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.
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.