In vitro culture and epigenetic variation in northern berry crop

Abstract

Vaccinium vitis-idaea L., commonly known as lingonberry and also popular as partridgeberry, is a nutrient-rich, antioxidant-packed "superfruit" with immense commercial potential. Native to boreal regions across North America, Eurasia, Alaska, and Canada, lingonberries have long been valued by indigenous communities. Despite their health-promoting properties, large-scale cultivation of lingonberries remains limited due to their woody recalcitrant nature and poor proliferation via traditional or conventional propagation methods. My research explores somatic embryogenesis (SE) as a viable micropropagation strategy for lingonberry. For the first time, a successful SE protocol was established in two lingonberry genotypes using thidiazuron (TDZ) on a semi-solid medium. The role of TDZ in influencing redifferentiation and antioxidant activity was systematically studied. Regenerated plants were not only genetically identical but also demonstrated altered phytochemical profiles, particularly increased levels of flavonoids, anthocyanins, and antioxidants, when compared to their donor plants. To understand the underlying epigenetic mechanisms, global DNA methylation patterns were analyzed using UHPLC-MS/MS, a rapid and highly accurate technique. The in vitro SE-regenerated plants exhibited higher methylation levels compared to the ex vitro plants. Interestingly, even with reduced methylation in ex vitro, the elevated phytochemical levels (of catechin) persisted, suggesting the presence of epigenetic memory or biological imprint. To validate this observation, enzymatic methyl sequencing (EM-Methyl seq), a more precise alternative to bisulfite sequencing, was applied for the first time in lingonberry to confirm methylation pattern changes in CpG, CHG and CHH sites. The final chapter addresses hyperhydricity, a frequent physiological disorder in tissue culture, by identifying its anatomical, morphological, biochemical indicators and proposing mitigation strategies. Gene expression analysis was conducted to identify the genetic basis of hyperhydricity, focusing on which genes are responsible for its onset and how their expression patterns differ between hyperhydric and non-hyperhydric shoots. The study also examined how beneficial genes, such as those involved in antioxidant activity and defense responses, become upregulated under hyperhydric conditions. Insights from this analysis contribute to understanding how hyperhydricity can be mitigated using potassium silicate, with the aim of promoting recovery and restoring normal physiological function in hyperhydric shoots. Together, these findings enhance the understanding of in vitro culture, regeneration, and epigenetic stability in lingonberry, advancing its potential as a sustainable, high-value crop for agriculture, nutrition, and human health.