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.
Shoot tips excised from shoot culture of Salvia officinalis were encapsulated in 2% or 3% (w/v) sodium alginate and exposed to 50 mM calcium chloride for complexation. Immediately or after 6, 12 or 24 weeks of storage at 4°C, the synthetic seeds were cultured for 6 weeks on half-strength MS medium supplemented with indole-3-acetic acid (IAA) (0.1 mg/l) and solidified with 0.7% agar. The frequency of shoot and root emergence from encapsulated shoot tips was affected by the concentrations of sodium alginate and additives in the gel matrix (sucrose, gibberellic acid, MS nutrient medium) as well as duration of storage. The frequency of shoot and root induction of non-stored synthetic seeds was highest with shoot tips encapsulated with 2% sodium alginate containing 1.5% sucrose and 0.5 mg/l gibberellic acid (GA3). Shoot tips maintained their viability and ability to develop shoots even after 24 weeks of storage when they were encapsulated in 3% alginate with 1/3 MS medium, sucrose (1.5%) and GA3 (0.25 mg/l). Root formation tended to decrease with storage time. Overall, 90% of the plantlets derived from stored and non-stored synthetic seeds survived in the greenhouse and grew to phenotypically normal plants. This procedure can enable the use of synthetic seed technology for germplasm conservation of S. officinalis, a plant species of high medical and commercial value.