Abstract
The application of plant suspension culture to produce valuable compounds, such as the triterpenoids oleanolic acid and ursolic acid, is a well-established alternative to the cultivation of whole plants. Cambial meristematic cells (CMCs) are a growing field of research, often showing superior cultivation properties compared to their dedifferentiated cell (DDC) counterparts. In this work, the first-time establishment of O. basilicum CMCs is demonstrated. DDCs and CMCs were cultivated in shake flasks and wave-mixed disposable bioreactors (wDBRs) and evaluated regarding triterpenoid productivity and biomass accumulation. CMCs showed characteristic small vacuoles and were found to be significantly smaller than DDCs. Productivities of oleanolic and ursolic acid of CMCs were determined at 3.02 ± 0.76 mg/(l*d) and 4.79 ± 0.48 mg/(l*d) after 19 days wDBR cultivation, respectively. These values were consistently higher than any productivities determined for DDCs over the observed cultivation period of 37 days. Elicitation with methyl jasmonate of DDCs and CMCs in shake flasks resulted in increased product contents up to 48 h after elicitor addition, with the highest increase found in CMCs at 232.30 ± 19.33% (oleanolic acid) and 192.44 ± 18.23% (ursolic acid) after 48 h.
Highlights
The perennial herb Ocimum basilicum, commonly known as basil, is valued worldwide for its culinary uses, but its applications go well beyond that
To assess productivity in wave-mixed disposable bioreactors (wDBRs), a BIOSTAT RM 20 basic rocker (Sartorius AG, Göttingen, Germany) with 2 l Flexsafe RM basic SC bags was used. 1 g/l cell wet weight (CWW) dedifferentiated cells (DDCs) or Cambial meristematic cells (CMCs) from precultures were incubated in their respective liquid media for 37 days at 7° tilt, 20 rpm, 50 ml/min aeration with ambient air and 28 °C in darkness. 1 ml samples containing medium and biomass were drawn in a 3 days-rhythm starting after 4 days
Abundance of small vacuoles and generally small cell size compared to DDCs from the same species are distinguishing features widely described for CMCs (Lee et al 2010; Song et al 2019; Zhou et al 2015).To confirm the observed structures as small vacuoles in CMCs, they were stained with neutral red, which penetrates the cell wall and membranes, accumulating in vacuoles (Fig. 1c)
Summary
The perennial herb Ocimum basilicum (family Lamiaceae), commonly known as basil, is valued worldwide for its culinary uses, but its applications go well beyond that. An interesting type of plant cell culture are cambial meristematic cells (CMC), which are derived from vascular cambium. These cells were described to grow at a faster rate, aggregate less and accumulate more product than dedifferentiated cells (DDCs) of the same plant (Ochoa-villarreal et al 2015). CMCs were described for Taxus cuspidata (Lee et al 2010), Catharanthus roseus (Moon et al 2015; Zhu et al 2018) and Tripterygium wilfordii (Song et al 2019) among others, but not yet for O. basilicum
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