An effective procedure for producing transformed Centaurium erythraea plants from synthetic seeds is described. Explants were encapsulated in 3% sodium alginate with 3% sucrose. Encapsulated hairy roots were cultured on half-strength Murashige and Skoog (1/2 MS) or Woody Plant (WPM) agar-solidified regeneration media supplemented with 6-benzylaminopurine (BAP) or without the cytokinin. The use of WPM with 0.5 mg/L of BAP gave the best shoot formation frequency (86%) and mean number of shoots (15) per root segment. Shoots rooted with 97% frequency on 1/2 MS without growth regulators. Encapsulated shoot buds were cultured on onethird- strength MS agar medium (1/3 MS) supplemented with indole-3-butyric acid (IBA) (0.05 mg/L). The plantlet conversion frequency was 32%. The encapsulated hairy roots and shoot buds were stored for 4, 6 or 14 weeks at 4°C. Synthetic seeds encapsulated with 3% sodium alginate with 3% sucrose stored at 4°C remained viable for 6 weeks but their developmental parameters significantly decreased. Adding nutrient medium and growth regulator to the alginate matrix increased plantlet recovery from both non-stored and stored synthetic seeds: synthetic seeds retained their viability and ability to form plantlets even after 14 weeks of storage. Regenerated transformed plantlets of C. erythraea were acclimatized in the greenhouse.
Abstract Rehmannia glutinosa hairy roots were used to evaluate the effect of methyl jasmonate (MeJa) and salicylic acid (SA) on increase of root biomass and production of iridoids (catalpol, harpagide) and phenylethanoids (verbascoside and isoverbascoside). The elicitors were added to 23-day-old culture separately at concentrations between 50 and 200 μM or in combinations at concentrations of 50 and 100 μM. Roots were harvested 72 h and 120 h after elicitation. The type of elicitor, its concentration and exposure time were found to strongly affect the content of each analyzed compound. A 72-hour treatment with 200 μM MeJa was the most effective in increase of verbascoside content (60.07 mg·DW−1 equivalent to 845.45 mg·L−1) and isoverbascoside (1.77 mg·DW−1 equivalent to 24.94 mg·L−1): these respective amounts were roughly 10- and 6.4-fold higher than the control values (unelicited roots). Exposure to 150 μM MeJa provided optimal harpagide content after 72 hours (0.136 mg·DW−1; 7.5-fold increase compared to the control), and catalpol content after 120 hours (up to 2.145 mg·DW−1). The combination of MeJa and SA also resulted in higher levels of secondary metabolites compared to the control culture, although these levels were lower than those observed for MeJa alone at the optimal concentration and exposure time. SA alone was less efficient in enhancing metabolite production than MeJa.
Centaurium erythraea plants obtained by indirect organogenesis are described in the paper. The plants were initiated from a single adventitious shoot regenerated from callus derived from the cotyledon of a 30-day-old seedling. The shoot was multiplied on MS medium supplemented with IAA (0.1 mg·L-1) and BAP (1.0 mg·L-1). The multiplication rate (28 shoots per culture within 4 weeks) was highest at the first subculture and decreased in further subcultures. The shoots were rooted on MS medium. The effect of IBA (0.1 mg·L-1) on the number of shoots forming roots differed depending on the composition of the basal medium (MS). The rooted shoots were transplanted to soil and grown in a greenhouse with 90% effectiveness. RAPD analysis was done with adventitious shoots of C. erythraea from in vitro culture. In shoots and whole plants regenerated from the callus tissue, secoiridoid content was determined by the HPLC method. We showed significant differences in morphology (leaf size, fresh and dry weight and height of plants) and changes in the DNA profiles as compared to earlier reports for shoot tip-derived shoots and plants of C. erythraea, but the two groups of plants biosynthesized the same qualitative pattern and similar levels of secoiridoids, up to 150 mg·g-1 dry weight; the increased biomass of plants regenerated from callus tissue makes them a better source of secondary metabolites.