The influence of cryopreservation via encapsulation-dehydration on growth kinetics, embryogenic potential and secondary metabolite production of cell suspension cultures of Gentiana capitata Buch.-Ham. ex D.Don and Gentiana decumbens L.f.

Some species of Gentianaceae are an abundant source of valuable, much demanded pharmacologically active secondary metabolites. Embryogenic cell suspensions may provide both raw material for secondary metabolite extraction and somatic embryos (SE), protoplasts, and cells for plant biotechnology. Their cultures can be maintained for a long time with cryopreservation. Encapsulation-dehydration has been reported as a reliable technique for preserving the viability and recovery of embryogenic cells. Nonetheless, the elevated sucrose concentrations in the culture medium during preculture and dehydration may alter the embryogenic competence of cells, their genetic stability and metabolite biosynthesis. Therefore, the objectives of this study were to evaluate the effect of sucrose concentrations in the medium and cryopreservation on the growth kinetics, embryogenic potential and secondary metabolite production of cotyledon-derived cell suspensions of Gentiana capitata and Gentiana decumbens. The growth kinetics, the number of SE regenerated, and the 2 C DNA content were assessed for cell suspensions cultured on liquid Murashige and Skoog medium with 3% and 6% sucrose, either encapsulated in calcium alginate or non-encapsulated, either cryopreserved via encapsulation-dehydration or non-cryopreserved. The secondary metabolites were analyzed using UHPLC-DAD-IT-MS/MS. The biomass growth rate was always higher on medium with 3% sucrose than on medium with 6% sucrose irrespective of the gentian species, with the maximum growth rate of 146 ±7% for G. capitata and 116 ±22% for G. decumbens at 4 weeks. The growth rate of suspension culture from cryopreserved plant material was always lower and after 4 weeks being 28±5% and 33±3% for G. capitata and G. decumbens, respectively. Prior to encapsulation the SE productivity of G. capitata cell suspension was on average 7.5 times greater than that of G. decumbens for cultures from medium with 3% of sucrose and on average 4.6 times greater for cultures from medium with 6% sucrose. In both species cell suspensions cultured on medium with 6% of sucrose produced more somatic embryos (3.7±0.6 and 0.8±0.2 per 50 mg of cell suspension of G. capitata and G. decumbens, respectively) than those from MS3 medium (3.0±0.7 and 0.4±0.2 per 50 mg cell suspension of G. capitata and G. decumbens, respectively). Variation in the 2C DNA content was detected in G. decumbens tissues treated with higher sucrose concentrations. The secondary metabolite composition in cell suspensions differed from that in plants grown in vitro. Cell aggregate extracts did not include secoiridoids typical of Gentianaceae, but some benzophenone and isovitexin derivatives (flavonoids) previously undetected in these species.