Light and Auxin Choices Steer Medicine-Making Power of White Wormwood Cell Cultures

A humble shrub that grows across south-eastern Europe may hold a lesson for how laboratories of the future manufacture plant-based medicines. Artemisia alba Turra, an essential-oil-bearing wormwood long used in Mediterranean folk medicine as a tonic and digestive aid, produces a rich arsenal of protective chemicals: volatile terpenes in its aromatic oils, and non-volatile polyphenols such as flavonoids and phenolic acids that show anti-inflammatory, antimicrobial, antioxidant and even pro-apoptotic activity in laboratory preparations. A new study published in the journal BioRisk has now mapped, in fine biochemical detail, how light and synthetic growth hormones control just how much of these valuable polyphenols its cells will make when grown entirely outside the plant.

The research, carried out on non-differentiated cell aggregate cultures initiated from sterile-grown Artemisia alba plants, set out to answer a deceptively simple question: if you strip the plant of its organized leaves, stems and roots and grow its cells in liquid flasks, can you still coax it into industrial-scale biosynthesis, and which recipe of light and hormones gets you the most product? The answer, according to the team, is a resounding yes, with important caveats about which tissues to start from and which chemical signals to send.

The experimental design was elegant in its symmetry. Leaf and root explants were placed into liquid Murashige and Skoog medium supplemented with the cytokinin benzyladenine and one of two auxins, indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA). Half the cultures were grown under a 16/8-hour light-dark photoperiod, half in complete darkness, producing eight distinct experimental lines that were tracked over six months and three passages. The researchers measured fresh-to-dry weight ratios, examined the cultures under a stereomicroscope, and quantified stress markers including malondialdehyde, a product of lipid peroxidation, and hydrogen peroxide, alongside spectrophotometric assays of total phenolics and flavonoids.

The morphological story unfolded in stages. Two months after induction, remnants of differentiated organs and tissues were still visible, more so in root-derived lines and in cultures treated with NAA. Light-grown cultures displayed intense green pigmentation, a visual signature of photosynthetic pigment formation, while dark-grown lines were etiolated and pale. By four months, the degree of differentiation had dropped markedly, and by six months all eight lines were completely de-differentiated, forming cell aggregates that were larger in leaf-derived lines and smaller but denser in root-derived ones.

The choice of auxin left a clear physical fingerprint on the cultures. IBA-treated lines formed bulky cell aggregates that were loose, brittle and less dense, and they de-differentiated more readily. NAA-treated lines resisted de-differentiation longer, particularly those from root explants, but once stabilized they produced smaller, significantly more compact aggregates with better dry biomass accumulation, reflected in a lower fresh-to-dry weight ratio. Root-derived lines also showed persistent hyperhydricity, an excess water content in the early stages, and sporadic root morphogenesis that survived several passages, suggesting they were harder to fully convert into a homogeneous cell culture.

The stress biochemistry told an equally coherent story. Light-grown lines showed elevated levels of both malondialdehyde and hydrogen peroxide compared with their dark-grown counterparts, a pattern the authors attribute to reactive oxygen species generated by ongoing photosynthetic activity. Leaf-derived lines were especially affected: when grown in the light they retained some photosynthetic capacity and correspondingly high levels of both stress markers, but in darkness they lost their green pigmentation entirely and both markers dropped sharply, indicating less oxidative stress and less lipid peroxidation. Intriguingly, in light-grown root lines, high hydrogen peroxide levels coincided with lower malondialdehyde, hinting that the root tissue mounted an effective antioxidant defense that protected its membranes from oxidation.

But the headline result concerns the polyphenols themselves, and here the data converge on a striking conclusion. Cultures grown under the 16/8-hour photoperiod produced higher amounts of phenolic compounds than those kept in the dark, consistent with the idea that phenolics are part of the plant cell’s stress response and that light acts as a stressor. More unexpectedly, root-derived lines significantly outperformed leaf-derived ones in biosynthetic capacity, and within the root lines, NAA treatment boosted total phenolic content by 58 to 140 percent compared with IBA. Flavonoid levels followed the same pattern, with light-grown and NAA-treated root lines leading the pack.

The single best performer across all four measured parameters, lipid peroxidation, oxidative stress, phenolic productivity and flavonoid productivity, was the root-derived line treated with NAA and grown in the light, designated A2 ER_3NAA hv. Despite a watery start after induction, its hydration dropped substantially after the second passage, and it emerged as the most productive line for the target compounds. Its dark-grown NAA sibling, A2 ER_3NAA, was close behind. The authors suggest these lines could serve as the foundation for future high-yielding root-based production systems for Artemisia alba secondary metabolites.

Why would the tissue that resists de-differentiation the most also be the best chemical factory? The researchers propose that the elevated phenolic and flavonoid levels in root-derived lines may be linked to their impaired ability to fully de-differentiate. Flavonoids are known to be involved in controlling plant cell growth and differentiation, and they carry well-documented pharmacological properties, including modulation of enzymes such as cyclo-oxygenase and lipoxygenase, detoxification of carcinogens, and other health benefits. In other words, the very developmental stubbornness of the root cells may be the biochemical engine driving their medicinal output.

Beyond the specific case of white wormwood, the study carries a broader message for plant biotechnology: secondary metabolite productivity in vitro is not a fixed trait but a dynamic process tightly coupled to growth, development and the cell’s interaction with its environment. Light, hormone type and tissue of origin all interact to shape both the morphology of a culture and its chemical profile. For an industry increasingly interested in producing plant-derived pharmaceuticals in bioreactors rather than fields, the lesson from Artemisia alba is that the recipe matters as much as the ingredient, and that sometimes the most productive cells are the ones that refuse to forget what they were.

Subject of Research: Effects of light and auxin treatments on morphogenesis and polyphenolic compound productivity in in vitro cell aggregate cultures of Artemisia alba Turra

Article Title: Light and auxin treatments affect morphogenesis and polyphenolics productivity in Artemisia alba Turra cell aggregates in vitro

Article References: Pecheva, D., & Danova, K. (2022). Light and auxin treatments affect morphogenesis and polyphenolics productivity in Artemisia alba Turra cell aggregates in vitro. BioRisk, 17, 213-225. https://doi.org/10.3897/biorisk.17.77452

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77452

Keywords: Artemisia alba, plant cell culture, polyphenolics, flavonoids, auxin, NAA, IBA, secondary metabolites, in vitro biotechnology, oxidative stress, photoperiod, morphogenesis