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Punica granatum L. is an important horticultural fruit crop with high medicinal and economic value. Its rising commercial demand necessitates the production of high-quality planting material. Here, we describe an efficient protocol for direct organogenesis in Punica granatum L. cv. Kandhari Kabuli from mature leaf expiant. The optimized sterilization procedure for expiant includes sequential treatment with 70% ethanol (0.75 min), 0.2% Bavistin (15 min), and 0.5% sodium hypochlorite (2 min), which resulted in 83% axenic cultures. The accumulation of phenolics was effectively controlled by subculturing of leaf expiants three to four times at a regular interval of 24 h. The organogenic capability of leaf segments was investigated on full-strength Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs), including the cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) alone or in combination with anaphthaleneacetic acid (NAA). BAP promoted the greatest morphogenic response as compared to that from TDZ. However, the greatest frequency of shoot induction (43%) was achieved on MS medium supplemented with 10 BAP and 2.5 μM NAA under dark incubation for 2 wk. Furthermore, micro-shoot proliferation and elongation were achieved on multiplication medium consisting of MS medium supplemented with 9.0 μM BAP, 2.5 μM Kinetin (KN), and 0.5 μM gibberellic acid (GA₃) up to the third subculturing. However, further subculturing resulted in vitrification. A hormone-free medium containing 300 mg L⁻¹ activated charcoal (AC) was found to be effective to reduce vitrification and promote shoot multiplication. In vitro rooting was carried out on the ½ MS basal medium containing 500 mg L⁻¹ AC using shoots from different subculture passages. Successive subculturing tends to have a positive effect on in vitro rooting and increased rooting up to 70.62%. Well-rooted plantlets were acclimatized successfully in the small plastic pots containing sterilized sand and later shifted to the soil. This optimized protocol can be routinely used for rapid large-scale propagation of pomegranate and is a prerequisite for trait improvement via genetic engineering.

In order to induce in vitro axillary shoot proliferation from single-node expiants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 µM; 0.25 µM; 0.5 µM; 1.0 µM; 2.5 µM) for 8 wk followed by cultivation on hormone-free medium (AMO) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 µM, 15.0 µM, or 30.0 µM) followed by cultivation on AMO for 8 wk. The highest number of axillary shoots per expiant was achieved with 0.1-µM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-µM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-µM TDZ nor by those triggered by the 30.0-µM TDZ pulse treatment.

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Paeonia , a traditional ornamental flower and economically important crop, requires efficient propagation and targeted breeding for sustainable industrial application. Somatic embryogenesis provides a potentially transformative method for regenerating and genetically transforming Paeonia , offering an alternative route for rapid propagation and precision breeding. However, SE application in Paeonia remains challenging due to strong genotype dependence and tissue culture recalcitrance. This review summarizes recent progress in understanding the key factors affecting SE efficiency, including the decisive role of genotype, explant selection, plant growth regulator combinations, and culture conditions. Critical bottlenecks limiting large-scale application are discussed, particularly browning and hyperhydricity. Additionally, this review covers advances in genetic transformation technologies for Paeonia , including both stable transformation via Agrobacterium -mediated systems and transient transformation approaches such as Agrobacterium infiltration and virus-induced gene silencing (VIGS) for functional genomics studies. Although significant progress has been achieved, key challenges remain including low regeneration efficiency, incomplete transformation systems, and genotype-dependent recalcitrance across most commercial cultivars. We identify critical bottlenecks and propose that future research should integrate optimized culture systems with emerging gene editing technologies to facilitate the development of new Paeonia cultivars, thereby enhancing industrial scalability and market competitiveness.

Micrografting is a powerful biotechnological tool for the micropropagation of fruit trees and can be applied to clonal propagation of recalcitrant species, virus eradication, pathogen management, early screening of scion/rootstock compatibility, and breeding. In olive, however, this technique remains poorly explored, particularly regarding grafting onto fully clonal rootstocks. In this study, we developed an in vitro micrografting (IVM) protocol based entirely on clonally propagated material. Two cultivars: ‘Galega’ and ‘Arbequina’, and two genotypes: genotype 26 (G26), and Genotype 35 (G35), were selected based on their vigor and rooting ability. IVM was optimized by testing different combinations of scion and rootstock length and diameter. Morphological variations, including callus formation, wound healing, and vascular connection, were evaluated in both homografting (Galega) and heterografting combinations. Histological analyses were performed at 7, 14, 21, 30, 45, and 60 days after micrografting (DAM) to characterize graft union development. Wounding occurred as early as 7 DAM, while between 14 and 21 DAM, parenchymatous cells proliferated and differentiated into tracheid cells, indicating the wound healing process. By 30–45 DAM, vascular continuity was re-established, confirming successful graft formation. Overall, these results provide a detailed temporal and anatomical characterization of graft union development and support the optimization and scaling-up of IVM for nursery production. Furthermore, this approach may contribute to sustainable strategies against emerging threats such as Xylella fastidiosa and may allow modulation of vegetative vigor through rootstock selection.

Egypt has encountered a significant sugar scarcity since 2023, due to a water crisis, which has reduced local sugar production, prompting a search for alternative sources. Stevia rebaudiana (Asteraceae) is a natural source of steviol glycosides, which are high-intensity, low-calorie sweeteners with increasing demand in food and pharmaceutical industries. Despite its potential as a water-efficient alternative to sugar crops, Egypt lacks optimized protocols for stevia propagation and secondary metabolite enhancement. This study aimed to develop an efficient in vitro regeneration system for a local stevia genotype using callus induction, as well as both direct and indirect micropropagation and to assess its impact on steviol glycoside accumulation by comparison with conventionally soil-grown plant. Explants were cultured on Murashige and Skoog (MS) media supplemented with varying concentrations of BAP, NAA, and kinetin to evaluate callus formation, shoot proliferation, and root development. Optimal conditions yielded significantly higher shoot regeneration frequencies (up to 93%) and shoot number per explant (up to 12.6). Regenerated plants were acclimatized with a survival rate exceeding 85%. Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) was used for metabolite profiling of the four studied stevia. A total of 18 compounds were detected across the four studied stevia samples, including 11 phenolic compounds, and 7 diterpenoids, primarily stevioside, rebaudioside A, and rebaudioside C. Metabolite quantification based on relative peak areas revealed that the direct micropropagation strategy yielded the highest levels of stevioside and rebaudioside A (13.17 and 5.71%, respectively), surpassing those in soil-grown plants, callus-derived and indirectly propagated samples. Multivariate data analysis was conducted to identify relationships among metabolite markers in the four studied stevia samples. The metabolite profiles of both soil-grown and regenerated through direct micropropagation stevia was found to be similar, with both being rich in steviol glycosides. Notably, the growth duration varied among the four studied stevia. The soil-grown and indirectly micropropagated stevia took 180 and 196 days to reach maturity, respectively while stevia regenerated via direct micropropagation took 140 days, demonstrating a more rapid development. These findings demonstrated that direct micropropagation not only enhances growth but also conserves metabolic integrity, and highlights it as an ideal strategy for scalable production of sweetener under resource-restricted settings in arid and semi-arid regions.

Cell and tissue plant cultures are used either to save vulnerable species from extinction or to multiply valuable genotypes, or both, and are widely applied for economically important plant species. For medicinal plants, the use of in vitro technologies for the production of secondary metabolites and pathogen-free plants has been greatly developed. Two opposite aspects characterize the in vitro micropropagation of medicinal plants: maintaining genetic fidelity for the perpetuation and preservation of elites, and the identification and exploitation of somaclonal variations associated with new, useful traits. A balance between what is advantageous and what is undesirable is necessary, and this implies the identification of somaclonal variability at all levels, from the phenotypic to molecular ones. This review addresses the somaclonal variation arising from the in vitro multiplication of medicinal plants from three perspectives: cytogenetics, genetics, and epigenetics. The possible causes of the appearance of somaclones, the methods for their identification, and the extent to which they are desirable are presented comparatively for different plant species with therapeutic properties. The emphasis is on the subtle changes at the genetic and epigenetic level, as it results from the application of methods based on DNA markers.

The paper is devoted to the optimization of one of the factors influencing the success of micropropagation – the choice of the optimal part of vine to take explant, size of the explant and the method of its exposure in a test tube. The studies have confirmed that the place, where micro-cutting was taken, its size and its orientation during planting can affect the speed of growth processes and the survival rate of plants. The experiment was put on the Purple Early grapevine variety. The part of vine for taking micro-cutting, best size of explant and the method of its orientation in the test tube are established.