Osmotic priming-induced cryotolerance uncovers rejuvenation of grapevine cell cultures: morphogenetic changes and gene expression pattern highlighting enhanced embryogenic potential.

Cryopreservation is a reliable technique for the long-term storage and preservation of embryogenic cells, maintaining their viability without loss of their embryogenic capacity. However, the large-scale conservation of grapevine embryogenic lines in cryobanks remains limited. A significant challenge is understanding somatic cell rejuvenation. Here, we investigate the encapsulation/dehydration and encapsulation/vitrification for cryopreserving embryogenic material. Cell rejuvenation and enhanced embryogenic competence were observed after cryopreservation, as evidenced through structural cellular changes observed by histology and electron scanning microscopy. Results showed that cryopreserved samples of 110-Richter, Riesling, and Tempranillo using encapsulation/dehydration had better survival rates, averaging 81%, 62%, and 48%, respectively, while encapsulation/vitrification yielded lower survival rates, averaging 58%, 42%, and 32%, respectively. Cryopreservation also improved post-thaw recovery and regeneration efficiency assessed through regrowth of proembryogenic masses and somatic embryo conversion reaching 54-72% against 11-17% in control samples. Cryopreservation triggered changes in gene expression patterns and exhibited considerable increase at genotype-specific basis of 1.5- to 4.5-fold in SERK1, BBM, and WOX associated to embryogenic competence as well as in ChitIV and LEA involved in stress response. Membrane stability index, hydrogen peroxide, and proline contents were used as indicators of oxidative stress uncovering a key role of an osmotic trans-priming effect leading to cryotolerance. Our finding highlighted that cryopreservation enhances embryogenic capacity in senescent callus and probably acts as a screening process allowing safe maintenance of proembryogenic cells and promoting their recovery. This study provides a high throughput innovation to set up cryolines for cell rejuvenation of grapevine and other important plant species.