Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production

Abstract

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L^-1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L^-1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 μM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Keywords: HPLC; bellidifolin; osmotic stress; secondary metabolites; shoot culture.

Figure 1

Gentianella lutescens subsp. carpatica in a natural habitat on Povlen mountain (locality Razbojište), Serbia.

Figure 2

HPLC profile (λ = 260 nm) of methanol extract of G. lutescens. Peaks: 1—swertiamarin, 2—gentiopicrin, 3—mangiferin, 4—campestroside, 5—isoorientin, 6—demethylbellidifolin-8-O-glucoside, 7—swertisin, 8—bellidifolin-8-O-glucoside, 9—veratriloside, 10—demethylbellidifolin, 11—bellidifolin.

Figure 3

Chemical structures of secoiridoid and xanthone compounds identified in G. lutescens. 1—swertiamarin, 2—gentiopicrin, 3—mangiferin, 4—campestroside, 5—isoorientin, 6—demethylbellidifolin-8-O-glucoside, 7—swertisin, 8—bellidifolin-8-O-glucoside, 9—veratriloside, 10—demethylbellidifolin, 11—bellidifolin.

Figure 4

In vitro propagation of G. lutescens. (A) Open pod with immature seeds (bar = 5 mm), (B) shoot multiplication on BM + 0.2 mg L⁻¹ BA (bar = 10 mm), (C) root elongation in PGR-free ½ MS liquid medium after treatment of shoots with 5.0 mg L⁻¹ IBA for 2 days (bar = 10 mm).

Figure 5

The content of xanthones mangiferin (A), demethylbellidifolin-8-O-glucoside (DMB-8-O-glc) (B), bellidifolin-8-O-glucoside (bell-8-O-glc) (C), demethylbellidifolin (DMB) (D) bellidifolin (E) in five shoot lines of G. lutescens cultured on BM + 0.2 BA mg L⁻¹ for 35 days. Wild G. lutescens herb grown in nature was used as a control. Values are the means ± SE of four to six biological replicates (n = 4–6). Data of xanthones content in shoot lines were analyzed by one-way ANOVA (F). The values followed by different letters were significantly different according to Fisher’s LSD test at p ≤ 0.05; asterisk (*) indicate a significant difference of values between in vitro shoot lines and wild plants samples according to Student’s t-test at p ≤ 0.05.

Figure 6

The effect of increasing concentrations (58.4, 161.8, 175.2, and 233.6 mM) of sucrose (A–D) and sorbitol (E–H) on the growth of shoot cultures of G. lutescens (line 5) after 35 days of culture. The increasing concentrations of sucrose or sorbitol were added into control medium (BM + 0.2 mg L⁻¹ BA containing 58.4 mM sucrose). Values are the means ± SE of eight to ten biological replicates (n = 8–10). Data were analyzed by one-way ANOVA. Values followed by different letters are significantly different according to Fisher’s LSD test at p ≤ 0.05.

Figure 7

The effect of sucrose at increasing concentrations (58.4, 161.8, 175.2, and 233.6 mM) on the growth and flowering of shoot cultures of G. lutescens (line 5) after 35 days of culture (bar = 20 mm). The increasing concentrations of sucrose were added into control medium (BM + 0.2 mg L⁻¹ BA containing 58.4 mM sucrose). Arrows indicate floral bud development. Right—shoot with normally developed flower regenerated on the medium with 175.2 mM sucrose (bar = 5 mm).

Figure 8

The effect of increasing concentrations (58.4, 161.8, 175.2, and 233.6 mM) of sucrose (A–E) and sorbitol (F–J) on the content of xanthones mangiferin, demethylbellidifolin-8-O-glucoside (DMB-8-O-glc), bellidifolin-8-O-glucoside (bell-8-O-glc), demethylbellidifolin (DMB) and bellidifolin in shoots cultures of G. lutescens (line 5) after 35 days of culture. The increasing concentrations of sucrose or sorbitol were added into control medium (BM + 0.2 mg L⁻¹ BA containing 58.4 mM sucrose). Values are the means ± SE of four to eleven biological replicates (n = 4–11). Values denoted by the same letter are not significantly different according to the Fisher’s LSD test at p ≤ 0.05 following one-way ANOVA.

Figure 9

Effect of elicitors jasmonic acid (JA), methyl jasmonat (MeJA) and salicilic acid (SA) at increasing concentrations (100, 200, 300, and 500 µM) on the growth index and xanthone production of shoot cultures of G. lutescens (line 5). Growth index (A), mangiferin (B), DMB-8-O-glc (C), bell-8-O-glc (D), DMB (E), and bellidifolin (F) content. Data represent means ± SE of four to seven biological replicates (n = 4–7). Values denoted by the same letter are not significantly different according to the Fisher’s LSD test at p ≤ 0.05 following ANOVA multifactorial analysis.