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 production is a suitable method for the rapid, mass, and uniform proliferation of Satureja khuzistanica Jamzad, a valuable medicinal plant of the Lamiaceae family. In this study, the encapsulation of micro-cuttings of S. khuzistanica was investigated using sodium alginate (SA). In order to determine the best conditions for encapsulation in the S. khuzistanica plant, different concentrations of SA and CaCl 2 .2H 2 O, different types of explant and matrix culture media, different types and concentrations of plant growth regulator, and different cold storage conditions of synthetic seeds were tested. Also, the genetic stability of regenerated plants from synthetic seeds using the ISSR molecular marker as well as the content of rosmarinic acid in synthetic seed derived plants were evaluated in this study. The use of 3% SA and 100 mM CaCl 2 .2H 2 O was found to be the best composition for gel complexation. Encapsulation of nodal segments with 1/2 MS medium containing 2.5 µM BAP 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 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 0.45 mg/L 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 to the plant. 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 two 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 signi cantly enhanced the rosmarinic acid content up to 7.77 times the natural seed derived plants. Genetic restoration programs, short-term storage, and germplasm distribution in S. khuzistanica plants could all bene t from the encapsulation regeneration strategy reported here.

In Vitro Preservation of Somatic Seeds and Nonencapsulated Hemp Shoot Tips

“…Synthetic seed technology has been also used for germplasm conservation and multiplication. The choice of the initial explants and encapsulating agent and matrix, the addition of growth regulators and nutrients to the capsules, and the choice of experimental conditions substantially influence the success of synthetic seed production, as well as their storage and regeneration [3]. Prepared, encapsulated synthetic seeds can be stored at low temperatures.…”