Alginate encapsulation of micro-cuttings in endangered Satureja khuzistanica species: a promising method for obtaining genetically stable plants with high rosmarinic acid content

Abstract

Synthetic seed technology is a suitable approach for the rapid, and uniform mass production of Satureja khuzistanica Jamzad, a valuable medicinal plant of the Lamiaceae family. In this study, the encapsulation of micro-cuttings of S. khuzistanica using sodium alginate (SA) was investigated. In order to determine the best conditions for encapsulation, different concentrations of SA and CaCl2·2H2O, different types of explants and matrix culture media, different types and concentrations of plant growth regulators, and different cold storage conditions of synthetic seeds were tested. Also, the genetic stability of regenerated plants from synthetic seeds using the inter simple sequence repeat (ISSR) molecular marker as well as the content of rosmarinic acid in synthetic seed-derived plants were evaluated. The use of 3% SA and 100 mM CaCl2·2H2O was found to be optimal for gel complexation. Encapsulation of nodal segments with 1/2 Murashige and Skoog (MS) medium containing 2.5 µM 6-Benzylaminopurine resulted in the highest regrowth percentage (72.22%) and regrowth rate (0.173). Pre-culture of nodal segments in 1/2 MS medium containing 5 µM indole-3-butyric acid (IBA) and 0.2% activated charcoal for 10 days and the use of 2.5 µM IBA in the rooting medium resulted in the highest rooting percentage (88.88%) in synthetic seed-derived plants. The highest regrowth percentage (61.11%) and regrowth rate (0.173) of synthetic seeds were obtained using 2.0 µM thidiazuron (TDZ) in MS liquid medium. The placement of germinated seeds on the coco peat substrate resulted in the highest conversion rate (61.11%) of synthetic seeds. Also, storage of encapsulated nodal segments at 4 °C in MS culture medium, compared to cold storage without using MS medium, resulted in better regrowth of synthetic seeds. The highest regrowth percentage (44.44%) and regrowth rate (0.092) for cold-stored synthetic seeds occurred after 2 weeks. The genetic stability testing by ISSR molecular marker showed that synthetic seed-derived plantlets were genetically similar to their mother plants. Also, encapsulated nodal segments and shoot tip-derived plants significantly enhanced the rosmarinic acid content up to 7.77 times that of the natural seed-derived plants. Genetic restoration programs, short-term storage, and germplasm distribution in S. khuzistanica plants could all benefit from the encapsulation regeneration strategy reported here.