Romania has a relatively high diversity of plant species, including 3829 vascular and 979 non-vascular spontaneous plant taxa (species and subspecies). Due to uncontrolled harvesting as well as other causes, including climate change and ecological collapse, the speed of species extinction and the narrowing of the genetic base of plant resources has been reported as a critical issue. Therefore, the national Red List of Romanian flora includes 1453 threatened taxa, of which 95 are endemic and 90 subendemic. Many of these have high ornamental, medicinal–cosmetic, and/or aromatic properties. The high extinction risk of these valuable plants has stimulated both the reconsideration of their vital importance as genetic resources and interest in finding effective methods for conservation. Cultivating these phytogenetic resources in a human-controlled environment is of high importance for effective ex situ conservation, which can further serve sustainable exploitation needs and may facilitate in situ conservation actions. In vitro culture is a powerful tool for producing elite plants for cultivation for different purposes. This review summarizes the current knowledge on in vitro multiplication of 22 endemic and subendemic native plants of Romania, examining the materials used, the treatments applied, and the results obtained in each stage of the micropropagation protocol (culture initiation, proliferation, rooting, and acclimatization). The findings from the reviewed studies are presented in a comparative way, and the potential of plant tissue culture in conservation and sustainable exploitation of these Romanian species is outlined.

The aim of this study was to elaborate an efficient in vitro multiplication protocol for Mentha piperita L. (peppermint) and to perform a comparative evaluation of some biochemical compounds in plants regenerated by micropropagation and conventional method. The use of a plain Murashige and Skoog (MS) basal medium favored the induction of regenerative processes, the percentage of explants that started to grow four weeks after inoculation being 92%. The highest multiplication rate (7.12 shoots/explant) and the highest average shoot length (8.11 cm) were obtained on the MS medium supplemented with 1 mg/L benzylaminopurine, when nodal fragments were used as explants. The rooting phase was not necessary, the shoots developing roots on the multiplication medium. The acclimatization rate of in vitro regenerated plants to ex vitro conditions was 96%. Although biochemical investigations revealed some differences between in vitro regenerated plants and those obtained by conventional methods, the results obtained show that micropropagation can be used successfully to obtain high-quality peppermint biological material, a potential source of bioactive compounds with therapeutic effect.

This study evaluated biomass accumulation and phenolic compound production in seven Hypericum species (H. androsaemum, H. calycinum, H. hirsutum, H. kalmianum, H. olympicum, H. perforatum, and H. triquetrifolium) cultivated in vitro under varying growth regulator treatments and culture periods. Shoots were grown on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) or meta-topoline (mT) and analyzed after 40 and 60 days. MS medium supplemented with 0.2 mg/L BA was the most effective condition for promoting biomass across all species, with shoot fresh weight increasing significantly at 60 days, particularly in H. olympicum, H. perforatum, and H. triquetrifolium. High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) identified 13 phenolic compounds, including flavonols, hydroxycinnamic acids, anthocyanins, phloroglucinols, and naphthodianthrones. Phenolic profiles were species-specific and influenced by culture period. H. kalmianum accumulated the highest total phenolic content (37.6 mg/g DW), while H. olympicum was the top producer of hypericin and pseudohypericin. These results highlight the crucial role of culture conditions in regulating both biomass and phytochemical production and provide a promising approach for producing bioactive metabolites in Hypericum species through in vitro systems.

Gelling agents are widely used to solidify plant tissue culture media, yet differences among commercial products may influence the medium’s physical properties and in vitro development of explants. The aim of this study was to characterize the rheological behavior of six gelling agents (Daishin agar, Gelcarin, Gelrite, Microagar, Phytoagar, and Plant agar) and to examine it in parallel with in vitro performance in Hypericum perforatum, Mentha × piperita, and Stevia rebaudiana. Rheological measurements were performed under steady shear by recording apparent viscosity and shear stress across 5–300 s⁻¹. Daishin agar showed the highest apparent viscosity (49,028.95 ± 128 mPa·s), whereas Gelrite exhibited the lowest viscosity (7826.75 ± 98 mPa·s). Plant responses were evaluated after four weeks on PGR-free Driver and Kuniyuki Walnut (DKW) medium by assessing shoot growth, rooting parameters, and shoot water content. In H. perforatum, the longest shoots were obtained on Gelrite (3.92 ± 0.34 cm), accompanied by the highest rooting percentage (95%). In M. × piperita, Gelcarin produced the longest shoots (8.20 ± 0.55 cm) and the highest number of roots per explant (9.75). In S. rebaudiana, Gelcarin promoted superior root elongation (2.86 ± 0.16 cm) and enhanced shoot growth, while Plant agar also supported favorable shoot development. Shoot water content ranged between 74% and 90%, depending on species and gelling agent. These findings highlight the practical relevance of considering low-shear rheological properties when comparing gelling agents for improving the consistency of in vitro culture media.

This study aimed to develop an efficient micropropagation protocol for Plectranthus amboinicus (Lour.) Spreng, evaluate the genetic fidelity of in vitro-regenerated plants using RAPD molecular markers, and perform a biochemical assessment of micropropagated material compared with acclimatized plants. For the initiation of in vitro cultures, explants (uninodal fragments) were inoculated on Murashige and Skoog (MS) medium without plant growth regulators (PGRs). Various plant growth regulator treatments were tested to evaluate the in vitro regenerative potential of the explants. The highest multiplication rate (3.43 ± 0.21 shoots per explant) was obtained on MS basal medium supplemented with 3 mg/L 6-benzylaminopurine (BAP) and 3 mg/L gibberellic acid (GA₃), while the greatest shoot length (3.22 ± 0.22 cm) was achieved on MS medium containing 1 mg/L BAP and 3 mg/L GA₃. Indole-3-butyric acid (IBA) (0.5 mg/L), applied alone or in combination with GA₃ (3 mg/L), was the most effective treatment for inducing a well-developed root system, facilitating successful acclimatization of regenerated plants (100% survival rate). In vivo rooting of shoots simultaneously with acclimatization was also tested. The best results (100% rooting rate) were obtained using perlite or Jiffy peat pellets as the rooting substrate, and the shoots were treated with Radi Stim. Molecular analyses confirmed the genetic fidelity of the micropropagated plants. Biochemical analyses revealed higher levels of phenolic compounds and flavonoids in in vitro-cultured plants than in acclimatized plants. These results validate the micropropagation protocol morphologically, genetically, and biochemically, and highlight in vitro culture as an efficient approach for producing high-quality P. amboinicus plant material and for the sustainable production of valuable bioactive compounds.