In Vitro Strategy for the Enhancement of the Production of Bioactive Polyphenols in Transformed Roots of Salvia bulleyana

Abstract

The underground parts of Salvia bulleyana, a rare Chinese plant species, have long been used in traditional Chinese medicine. The Rhizobium rhizogenes-transformed root culture obtained from this plant might be a promising novel source of valuable phenolics, including rosmarinic acid. The present study identifies for the first time, the optimal growth conditions of S. bulleyana hairy roots regarding production efficiency. The comprehensive optimization comprised cultivation in different basal media (B5, SH, MS, and WP) with full- and half-strength macro- and microelements, different vitamin contents (full, half, one-quarter part, and without) and sucrose concentrations (2, 3, 4, 5%), and under different light conditions: in dark, under blue LED (λ = 430 nm), red LED (λ = 670 nm), mixed blue and red LED (30%:70%), and white LED (390-670 nm). Hairy root growth and bioactive compound accumulation were also detailed every five days over the 50-day culture cycle. The optimal conditions were determined using a technique for order preference by similarity to the ideal solution (TOPSIS). The most efficient combination for root growth and polyphenol content was found to be ½SH liquid medium with half vitamin concentration and 3% sucrose when grown in the dark. The biomass yield during the growth cycle was 6.1 g (fresh weight-FW) and 0.92 g (dry weight-DW) on one Erlenmeyer flask: a 14.3-fold increase in FW and 16.1-fold increase in DW in relation to the inoculum. The highest mean total phenolic content was 93.6 mg/g DW including about 70 mg/g DW rosmarinic acid, reached on day 40 of culture; compared to roots of two-year-old plants grown under field conditions, the total phenolic acid content was four times higher and rosmarinic acid eight times higher. The obtained results place the investigated culture among the best hair root cultures for rosmarinic acid production.

Keywords: TOPSIS method; basal medium selection; genetic transformation; light spectra; plant biotechnology; rosmarinic acid; salvianolic acid K.

Figure 1

Growth indexes of transformed roots of S. bulleyana received for fresh (GI FW) and dry (GI DW) weight during cultivation in different media for five weeks. The results are mean values ± SE. Different letters indicate statistical differences between samples separately for FW and DW.

Figure 2

Transformed roots of S. bulleyana C4 clone cultivated in different media after five weeks (scale 1 cm).

Figure 3

Growth indexes of transformed roots of S. bulleyana received for fresh (GI FW) and dry (GI DW) weight during cultivation in SH and ½SH medium supplemented with different concentrations of vitamins (FV—full vitamin, ½FV—half strength of vitamins, ¼FV—quarter of strength of vitamins, 0V—without vitamins) for five weeks. The results are mean values ± SE. Different letters indicate statistical differences between samples separately for FW and DW.

Figure 4

Growth indexes of transformed roots of S. bulleyana received for fresh (GI FW) and dry (GI DW) weight during cultivation in ½SH medium with ½ vitamin concentration supplemented with different sucrose concentrations (2–5%) for five weeks. The results are mean values ± SE. Different letters indicate statistical differences between samples separately for FW and DW.

Figure 5

Growth indexes of transformed roots of S. bulleyana received for fresh (GI FW) and dry (GI DW) weight during cultivation in ½SH medium with ½ vitamin concentration under different light conditions (W—white, M—mixed, B—blue, R—red, D—dark) for five weeks. The results are mean values ± SE. Different letters indicate statistical differences between samples separately for FW and DW.

Figure 6

Time course of fresh and dry weight of S. bulleyana transformed roots cultivated for 50 days in ½SH liquid medium with ½ vitamin concentration. The results are mean values ± SE.

Figure 7

Time course of polyphenolic acid content in S. bulleyana transformed roots cultivated for 50 days in ½SH liquid medium with ½ vitamin concentration. CA—caffeic acid, RAH—rosmarinic acid hexoside, SAE—salvianolic acid E, RA—rosmarinic acid, SAK—salvianolic acid K, CAD I—caffeic acid derivative I, MR—methyl rosmarinate, SAF I—salvianolic acid F isomer I, SAF II—salvianolic acid F isomer II. The results are mean values ± SE.