Effect of growth medium composition on the viability and genetic stability of Chrysanthemum × morifolium Ramat. explants under in vitro cold storage conditions

Cultivation of plants at low positive temperatures is promising for the in vitro preservation of valuable Chrysanthemum × morifolium Ramat. varieties. The study aims to examine the effect of chlorocholine chloride concentrations in a specialized medium on the viability and the physiological and morpho-anatomical parameters of chrysanthemum plants (Excel, Sheer Purple, and William Seward varieties), as well as their genetic stability after 12 months of cold storage under in vitro gene bank conditions. In vitro microshoot tips (without leaves) were placed on a 1/4 Murashige and Skoog medium supplemented with 60.0 g/L sucrose and 0.2–0.8 g/L chlorocholine chloride. The explants were stored in cooling chambers at a temperature of 4 °C. After 12 months of preservation, a reduction was observed in growth kinetics with increasing concentration of chlorocholine chloride in the medium. The viability of explants decreased from 96 to 91% at higher concentrations of chlorocholine chloride. The effective photochemical quantum yield of photosystem II decreased more than twice under cold storage conditions as compared to the control. Several histological features were identified in the leaves of in vitro explants cold-stored in the gene bank: decreased in leaf thickness and mesophyll density in leaf blades, degradation of palisade tissue, and a high degree of transpiration regulation. The SSR and ISSR analyses were used to show that the microshoots of the examined chrysanthemum varieties were identical to the original plants following in vitro cold storage. The conducted studies revealed that the presence of 0.6–0.8 g/L chlorocholine chloride in the medium reduces the growth kinetics of explants while maintaining their high viability and genetic stability.