Plant tissue culture and molecular techniques for northern berry crop improvement

Vaccinium species, black huckleberry (Vaccinium membranaceum) and half-highbush blueberry hybrids, are rich in bioactive compounds and are vital for their commercial and medicinal applications. The challenge of hyperhydricity effects significant economic and physiological problems, affecting plant health, growth, and propagation efficiency. Our study explored various aspects of hyperhydricity in micropropagated plantlets using advanced bioreactor systems, such as stationary (Growtek) and temporary immersion (RITA). The Growtek system demonstrated higher shoot proliferation (up to 76%) but exhibited the highest hyperhydricity rates, particularly in certain cultivars (e.g., HB₂ at 72.34 ± 2.45%). Scanning electron microscopy (SEM) and histological analyses revealed pronounced structural changes, including closed stomata, enlarged intercellular spaces, and disrupted vascular bundles with immature sieve elements in hyperhydric plantlets. Tissue histology highlighted a compressed adaxial epidermis and irregular mesophyll cell morphology. Biochemical profiling indicated elevated phenolic content and catalase activity in hyperhydric plants, while non-hyperhydric greenhouse-grown plants displayed higher antioxidant activity and higher levels of anthocyanins and proanthocyanins. Using ¹H NMR, distinct metabolite profiles were identified, with hyperhydric plantlets exhibiting strong signals of catechin (C₁₅H₁₄O₆) and other untargeted metabolites, such as quercetin-3-O-glucuronide and chlorogenic acid, demonstrating enhanced stress response. This underscores the potential adaptive mechanisms in hyperhydricity, marked by unique metabolic changes. DNA methylation analysis provided comprehensive insights into epigenetic regulation under hyperhydric conditions. The genome wide bisulfite sequencing highlighted shifts in methylation patterns, with increased mCG and mCHG methylation and reduced mCHH levels in hyperhydric plantlets compared to controls. These changes were particularly evident in transposable element-rich regions, correlating with reduced gene density and altered regulatory pathways. Gene Ontology (GO) analysis indicated significant changes in biological, cellular, and molecular functions, suggesting that hyperhydricity induces stress-related epigenetic modifications that potentially impact gene expression and adaptive responses. This study emphasizes the importance of understanding DNA methylation in relation to hyperhydricity to develop strategies for micropropagation.