Micropropagation Protocol and Genetic Stability of the Salix myrtilloides Plants Cultivated In Vitro

SIMPLE SUMMARY: Salix myrtilloides is a relict species endangered with extinction in many Central European countries. To save the populations on the southern border of its range, in vitro propagation was used for plants production because it allows one to obtain a lot of new plants in a relatively short time without damaging the existing ones. We collected shoot pieces and multiplied them in a growing media, which contained nutrients and growth regulators. When we produced enough daughter plants, we planted them into soil and hardened them to natural conditions. Based on the conducted genetic analysis and flow cytometry, we stated that obtained plants are genetically identical to the mother ones. The conducted research confirmed that tissue culture may be used to propagate the endangered S. myrtilloides species and that the obtained plants may be used to establish new populations or to strengthen the existing ones. ABSTRACT: Salix myrtilloides L. is a relict species, threatened with extinction in many European countries. To prevent the loss of the species, tissue culture was established to produce plant material for reintroduction in natural habitats. Micropropagation was chosen as a method to obtain new plants. S. myrtilloides shoots were disinfected with NaOCl, AgNO(3), or with a two-step disinfection with NaOCl, and then placed on MS medium supplemented with BA at 1 mg·dm(−3) and IBA at 0.1 mg·dm(−3). Regenerated shoots were cultivated in presence of BA, KIN, and 2iP to select the treatment with the highest multiplication rate. The obtained plants were acclimatized to ex vitro conditions. Inter-simple sequence repeat (ISSR) and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. The best disinfection results were obtained when explants were treated with 1.5% NaOCl for 20 min. The highest multiplication rate and good quality plants were noted in the control media, without growth regualtors and in presence of kinetin at 0.5 mg·dm(−3). Flow cytometry and ISSR analyses confirmed genetic stability in plantlets, which indicated the possibility to use the in vitro obtained plants for reintroduction.