Impact of pre-culture on short- and long-term in vitro recovery of the biosynthetic potential and enzymatic and non-enzymatic antioxidant defense of Hypericum rumeliacum Boiss. after cryostorage

Abstract

Due to its high hypericin and pseudohypericin in vitro biosynthetic capacity, the Balkan endemic Hypericum rumeliacum was selected as a prospective candidate for long-term preservation of valuable medicinal plant germplasm. Initial cryopreservation experiments were previously conducted based on the successful protocol established and reported for the widely studied H. perforatum. This is the first report on the impact of pre-culture duration on the short- and long-term in vitro recovery of the biosynthetic potential and antioxidant defense system of H. rumeliacum cryopreserved by vitrification. Cryopreservation did not impair the phenolics and flavonoids production of the regenerated plants. Moreover, hypericin and pseudohypericin levels even increased substantially in one of the regenerated lines, reaching yields from 0.107 and 0.752 mg g−1 DW in the control up to 0.277 and 1.112 mg g−1 DW for hypericin and pseudohypericin, respectively. However, the physical injury stress of the pre-culture treatment manipulations affected the physiological status of regenerants in a time dependent manner. Within 6 months after thawing, regenerants with the highest oxidative stress after pre-culture, were characterized with an augmentation of antioxidant metabolites such as phenolics, flavonoids, glutathione and ascorbic acid as well as increased antioxidant enzymatic activities in comparison with both the non-frozen control and the regenerants with the lowest pre-culture oxidative stress. Then, after 18 months of recovery, the same first H. rumeliacum group displayed a marked drop of enzymatic antioxidant activity as compared with the other groups of plants. Further research is needed to target oxidative stress alleviation to optimize H. rumeliacum cryopreservation protocol.