The aim of this study was to evaluate the content and profile of the phenolic compounds (PCs) and antioxidant properties of field-grown leaves, in vitro leaves and in vitro callus cultures of the blackberry ‘Čačanska Bestrna’ and blueberry ‘Toro’. In vitro shoots of the selected genotypes were grown either on original Murashige and Skoog (MS) medium containing 1 mg/L BA, 0.1 mg/L IBA and 0.1 mg/L GA₃ (‘Čačanska Bestrna’) or on MS medium with macroelements reduced to ½, 2 mg/L zeatin and 0.2 mg/L IAA (‘Toro’). Callus cultures were induced from in vitro leaves and established on MS medium with 2 mg/L BA and 2 mg/L 2,4-D (‘Čačanska Bestrna’) or MS medium with half strength macroelements, 2 mg/L BA, 2 mg/L 2,4-D and 1 mg/L NAA (‘Toro’). Total phenolic (TPC) and flavonoid content (TFC) were the highest in blueberry leaves, whereas low TPC and TFC values were obtained in callus cultures of both cultivars. A higher content of PCs in blueberry leaves compared to blackberry leaves was determined by the UHPLC-DAD MS/MS technique. Quercetin derivatives and phenolic acids were the dominant PCs in the leaves of both berries, whereas gallocatechin was present in a significant amount in blueberry leaves. Callus cultures of both berries had a specific PC profile, with none detected in the leaves except quercetin-3-O-glucoside and quercetin-3-O-rutinoside. Blackberry leaves showed the best antioxidant properties as estimated by ferric reducing power (FRP), ABTS^•+ and DPPH^• scavenging activity assays. Callus cultures of both berries exhibited three to five times lower ABTS^•+ and ten to seventeen times lower DPPH^• scavenging activity compared to corresponding leaves. The analyzed leaves and callus cultures can be a good source of PCs with good antioxidant properties and specific phenolics, respectively, for applications in the food and pharmaceutical industries.

A study was conducted to investigate the effect of different storage periods and temperatures on pollen viability in vitro and in vivo in plum genotypes ‘Valerija’, ‘Čačanska Lepotica’ and ‘Valjevka’. In vitro pollen viability was tested at day 0 (fresh dry pollen) and after 3, 6, 9 and 12 months of storage at four different temperatures (4, −20, −80 and −196 °C), and in vivo after 12 months of storage at distinct temperatures. In vitro germination and fluorescein diacetate (FDA) staining methods were used to test pollen viability, while aniline blue staining was used for observing in vivo pollen tube growth. Fresh pollen germination and viability ranged from 42.35 to 63.79% (‘Valjevka’ and ‘Čačanska Lepotica’, respectively) and 54.58 to 62.15%, (‘Valjevka’ and ‘Valerija’, respectively). With storage at 4 °C, pollen viability and germination decreased over the period, with the lowest value after 12 months of storage. Pollen germination and viability for the other storage temperatures (−20, −80 and −196 °C) were higher than 30% by the end of the 12 months. Pollination using pollen stored at 4 °C showed that pollen tube growth mostly ended in the lower part of the style. With the other storage temperatures, pollen tube growth was similar, ranging between 50 and 100% of the pistils with pollen tubes penetrated into the nucellus of the ovule in the genotype ‘Čačanska Lepotica’. The results of these findings will have implications for plum pollen breeding and conservation.

The Balkan Peninsula is a European biodiversity hotspot, home to 6,500 native vascular plant species, many of which are endemic. The region has diverse range of climates and complex topography, creating conditions that suit many woody ornamental, fruit, and forest species. Nevertheless, climate change, habitat destruction, invasive species, plant diseases, and agricultural practices threaten natural ecosystems and cultivated species. Many Balkan countries have addressed these challenges using advanced biotechnological approaches, including micropropagation, in vitro conservation, and in vitro selection for stress-tolerant genotypes. This paper provides a comprehensive overview of in vitro plant biotechnology progress in the Balkan countries from the beginning of the 21^st century to the present, with a focus on woody horticultural and forest species. The primary objectives of scientific research include optimizing media composition for all components and micropropagation stages, as well as effective initial explant selection. In addition, temporary immersion bioreactors, synthetic seed technology, and cryopreservation techniques have been explored to enhance plant production and conservation. Scientific research for woody ornamentals and fruit tree species has progressed in Albania, Bulgaria, Croatia, Greece, and Serbia while remaining limited in other Balkan countries. Forest tree production research is currently conducted in Bulgaria, Greece, and Serbia, with labs focusing on micropropagation and ex situ conservation. In addition to advances in scientific research, several commercial companies operate in the Balkans, with establishments in Serbia, Greece, Bulgaria, and North Macedonia, that produce in vitro-derived planting material for fruit trees and woody ornamental plants. Despite this progress, research in the Balkan countries remains fragmented, emphasizing the need to strengthen regional collaboration and knowledge exchange to promote agricultural development processes and biotechnological applications in this region. This review represents the first exploration of this topic in the Balkans and successfully unites researchers from several countries. It highlights key scientific advances in in vitro biotechnology for woody plants, identifies challenges, and proposes solutions. The value of interdisciplinary collaboration is emphasized through the optimization of in vitro methodologies, the promotion of germplasm conservation, and the sustainable use of plant genetic resources in the Balkans.

‘Crvena Ranka’ is one of the oldest autochthonous plum cultivars in Serbia. However, it is endangered due to the long-term pathogen pressure and continuous suppression by commercially important cultivars and therefore faced with genetic erosion. This study was carried out to investigate the suitability of two efficient and simple cryopreservation methods using aluminium cryo-plates for its conservation. Precultured shoot tips of this genotype were embedded in alginate gel into cryo-plates wells. Osmoprotection was performed using loading solution with 1.9 M glycerol and 0.5 M sucrose (30 min at room temperature). In the V cryo-plate protocol, explants were dehydrated at room temperature for 20 or 40 min with PVS A3 (37.5% glycerol, 15% dimethylsulfoxide, 15% ethylene glycol and 22.5% sucrose) or with PVS3 (50% glycerol and 50% sucrose) for 60 min. In the D cryo-plate protocol, desiccation for 2, 2.5 or 3 h was performed over silica gel. Then, the cryo-plates were directly immersed into liquid nitrogen. Unloading was done in MS medium containing 0.8 M sucrose (30 min at room temperature). In the V cryo-plate procedure regrowth of cryopreserved explants dehydrated with PVS A3 was between 50% and 51.9%, while in those dehydrated with PVS3 it was 66.7%. As for the D cryo-plate method, regrowth of cryopreserved explants ranged between 30-40%. After regrowth, shoots were successfully multiplied and rooted. Results prove the feasibility of these new cryogenic methods for a long-term storage of this valuable Prunus genotype.

The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were briefly pre-cultured for 1 day at 23 °C in the dark on a medium containing 0.3 M sucrose and then embedded in calcium alginate gel within the wells of the aluminum cryo-plates. In the V cryo-plate protocol, dehydration was carried out at room temperature using the following vitrification solutions: original plant vitrification solution 2 (PVS2) and 90% PVS2 solution (for 20 and 40 min) and plant vitrification solution 3 (PVS3) (for 60 and 80 min). In the D cryo-plate protocol, desiccation was performed for 2, 2.5, or 3 h over silica gel at 23 °C. The effect of different treatments was evaluated by monitoring the regrowth of both non-frozen and cryo-preserved explants. After cryo-preservation, five genotypes achieved regrowth rates over 40% in at least one of the applied protocols, while two genotypes showed regrowth rates of around 10%. A significant improvement in regrowth success for all genotypes using both cryo-plate methods was achieved by pre-culturing shoot tips for 7 days on a medium containing 0.5 M sucrose in complete darkness at 4 °C. Shoots regenerated from cryo-preserved explants were further monitored in vitro. By the third subculture, they had not only regained but had even exceeded the multiplication capacity (index of multiplication, length of axial, and lateral shoots) of shoots regenerated from dissection controls. Following multiplication, the cryo-preserved shoots were successfully rooted and rooting ability was assessed by monitoring the percentage of rooting, number and length of roots, and height of rooted plantlets.

Although nanotechnology is increasingly applied in plant tissue culture in many parts of Europe, its use in the Balkans remains limited, revealing a regional gap with untapped potential for advancing in vitro propagation and preservation of woody plant species. Building upon a recently published regional review covering 2001–2024, which analyzed in vitro biotechnology progress in nine Balkan countries, this paper introduces the concept of a “nano gap”, referring to the limited connection between existing nanotechnology research potential and its use in in vitro woody plant biotechnology. In Serbia, Greece, Bulgaria, Croatia, and Albania, significant progress has been made in optimizing micropropagation and in vitro conservation strategies by introducing temporary immersion systems, synthetic seed technology, adapting genotype-specific sterilization and multiplication protocols, and modifying established cryopreservation methods for regional woody species. However, the integration of nanotechnology into these systems remains largely unexplored. To date, there are no published results or validated applications for nano-enhanced media or nanoscale delivery systems for micropropagation and in vitro conservation of woody species. The limited integration of nanotechnology may be due to insufficient funding, lack of specialized infrastructure, and limited interdisciplinary expertise. Nevertheless, many Balkan countries possess growing capacities in nano-applications within agriculture and environmental sciences and are ready to advance toward interdisciplinary research and innovation. By mapping both scientific readiness and structural barriers, this review provides a strategic framework for bridging the “nano gap” and offers a novel regional perspective with broader implications for European research policy, sustainable agriculture, biodiversity preservation, and green innovation.