Slow-growth storage of Quercus robur

Shoots derived from an 800-year-old <i>Q. robur</i> tree (Rogalin Landscape Park, Poland), previously established and cultured <i>in vitro</i> (maintenance/multiplication culture medium: 3.5 μM 6-benzylaminopurine [BAP] + 30 g L⁻¹ sucrose + 7 g L⁻¹ agar) under a light intensity of 80 μmol m⁻² s⁻¹ of photosynthetically active radiation (PAR), a photoperiod of 16 h light/8 h dark, and a temperature of 21 °C (growth room condition), were used in this study. Approximately 2.5 cm-long shoots were transferred to 372 mL polycarbonate flasks (PTL-100™, PhytoTech Labs, Lenexa, U.S.) containing culture medium supplemented with 40 g L⁻¹ sucrose and 7 g L⁻¹ agar. The experimental treatments consisted of two BAP concentrations (3.5 and 7.0 μM) combined with two levels of activated charcoal (AC) (0 and 2 g L⁻¹). Following transfer to the treatment media, the plant material was maintained in a growth room at 21 °C for seven days, then placed in darkness at 15 °C for 48 hours. Subsequently, the explants were cultured in the dark under SG conditions in growth chambers (MIR-253 incubator, Sanyo, Osaka, Japan) at either 3 °C or 10 °C. To monitor morphophysiological changes during and after SG, samples from each treatment were collected every 45 days over a total period of 135 days. Time zero was defined as the first day of culture at 3 °C or 10 °C. At each storage interval (45, 90, and 135 days), a subset of explants from each treatment was transferred to maintenance culture medium at 21 °C (growth room condition) and cultivated for 45 days to assess whether organogenic capacity was retained (RG phase). To evaluate the effects of the treatments on the plant material, physiological analyses were performed both immediately after each storage period and after 45 days of RG. The physiological status of the explants was assessed based on growth parameters (fresh mass and number of new shoots), as well as proline, starch, and photosynthetic pigment contents, and photosynthetic apparatus efficiency (JIP-test). Additionally, the hormonal metabolism profile was determined after the RG period. Plants cultured on standard maintenance medium for 45 days at 21 °C, without exposure to SG conditions, served as controls.