Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm

SIMPLE SUMMARY: Optimization of regrowth conditions is critical for the successful cryopreservation of in vitro plant germplasm. This review discusses the five major strategies available at the regrowth stage for improving plant material performance after cryopreservation. These strategies involve physical factors (modification of osmotic environment, light/dark conditions, and light quality) and chemical factors (recovery medium composition, application of exogenous additives, and the influence of plant growth regulators depending on the type of cryopreserved materials). This summary is meant to serve as a guideline for choosing the most suitable regrowth conditions for plant germplasm to be cryopreserved. We also propose the combinations of factors that may benefit the recovery of cryopreservation-sensitive species and types of materials. ABSTRACT: Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied. The conditions at the recovery stage play a vital role in supporting material regrowth after cryopreservation and, when optimized, may shift the life-and-death balance toward a positive outcome. In this contribution, we provide an overview of the five main strategies available at the recovery stage to improve post-cryopreservation survival of in vitro plant materials and their further proliferation and development. In particular, we discuss the modification of the recovery medium composition (iron- and ammonium-free), exogenous additives to cope with oxidative stress and absorb toxic chemicals, and the modulation of medium osmotic potential. Special attention is paid to plant growth regulators used at various steps of the recovery process to induce the desired morphological response in cryopreserved tissues. Given studies on electron transport and energy provision in rewarmed materials, we discuss the effects of light-and-dark conditions and light quality. We hope that this summary provides a helpful guideline and a set of references for choosing the recovery conditions for plant species that have not been cryopreserved. We also propose that step-wise recovery may be most effective for materials sensitive to cryopreservation-induced osmotic and chemical stresses.