Centaurea kilaea Boiss. is an endangered perennial species from the Asteraceae family, primarily distributed on the coastal dunes of northwest Türkiye. This study establishes the first efficient in vitro propagation and callogenesis protocols to support biodiversity conservation and reintroduction efforts for this species. Seeds and nodal explants were cultured on Schenk Hildebrandt medium supplemented with various concentrations of cytokinins and auxins. The results demonstrated that 1.0 mg L− 1 forchlorfenuron (CPPU) was the most effective treatment for seed germination, achieving a rate of 80
Hemidesmus indicus (Indian sarsaparilla) is a source of several pharmaceutically importantroot-specific secondary metabolites such as 2-hydroxy-4-methoxy benzaldehyde, 2-hydroxy-4-methoxy benzoic acid, isovanillin, and lupeol, which has increased demand for root biomass. The present study was conducted to improve root biomass production in H. indicus using indole-3-butyric acid (IBA) pulse treatment of in vitro-raised shoots and a hydroponic culture system. Pulse treatment of shoots with 400 mg L− 1 IBA, followed by 12 weeks of hydroponic culture, yielded optimal rooting (52.0 roots per shoot, mean root length 79.0 cm) and the highest root biomass (8.3 g fresh weight; 1.25 g dry weight per shoot). The root anatomical studies demonstrated that the cork, phellogen, phelloderm, ground tissues, and vascular elements were well-developed in these roots compared to the control and other concentrations (100 to 300 and 500 mg L− 1) of IBA treatments. These findings demonstrate the feasibility of scaling up hydroponic adventitious root cultures of H. indicus for the production of bioactive compounds, offering a sustainable alternative to wild harvesting and mitigating pressure on natural populations.
Polyscias fruticosa and P. balfouriana are medicinal plants that produce valuable oleanane-type triterpene glycosides, including polysciosides, compounds with demonstrated antimicrobial, anti-inflammatory, antihistamine, immunomodulatory, and adaptogenic effects. However, the natural supply of these species is limited and insufficient to meet the growing demand for their bioactive compounds. In this study, adventitious (non-transgenic) root cultures of both species were established as sustainable platforms for producing biomass and bioactive metabolites. The root cultures grew well in liquid medium, reaching maximum dry biomass of 19.74 g/L for P. fruticosa and 8.76 g/L for P. balfouriana. UPLC-ESI-TOF-MS profiling revealed that the adventitious roots synthesized a diverse spectrum of oleanane-type triterpene glycosides, including polysciosides, ladyginosides and their isomers, chikusetsusaponin IVa and others. The total triterpene glycoside content was 5.15 mg/gDW in P. fruticosa and 2.70 mg/gDW in P. balfouriana roots. The roots also exhibited distinct fatty acid profiles, characterized by higher proportions of palmitic acid (26–29
Quillaja lancifolia D. Don (current correct taxonomic designation of the synonym Q. brasiliensis (A. St.-Hil. Tul.) Mart.) is a promising source of triterpenoid saponins of high biotechnological value, including QS-21, a potent immunological adjuvant used in human vaccine formulations, producible in cell suspension cultures. Herein, an efficient Agrobacterium tumefaciens–mediated genetic transformation protocol for generating transgenic calli of Q. lancifolia was established. Transformation was performed using A. tumefaciens EHA105 harboring the binary vector pH7WG2D-GUS containing the β-glucuronidase (GUS), the green fluorescent protein (GFP), and hygromycin phosphotransferase (hpt) genes. Under the established conditions (OD₆₀₀ = 0.6, 30 min infection, 72 h co-cultivation), hygromycin-resistant callus sectors expressing reporter genes were consistently obtained on hygromycin-containing culture medium. Transgene presence was confirmed by PCR amplification of the hpt gene from genomic DNA. Histochemical GUS staining and GFP fluorescence were shown after confirmation of Agrobacterium elimination by successive subcultures on meropenem-containing medium, followed by cultivation on antibiotic-free conditions. The overall transformation efficiency reached 41.6 ± 4.6
Curcuma sumatrana Miq. (Koenih Rimbo) is an endemic wild turmeric species from West Sumatra, Indonesia, valued in traditional medicine for its health benefits. However, increasing anthropogenic pressure has led to decline in natural populations, necessitating the development of efficient propagation protocols to support both conservation and bioprospecting. This study establishes a five-stage in vitro propagation framework using rhizome bud-derived shoots on Murashige and Skoog (MS) medium: (1) shoot initiation using 6-benzylaminopurine (BAP) and silver nitrate (AgNO3), (2) shoot multiplication using thidiazuron (TDZ) and BAP, (3) microrhizome induction using sucrose and BAP, (4) root induction using 1-naphthaleneacetic acid (NAA) and varying MS medium strengths, and (5) acclimatisation. Results indicated that BAP improved shoot development regardless of AgNO3; specifically, ≤ 4 mg L-1 BAP maximised shoot height (4.85 ± 0.37 cm) and leaf number (2.25 ± 0.25). Shoot multiplication was enhanced by supplementing the medium with 0.5 mg L-1 TDZ (6.07 ± 0.57 shoots). Microrhizome induction was optimised using 60 g L-1 sucrose (0.70 ± 0.07 cm diameter) or 4 mg L-1 BAP (0.68 ± 0.09 cm), however, both treatments slightly reduced overall morphological vigor. The rooting stage with 0.25 mg L-1 NAA in half-strength MS medium yielded the most roots (4.00 ± 0.20). Acclimatised plantlets exhibited a high survival rate (85–95
A novel, low-cost protocol was developed to induce in vitro microrhizomes in ginger (Zingiber officinale Rosc.) cv. Himgiri by optimizing culture conditions and medium components. Different carbon sources, including sucrose, glucose, and table sugar; cytokinin-auxin combinations; photoperiods; and alternative gelling agents were evaluated. An optimized medium (MIM12), containing 70 g/L sucrose, 3 mg/L BA, and 0.5 mg/L NAA, resulted in the earliest induction and the highest frequency of healthy microrhizomes. Comparable responses were obtained using a low-cost formulation containing 60 g/L table sugar (LCMIM12). Further cost reduction of 66.22
This review discusses the challenges and effective approaches for sustainable delivery of plant secondary metabolites through plant cell tissue and organ culture. Special attention has been given to utilizing the species’ indigenous biosynthetic capacity without performing genetic manipulations. Plant metabolic profile is a genetically pre-determined trait, bound to the particular taxon on the family, genus, species or even subspecies level. However, its qualitative and quantitative characteristics are surprisingly variable and in dynamic interplay with the environment (in terms of temperature, light regime, soil characteristics, climatic and geographic conditions, etc.). Secondary metabolites production, accumulation and translocation relate also to the presence of highly specialized anatomical structures, thus being strongly affected by growth, development and morphogenesis of the plant individual. This is the reason why modification of cultivation parameters in vitro can affect secondary metabolites production and is even capable to induce the accumulation of compounds not always detectable in randomly in situ collected plant material. The present review summarizes the groups of key cultivation factors for affecting secondary metabolites production in biotechnologically cultivated plant material. Classical optimization approaches, utilized in commercial delivery of bioactive secondary metabolites have also been summarized.
Bursaphelenchus xylophilus is a pathogen that causes devastating mortality in pine forests, characterized by high virulence, rapid transmission, and difficulty for control. Exploring resistance genes and resistance mechanisms are effective strategies for addressing pine wilt disease. In this study, a pathogen induced gene, PtCRK2, was cloned from the pathogen-resistant line of Pinus tabuliformis, which is 1,188 bp in length and encodes a receptor kinase. The gene was introduced into P. tabuliformis callus. Triphenyltetrazolium chloride (TTC) staining assay revealed that the PtCRK2-overexpression callus and control performed better under regular conditions without significant difference. When subjected to the treatment of B. xylophilus, the pinus callus displayed the color fading, indicating the cell activity decreased. The PtCRK2-overexpression calli performed better than the control with the callus color redder. Measurement of triphenyltetrazolium formazan (TTF) content showed 1.84-fold higher in the transgenic calli than the control. The physiological index tests showed that the relative electrical leakage and MDA content of the control were 1.33-fold and 1.46-fold higher than the PtCRK2-overexpression callus, respectively. All these findings indicated that PtCRK2 gene significantly enhanced the resistance of P. tabuliformis against B. xylophilus and can serve as an effective gene resource for molecular breeding.
Mosses are non-vascular plants widely distributed across multiple habitats and capable of thriving in extreme environments, such as Antarctica and the Arctic. However, knowledge of the genetic, metabolic, and molecular diversity of polar species remains limited, necessitating a better understanding of their mechanisms of response to harsh conditions. In this context, axenic in vitro cultivation under laboratory conditions is a powerful tool for plant sciences to elucidate physiological and biochemical responses. Nevertheless, in vitro cultivation of polar mosses can be challenging, as most species lack viable sporophytes or are predominantly sterile. Alternatively, gametophores can be used; however, many fail to withstand surface decontamination or are subsequently overrun by microorganisms. To establish an efficient and replicable decontamination method, we compared the efficacy of treatments applied to field‑collected gametophores from three sites and pre‑cultured leafy shoots of Warnstorfia sarmentosa (Wahlenb.) Hedenäs as a model species, using different concentrations of sodium dichloroisocyanurate (NaDCC) and sodium hypochlorite (NaClO). Explants derived from pre-cultured tissue achieved an overall success rate of 64.1
In this study, the effects of 2, 3, 4, 5 and 6 weeks of incubation period (IP) in floating perlite bed (FP) for ex vitro rooting and acclimatization of ‘Viking’ chokeberry microshoots were investigated on plant development, leaf anatomical features and chlorophyll contents. The effects of IPs on ex vitro rooting of microshoots were not significant and the mean rooting percentage was determined as 89.4
Handroanthus impetiginosus ‘Naples White’, a white-flowered cultivar of H. impetiginosus in Bignoniaceae, is valued for its conspicuous large white paniculate inflorescences in early spring. However, the scarcity of stock plants and extremely low fruit set limit conventional propagation. Here, in vitro regeneration protocols of H. impetiginosus ‘Naples White’ were established from apical stem segments, nodal stem segments, cotyledonary nodes, hypocotyls and root segments of aseptic seedlings. The optimum medium for axillary bud multiplication was Murashige and Skoog (MS) medium supplemented with 4.96 µM meta-topolin riboside (mTR) and 0.049 µM indole-3-butyric acid (IBA), giving a multiplication coefficient of 5.14. Hypocotyl explants cultured on half-strength Woody Plant Medium (1/2 WPM) containing 2.07 µM meta-topolin (mT) and 2.22 µM 6-benzylaminopurine (BAP) achieved an adventitious shoot induction frequency of 50.00
Chusquea mimosa, a rare woody bamboo endemic to the Brazilian Atlantic Forest, faces significant conservation constraints due to irregular flowering, limited seed availability, and the absence of efficient vegetative propagation protocols. This study aimed to develop an integrated strategy combining seed cryopreservation and in vitro propagation to support ex situ conservation and large-scale multiplication. Seeds were dehydrated for 18 h, reducing moisture content from 22.8
We established an efficient in vitro regeneration protocol for Paeonia ostii ‘Feng Dan’ based on large-scale proliferation of meristematic nodules (MNs) in suspension culture. The results showed that segmented nodules with a diameter of 3 mm were more suitable as explants than those of 6 mm. With the increase of auxin concentration in liquid medium, number of nodules decreased. The maximum number of nodules (183.33) was obtained in liquid Murashige and Skoog (MS) medium at 150 revolutions per minute (rpm) on a rotary shaker. Furthermore, the optimal percent of leaf cluster (48.89
Propagation systems based on somatic tissues or cells of elite material is a powerful approach in plant cultivation; however, comparable effective and controllable systems remain limited for kelps, an economically important group of marine macroalgae. In this study, we present a method for the controlled and sustained propagation of the commercial brown alga Undaria pinnatifida using protoplast-derived aposporous filaments (PDAFs). We found that green LED light promotes the formation and maintenance of PDAFs, while sporophyte regeneration is successfully triggered under dichromatic (red plus blue) light. PDAFs produced under green light yielded significantly more sporophytes than those from white or red-light conditions. Optimal and sustained sporophyte production was achieved using 500–750 filaments/mL, light intensities of 40–80 µmol photons m−² s− 1, and subcultures younger than four months with at least six weeks of regrowth. Comparative nuclear DNA content analysis using haploid and diploid reference materials indicated that both PDAFs and regenerated sporophytes exhibited relative DNA content consistent with diploid levels. Furthermore, cryopreservation using proline-based cryoprotectants resulted in high post-thaw viability (73–76
Lychnis flos-cuculi L. is a plant species valued for its rich content of phenolic compounds and traditional therapeutic applications; however, the decline of natural populations necessitates the development of alternative biomass sources. The present study investigated the effect of biotic elicitation with Evernia prunastri (L.) Ach. thallus extract and usnic acid on phenolic compound accumulation in in vitro shoot cultures of L. flos-cuculi. Shoots were cultivated in liquid Murashige and Skoog medium under shake-flask conditions and treated with E. prunastri thallus extract (1 mg/mL–3 mg/mL) or (+)-usnic acid previously isolated from E. prunastri (6.25–25 µM) for 24, 72, 144, and 192 h. The concentrations of selected phenolic acids and flavonoids were determined by HPLC-DAD. The elicitation treatments did not adversely affect shoot morphology or physiological status; however, biomass growth generally decreased with increasing elicitor concentration and exposure time. Four phenolic acids (caffeic, ferulic, p-coumaric, and protocatechuic acids) and three flavonoids (apigenin, luteolin, and vitexin) were identified in the shoot biomass. The highest phenolic acid accumulation following elicitation with E. prunastri thallus extract was confirmed after 192 h at 3 mg/mL, particularly for p-coumaric acid (14.21 mg/100 g DW). Usnic acid proved to be an effective elicitor of flavonoid biosynthesis, resulting in the highest apigenin content (17.81 mg/100 g DW) after 192 h treatment with 25 µM usnic acid. The results demonstrate that biotic elicitation with lichen-derived compounds effectively enhances the production of valuable phenolic metabolites in L. flos-cuculi in vitro cultures. Therefore, agitated shoot cultures represent a promising sustainable source of bioactive compounds for pharmaceutical and biotechnological applications. Biotic elicitation with Evernia prunastri extract and usnic acid enhanced the accumulation of phenolic acids and flavonoids in Lychnis flos-cuculi in vitro shoot cultures.
DNA-free genome editing offers a route to improve ornamental crops without stable integration of foreign DNA, yet its use remains constrained by genotype dependence, limited regeneration capacity, and the difficulty of converting transient edits into recovered edited plants. We define DNA-free editing narrowly as the delivery of preassembled CRISPR/Cas ribonucleoproteins (RNPs) or other non-integrating reagents, such as mRNA, into recipient cells, and we distinguish it throughout from DNA-based editing, which relies on stable or transient expression of nuclease-encoding constructs. Progress in ornamentals is synthesized through a protoplast-centred yet delivery-aware framework that links three sequential stages: recovery of physiologically competent cells, transient editing performance, and regeneration with reliable edit validation. Primary DNA-free evidence in ornamentals remains confined to a few tractable systems. Petunia × hybrida provides the strongest end-to-end RNP evidence, whereas Dianthus caryophyllus provides edited-callus evidence and candidate sequence-variant evidence following tissue electroporation. DNA-based studies are treated only as explicitly labeled comparators that define trait targets rather than as DNA-free evidence. We compare protoplast transfection with emerging alternatives, including tissue electroporation and nanomaterial-, viral-, and mRNA-based delivery, and argue that progress will depend less on editing chemistry than on integrating delivery, tissue culture, regeneration, and validation into genotype-responsive workflows.
Efficient micropropagation protocols were developed for Tripleurospermum conoclinium, T. heterolepis, and T. repens, three medicinal, endemic species of Türkiye, to support their ex situ conservation and sustainable preservation. The effects of different cytokinin–auxin combinations (6-benzylaminopurine (BAP, 0.25, 0.5 and 1 mg L⁻¹), kinetin (KIN, 0.25, 0.5 and 1 mg L⁻¹), 6-(γ,γ-dimethylallylamino)-purine (2iP, 0.25, 0.5 and 1 mg L⁻¹) with indole-3-butyric acid (IBA, 0.1 mg L⁻¹)) and naphthaleneacetic acid (NAA, 0.1 mg L− 1) on shoot proliferation and elongation were evaluated on Murashige and Skoog (MS) medium. The highest shoot numbers were obtained on MS medium supplemented with 0.25 mg L⁻¹ BAP in combination with low NAA concentrations, reaching 3.20 shoots per explant in T. conoclinium and 5.25 shoots per explant in T. heterolepis, whereas T. repens produced a maximum of 3.80 shoots per explant under optimized PGR treatments. Shoot elongation was favored by kinetin or BAP combined with indole-3-butyric acid. Root induction was strongly auxin-dependent, with indole-3-butyric acid enhancing root number and secondary root formation, yielding up to 6.10 roots per shoot in T. conoclinium, 2.19 roots per shoot in T. heterolepis, and 4.95 roots per shoot in T. repens. Successfully rooted plantlets were acclimatized under ex vitro conditions, exhibiting high survival rates (82–95
The acclimatization of micropropagated strawberry plantlets (Fragaria × ananassa ‘Camarosa’) is a key stage often associated with challenges in photosynthetic stability and adaptation during the transition from in vitro to ex vitro conditions. In this study, essential oils (EOs) from Thymus capitatus (thyme) and Rosmarinus officinalis (rosemary) were evaluated as natural biostimulants to enhance plantlet performance. The MS rooting medium was supplemented with rosemary or thyme EO at 10 or 100 ppm, and a total of 16 PhotosynQ parameters were measured to evaluate photoprotection, chloroplast proton motive force (pmf), and electron flow (LEF), as well as environmental and leaf structural traits. Thyme EO treatments significantly outperformed non-supplemented (T0) and auxin-only (T+) controls, consistently improving energy dissipation, pmf-related indices, and non-photochemical quenching (NPQ), thereby stabilizing photosystems. Rosemary EO provided moderate but consistent improvements. Notably, 10 ppm thyme EO induced strong but energy-demanding photoprotection, whereas 100 ppm established a diverse and efficient photoprotective strategy, optimally balancing photochemical performance with metabolic efficiency. EO treatments also decoupled photosynthetic performance from energy-costly thermal dissipation, outperforming auxin-only controls. These results demonstrate that thyme EO, particularly at higher concentrations, is an effective bioactive supplement for the rooting medium, enhancing physiological resilience, photoprotection, and successful acclimatization of micropropagated strawberry plantlets. This study highlights the potential of EO-based priming as an eco-friendly strategy to optimize micropropagation outcomes and support sustainable plant production practices. Thyme essential oil priming enhances micropropagated strawberry plantlet acclimatization by improving photosynthetic efficiency, photoprotection and energy balance, offering a sustainable, eco-friendly strategy for in vitro-to-ex vitro transition.
Potato (Solanum tuberosum L.) is an economically important staple crop worldwide, but its productivity and quality are limited by its complex tetraploid genome and increasingly severe biotic and abiotic stresses. Emerging genome-editing technologies, including engineered nucleases (ZFNs, TALENs), CRISPR/Cas-based systems, and post-genome-editing precision tools (base editors, prime editors), offer promising approaches to accelerate precision breeding in potatoes. We review the current state and applications, focusing on delivery strategies that enable safe, DNA-free, transgene-free edits of polyploid germplasm; base and prime editing for seamless edits at the single-nucleotide and templated sequence levels; and the integration of miRNA profiling and multi-omics (genomics, transcriptomics, proteomics, metabolomics) to prioritize targets and validate phenotypes. This review summarizes examples of reduced tuber browning, modified starch characteristics, and editing of susceptibility loci for late blight and viral resistance, as well as technical challenges specific to potato, such as allele identification in tetraploids, editing efficiency, and bystander edits. This is complemented by a discussion of simplified regulatory, biosafety, and ethical considerations, with actionable recommendations for straightforward, DNA-free workflows, standardized off-target and allele-level analyses, and transparent reporting. Finally, this review identifies key research priorities to facilitate the rapid translation of genome-editing technologies into resilient, safe, and valuable potato varieties.
The genus Lathyrus comprises several important multipurpose and resilient legume species, serving as a valuable reservoir of genetic diversity for stress tolerance and nutritional enhancement. However, its wild species are underutilized in breeding due to asynchronous flowering, short pollen longevity, and lack of standardized germination and conservation protocols. The present study is a novel attempt to optimize the in vitro pollen germination protocol and to develop a reliable cryopreservation strategy for six Lathyrus species (L. pseudocicera, L. odoratus, L. tingitanus, L. annuus, L. aphaca and L. cicera). Pollen exhibited strong species-specific responses to sucrose concentration, with maximum germination recorded on 15
