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Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to “native” hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages.

Plants generally have the highest regenerative ability because they show a high degree of developmental plasticity. Although the basic principles of plant regeneration date back many years, understanding the cellular, molecular, and physiological mechanisms based on these principles is currently in progress. In addition to the significant effects of some factors such as medium components, phytohormones, explant type, and light on the regeneration ability of an explant, recent reports evidence the involvement of molecular signals in organogenesis and embryogenesis responses to explant wounding, induced plant cell death, and phytohormones interaction. However, some cellular behaviors such as the occurrence of somaclonal variations and abnormalities during the in vitro plant regeneration process may be associated with adverse effects on the efficacy of plant regeneration. A review of past studies suggests that, in some cases, regeneration in plants involves the reprogramming of distinct somatic cells, while in others, it is induced by the activation of relatively undifferentiated cells in somatic tissues. However, this review covers the most important factors involved in the process of plant regeneration and discusses the mechanisms by which plants monitor this process.

Clonal propagation of elite, disease-free coconut palms is a promising technique for producing uniform planting material with high yield and disease resistance. Over the past few decades cloning of coconut has been attempted in a number of laboratories worldwide; however, success has been limited. In the present study, immature inflorescences of 2–12 cm size were collected from West Coast Tall variety and the rachilla segments were cultured on four different media combinations in dark condition. White translucent outgrowths were maximum in Y3 medium supplemented with 4.54 μM 2,4-dichlorophenoxyacetic acid (92%) followed by medium 72 with 41.4 μM picloram, 61.8 μM putrescine and 4.54 μM thidiazuron (TDZ) (87%). After eight weeks in dark, shoot-like outgrowth was noticed more in Y3 III (65%) followed by Y3 I. After eight months dark incubation, the cultures were transferred to 1/2 Murashige and Skoog (MS) with two hormone combinations and high frequency of multiple shoot formation was noticed in 1/2 MS with 5.37 μM naphthalene acetic acid (NAA) and 4.44 μM 6-benzylaminopurine (BAP). Maximum shoot development was observed Y3 medium fortified with 5 μM 2-isopentenyl adenine (2ip) and 5 μM BAP. The individual shoots after development of 3–4 leaves were transferred to 1/2 Y3 medium supplemented with 5.37 μM NAA and 24.6 μM indole-3-butyric acid (IBA), and root initiation was observed in 39.28% plantlets. Start codon targeted (SCoT) profiling based on banding pattern of PCR-amplified products confirmed the clonal fidelity of in vitro regenerated coconut plantlets. The study indicates the possibility of developing an in vitro regeneration protocol for coconut using immature inflorescence explants.

Overexpression of csi-miR156a enhances the somatic embryogenesis capability of citrus through downstream repression of CsSPL3 and CsSPL14. Abstract miR156 is a highly conserved plant miRNA and has been extensively studied because of its versatile roles in plant development. Here, we report a novel role of miR156 in regulating somatic embryogenesis (SE) in citrus, one of the most widely cultivated fruit crops in the world. SE is an important means of in vitro regeneration, but over the course of long-term sub-culturing there is always a decline in the SE potential of the preserved citrus embryogenic callus, and this represents a key obstacle for citrus biotechnology. In this study, the SE competence of citrus callus of wild kumquat (Fortunella hindsii) was significantly enhanced by either overexpression of csi-miR156a or by individual knock-down of the two target genes, CsSPL3 and CsSPL14, indicating that the effect of miR156-SPL modules was established during the initial phases of SE induction. Biological processes that might promote SE in response to miR156 overexpression were explored using RNA-seq, and mainly included hormone signaling pathways, stress responses, DNA methylation, and the cell cycle. CsAKIN10 was identified as interacting protein of CsSPL14. Our results provide insights into the regulatory pathway through which miR156-SPL modules enhance the SE potential of citrus callus, and provide a theoretical basis for improvement of plant SE competence.

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla ‘Hyd1’ was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla ‘Hyd1’.

Microbial contamination is a serious problem in temporary immersion systems (TIS) during commercial micropropagation. The use of adequate doses of silver nanoparticles (AgNPs), formulated as Argovit™, is an alternative to reduce the contamination indices and promote development in plants. The aim of this study was to evaluate the antimicrobial and hormetic effects of Argovit on in vitro regeneration of vanilla (Vanilla planifolia) using a TIS. In vitro regenerated shoots were grown in Murashige and Skoog (MS) liquid medium with Argovit at five different concentrations (0, 25, 50, 100 and 200 mg/l) using a temporary immersion bioreactor system (RITA®). At 30 days of culture, contamination percentage was evaluated and shoot regeneration and length were used to determine the hormetic response. Analysis of macro and micronutrient contents was performed. In addition, the effect of Argovit on total phenolic content (TPC), reactive oxygen species (ROS) production, antioxidant capacity (ORAC) and lipid peroxidation (LP-MDA) was determined. Results showed that bacterial contamination was reduced at 50, 100 and 200 mg/l of Argovit. Growth stimulation was observed at 25 and 50 mg/l of Argovit, while significant inhibition was detected at 100 and 200 mg/l of Argovit. Mineral nutrient analysis revealed changes in macro and micronutrient concentrations exerted by Argovit. Moreover, the presence of Argovit induced the production of ROS and increased total phenolic content, antioxidant capacity and lipid peroxidation with a dose-dependent effect. Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNPs-induced hormesis for vanilla. Therefore, the addition of 50 mg/l of Argovit in the culture media had an antimicrobial and hormetic effect. Use of Argovit could be an efficient strategy for commercial micropropagation of vanilla and other species.

Plants, known for their immobility, employ various mechanisms against stress and damage. A prominent feature is the formation of callus tissue—a cellular growth phenomenon that remains insufficiently explored, despite its distinctive cellular plasticity compared to vertebrates. Callus formation involves dedifferentiated cells, with a subset attaining pluripotency. Calluses exhibit an extraordinary capacity to reinitiate cellular division and undergo structural transformations, generating de novo shoots and roots, thereby developing into regenerated plants—a testament to the heightened developmental plasticity inherent in plants. In this way, plant regeneration through clonal propagation is a widely employed technique for vegetative reproduction. Thus, exploration of the biological components involved in regaining pluripotency contributes to the foundation upon which methods of somatic plant propagation can be advanced. This review provides an overview of the cellular pathway involved in callus and subsequent de novo shoot formation from already differentiated plant tissue, highlighting key genes critical to this process. In addition, it explores the intricate realm of epigenetic regulatory processes, emphasizing the nuanced dynamics of DNA methylation that contribute to plant regeneration. Finally, we briefly discuss somaclonal variation, examining its relation to DNA methylation, and investigating the heritability of epigenomic changes in crops.

Hairy root (HR) culture, a successful biotechnology combining in vitro tissue culture with recombinant DNA machinery, is intended for the genetic improvement of plants. This technology has been put to use since the last three decades for genetic advancement of medicinal and aromatic plants and also to harvest the economical products in the form of secondary metabolites that are significantly important for their ethnobotanical and pharmacological properties. It also provides an efficient way out for the quicker extraction and quantification of the valuable phytochemicals. The current review provides an account of the in vitro HR culture technology and its wide-scale applications in the field of research as well as in pharmaceutical industries. Different facets of HR with respect to the culture establishment, phytochemical production as well as research investigations concerning the areas of gene manipulation, biotransformation of the secondary metabolites, phytoremediation, their industrial utilisations and different problems encountered during the application of this technology have been covered in this appraisal. Eventually, an idea has been provided on HR about the recent trends on the progress of this technology that may open up newer prospects in near future and calls for further research and explorations in this field.Key points• Genetic engineering–based HR culture aims towards enhanced secondary metabolite production.• This review explores an insight in the HR technology and its multi-faceted approaches.• Up-to-date ground-breaking research applications and constraints of HR culture are discussed.

Tissue regeneration upon wounding in plants highlights the developmental plasticity of plants. Previous studies have described the morphological and molecular changes of secondary vascular tissue (SVT) regeneration after large-scale bark girdling in trees. However, how phytohormones regulate SVT regeneration is still unknown. Here, we established a novel in vitro SVT regeneration system in the hybrid aspen (Populus tremula × Populus tremuloides) clone T89 to bypass the limitation of using field-grown trees. The effects of phytohormones on SVT regeneration were investigated by applying exogenous hormones and utilizing various transgenic trees. Vascular tissue-specific markers and hormonal response factors were also examined during SVT regeneration. Using this in vitro regeneration system, we demonstrated that auxin and cytokinin differentially regulate phloem and cambium regeneration. Whereas auxin is sufficient to induce regeneration of phloem prior to continuous cambium restoration, cytokinin only promotes the formation of new phloem, not cambium. The positive role of cytokinin on phloem regeneration was further confirmed in cytokinin overexpression trees. Analysis of a DR5 reporter transgenic line further suggested that cytokinin blocks the re-establishment of auxin gradients, which is required for the cambium formation. Investigation on the auxin and cytokinin signalling genes indicated these two hormones interact to regulate SVT regeneration. Taken together, the in vitro SVT regeneration system allows us to make use of various molecular and genetic tools to investigate SVT regeneration. Our results confirmed that complementary auxin and cytokinin domains are required for phloem and cambium reconstruction.

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 μM Zeatin (ZT) + 0.57 μM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 μM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including (LOC107843874, LOC107843885), (LOC107848380, LOC107862455), (LOC107870540), (LOC107839518), (LOC107846008), (LOC107852624), (LOC107841020), (LOC107839415), (LOC107843441), and (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.