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. 2022 Aug 5;11(8):1526.
doi: 10.3390/antiox11081526.

Linum lewisii Adventitious and Hairy-Roots Cultures as Lignan Plant Factories

Roméo Arago Dougué Kentsop  1 , Roberto Consonni  2 , Michela Alfieri  2 , Marina Laura  3 , Gianluca Ottolina  2 , Iride Mascheretti  1 , Monica Mattana  1
Affiliations

Linum lewisii Adventitious and Hairy-Roots Cultures as Lignan Plant Factories

Roméo Arago Dougué Kentsop et al. Antioxidants (Basel). .

Abstract

Plants synthesize specific secondary metabolites for survival, reproduction, environmental resilience, and defense. Among them, lignans are a class of polyphenols with several bioactive properties: chemopreventive, anti-inflammatory, antiviral, and antioxidant. These compounds are often extracted from field-grown plants with very low yields. To overcome these constraints, in vitro tissue cultures provide a tool to optimize large-scale production. Moreover, the use of elicitation to increase secondary metabolite production is gaining importance. The aim of this work was to develop adventitious (ARL) and hairy roots (HRL) from Linum lewisi, a species able to synthesize arylnaphthalene lignans such as justicidin B. The ARL and HRL were obtained for the first time and characterized for their phenol content, antioxidant activity, and the production of justicidin B after treatments with several elicitors and precursor feeding. Through NMR spectroscopy, other four lignans were highlighted and identified in the roots extracts. A pilot-scale bioreactor was adopted to assess the suitability of the developed root cultures for future large-scale production. The ARL and HRL cultures showed a justicidin B production higher than other Linum species cultures described up to now (75.8 mg/L and 82.2 g/L), and the production more than doubled after elicitation with MeJA.

Keywords: antioxidant capacity; elicitation; flax; justicidin B; lignans; precursor feeding; tissue cultures.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Biomass of L. lewisii hairy and adventitious root cultures expressed as gram (g) of FW per flask; (B) total phenol content expressed as µg GAE/mgDW; (C) radical scavenging activity expressed as % inhibition DPPH/mgDW. From HRL1 to HRL7: hairy root lines, ARL1 and ARL2: adventitious root lines. The analyses were performed on roots extracts after six weeks of growth. Each bar represents the average of three biological replicates ± SD.
Figure 2
Figure 2
(A) Picture of HRL6 and ARL1 cultures at 0, 4, and 6 weeks. (B) Growth kinetic of Linum lewisii HRL6 and ARL1 cultures. FW: fresh weight expressed as grams (g) per flask. Each value represents the average of three biological replicates ± SD. Student’s t-test was applied. (C) Accumulation of justicidin B in HRL6 and ARL1 extracts expressed as mg of justicidin B.
Figure 3
Figure 3
1H NMR spectrum of isolated isojusticidin B (left) containing traces of secoisolariciresinol (right). The blue marks belong to unassigned compounds.
Figure 4
Figure 4
1H NMR spectrum of tuberculatin.
Figure 5
Figure 5
1H NMR spectrum of 7-O-β-d-xylofuranosyl-(1→5)-O-β-d-xylofuranosyl-diphyllin.
Figure 6
Figure 6
1H NMR spectrum of 7-O-β-d-xylofuranosyl-(1→5)-O-β-d-xylofuranosyl-(1→5)-O-β-d-glucosyl-diphyllin.
Figure 7
Figure 7
HRL6 and ARL1 cultures, control (Ctrl), and treated tissues with the elicitors and/or precursor feeding (left panel). Biomass accumulation of HRL6 and ARL1 expressed as FW (grams) in non-treated (Ctrl) and treated with MeJA, COR, SA, FA, MeJA + FA, and COR + FA (right panel). Each bar represents the average of three biological replicates ± SD.
Figure 8
Figure 8
Total phenolic and radical scavenging activity in Linum lewisii root cultures non-treated (control) and treated with methyl jasmonate (MeJA), coronatine (COR), 50 µM (SA 50 µM), and 100 µM (SA 100 µM) salicylic acid, ferulic acid (FA), MeJA and FA (MeJA + FA) and COR and FA (COR + FA). (A) Total phenol content expressed as µg of gallic acid equivalent (GAE) per mg dry weight (DW). (B) Radical scavenging activity expressed as percentage of inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) per mg DW. Each bar represents the average of three biological replicates ± SD. Student’s t-test was applied: * indicates significance of treated samples versus control per each tissue (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001); apex letters indicate significance of the same treatment between the two tissue cultures, a: control, b: MeJA, c: COR, d: SA 50, e: SA 100, f: FA, g: MeJA + FA, h: COR + FA (a p ≤ 0.05; aa p ≤ 0.01, aaa p ≤ 0.001).
Figure 9
Figure 9
Accumulation of justicidin B in Linum lewisii root cultures extracts. (A) justicidin B expressed as mg/DW. (B) Justicidin B expressed as mg/flask. Each bar represents the average of three biological replicates ± SD. Student’s t-test was applied: * indicates significance of treated samples versus the respective control per each tissue (** p ≤ 0.01; *** p ≤ 0.001).
Figure 10
Figure 10
Linum lewisii HRL6 in bioreactor after four weeks of growth.
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