Abstract
The increasing demand for medicinal plants in Kazakhstan, driven by health consciousness and limited access to conventional medicines in remote regions, highlights the need for sustainable cultivation and conservation strategies. Despite the high pharmacological value of milk thistle (Silybum marianum), challenges related to long-term seed preservation remain inadequately addressed. Therefore, developing efficient cryopreservation techniques is essential to ensure genetic stability, prolonged seed viability, and reliable resource availability for medicinal and industrial use. This study aimed to evaluate the factors determining the success of cryopreservation of Silybum marianum seeds, including the freezing method, thawing regimen, and germination conditions. Initial seed moisture and weight were determined, and Silybum marianum seeds were stored in liquid nitrogen (−196°C) for one month. Seeds were cryopreserved in cryotubes and in aluminum foil; seeds stored using traditional methods served as controls, and viability was evaluated through germination and germination energy. Prior to cryopreservation, the seed moisture content of Silybum marianum was adjusted to 3.2%. The study demonstrated that utilizing plastic cryovials yielded significantly higher preservation efficiency compared to aluminum foil packaging. Counter to conventional cryobiological paradigms, these ultradry seeds exhibited enhanced post-thaw viability when subjected to slow warming at room temperature, rather than rapid thawing in a water bath. Ambient light conditions emerged as the most critical determinant for post-cryogenic recovery. Specifically, dark incubation facilitated high and synchronized germination, whereas light exposure severely impeded embryo metabolism, thereby substantiating the pronounced negative photoblastism of this species. In the absence of illumination, seedlings underwent skotomorphogenesis, developing etiolated structures that successfully transitioned to active photosynthesis upon subsequent exposure to light. Consequently, the optimal strategy for safeguarding the Silybum marianum gene pool involves the cryopreservation of ultradry seeds within plastic cryovials coupled with ambient-temperature slow thawing. To accommodate the species' distinct light-inhibited germination behavior, a novel two-stage regeneration protocol is proposed, incorporating initial dark incubation followed by light-induced greening of etiolated seedlings. Furthermore, an increased sowing depth of 5–8 cm is recommended for open-field cultivation.