction. Following ascorbic acid treatment, the productivity of xanthotoxin and bergapten in a root clone harbouring a pathogenic gene was 2.8 and 3.7 times greater than that recorded in wild-type roots, respectively. These results indicate that root cultures possessing pathogenic rol genes offer a potential means to produce furanocoumarins.
Key words: Agrobacterium rhizogenes, furanocoumarin induction, Glehnia littoralis, root clone.Plant Biotechnology 28, 317-321 (2011) DOI: 10.5511/plantbiotechnology.11.0330a
Metabolomics NoteAbbreviations: AsA, ascorbic acid; BMT, bergaptol O-methyltransferase; CTAB, cetyltrimethylammonium bromide; DMSO, dimethyl sulfoxide; DW, dry weight; HPLC, high performance liquid chromatography; IBA, indole-3-butyric acid; MeJA, methyl jasmonate, MS, Murashige and Skoog; PAL, phenylalanine ammonia-lyase; RT-PCR, reverse transcription-polymerase chain reaction; SA, salicylic acid; UBI, ubiquitin; WT, wild type; YE, yeast extract. This article can be found at http://www.jspcmb.jp/ Published online June 10, 2011 a wild-type (WT) root for 6 weeks. The number and length of roots formed from each root tip was counted each week, and the weight was measured at 6 weeks. Root segments started to form a callus during the initial 2 weeks, although the extent of callus formation varied across clones. Thereafter, root differentiation from the callus began, followed by root elongation, leading to differing extents of root propagation in the clones. In brief, 3 clones (Gl2, Gl33, and Gl37) were rich in callus, resulting in higher weights than that recorded in other roots (Figure 1), but fewer roots were produced than WT (Figure 2A). However, one clone (Gl6) produced the highest number of roots (31Ϯ5 per segment) after 6 weeks, with a value almost double that of WT roots (16Ϯ4 per segment; Figure 2A). The number of roots formed by other clones, including Gl4, was similar to that recorded for WT roots. With respect to root length, Gl6 formed the longest root (11.4Ϯ2.0 mm), followed by Gl4 (8.7Ϯ2.1 mm), while other clones, including WT (5.7Ϯ0.7 mm), showed similar growth after 6 weeks ( Figure 2B).Since differences among clones appeared to be associated with the nature of the T-DNA insert present in each individual root clone, the insertion and expression of T-DNA was analysed by PCR and RT-PCR, respectively. There are 2 types of T-DNA on Ri-pla...