Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

Abstract

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 lM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 lM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC-ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g -1 dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g -1 dry wt. and 9.97 mg g -1 dry wt., respectively) comparable to those found in root tubers.

Establishment of Tripterygium wilfordii Hook. f. Hairy Root Culture and Optimization of Its Culture Conditions for the Production of Triptolide and Wilforine

“…For the induction of hairy roots, A. rhizogenes strain A4 had a higher induction rate than ATCC 15834, although both could induce hairy roots. This result is consistent with previous reports that different types of Agrobacterium strains of A. rhizogenes would have a different efficiency in inducing hairy roots [8,20]. For both inducing and maintenance of hairy roots in T. wilfordii, we found that 1/2MS was better than the other media.…”

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS

“…4.5 μg/g DW. In other experiments with hairy root cultures of this species [ 8 , 9 ], the amounts of iridoids in the biomass from the in vitro cultures were also considerably lower than in the raw material from the soil-grown plant, and were as follows: 0.32 mg/g DW (Hpd [ 55 ]) and 0.66 mg/g DW (total iridoid content [ 56 ]). Those results, and also our own results on the shoot cultures of M .…”

Phenylpropanoid Glycoside and Phenolic Acid Profiles and Biological Activities of Biomass Extracts from Different Types of Verbena officinalis Microshoot Cultures and Soil-Grown Plant

“…Very high concentrations of verbascoside were reported in suspension cultures of Buddleja cordata (11.6 g/100 g DW) [ 47 ], Plantago media (9.16 g/100 g DW) [ 48 ], and cultures of Cistanche salsa (689 mg/L) [ 49 ] and H. procumbens maintained in pulse-aerated column bioreactors (165.42 mg/L/day) [ 50 ]. Depending on the cultivation method, high levels of verbascoside (from 2.2 to 6.0 g/100 g DW) as well as high levels of isoverbascoside (maximum content: from 0.8 to 1.8 g/100 g DW) were obtained in the hairy root cultures of Rehmannia glutinosa [ 51 , 52 , 53 ]. In the microshoot cultures of Scutellaria baicalensis maintained on MS medium, the content of verbascoside reached 830.9 mg/100 g DW [ 54 ], in the microshoot cultures of Scutellaria altissima it was 0.5 g/100 g DW [ 55 ] and in microshoot cultures of Scutellaria lateriflora maximal content on MS medium was 381.73 mg/100 g DW [ 56 ].…”