Oldenlandia affinis subsp. suspension

Doffek, Benjamin, Huang, Yvonne, Huang, Yen-Hua, Chan, Lai Yue, Gilding, Edward K., Jackson, Mark A. & Craik, David J., 2022, Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming, Phytochemistry (113053) 195, pp. 1-10 : 4

2.3. Chemical elicitation of O. affinis suspension cells to recover expression of kalata B1

Media compositions tested for suspension cultures.

Given that cyclotide profiles have previously been shown to differ between in vitro and field grown O. affinis ( Mylne et al., 2010) , we were interested to determine the effect that chemical elicitors may have on restoring the expression of cyclotides in O. affinis suspension cells ( Fig. 4 ). A series of treatments representing inputs from biotic (methyl jasmonate, MeJA) and abiotic (NaCl) stresses were applied to O. affinis suspension cell cultures. These treatments were: 3 μM MeJA, 50 μM MeJA, 100 μM MeJA, 3 mM NaCl, 50 mM NaCl, 200 mM NaCl. The only elicitor treatment that consistently and significantly induced accumulation of cyclotides was NaCl (3 mM final concentration). However, only the levels of existing suspension cell expressed cyclotides (kB23 and cyO2) were improved ( Fig. 4A ). Using light microscopy, suspension cells treated with 3 mM NaCl displayed normal morphology at day 1 but at day 7 the cells were noticeably distended as were cells treated with 50 mM NaCl ( Fig. 4B–D ). At 200 mM NaCl cell death had occurred within 4 days (data not shown). Additional work is required to determine the molecular basis of this cyclotide upregulation, which might be caused by changes in osmotic pressure or upregulation of genes associated with plant cell death, including that of AEPs ( Hara-Nishimura et al., 2005).

Figures

Fig. 4. Inducibility of cyclotide accumulation in O. affinis suspension cells. A Cyclotide accumulation profiles in O. affinis suspension cells exposed to biotic (methyl jasmonate, MeJA) and abiotic (NaCl) signals. Replicate (n =3) cultures were assayed 1, 4 and 7 days after addition of inducer. Treatments highlighted by an asterisk are significantly different (P <0.05). Cell morphology of B day 1 cells in 3 mM NaCl, C day 7 cells in 3 mM NaCl and D day 7 cells in 50 mM NaCl.