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• Putrescine enhances microshoot regeneration in Turiaçu pineapple. • Nodular cluster induction boosts propagation efficiency. • Morphoanatomical insights shed light on cluster development. • Nodule clusters offer a promising method for pineapple propagation. Nodular clusters are a morphogenetic pathway used in the in vitro propagation of Bromeliaceae due to their high responsiveness in the regeneration of microshoots. Additionally, endogenous polyamines, especially putrescine, may contribute to the induction of these nodular clusters. However, the potential effects of this regulator on the regeneration of microshoots when applied to explants are unclear, especially in traditional pineapple cultivars such as "Turiaçu". Therefore, our objective here was to induce and characterize the morphoanatomy of nodular clusters in combinations of 6-benzylaminopurine (BAP) and putrescine (PUT) in "Turiaçu" pineapple. Leaf segments induced in culture medium supplemented with 4 μM naphthaleneacetic acid (NAA), 8 μM BAP, and 100 μM PUT resulted in an induction rate of 56 %, with the formation of microshoots via nodular clusters, increased length, and the number of leaves per microshoot. Morphoanatomical studies revealed that in the medium with 4 μM NAA, 8 μM BAP, and 100 μM PUT, the clusters presented a voluminous size, microshoots with leaves, and the development of new leaves along the base of the explant, and the formation of meristemoids with small cells arranged in a spiral. The results indicate that auxins and cytokinins combined with exogenous putrescine are efficient for inducing nodule clusters. Thus, exogenous putrescine improves the efficiency of microshoot regeneration by nodular clusters. [ABSTRACT FROM AUTHOR]

Date palm(Phoenix dactylifera L.) holds substantial agricultural, nutritional, and economic significance, particularly in arid and semi-arid regions. Traditional propagation methods, such as offshoot and seed propagation, face limitations including slow growth rates, limited availability of offshoots, and genetic instability, making the adoption of in vitro culture techniques essential for the mass propagation and genetic improvement of Date palm. This comprehensive review delves into the intricacies of in vitro culture protocols for Date palm, focusing on the development of multiple shoot, root, and organ cultures. The review meticulously examines the critical factors influencing each stage of in vitro propagation, including the selection of explants, optimization of culture media, and control of environmental conditions. The efficacy of different plant growth regulators (PGRs), such as cytokinins and auxins, in promoting shoot proliferation, root induction, and organogenesis is compared and analyzed, highlighting the optimal concentrations and combinations for successful tissue culture outcomes. For multiple shoot induction, the review discusses the use of shoot tips, axillary buds, and immature inflorescences, with media formulations like MS, B5, and WPM supplemented with specific PGRs such as BAP and kinetin. The process of root induction is examined, emphasizing the role of auxins like IBA and NAA in developing robust root systems and the influence of environmental factors like light and temperature. Organ culture techniques, particularly embryogenic callus induction and somatic embryogenesis, are explored, detailing the use of high auxin concentrations for callus formation and the transition to media with balanced PGRs for embryo maturation. Additionally, the review addresses the challenges of somaclonal variation and genetic stability in tissue culture, proposing strategies for monitoring and mitigating these issues through genetic and epigenetic analyses. The potential of integrating advanced biotechnological tools, such as CRISPR/Cas9, for precise genetic modifications is discussed, alongside the need for improved acclimatization protocols to enhance the survival rates of in vitro propagated plantlets. Future research directions are identified, focusing on minimizing somaclonal variation, optimizing acclimatization, exploring diverse explant sources, and scaling up production through automated culture systems. By synthesizing the latest research findings and offering detailed comparisons of various in vitro protocols, this review aims to provide a valuable resource for researchers and practitioners seeking to enhance the efficiency and success of Date palm micropropagation, ultimately contributing to the sustainable cultivation and genetic improvement of this vital crop. [ABSTRACT FROM AUTHOR]

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications. [ABSTRACT FROM AUTHOR]

Momordica cochinchinensis (Lour.) Spreng. is a tropical dioecious plant belonging to the family, Cucurbitaceae also known as Gac, baby jack fruit, cochinchin gourd and sweet gourd. The plant is noted for its densely orange fruit, rich in nutrients, especially with 70 times more lycopene content than tomatoes. It possesses medicinal properties and the seeds are used in traditional Chinese medicine called Mubiezhi to treat fluxes, liver and spleen disorders, wounds and hemorrhoids. This study evaluated the effect of combination of pH, sucrose and BAP concentration on in vitro multiple shoot induction response of M. cochinchinensis according to guidelines of design of experiment. For those three factors, Response surface methodology (RSM) using the Box-Behnken design (BBD) was chosen and applied to the design matrix. The response shoot number/explant was evaluated by in vitro culture under variable conditions given in the design matrix. The response shoot number/explant was evaluated using in vitro culture under variable conditions given in the design matrix. The outcome of this method is an equation that calculates the shoot number as a function of pH, sucrose, and BAP concentration This study created response surface plots and contour plots for each factor, by analysing each factor's level of influence on the response shoot induction. [ABSTRACT FROM AUTHOR]

Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (Citrus x limon var. limon x Citrus latifolia var. latifolia). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5–2 mg L−1 benzyladenine (BA) + 0.5 mg L−1 gibberellic acid (GA3) + 0.25 mg L−1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L−1 BA + 0.5 mg L−1 kinetin (KIN) + 0.5 mg L−1 GA3 + 0.25 mg L−1 NAA. Rooting was best promoted by 1 mg L−1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study. [ABSTRACT FROM AUTHOR]

This study focuses on the endangered neo-endemic Baltic dunes species Linaria loeselii Schweigg. (Plantaginaceae), also known as Linaria odora (M. Bieb.). By utilizing in vitro cultures, we successfully germinated seeds collected in situ. Our method, which involved using media supplemented with 5 µmol/L 6-benzylaminopurine, led to the indirect regeneration of shoots after 60 days of culture in the dark, significantly increasing the number of progeny plants. Additionally, the medium supplemented with 2.85 μmol/L indole-3-acetic acid and 10.2 μmol/L paclobutrazol allowed rooting after 30 days of shoot fragments. This research provides a potential basis for developing Linaria loeselii introduction programs into the environment, thereby contributing to the conservation of this endangered species. [ABSTRACT FROM AUTHOR]

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AbstractMelatonin was discovered in plants in 1995, and so-called phytomelatonin. Its characteristic qualities as an antioxidant molecule and with amphiphilic nature make it an excellent candidate to use in plant biotechnological techniques. In plant physiology, melatonin has demonstrated with multiple studies great possibilities of application in crop improvement in aspects such as germination, rooting, plant growth, leaf-senescence, floration, fruit-set, parthenocarpy, fruit ripening and cut flower conservation, among others. In this work, we reviewed many studies on the possible role of melatonin to improve the different plant tissue culture goals, among them: the role of melatonin in callus culture induction and its effects in plant biomass production; the possible action of melatonin as an inductor or elicitor of different secondary metabolite generation in in vitro cultures; the interesting possibilities of melatonin as a protective agent (safener) against abiotic stressors present in in vitro cultures; the potential of melatonin in regulating plant organogenesis, modulating shooting/rooting responses; at last, the studies on the relevant role of melatonin in plant material cryopreservation and the improvement of cell transformation using A. tumefaciens have been discussed. [ABSTRACT FROM AUTHOR]

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies. [ABSTRACT FROM AUTHOR]

WUSCHEL-related homeobox (WOX) genes are plant-specific homeobox transcription factors and are known to play a key role during in vitro embryogenesis and organogenesis. To decipher the structural basis and genetic diversification of WOX family genes, this study performed a genome-wide analysis in Plantaginaceae. In the current study, Picrorhiza kurrooa and Antirrhinum majus (snapdragon; a model organism) of the Plantaginaceae family were used for in silico analysis of WOX gene family members. The physiochemical properties, subcellular localization, and phylogenetic analysis in P. kurrooa were evaluated. Further, to understand gene structure, chromosomal localization, and synteny, A. majus along with Arabidopsis thaliana was used due to unavailability of chromosome-level resolved genome in P. kurrooa. Five WOX and one WUS members were identified in the P. kurrooa genome while eight WOX and eight WUS members were identified in A. majus distributed in five clades. Phylogenetic analysis revealed that PkWOX and AmWOX showed greater similarity to each other, as do PkWUS and AmWUS, compared to AtWOX and AtWUS. Further, the tree was separated in three different clades, including ancient, intermediate, and WUS clade. Chromosomal localization of WOX and WUS genes in A. majus revealed their distribution between 2 and 7 chromosomes while chromosome 1 and 8 did not contain any member. Expression analysis of P. kurrooa WOX and WUS genes showed differential expression during the different stages of in vitro organogenesis (mother leaf, callus initiation, callus proliferation, and regeneration) and different temperature gradients (25°C, 15°C, and 10°C). Taken together, this study provided significant insight into the developmental and stress responsiveness of WOX gene family members in P. kurrooa, which can be further explored as promising candidates. [ABSTRACT FROM AUTHOR]

Chinese artichoke (Stachys sieboldii [Miq.]) is a popular healthcare food owing to its high contents of stachyose. Viral infections have caused the severe degeneration of germplasm in this plant species. An efficient virus elimination and micropropagation system is required to produce Chinese artichoke plants that are free of viruses. A protocol for virus elimination in Chinese artichoke was established by comparing the potential for growth of the terminal buds using shoot apices as starting materials, in media with or without plant growth regulators (PGRs) and various concentrations of sucrose. Murashige and Skoog (MS) medium without any PGR performed better than that supplemented with PGRs as indicated by a higher rate of survival of shoot apices, higher plant height, more root numbers and adventitious buds, and increased biomass (P < 0.05). An experiment to further optimize the sucrose concentration demonstrated that MS medium with 5.0% sucrose (w/v) efficiently induced the growth of both roots and shoots, thus, achieving the efficient micropropagation of Chinese artichoke within 4 wk. Compared with the mother plants in which viruses had not been eliminated, the virus-free plants had significantly higher numbers of tubers, increased yield, and a higher content of stachyose (P < 0.05). Higher contents of endogenous hormones were detected in Chinese artichoke, which may explain the efficient micropropagation without the use of exogenous PGRs. This simple protocol enabled the production of virus-free Chinese artichoke to enhance the yield of tubers and high content of stachyose. [ABSTRACT FROM AUTHOR]

Petiole segments of an economically ornamental plant Oxalis triangularis A.st.-Hil have been used in the present study for rapid, reliable and systematic in vitro propagation. The morphogenic effect of a-Naphthalene acetic acid (NAA) at various concentrations (0.5 - 3.0 mg/L) was studied individually or in combination with 6-benzylaminopurine (BAP). Superior multiplication rates were achieved on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BAP. Of the tested combinations, maximum shoot regeneration (100.00 %), mean shoot number (14.60 ± 0.37 shoots per segment), and shoot length (18.11 ± 0.30 mm) were recorded on MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BAP after 6 weeks of incubation. Half-strength MS medium augmented with 1.0 mg/L indole-3-butyric acid (IBA) was the optimal medium for in vitro root induction, (24.60 ± 0.37 roots per shoot with 9.10 ± 0.32 mm in length) after 6 weeks of incubation. The regenerated plantlets showed 100 % survival success during acclimatization and exhibited normal growth characteristics and morphology. This optimized protocol is cost-effective and highly efficient for the rapid mass propagation of Oxalis triangularis A.st.-Hil. [ABSTRACT FROM AUTHOR]

Aquatic plants are grown in aquaria for their beauty and to maintain the quality of water. The explants of Lobelia cardinalis L. were obtained from the Aquatic Plant Center Co., Ltd., Thailand. In vitro clonal propagation offers alternative strategy for its rapid clonal multiplication. In this study, multiple and rapid propagations of Lobelia cardinalis L. with tissue culture techniques were aimed. Highly efficient and repeatable in vitro regeneration protocol was established from nodal segments of Lobelia cardinalis L. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation, number of leaves and rooting of shoots to obtain complete plantlets of Lobelia cardinalis L. Among 2 PGRs such as 6-benzylaminopurine (BAP) and a-Naphthalene acetic acid (NAA) tested for multiple shoot induction. Regenerated shoots had a higher number of shoots on MS medium contained cytokinins with auxins. The maximum shoot proliferation (100 %), number of shoots per explant (6.20 ± 0.51 shoots), shoot length (11.67 ± 0.69 mm) and number of leaves per explant (58.10 ± 0.38 leaves per explant) were observed in MS medium supplemented with 1.0 mg/L BAP and 2.0 mg/L NAA. Rooting was achieved within 3 to 4 weeks on all the rooting media, but MS medium augmented with 1.0 mg/L indole-3-butyric acid (IBA) produced the maximum number of strong and healthy roots (11.70 ± 0.27 roots). The regenerated complete plantlets with healthy roots and shoot system were transferred to 3.5 cm diameter black net plastic pots containing rockwool medium and successfully acclimatized with a survival rate of 100 % in the greenhouse and no phenotypic variations were observed among regenerated plants. The success of this protocol offers highly efficient method of the plant multiplication, which would be beneficial for plant tissue culturist, and nursery people where regular supply of plants is a large number required. This protocol can be utilized for commercial scale propagation. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum Royle, also known as Podophyllum emodi Wall, holds significant ecological, ornamental, and medicinal values. However, it has become endangered due to overexploitation, prolonged seed dormancy, slow natural regeneration, and climate change. This study developed an efficient in vitro protocol for callogenesis and micropropagation of P. hexandrum to conserve germplasm in in vitro conditions. Callus formation from various plant parts, including the leaf, stem, rhizome, radicle, and cotyledon, was induced using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The combination of benzyladenine at 1 mg L−1 and 4-dichlorophenoxy acetic acid at 3 mg L−1 was optimal for biomass production, yielding 215.88 ± 0.31 mg, with growth per gram at 8.32 ± 0.32 and a growth rate of 13.62 ± 0.25 mg/day on MS medium. For shoot proliferation, benzyladenine (3.5 mg L−1) and naphthalene acetic acid (0.5 mg L−1) combined with activated charcoal showed the highest shoot induction percentage per explant. For shoot regeneration from calluses, 6-benzylaminopurine (0.5 mg L−1) and thidiazuron (2 mg L−1) were most effective, producing superior shoot length, number of regenerations, and regeneration percentage. Root induction was successful with α-naphthalene acetic acid supplementation (0.5 to 1.5 mg L−1) in MS medium, resulting in the highest number per explant (4.08 ± 0.08), length (5.45 ± 0.15 cm), and rooting rate (87.00 ± 1.66%) of roots in plantlets. Subculturing for callus culture was performed every 28 days for up to four subcultures to prevent nutrient depletion and toxic metabolite accumulation, ensuring tissue health and viability. Continuous subculturing of callus on MS medium maintained healthy P. hexandrum germplasm in vitro. Overall, this micropropagation protocol provides a rapid system for conserving P. hexandrum germplasm. [ABSTRACT FROM AUTHOR]

The safe management of sewage waste is a current concern due to population growth and waste production. Biosolids, derived from sewage sludge treatment, are globally used as organic fertilizers, aligning with Sustainable Development Goal 6 for resource recycling. However, biosafety concerns arise due to the presence of metals and microplastics in biosolids, potentially impacting soil and water. This study investigated biosolids’ use for in vitro cultivation of Bowdichia virgilioides Kunth. Results indicate that while biosolids can replace traditional nutritional media, balancing their concentration is crucial for optimizing plant growth. The WPM (Wood Plat Medium) remains essential for in vitro cultivation, but substituting it with biosolids at concentrations of up to 2 g L− 1 is feasible, providing similar plant development compared to the WPM medium. However, when combined, there is a complex and challenging interaction between biosolids and the culture medium. [ABSTRACT FROM AUTHOR]

Lavandula stoechas subsp. luisieri and Pterospartum tridentatum are two valuable aromatic and medicinal plants. Their biometric and morphological parameters, such as the number of new shoots, length of the longest shoot, multiplication rate, and fresh weight, were evaluated using the multiplication MS medium protocol. The rooting protocols involved immersing the explants in IBA (1 g L−1) and a commercial IBA (3.3 g L−1) preparation (Clonex®). Slow-growth conservation assays were carried out using two different sucrose concentrations (15 g L−1 and 30 g L−1), and a control, with the cultures kept at 4 °C for 12 months. The multiplication rate for L. stoechas subsp. luisieri was 6.8, and that of P. tridentatum was 13.3, achieved using the MS medium supplemented with 0.2 mg L−1 BAP, 1 mg L−1 BAP, and 0.5 mg L−1 IBA. The application of Clonex® showed the best ex vitro rooting results in L. stoechas subsp. luisieri (77%) and P. tridentatum (90%). In the slow-growth conservation assays, at 4 °C, in darkness for 12 months, an excellent survival rate was achieved in L. stoechas subsp. luisieri (>80%) and P. tridentatum (>90%), even at the reduced sucrose concentration. This study demonstrates the effectiveness of in vitro multiplication and ex vitro rooting protocols for two valuable aromatic and medicinal plants. These findings are significant for the ex situ conservation of these species, as they provide effective long-term preservation and utilization strategies. [ABSTRACT FROM AUTHOR]

The demand for bioactive secondary metabolites of natural origin is increasing every day. Micropropagation could be a strategy to respond more quickly to market demands, regardless of seasonality. This research aims to evaluate in vitro-grown plants of two hop varieties, namely Columbus and Magnum, as a potential source of bioactive compounds. The extracts were characterized in terms of total phenolic content by a Folin–Ciocalteu assay and antioxidant capacity by DPPH•, ABTS+, and FRAP assays. The bioactive compound profile of the extracts from both varieties was determined by using UPLC-ESI-QqQ-MS/MS. The results confirmed richness in (poly)phenols and other secondary metabolites of the in vitro-grown hop plantlets. Thirty-two compounds belonging to the major families of phytochemicals characteristic of the species were identified, and twenty-six were quantified, mainly flavonoids, including xanthohumol and isoxanthohumol, phenolic acids, as well as α- and β-acids. This study confirms the validity of in vitro-derived hop plantlets as source of bioactive compounds to be used in the nutraceutical, pharmaceutical, and food industries. [ABSTRACT FROM AUTHOR]

The large-leaved Homalomena (LLH, Homalomena pendula) represents an endangered medicinal plant species within Vietnam, primarily attributed to its recognized tonic properties. Despite its imminent threat of extinction within Vietnamese ecosystems, the development of a robust protocol for molecular species identification and large-scale propagation of LLH remains absent. Consequently, we present the first conservation endeavor for LLH based on plant micropropagation techniques, with plant materials validated through anatomical observations and DNA barcoding (rbcL). Our investigation yielded five rbcL sequences specific to LLH, serving as the current best barcode for LLH identification and thereby facilitating forthcoming taxonomic endeavors. Optimization of in vitro culture conditions revealed that the Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine, 0.5 mg/L α-naphthaleneacetic acid, and 60 g/L mashed potato, alongside the incorporation of 0.5 mg/L indole-3-butyric acid to the basal MS medium, yielded optimal outcomes for shoot proliferation and root development, respectively. After successful micropropagation, acclimatization of rooted plantlets to a substrate comprising soil, coconut coir, and rice husk (in a 1:1:1 ratio) culminated in a 100% survival rate among the plants.Key message: The first established protocol for species identification and micropropagation of H. pendula. [ABSTRACT FROM AUTHOR]

Use of meta-Topolin (mT) has significantly affected tissue culture results in several plant species. In this study, a fast and efficient micropropagation protocol for Withania somnifera was standardized. Also, a comparison was made between the effect of using M.S. media supplemented with mT and M.S. media supplemented with different concentrations of B.A.P. + AdSO4 on the in vitro regeneration of W. somnifera. M.S. media supplemented with I.B.A. was used to obtain a healthy and advantageous root system. The resulting plantlets were analyzed for their total chlorophyll and protein content. The comparison revealed that 2.5 mg/l mT supplemented M.S. media showed better results with 88.9 ± 0.42% bud breaking percentage, a shoot multiplication rate of 22.4 ± 4.16 shootlets per explants, total chlorophyll content of 0.887 ± 0.004 mg/g and protein content of 25.67 ± 0.25%. Healthy adventitious root systems were observed in regenerated shootlets inoculated in M.S. media supplemented with 2.0 mg/l I.B.A. During hardening, 80% of plants treated initially with mT survived. Whereas treatment with M.S. media supplemented with 1.0 mg/l B.A.P. + 50 mg/l AdSO4 resulted in bud breaking percentage of 61.1 ± 0.09%, a shoot multiplication rate of 15.8 ± 7.81 shootlets per explant, total chlorophyll content of 0.7194 ± 0.0055 mg/g and protein content of 23.33 ± 0.25%. During hardening, 78% of plantlets treated initially with B.A.P. + AdSO4 survived. Therefore, the study concludes that mT may be used as an alternative source of cytokinin for obtaining healthy plantlets with a higher rate of plant regeneration in W. somnifera.Key message: The study carried out to standardize a viable protocol for meta-Topolin as alternative source of cytokinins in micropropagation for Withania somnifera and to compare the effects using different phytohormones. [ABSTRACT FROM AUTHOR]

Eucheuma denticulatum and Kappaphycus alvarezii are two of the most commercially important, cultivated carrageenophytes in Southeast Asia. Their large-scale farming through repeated vegetative propagation and virtual monocropping has resulted in a variety of phyconomic issues. The efficacy of two European, commercial liquid extracts from the brown seaweeds, i.e., Ascophyllum nodosum (ANE), a fucoid, and Laminaria digitata (LDE), a kelp, on the direct formation of axes and survival of these two eucheumatoids was examined using tissue culture techniques. Seaweed apical sections (2–3 mm long) were cultured for 45 days, with different concentrations of ANE and LDE (i.e., 0.005, 0.05, 0.5, 1.5, and 2.5 mL L−1) and compared with a control (UV-filtered seawater). Lower concentrations (< 1.5 mL L−1) of both liquid extracts promoted morphogenesis and growth of E. denticulatum and K. alvarezii in tissue culture, with the dilution rate of 0.05 mL LDE L−1 resulting in a high percentage survival (i.e., 95.3 ± 0.9%), formation of direct axes (i.e., 95.3 ± 0.9%), and the longest length (i.e., 10.0 ± 0.3 mm) for E. denticulatum after a typical 45-day culture period. A 0.5 mL ANE L−1 concentration was found to be beneficial for the tissue culture of K. alvarezii, given the relatively high final survival and direct axes formation (i.e., 96.2 ± 2.2%) and axes length (i.e., 8.2 ± 0.1 mm). This study supports the application of these temperate, brown seaweed-derived extracts as a phycobiostimulant enrichment in eucheumatoid micropropagation for mass production of plantlets for out-planting purposes. [ABSTRACT FROM AUTHOR]

Zani WNWM, Sidik NJ, Isa NAM, Awal A, Osman NI, Alias N, Nordin MK. 2024. Influence of media variations and growth regulators on in vitro propagation of Dendrocalamus asper bamboo. Nusantara Bioscience 16: 237-244. This study reports the effects of different types of media and combinations of plant growth regulators on the in vitro propagation of Dendrocalamus asper (Schult.f.) Backer bamboo species, a species known for its economic significance and challenges in traditional cultivation. The experiment was initiated through the cultivation of in vitro nodal segments as explants in various strengths (half-strength and full-strength) of Murashige and Skoog's (MS) and Vacin & Went (VW) media, supplemented with 1.0 mg L-1 6-benzylaminopurine (BAP) hormone. Optimal results were achieved in full-strength MS media with 1.0 mg L-1 BAP, with the highest shoot number (5.6 shoots) and length (2.14±0.18 cm), surpassing outcomes in VW media. Shoot multiplication in full-strength MS media, with varying BAP and indole-3-butyric acid (IBA) hormone combinations (0.5, 1.0, 2.0, and 4.0 mg L-1), was conducted. The combination of 4.0 mg L-1 BAP and 0.5 mg L-1 IBA yielded the highest number of shoots (5.17±3.97), while supplementation with 4.0 mg L-1 IBA alone resulted in the longest shoot with 1.89±1.15 cm. These findings underscore the significance of tailored conditions for optimal in vitro propagation of this species. Further investigations could explore additional factors influencing the propagation process for better refinement of bamboo cultivation techniques. [ABSTRACT FROM AUTHOR]

Abstract Background Kale, a versatile cruciferous crop, valued for its pro-health benefits, stress resistance, and potential applications in forage and cosmetics, holds promise for further enhancement of its bioactive compounds through in vitro cultivation methods. Micropropagation techniques use cytokinins (CKs) which are characterized by various proliferative efficiency. Despite the extensive knowledge regarding CKs, there remains a gap in understanding their role in the physiological mechanisms. That is why, here we investigated the effects of three CKs – kinetin (Kin), 6-benzylaminopurine (BAP), and 2-isopentenyladenine (2iP) – on kale physiology, antioxidant status, steroidal metabolism, and membrane integrity under in vitro cultivation. Results Our study revealed that while BAP and 2iP stimulated shoot proliferation, they concurrently diminished pigment levels and photosynthetic efficiency. Heightened metabolic activity in response to all CKs was reflected by increased respiratory rate. Despite the differential burst of ROS, the antioxidant properties of kale were associated with the upregulation of guaiacol peroxidase and the scavenging properties of ascorbate rather than glutathione. Notably, CKs fostered the synthesis of sterols, particularly sitosterol, pivotal for cell proliferation and structure of membranes which are strongly disrupted under the action of BAP and 2iP possibly via pathway related to phospholipase D and lipoxygenase which were upregulated. Intriguingly, both CKs treatment spurred the accumulation of sitostenone, known for its ROS scavenging and therapeutic potential. The differential effects of CKs on brassicasterol levels and brassinosteroid (BRs) receptor suggest potential interactions between CKs and BRs. Conclusion Based on the presented results we conclude that the effect evoked by BAP and 2iP in vitro can improve the industrial significance of kale because this treatment makes possible to control proliferation and/or biosynthesis routes of valuable beneficial compounds. Our work offers significant insights into the nuanced effects of CKs on kale physiology and metabolism, illuminating potential avenues for their application in plant biotechnology and medicinal research.

Light-emitting diodes (LEDs) have become an alternative light source to the fluorescent lamp (FL) for the maintenance of plant tissue cultures due to their low heat emission, low power consumption and the ability to fine-tune the light spectrum. In this study, the effect of LEDs on the growth and leaf stomata features of in vitro cultivated raspberries (Rubus idaeus L. 'Lloyd George') was examined. The plantlets were grown in vitro under a lighting system based on the Philips GreenPower LED research module. Four groups of LEDs emitting white (W), red (R), blue (B), and mixed (W:R:B: far red = 1:1:1:1) lights and FL (control) were used. As a second control (marked as EV), plants grown in the multiplication and rooting stage under fluorescent lamps and then acclimatized ex vitro in a greenhouse for 90 days in natural light were included. Relative growth rate (RGR), protein content as well as stomata morphology and density of the plantlets were analysed after three passages under corresponding light treatment. The results show that different LEDs specifically affect the growth and size of leaf stomata and density of in vitro cultured raspberry plants and can be applied as an effective modulator of morphogenesis during micropropagation. The combination of white, blue, red and far red LED stimulated the accumulation of biomass and proteins, as well as the formation of a higher number of stomata on the lower surface of the leaves, which would be a prerequisite for more effective control of water loss from plantlets during the ex vitro acclimatization.

Background: Kale, a versatile cruciferous crop, valued for its pro-health benefits, stress resistance, and potential applications in forage and cosmetics, holds promise for further enhancement of its bioactive compounds through in vitro cultivation methods. Micropropagation techniques use cytokinins (CKs) which are characterized by various proliferative efficiency. Despite the extensive knowledge regarding CKs, there remains a gap in understanding their role in the physiological mechanisms. That is why, here we investigated the effects of three CKs – kinetin (Kin), 6-benzylaminopurine (BAP), and 2-isopentenyladenine (2iP) – on kale physiology, antioxidant status, steroidal metabolism, and membrane integrity under in vitro cultivation. Results: Our study revealed that while BAP and 2iP stimulated shoot proliferation, they concurrently diminished pigment levels and photosynthetic efficiency. Heightened metabolic activity in response to all CKs was reflected by increased respiratory rate. Despite the differential burst of ROS, the antioxidant properties of kale were associated with the upregulation of guaiacol peroxidase and the scavenging properties of ascorbate rather than glutathione. Notably, CKs fostered the synthesis of sterols, particularly sitosterol, pivotal for cell proliferation and structure of membranes which are strongly disrupted under the action of BAP and 2iP possibly via pathway related to phospholipase D and lipoxygenase which were upregulated. Intriguingly, both CKs treatment spurred the accumulation of sitostenone, known for its ROS scavenging and therapeutic potential. The differential effects of CKs on brassicasterol levels and brassinosteroid (BRs) receptor suggest potential interactions between CKs and BRs. Conclusion: Based on the presented results we conclude that the effect evoked by BAP and 2iP in vitro can improve the industrial significance of kale because this treatment makes possible to control proliferation and/or biosynthesis routes of valuable beneficial compounds. Our work offers significant insights into the nuanced effects of CKs on kale physiology and metabolism, illuminating potential avenues for their application in plant biotechnology and medicinal research. [ABSTRACT FROM AUTHOR]

Advancements in micropropagation techniques have made it easier to produce large numbers of cannabis clones, but these methods may also introduce genetic instability over successive generations. This instability often manifests as somaclonal variation, characterized by the progressive accumulation of genetic mutations or epigenetic alterations with each subculture. In this study, we examined how mutations accumulate in cannabis clones subjected to 6–11 subcultures. Using genotyping-by-sequencing, we identified 9405 polymorphic variants across 70 clones. The analysis revealed a correlation between the number of subcultures and the frequency of these mutations, revealing that genetic changes accumulate over successive subcultures despite clones sharing the same chronological age. Furthermore, we evaluated the functional impacts of accumulated mutations, with particular attention to implications on gene function and overall plant health. While rare, 14 high-impact variants were identified in genes that are important for plant development. Notably, six variants were also found in genes related to cannabinoid and terpene synthesis pathways, potentially affecting the plant's biochemical composition. These findings highlight the need for genetic assessments in micropropagation protocols, impacting plant breeding and conservation. Understanding genetic variations in clonally propagated plants optimizes practices for stability. Crucial for cannabis and horticultural plants, it emphasizes techniques to prevent genetic decay and ensure viability. [ABSTRACT FROM AUTHOR]

Plant biotechnology has become a viable strategy for agriculture, its applications include strategies aimed at obtaining and maintaining pathogen-free cultivars, genetic improvement (anther cultivation, embryo rescue, etc.) and micropropagation, used to obtain a greater number of individuals in less time and reduced spaces. Each of its phases depends largely on an adequate culture medium, since the growth, development and morphogenesis of the explant is mainly due to its chemical composition. For the production of garlic "seed" (Allium sativum L.), all phases are carried out in a growth chamber under automatically controlled aseptic and artificial conditions and at a temperature ranging between 22-25 °C. Somatic embryogenesis allows complete plants to be regenerated from a single cell. Garlic is an annual plant that only reproduces asexually, which is why bacteria, fungi and mainly viruses, which contribute to a 50-80 % decrease in its yield, affect it. An alternative to avoid these problems is the use of chemotherapy, thermotherapy or the combined use of one of these techniques with the cultivation of meristems, to obtain plants of high biological quality. Depending on the behavior of the culture during its in vitro stage, its response will be in the acclimatization phase and later in field conditions. This work was carried out with the objective of deepening the knowledge of some biotechnological techniques that are applied in the cultivation of garlic. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the impact of varying gamma irradiation doses on banana scalps' survival and regeneration potential, specifically focusing on their interaction with different culture media components. Banana scalps, derived from a selected cultivar (Calcutta-4), were exposed to gamma irradiation doses ranging from 10 Gy to 70 Gy. Following irradiation, the scalps were transferred to fresh culture media, and their survival rates and regeneration potential were assessed. The findings revealed a clear inverse relationship between radiation dose and scalp survival. Lower doses of 10 Gy and 20 Gy exhibited higher survival rates when cultured on a modified MS medium. In comparison, higher doses, such as 50 Gy and 60 Gy, led to a significant decrease in survival. The optimal gamma irradiation dose for both scalp survival and successful regeneration fell within the range of 30 Gy to 40 Gy when using a medium supplemented with lower concentrations of BAP and Ads (1.5 mg/l to 2.0 mg/l BAP + 200 mg/l to 300 mg/l Ads). Furthermore, the study demonstrated that the surviving irradiated scalps exhibited a high potential for shoot regeneration when cultured on an MS medium with 3.0 mg/l BAP + 400 mg/l Ads. Gamma irradiation facilitated the initiation of new shoots, and the specific concentration of plant growth regulators and media composition influenced the response. These findings have significant implications for optimizing tissue culture techniques and improving crop production in banana cultivars and are crucial for advancing banana farming practices. [ABSTRACT FROM AUTHOR]

• Investigation of strategies for enhanced biosynthesis of bioactive compounds. • Pathway for the biosynthesis of glucosinolates, isothiocyanates, thiocarbamates, and omoringone. • Commercialization challenges addressing, contamination, propagation and factors impacting yield and quality. • Role in green synthesis of nanoparticles with antimicrobial, antioxidant, and other potential applications. Moringa oleifera Lam., also known as the "drumstick tree" or "miracle tree,'' has a wide range of applications in both traditional and modern medicine due to presence of specific bioactive compounds. This review investigates the chemical composition of the plant, the mechanisms involved in the biosynthesis of bioactive compounds, and strategies for increasing their production. Glucosinolates, isothiocyanates, and other secondary metabolites are among the important bioactive compounds found in Moringa , which have shown anti-inflammatory, antimicrobial, antioxidant, and anticancer properties. These findings are gaining popularity for their potential applications in medicine, nutrition, and industry. This paper discusses the challenges of commercializing Moringa while focusing on issues related to contamination, propagation methods, environmental factors that influence yield and novel use of M. oleifera for the green synthesis of metallic and nonmetallic nanoparticles such as silver, zinc, copper, iron, gold, chitosan and others. These nanoparticles have antimicrobial, antioxidant, and photocatalytic properties, making them useful for applications in healthcare, environmental remediation, and other fields. Finally, M. oleifera is a versatile and resourceful plant with the ability to resolve a wide range of health concerns and provide long-term viable alternatives to a wide range of medicinal and therapeutic applications. [ABSTRACT FROM AUTHOR]

This research focuses on the seasonal patterns of the ecophysiological recovery of four olive cultivars (Arbequina, Coratina, Frantoio, and Urano), both micropropagated and self-rooted, grown in a mature, irrigated, super high-density (SHD) orchard under Mediterranean conditions (Southern of Italy). The aim was to observe the impact of the micropropagation method on the ecophysiological responses. Ecophysiological parameters, including leaf water potential (LWP), stomatal conductance (gs), net photosynthetic rate (Pn), and transpiration rate (E) were assessed. Self-rooted trees consistently exhibited superior gs, E, and Pn recovery compared to the micropropagated ones. 'Arbequina' maintained elevated levels of Pn under water-deficit conditions. 'Coratina' exhibited increases in gs and E after irrigation. 'Frantoio' demonstrated recovery capabilities, with lower LWP and higher Pn under stress. 'Urano' micropropagated trees achieved higher gs values in mid-summer, while self-rooted trees sustained higher Pn later in the season. This field research highlighted the important role of the propagation method in optimizing the physiological performance of olive cultivars in SHD orchards. Furthermore, it highlighted the necessity of long-term studies on the effects of propagation methods and their interactions with other farming practices. [ABSTRACT FROM AUTHOR]

Zuccagnia punctata Cav. (“jarilla poposa”) is a monotypic argentine native medicinal plant used by indigenous communities for the treatment of several pathologies. The pharmacological properties have been scientifically validated and its potential use in phytocosmetic and pharmaceutic industry as antiaged, hypoglycemic, anti-inflammatory, antitumoral, antioxidant, antibacterial and antimicotic has been previously demonstrated. However, the species has a low propagation rate in natural conditions and has been included in the preliminary red list of endangered plants in category 3. Therefore, it is necessary to develop methodologies leading to its conservation and propagation to achieve its sustainable use. The objective of this work is to perform a micropropagation protocol for Z. punctata species for the sustainable use and conservation of this species. Furthermore, the study evaluated also the impact of “in vitro” propagation on the content of bioactive compounds of ethanolic extracts obtained from Z. punctata plants obtained “in vitro” with collected wild plants. Seeds have been used as mother material. The seed germination to obtain explants were performed on Murashige and Skoog medium (MS) without plant growth regulators (PGRs) containing sucrose, agar, and ascorbic acid. The same medium was employed for the multiplication of shoots. “In vitro” rooting was achieved in liquid and semisolid medium, and a higher yield was obtained in the latter. The plants were transferred into commercial substrate and acclimatized in a greenhouse. The micropropagated plants (MP) had a high content of total phenolic compounds similar to those obtained from wild adult plants (WAP) collected in the Monte Desert. The flavonoid content of MP was higher than that of WAP. The main chalcones identified in WAP, 2′,4′ dihydroxychalcone (DHC) and 2′,4′-dihydroxy-3′-methoxychalcone (DHMC) were also present in the MP with a rate 1/1, and 1/3 in WAP. The extracts obtained from the MP showed antioxidant, antiinflammatory and antimicrobial activity, similar to WAP. The “in vitro” propagation and acclimatization of Z. punctata constitutes a fundamental tool to achieve large-scale multiplication of this species in the future and the sustainable production of bioactive compounds with commercial applications. Reimplantation in the natural habitat could prevent the loss of this medicinal genetic resource. [ABSTRACT FROM AUTHOR]

The study aimed to find an accurate propagation program for the rootstock of peach Prunus persica cv. Garnem. The apical shoots and nodes of this rootstock were used as explants for the stages of initiation and multiplication, and they were planted on MS medium after adding different concentrations of Active Charcoal (AC), Polyvinyl Pyrrolidone (PVP), and Benzyl adenine (BA). The shoots were planted for the purpose of rooting on MS medium containing different concentrations of salts (1/4 MS, 1/2 MS and full MS salts) with different concentrations of IBA. In the establishment phase, the apical shoots grown on medium supplied with AC were given the highest response rate, the highest average length of plantlet, and the highest number of leaves. The best multiplication of explants in terms of the number of shoots and their length were obtained when cultivated on a medium supplemented with BA at 1 and 2 mg.L-1, whereas the application of BA at 1 mg. L-1 to the cultivated nodes gave the highest number of leaves. The best rooting percent was obtained when plantlets were s treated with 2 mg. L-1 was applied to 1/4 MS medium. The highest number of roots was obtained from plantlets grown in 1/2 MS medium treated with IBA at 3 mg.L-1, whereas the longest root was obtained from plantlets grown in 1/2 MS medium treated with 1 mg.L-1 IBA. [ABSTRACT FROM AUTHOR]

One of the important sources of human selenium intake is vegetables, so increasing of selenium in edible parts of plants, biofortification, provides an effective approach to reduce selenium deficiency. Origanum vulgare L. is an important medicinal plant which is also used as a valuable spice. The present study was done to evaluate the ability of selenium accumulation of micropropagated O. vulgare under sodium selenate and/or salicylic acid treatment. Micropropagation of O. vulgare was done from shoot-tips as explants on modified Murashige & Skoog (MS) medium. After 1 month, rooted plantlets were transferred to modified Murashige & Skoog solidified medium containing sodium selenate (0, 25 and 50 µM) and/or salicylic acid (0 and 100 mgL−1). After 4 wk, growth, selenium accumulation, biochemical parameters and oxidative stress indicators were determined. The results showed that the plantlets were able to accumulate 16.1 µg g−1 DW and 28.27 µg g−1 DW selenium in shoots at 25 and 50 µM selenate sodium concentrations respectively. Both concentrations of selenium increased shoot dry weight, length of shoot and root, soluble sugars and total protein content, peroxidase and superoxide dismutase activity. On the contrary, malondialdehyde content decreased under selenium treatment. It was found that by increasing selenium concentration the content of total protein, H2O2 and antioxidant enzymes activity increased significantly. Application of salicylic acid in combination with selenium improved growth but did not affect on selenium accumulation. The obtained results can be considered as the first report of biofortification of O. vulgare under tissue culture technique. [ABSTRACT FROM AUTHOR]

This study aimed to develop a suitable micropropagation protocol for black mulberry (Morus nigra L.), which cannot be easily clonally propagated like the other mulberry species. Clonal propagation of this species is gaining importance due to its health benefits and utilization in pharmacology. The effects of the culture media and cytokinin concentrations on the micropropagation of black mulberry were investigated. Single-axillary bud explants grown on Murashige and Skoog medium, Driver and Kuniyuki walnut medium, and Nas and Read medium (NRM) containing 0.25 mg L-1 of benzyladenine (BA) plus 0.01 mg L-1 of indole-3-butyric acid (IBA) were compared to cultures grown on NRM with thidiazuron (TDZ), meta-Topolin (mT), or BA at concentrations of 1.25, 2.5, 5.0, or 7.5 mg L-1 plus 0.01 mg L-1 of IBA. The culture medium had a significant effect on the shoot length, which was greater on the NRM (3.3 shoots per explant) compared to the other media. Shoot multiplication was similar on all of the tested media. TDZ negatively affected shoot formation and triggered rosette shoots and/or large callus formation. BA and mT promoted both shoot proliferation and elongation. Around 84% of the micro shoots rooted and the rooted shoots were successfully acclimatized to ex vitro conditions. [ABSTRACT FROM AUTHOR]

Genetic variability induced by cell and tissue culture techniques provides a new source of diversity for improvement of ornamental plants species. For this purpose, the effect of Thidiazuron (TDZ) on callus induction and number of subcultures of Salvia splendens was studied. The results showed that the MS medium supplemented with TDZ (0.5 mg/l) and Indole Acetic Acid IAA (0.1 mg/l) produced the highest percentage for explants producing callus (60%). For shoot multiplication, the best results were achieved when shoot tips were cultured on MS medium supplemented with TDZ (0.2 mg/l) alone. For subculture the same concentration of TDZ (0.2 mg/l) in media of eleven subcultures were performed at 21-day intervals over a total period of 231 days. For molecular variation detection, SCoT molecular markers were employed. Five SCoT primers were used and a total of 24 bands were obtained. The results showed high level of polymorphism between parent and their regenerated plants (subculture No. 11). These markers revealed distinctly different patterns of DNA fragments in all somaclones, and some distinctive bands were linked to the regenerated plants. For rooting, the best rooting percentage (100.00%), highest number of roots/shoot (7.00) and root length (2.58 cm) were observed when the shoots were cultured on MS hormonefree medium supplemented with 15 g sucrose/l. Plantlets were successfully acclimatized on Peat moss + perlite (1:1) with a survival rate of 100 %. [ABSTRACT FROM AUTHOR]

Plants are the sources of valuable biomass that are being currently used in many areas. It is important to produce high biomass for efficient commercial production. Amongst the many factors that affect in vitro propagation of plants, changing or enriching the media composition is one of the commonly used techniques in micropropagation of plants. Biochar is a solid product obtained from organic wastes and because of its rich composition, it has many beneficial effects on plants. In our study, Lavandula officinalis plantlets were subjected to two types of biochars (Geocharged biochar and Biorfe biochar) at 0.5 and 2 g/L concentrations and their effects were investigated by means of plant growth, biomass accumulation and biochemical composition. The results showed that 0.5 g/L concentration of biochar had better effects than 2 g/L concentration and except for biochemical composition, biochar type had no significant effect on plant growth and biomass accumulation. Mean root dry weights and multiple shoot formations/explant enhanced up to 3.7 and 4.17 times higher than the control at 0.5 g/L concentration. Explant browning was also detected lower in biochar-applied media. The differences between biochemical accumulations of different media were also found statistically significant. The total concentrations of phenolics and flavonoids and radical scavenging activities were detected lower when biochars were applied. The total antioxidant concentration was higher in the control group. These findings showed that biochars lowered the negative effects of the culture conditions for L. officinalis plantlets. [ABSTRACT FROM AUTHOR]

Fluorinated cytokinins have emerged as promising alternatives to traditional cytokinins in Phalaenopsis plant tissue culture, offering enhanced stability and bioactivity. However, their metabolic fate and impact on endogenous cytokinin profiles remain largely unexplored. This study builds upon previous research to investigate the comparative metabolism of the traditional cytokinin 6-Benzylaminopurine (BA) with the successful alternatives 6-(3-fluorobenzylamino)purine (FmT) and 6-(3-fluorobenzylamino)purine 9-riboside (FmTR). Additionally, this study examines the impact of another crucial factor, the use of ventilated versus closed containers, on metabolic processes. The results revealed the distinct metabolic profiles associated with each treatment, highlighting the complex interplay between exogenous and endogenous cytokinin levels. This study is the first to investigate the effects of these stable, synthetic, and exogenous cytokinins on the naturally occurring cytokinin levels and their metabolites in micropropagated Phalaenopsis. Additionally, we proposed an alternative inactivation pathway involving the conversion of FmTR and BA to pT and pTR. These findings provide valuable insights into the intricate relationship between cytokinin metabolism and plant growth under in vitro conditions. [ABSTRACT FROM AUTHOR]

Microsorum pteropus 'Windeløv' is a water fern from Asia commonly used in aquarium landscapes. This study aimed to develop a mass production method through the induction of green globular bodies (GGBs) from leaf explants of M. pteropus. Surface sterilization was performed on adventitious buds on the fronds of M. pteropus 'Windeløv' as explants. The results showed that the optimal explant disinfection condition was 1% sodium hypochlorite solution for 20 min, achieving a successful rate of 87.5 ± 9.6%. The most effective GGB induction was achieved with MS medium supplemented with 5.0 mg/L of NAA (1-Naphthaleneaceticacid) and 0.5 mg/L of 6-BA (6-benzylaminopurine), producing the highest number and size of GGBs, with a mean value of 4.0 ± 0.8 GGBs per explant and a diameter of 2.45 ± 0.61 mm after 8 weeks of culture. The regeneration of multiple sporophytes from the GGBs was only observed under hormone-free MS medium, with a mean value of 34.2 ± 7.3 sporophytes per GGB after 6 weeks of culture. Subsequently, rootless and separated sporophytes could be directly transplanted into the aquarium with a 100% survival rate. [ABSTRACT FROM AUTHOR]

The endangered plant species Adenophora liliifolia faces threats to its survival in the wild, necessitating the development of effective micropropagation techniques for potential reintroduction efforts. This study demonstrates that Adenophora liliifolia effectively reproduces on MS synthetic medium with diverse plant growth regulators (PGR) and natural extracts, facilitating swift micropropagation for potential future reintroduction endeavors. It highlights the substantial impact of PGR composition and natural extracts on the growth and development of A. liliifolia. The ideal growth medium for A. liliifolia was determined to be ½ MS with specific treatments. Additionally, incorporating silver nitrate (AgNO3) at 5 mg L−1 into the medium led to enhanced root formation and shoot length, albeit excessive concentrations adversely affected root development. Varying concentrations of NAA significantly affected different plant growth parameters, with the 0.1 mg L−1 treatment yielding comparable plant height to the control. Moreover, 50 mL L−1 of coconut water bolstered root formation, while 200 mL L−1 increased shoot formation during in vitro propagation. However, elevated doses of coconut water (CW) impeded root development but stimulated shoot growth. Experiments measuring chlorophyll a + b and carotenoid content indicated higher concentrations in the control group than differing levels of applied coconut water. Optimizing pH levels from 6.8–7 to 7.8–8.0 notably enhanced plant height and root formation, with significant carotenoid accumulation observed at pH 6.8–7. Soil samples from A. liliifolia's natural habitat exhibited a pH of 6.65. Ultimately, the refined in vitro propagation protocol effectively propagated A. liliifolia, representing a pioneering effort and setting the stage for future restoration initiatives and conservation endeavors. [ABSTRACT FROM AUTHOR]

Garlic has been successfully propagated using various micropropagation methods both through direct and indirect organogenesis. In vitro micropropagation from the inflorescence, root tip somatic embryogenesis, shoot multiplication in liquid cultures, shoot tip culture, meristem culture, stem-disc dome culture, stem-disc culture has been reported. Therapeutic techniques have been deployed to produce planting material devoid of viruses. This review comprehends various factors affecting morphogenesis and propagule production in garlic. [ABSTRACT FROM AUTHOR]

Plasma technology, relatively new in the fields of biomedicine, agriculture, and ecology, is the subject of intensive research as a prospective means of decontamination of various microorganisms (bacteria, viruses, and fungi). The objectives of the present study were to follow the effect of cold atmospheric plasma (CAP) treatment on in vitro grown plum plants (Prunus domestica L. 'Kyustendilska sinya' cv.) and the possibility of eradicating or inactivating plum pox virus (PPV) causing Sharka disease by CAP. The source tree is naturally co-infected by PPV (both M and D strains). In the experiments, two different plasma sources were used. First, a surface-wave-sustained Argon plasma torch and second, an underwater diaphragm discharge. For the treatments, nodal segments (10 mm in length) from in vitro cultured plum plants with or without one leaf were prepared. Apical shoots from treated plants (PPV-positive and negative clones as well non-treated controls) were cultivated in vitro for four passages. Then they were rooted and acclimatized to ex vitro conditions, and their virus status was observed periodically for more than 3 years after treatment for the appearance of Sharka symptoms. All plants, acclimatized to ex vitro conditions, were tested for PPV by immune capture–reverse transcription–polymerase chain reaction (IC-RT-PCR). As a first step in understanding the plasma treatment of living plants, a plasma treatment variant causing no damage must be established; this has been done in our previous works. Treatment of plants by plasma with parameters that have been carefully selected leads to better development than the non-treated plants. In the treated in vitro plants, no significant differences were found in the number and length of shoots compared to the control plantlets. In ex vitro acclimated plants, greater stem length was reported, but no differences in leaf number were observed. No significant differences in growth were recorded between the control and plants that were treated twice or three times. At this stage, 3 years after ex vitro cultivation in a greenhouse, Sharka symptoms were not registered on treated in vitro negative PPV plants, and the virus was not detected by IC-RT-PCR. Very mild symptoms were showing in CAP-treated PPV-positive plants. Development of typical Sharka symptoms on non-treated controls were observed. [ABSTRACT FROM AUTHOR]

The influence of the temperature and different light qualities emitted by light-emitting diodes (LEDs) and fluorescent lamps (Fl) on the micropropagation of the tree fern Cyathea delgadii was evaluated. The most efficient somatic embryo production was obtained on internode explants at 22 °C. The optimal temperature range for obtaining well-developed plants was 24–28 °C. This stimulated the elongation and development of the first leaf and the formation of the next leaf and roots primordia. Temperatures lower than 24 °C and higher than 28 °C inhibited the formation of young sporophyte organs and delayed their development. The RBUV (35% red, 15% blue, and 50% UV) and B (100% blue) lights and Fl light were beneficial for the sporophyte production on internode explants. However, plants obtained under RBUV light were undeveloped. The white LED light stimulated the number of explants capable of gametophyte production and development. The RB light (70% red and 30% blue) enhanced the number of roots of newly-formed plants. Most of the LED lights tested had a good impact on root elongation compared to Fl light and constant darkness. The R light (100% red) benefits leaf development and elongation. Research shows that temperature and LED lightning play a significant role in the process of morphogenesis in C. delgadii, significantly affecting the embryogenic competence of somatic cells and the development of sporophytes.Key message: A wide range of temperatures and light qualities have been investigated for optimizing the efficiency of in vitro morphogenesis of the tree fern Cyathea delgadii. [ABSTRACT FROM AUTHOR]

In vitro propagation, is becoming the predominant method for blackberry propagation due to its advantages compared to agamic traditional propagation methods. Synthetic seed technology represents a promising approach to further enhance the productivity of in vitro propagation facilitating the exchange of plant materials among laboratories and contributing to germplasm conservation efforts. This study aimed to establish an optimal protocol for the storage and sowing of synthetic blackberry seeds obtained through the encapsulation of clump bases. The synthetic seeds were sown without storage (Control) and after storage periods of 30, 60, and 120 days at 4 °C and 25 °C in the dark, in three different substrates (agarised, perlite, and potting). After forty-five days from sowing viability, regeneration rate, shoot and root numbers and lengths, as well as fresh and dry weights of the plantlets, were assessed. Results indicated that agarised substrate consistently exhibited favourable outcomes, with sustained regeneration rates and robust plantlet development even after prolonged storage at 4 °C. Synthetic seeds sown in perlite and potting substrates demonstrated enhanced regeneration rates following storage at 4 °C for 60 and 120 days. On the contrary, storage at 25 °C resulted in a notable decline in regeneration rate, highlighting its inadequacy for blackberry synthetic seed conservation purposes. These findings underscore the importance of sowing substrate selection and storage temperature in optimizing the storage and sowing protocols for synthetic blackberry seeds.Key message: A temperature storage of 4 °C and an agarised sowing substrate guarantee high regeneration rates for blackberry synthetic seeds up to 120 days of storage. [ABSTRACT FROM AUTHOR]

Bamboos are perennial, arborescent, monocarpic, and industrially important non-timber grasses that are used as a pristine source of inorganic nutrients. However, conventional vegetative propagation methods demonstrated inadequate multiplication potential. This study investigates how Bambusa balcooa’s in vitro growth, photosynthetic pigment content, and antioxidant capacity were affected by citrate- and cetyltrimethylammonium bromide-coated gold nanoparticles (AuNPs). Further, to unravel the regulatory mechanism underlying gold nano-elicitation and in vitro plant behavior, we conducted RNA sequencing of non-treated control, 400 µM citrate-AuNPs-treated, and 600 µM CTAB-AuNPs-treated plantlets. Numerous morphological, physiological, and biochemical parameters were observed to be variably impacted along the citrate- and CTAB-coated AuNPs concentration gradient (200–600 µM). B. balcooa in vitro shoots supplemented with Murashige and Skoog medium enriched with 6-benzylaminopurine, naphthaleneacetic acid, and 400 µM citrate-AuNPs displayed statistically significant shoot proliferation, photosynthetic pigment accumulation, and antioxidant activities. Contrarily, a decline in growth parameters was observed in MS media supplemented with BAP, NAA, and 600 µM CTAB-AuNPs. Transcriptome profiling revealed various differentially expressed genes (DEGs) and metabolic pathways associated with nano-elicitation and plant growth. Furthermore, identifying genes (such as Glyoxalase, Expansin, and ZAT) governing in vitro proliferation and oxidative stress responses could enhance our understanding of the mechanisms underlying AuNPs’ ability to modulate various physiological and biochemical activities during micropropagation. Therefore, gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the exploration of DEGs involved in the in vitro modulations regulated by AuNPs offer novel insights into the molecular mechanisms governing nano-elicited plant organogenesis more comprehensively.Key message: In vitro elicitation of differentially coated gold nanoparticles improves the biomass, photosynthesis, and antioxidant potential of Bambusa balcooa. RNA sequencing reveals transcriptional reprogramming in response to nano-elicitation. [ABSTRACT FROM AUTHOR]

Tissue culture is preferred for solving the shortcomings of low efficiency of conventional propagation in tree peony (Paeonia sect. Moutan), an economically important woody plant in China with various uses. Paeonia ostii ‘Feng Dan’ is one of the varieties with strong adaptability and high oil productivity. However, in vitro regeneration via organogenesis is hard to obtain. For instance, the indirect organogenesis from callus is a lengthy process, and the direct organogenesis is lacked. This study presents a protocol of direct organogenesis using cotyledon explant for in vitro regeneration of P. ostii ‘Feng Dan’, and includes two pathways. The explants need pretreated in the optimal dedifferentiation induction medium (DIM) [Murashige and Skoog (MS) medium with 2.27 µM thidiazuron (TDZ) + 5.37 µM α-naphthylacetic acid (NAA)] for 10 days, and then the cotyledons without callus induced were transferred to differentiation medium (DM) with 6 subcultures, 90 days in total. The best DM for the frequency of leaf clusters induction (66.67%) was Woody plant medium (WPM) containing 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 4.54 µM TDZ, and for the frequency of MNs induction (41.67%) was WPM containing 2.02 µM CPPU + 2.27 µM TDZ. For two pathways, mean number of regenerated shoots (≈ 4 shoots per explant), the frequency of root induction (≈ 50%) and the frequency of survival (≈ 40%) were similar. Histological studies confirmed the process of direct organogenesis, and revealed that shoots and MNs were originated from the increased division of meristematic cells under cortical tissue, as well as from actively dividing meristematic cells around the vascular center. Moreover, shoots regenerated through MNs differentiation were originated from the epidermal and subepidermal cells. This study is supplements in the field of in vitro regeneration in tree peony, and is an improvement to current protocols of clonal micropropagation, at the same time facilitating fundamental studies of developmental biology and genetic transformation.Key message: Plant regeneration through two pathways of direct organogenesis using cotyledon explant was first presented in Paeonia ostii ‘Feng Dan’. [ABSTRACT FROM AUTHOR]

Haplophyllum virgatum var. virgatum is an Iranian endemic plant. Its only natural habitat is the Geno region in the Hormozgan province of Iran. This plant’s limited distribution and special habitat conditions prompted us to investigate the possibility of its micropropagation and different biological aspects. In this study, while evaluating seed germination and seedling production of H. virgatum in two Murashige and Skoog (MS) and Gamborg (B5) culture media, shoot multiplication and rooting of single-node shoot explants of this plant were also investigated in the MS culture medium containing different concentrations of growth regulators. Flavonoids are part of the medicinal metabolites of different Haplophyllum species. Here, the flavonoid elicitation in the proliferated shoots of H. virgatum under 0, 100, and 150 µM methyl jasmonate (MeJA) treatments was done. The results demonstrated that the MS culture medium was more suitable than B5 for seed germination and natural growth of seedlings. Moreover, for H. virgatum shoot multiplication, one mg/L kinetin was more appropriate compared to other concentrations of this cytokinin (0.5, 1.5, 2, 2.5, and 3 mg/L) and the corresponding concentrations of benzyl amino purine. Rooting was achieved only in a limited number of shoots, so it needs further investigation. Meanwhile, MeJA increased total phenolic, total flavonoid, and rutin (the main flavonoid of Rutaceae) contents in the proliferated shoots. This research provided new information about the micropropagation of H. virgatum and flavonoid elicitation by MeJA in this plant.Key message: For shoot multiplication of Haplophyllum virgatum, MS medium containing one mg/L kinetin showed the best results. Methyl jasmonate increased the phenolic and flavonoid contents of its proliferated shoots. [ABSTRACT FROM AUTHOR]

Withania somnifera (Ashwagandha) is a valuable therapeutic plant used in many Ayurvedic drugs marketed for health restoration. However, its commercial production faces challenges due to poor seed viability, low germination rates, and the absence of standardized propagation methods. Therefore, this investigation aimed to establish a fast and highly efficient method for direct shoot regeneration in W. somnifera by using the apical meristem perforation technique, which is a novel approach. Initially, rapid seed germination was standardized by soaking the seeds in distilled water for varying durations (0 to 48 h). The highest rates of shoot initiation (90.40 ± 0.51%) and mean number of direct shoot regenerations (16.20 ± 0.50 per explant) were achieved on MS basal medium fortified with meta-topolin (mT) at 3.0 mg L−1. For optimal root development, shoots (3 to 4 cm) were cultured on half-strength Murashige and Skoog (MS) medium with 3.0 mg L−1 indole-3-butyric acid (IBA). Subsequently, well-rooted seedlings (90% survival rate) were acclimatized under glasshouse conditions in pots filled with a mixture of sandy loam and vermicompost (4:1). Anatomical examination of leaves and stems from micropropagated and acclimatized glasshouse plants revealed well-developed cuticles, differentiated mesophylls, and vascular bundles. Furthermore, the accumulations of withaferin A (1.26 ± 0.12 mg g−1), withanone (1.39 ± 0.18 mg g−1), and withanoside V (0.11 ± 0.04 mg g−1) in greenhouse-acclimated plant leaves were 1.88, 2.28, and 2.30 times higher, respectively, than in tissue culture samples (0.52 ± 0.07 mg g−1, 0.73 ± 0.02 mg g−1, and 0.05 ± 0.02 mg g−1). In conclusion, the described procedure offered an effective and straightforward method for large-scale production of W. somnifera plants for commercial metabolites. [ABSTRACT FROM AUTHOR]

Herein, an in vitro multiplication protocol for Prunus campanulata was evaluated. The effects of 15.0, 30.0, and 45.0 g L−1 sucrose and three flask sealing methods (PVC film, rigid polypropylene lid, and rigid lid with membrane) were analyzed at the multiplication, rooting, and acclimatization stages. For in vitro multiplication, shoot tips of approximately 1 cm in length containing two pairs of leaves from seedlings germinated in vitro in Woody Plant Medium (WPM) were used. Evaluations considered the multiplication of shoot apexes in three subcultures (at 40, 80, and 120 d) for survival, number of shoots, shoot length, number of leaves, and multiplication rate. In the rooting phase, the rooting percentage, number of roots, and root length were assessed. In the acclimatization phase under shaded and full sun conditions, seedling survival and biometric characteristics were evaluated, including stem diameter, height, and number of leaves. The addition of sucrose to the medium, at a concentration of 45.0 g L−1, associated with sealing using PVC plastic film, showed inadequate results for in vitro multiplication of P. campanulata. The addition of sucrose to the culture medium in concentrations ranging from 15.0 to 30.0 g L−1 favored in vitro rooting. The use of a permeable membrane for sealing enabled the growth of more vigorous shoots in terms of percentage of rooting and root length, and these seedlings stood out in the acclimatization phases with greater rates of survival and improved biometric characteristics. [ABSTRACT FROM AUTHOR]

An efficient in vitro micropropagation system for Platanus × acerifolia 'F03', a radiation mutant that has not flowered for 19 yr, has been established. Axillary buds were used as explants. After sprouting on Murashige and Skoog medium (MS) supplemented with 0.3 mg·L−1 6-benzylaminopurine (6-BA) together with 0.05 mg·L−1 α-naphthaleneacetic acid (NAA), the best medium for multiplication and elongation was MS basal medium supplemented with 0.1 mg·L−1 6-benzylaminopurine and with a multiplication rate of 3.43 ± 0.57. Plantlets over 1.5 cm could root on McCown Woody Plant Medium (WPM) supplemented with 0.01 mg·L−1 α-naphthaleneacetic acid and 7.0 g·L−1 agar with an ideal rooting rate and transplanting survival rate of 100 ± 0%. The optimum survival rate was possible when trays were covered with a thin plastic lid for more than 14 d. After transplanting, air humidity and temperature were crucial for young plant survival. The system established here could serve as a pathway for commercial production of the line and provide sterile leaf explants for regeneration and further genetic improvement of the species. [ABSTRACT FROM AUTHOR]

Medicinal applications of Plumbagozeylanica and its metabolites on various diseases and low viability and inconsistent germination of its seeds are the reasons behind the loss of its genetic diversity. Hence, an efficient protocol for the short-term storage of P.zeylanica synthetic seeds, which is an overexploited medicinally valuable plant, was developed. Initially, in vitro culture was performed from nodal explants to develop synthetic seeds from its proliferated shoots. Murashige and Skoog (MS) medium augmented with 0.5 mg L−1 6-benzylaminopurine (BAP) resulted in the best morphogenetic response. Thereafter, the developed synseeds were stored for 2 wk at a temperature of 10 or 25°C in different conditions and further evaluated for regeneration. Higher re-growth rate (80%) and the identical morphogenetic response were recorded for the P.zeylanica synthetic seeds, which were stored at a temperature of 10°C in dark condition after its storage period. As per the available literature, this is the first report pertaining to in vitro low-temperature storage of synthetic seeds of P.zeylanica and can further be utilized for the conservation of elite clones for the study of medicinally potent species. [ABSTRACT FROM AUTHOR]

This article describes benefits of the application of zinc oxide submicron particles (ZnO SMPs), zinc oxide nanoparticles (ZnO NPs) and ZnO NPs combined with silver NPs (ZnO + Ag NPs) in chrysanthemum micropropagation. Single node explants of Chrysanthemum × morifolium (Ramat.) Hemsl. 'UTP Burgundy Gold (UBG)' and 'UTP Pinky Gold (UPG)' were inoculated on the Murashige and Skoog (MS) medium and treated with 100 mg · L−1, 200 mg · L−1, or 400 mg · L−1 ZnO SMPs, ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O). Generally, the tested materials stimulated the growth and development of plantlets. In 'UBG', the most prominent treatments affecting increases in the number of leaves, micropropagation coefficient, shoot length and shoot FW/DW weight included 400 mg · L−1 ZnO SMPs and 100 mg · L−1 ZnO NPs (6% H2O). In 'UPG', the treatments with 200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) and 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) were the most successful. The latter treatment stimulated an intensive development of root systems in the two studied cultivars. High values of leaf area, perimeter and width were reported in both cultivars for 400 mg · L−1 ZnO + 1% Ag NPs (6% H2O). As compared to the control, the treated plants were characterised by a similar or, most often, lower content of chlorophylls and carotenoids. The randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) marker system analyses of the 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated chrysanthemums confirmed their genetic fidelity with the control plants. The obtained results can be implemented in the commercial large-scale production of chrysanthemums. [ABSTRACT FROM AUTHOR]