Beyond the Protocol: Revisiting the Critical Role of Donor Plants in Cryopreservation of Economically Important Clonal Crops.

Shoot tip cryopreservation is essential for the long-term conservation of plant genetic resources. It provides the only reliable method for establishing a long-term, readily available gene pool of clonally propagated crops and elite in vitro clones used in the pharmaceutical, food, and cosmetic industries. Still, its success is often limited by the inherent sensitivity of many species to the osmotic and chemical stresses imposed by concentrated cryoprotectant (vitrification) solutions and severe dehydration. The optimization of modern cryopreservation protocols primarily focuses on modifying shoot tip preculture, cryoprotectant treatments, or regrowth conditions, while frequently overlooking donor plant preconditioning or relegating it to a secondary role. However, the physiological state of in vitro plants from which apical or axillary shoot tips are extracted may hold the key to successful post-cryopreservation recovery, especially in cryo-sensitive taxa. This review revisits the critical role of donor plant vigor and induced stress tolerance in the cryopreservation of clonal crops by systematically evaluating preconditioning strategies, including cold acclimation, sucrose pretreatment, and the use of growth regulators and signaling molecules such as abscisic, jasmonic, and salicylic acids, involved in stress signaling and tolerance development. The beneficial physiological changes induced by donor plant pretreatment, such as reduced freezable water content and the accumulation of protective compounds, are discussed in the context of contemporary cryopreservation methods. The effects of culture conditions, including the roles of ammonium and nitrates, light quality, culture density and aeration, medium strength, culture age, and subculture duration, are also considered. We analyze how different treatments of in vitro donor plants improve shoot tip tolerance to osmotic and/or chemical toxicity imposed by specific cryopreservation methods to support a material-centered selection of a cryopreservation procedure. Future directions and potential approaches for integrating target donor plant preconditioning into modern cryopreservation protocols for shoot tips, particularly in stress-sensitive species, are discussed.