The Optimization of Medium Conditions and Auxins in the Induction of Adventitious Roots of Pokeweed (Phytolacca americana L.) and Their Phytochemical Constituents

Abstract

Pokeweed, Phytolacca americana L., is considered a widely spreading invasive plant, while saponin contents accumulated in the roots have pharmaceutical uses, such as rheumatism treatments and anti-inflammation. Adventitious root cultures are an important source of diverse secondary metabolites, which have significant applications in various fields. This study focused on the optimization of parameters for root induction using different medium states and treatments with auxins on a pokeweed leaf. Semisolid and liquid MS (Murashige and Skoog, 1962) media were supplemented with indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) at 0.5, 1, 2, and 4 mg/L. Root growth parameters, e.g., induction percentage, root numbers, length, and weight, were measured to determine the adventitious root induction efficiency. Total phenolic content, total flavonoid content, total saponin content, and antioxidant activity were recorded. Results showed that adventitious roots induced in semisolid MS medium supplemented with 0.5 mg/L NAA exhibited a high density of lateral roots. Appropriate medium state and auxin for adventitious root induction in pokeweed were determined as semisolid medium supplemented with 2 mg/L NAA. Considering phytochemicals, adventitious roots induced in liquid medium containing 0.5-1 mg/L NAA had the highest yield extract percentage. Additionally, adventitious roots cultivated in a liquid medium enriched with 1 mg/L NAA exhibited the highest phenolic and saponin contents. A principal component analysis (PCA) biplot and hierarchical cluster analysis (HCA) heatmap demonstrated different response patterns between semisolid and liquid media applied with NAA. The results of the semisolid media were grouped together due to high expression levels of the root induction parameters, while elevated phytochemical values were observed in the liquid media treatments. The results suggested two different media that provide the highest adventitious root induction efficiency and the greatest phytochemical contents: semisolid medium with 2 mg/L NAA and liquid medium with 1 mg/L NAA, respectively. These culture media can be applied to optimize adventitious root culture of pokeweed and in vitro phytochemical production.

Figure 1

Adventitious root induction from pokeweed leaf explants cultured on semisolid and liquid media containing NAA and IBA: (a) semisolid culture with 2 mg/L NAA; (b) leaf explants with adventitious roots from semisolid culture with 1 mg/L NAA; (c) semisolid culture with 1 mg/L IBA; (d) leaf explant with adventitious roots from semisolid culture with 1 mg/L IBA; (e) liquid culture with 0.5 mg/L NAA; (f) leaf explant with adventitious roots from liquid culture with 1 mg/L NAA; (g) liquid culture with 1 mg/L IBA; (h) leaf explant with adventitious roots from liquid culture with 1 mg/L IBA (yellow arrow = adventitious root; white arrow = lateral root; arrow head = root hair; all scales = 1 cm).

Figure 2

Length of adventitious and lateral roots (a, b) and fresh and dry weight of pokeweed roots (c, d) obtained from semisolid and liquid cultures with NAA and IBA. Data are represented by means and bars represent ± SE, with different letters indicating significant differences by two-way ANOVA and LSD (p  <  0.05).

Figure 3

PCA biplot showing the relation among parameters and their influence on the treatments (ARP = adventitious root induction percentage; ARN = adventitious root number; ARL adventitious root length; LRN = lateral root number; LRL = lateral root length; FW = fresh weight; DW = dry weight; YP = yield percentage; TPC = total phenolic content; TFC = total flavonoid content; TSC = total saponin content; DPPH = antioxidant activity by DPPH assay).

Figure 4

The HCA heatmap explaining the response patterns of growth and phytochemical constituents due to different culture treatments (ARP = adventitious root induction percentage; ARN = adventitious root number; ARL adventitious root length; LRN = lateral root number; LRL = lateral root length; FW = fresh weight; DW = dry weight; YP = yield percentage; TPC = total phenolic content; TFC = total flavonoid content; TSC = total saponin content; DPPH = antioxidant activity by DPPH assay).