Plant Cell Cultures Research Articles

Diversity of bioactive secondary metabolites produced by medicinal plants of Physalis angulata L. (Ciplukan)

Plants are a main source of various types of secondary compounds. Plant secondary compounds play a greater role in interacting with the environment than contributing to the growth and development. Therefore, different environmental conditions besides influencing growth can also affect the profile and concentration of secondary compounds. Ciplukan (Physalis angulata L.) has been known as medicinal plants. The medicinal properties are derived from bioactive secondary compounds especially withanolide and physalin. This study aims to identify the diversity of secondary metabolites found in in vitro callus and plants tissue of ciplukan. Withanolide and physalin profiles of callus tissues, cotyledonary shoot-derived plantlet and germinated seeds-derived plant, were evaluated by LC-MS analysis. The LC-MS analysis of methanol extract showed the diversity in the amount and type of withanolide and physalins. This study confirmed that in undifferentiated callus cultures and in vitro induced plantlet of P. angulata the biosynthesis activity was not altered and the accumulation sites of withanolides were not missing. However in vitro regenerated plant produced withanolides and physalins in higher number. Therefore, modification of plant cell culture system to improve withanolide including physalin production of P. angulata is a good future prospect.

Multi-fold enhancement in vitamin E (alpha-tocopherol) production via integration of bioprocess optimisation and metabolic engineering in cell suspension of sunflower

Alpha-tocopherol, a highly active form of the antioxidant vitamin E in humans, effectively scavenges free radicals and protects the cell membranes. The aim of our study was to enhance alpha-tocopherol production in sunflower cell suspension using elicitation, precursor addition and culture condition optimization. Among various elicitors and precursors studied, sodium chloride (50 mg/L) added as elicitor and phytol (50 mg/L) added as the precursor to an untransformed sunflower cell suspension, resulted in maximum productivity enhancement of alpha-tocopherol up to 2.3- and 1.9-fold, respectively compared to a control untransformed sunflower cell suspension (0.02 mg/L/d). In separate experiments, the volumetric productivity of alpha-tocopherol in the untransformed suspension was enhanced up to 2.1- fold (0.09 mg/L/d) under optimized conditions of light intensity (3500 lx), temperature (30 °C), initial pH (6.5) and inoculum size (6 g/L). We then extrapolated these strategies (elicitor and precursor addition, culture condition optimization) for implementation on a high alpha-tocopherol yielding metabolically engineered cell suspension overexpressing 4-hydroxyphenylpyruvate dioxygenase (TS4), which resulted in enhancement (1.9-, 1.45- and 2.72-fold respectively) in the volumetric productivity of alpha-tocopherol compared to the transformed cell suspension grown under control conditions (0.18 mg/L/d). Thus, integration of bioprocess optimization and metabolic engineering resulted in a multi-fold (up to 12-fold) enhancement in alpha-tocopherol productivity in comparison to the untransformed cells. Therefore, our study demonstrates that productivity enhancement strategies like bioprocess optimisation and metabolic engineering (developed separately in parallel) for a given system can be combined to achieve maximum productivity in lesser time and cost than a relay system of optimization in long-duration plant cell cultivations.

Cellular engineering of plant cells for improved therapeutic protein production.

In vitro cultured plant cells, in particular the tobacco BY-2 cell, have demonstrated their potential to provide a promising bioproduction platform for therapeutic proteins by integrating the merits of whole-plant cultivation systems with those of microbial and mammalian cell cultures. Over the past three decades, substantial progress has been made in improving the plant cell culture system, resulting in a few commercial success cases, such as taliglucerase alfa (Elelyso®), the first FDA-approved recombinant pharmaceutical protein derived from plant cells. However, compared to the major expression hosts (bacteria, yeast, and mammalian cells), plant cells are still largely underutilized, mainly due to low productivity and non-human glycosylation. Modern molecular biology tools, in particular RNAi and the latest genome editing technology CRISPR/Cas9, have been used to modulate the genome of plant cells to create new cell lines that exhibit desired “traits” for producing therapeutic proteins. This review highlights the recent advances in cellular engineering of plant cells towards improved recombinant protein production, including creating cell lines with deficient protease levels or humanized glycosylation, and considers potential development in the future.

An efficient direct screening system for microorganisms that activate plant immune responses based on plant\u2013microbe interactions using cultured plant cells

Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant–microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell–cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.

Direct protein delivery into intact plant cells using polyhistidine peptides.

Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8-His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

High Chromosomal Stability and Immortalized Totipotency Characterize Long-Term Tissue Cultures of Chinese Ginseng (Panax Ginseng).

Chinese ginseng (Panax ginseng C. A. Meyer) is a highly cherished traditional Chinese medicine, with several confirmed medical effects and many more asserted health-boosting functions. Somatic chromosomal instability (CIN) is a hallmark of many types of human cancers and also related to other pathogenic conditions such as miscarriages and intellectual disabilities, hence, the study of this phenomenon is of wide scientific and translational medical significance. CIN also ubiquitously occurs in cultured plant cells, and is implicated as a major cause of the rapid decline/loss of totipotency with culture duration, which represents a major hindrance to the application of transgenic technologies in crop improvement. Here, we report two salient features of long-term cultured callus cells of ginseng, i.e., high chromosomal stability and virtually immortalized totipotency. Specifically, we document that our callus of ginseng, which has been subcultured for 12 consecutive years, remained highly stable at the chromosomal level and showed little decline in totipotency. We show that these remarkable features of cultured ginseng cells are likely relevant to the robust homeostasis of the transcriptional expression of specific genes (i.e., genes related to tissue totipotency and chromosomal stability) implicated in the manifestation of these two complex phenotypes. To our knowledge, these two properties of ginseng have not been observed in any animals (with respect to somatic chromosomal stability) and other plants. We posit that further exploration of the molecular mechanisms underlying these unique properties of ginseng, especially somatic chromosomal stability in protracted culture duration, may provide novel clues to the mechanistic understanding of the occurrence of CIN in human disease.

Growth kinetics, metabolite yield, and expression analysis of biosynthetic pathway genes in friable callus cell lines of Rhodiola imbricata (Edgew)

The plant cell culture provides an efficient technique for growth and production kinetics studies of bioactive compounds present in plants. Therefore, the present study investigated, for the first time, to identify the specific culture days required for higher metabolite yield in Rhodiola imbricata. A transcript-level gene expression study was conducted to understand the molecular cues associated with metabolite biosynthesis. The leaf callus cell line showed an optimum growth rate (FW: 20 g/100 mL, DW: 1.21 g/100 mL), growth index (19.00), salidroside (3.68 mg/g DW, rosavin (0.21 mg/g DW and rosarin (0.08 mg/g DW) on 24th day of the incubation period. However, salidroside, rosavin, and rosarin metabolite yield followed the growth-dependent manner in both callus cell lines, while tyrosol and p-coumaric acid showed growth independent accumulation pattern. Moreover, the antioxidant study displayed maximum phenolic content (8.07 mg/g DW) on day 18, flavonoid content (9.73 mg/g DW) on day 3, and DPPH activity (IC50; 0.79 mg/mL) on day 24 in leaf callus cell line. The RT-qPCR analysis showed upregulation of PAL, 4-HPAAS, 4-HPAAR, and UDPGT genes associated with metabolite accumulation. Expression analysis revealed a positive correlation with the metabolites yield. Furthermore, the optimized parameter could be exploited for a higher yield of specific metabolite and sequential scale-up studies. This is the first-ever study on the specific growth phase and accumulation pattern of metabolites in the friable callus cell lines of Rhodiola imbricata. Similarly, molecular signatures were also investigated to elucidate key genes associated with salidroside and rosavins biosynthesis. Furthermore, current optimized parameters lay the foundation for a cell suspension study, scale-up, and year-round metabolite production.

Cellular Agriculture: Opportunities and Challenges.

Cellular agriculture is the controlled and sustainable manufacture of agricultural products with cells and tissues without plant or animal involvement. Today, microorganisms cultivated in bioreactors already produce egg and milk proteins, sweeteners, and flavors for human nutrition as well as leather and fibers for shoes, bags, and textiles. Furthermore, plant cell and tissue cultures provide ingredients that stimulate the immune system and improve skin texture, with another precommercial cellular agriculture product, in vitro meat, currently receiving a great deal of attention. All these approaches could assist traditional agriculture in continuing to provide for the dietary requirements of a growing world population while freeing up important resources such as arable land. Despite early successes, challenges remain and are discussed in this review, with a focus on production processes involving plant and animal cell and tissue cultures.

Machine learning tools can now help with improving Taxol production in plant cell cultures

Chemotherapeutic intervention for cancer care is an important step. One of the most effective chemotherapy agents in use today is Paclitaxel (PTX), sold under the common name Taxol and Oxanol. Due to its ability to inhibit microtubule formation in cells, PTX is effective at all stages of the cancer and is FDA approved for treatment of many types of cancer (ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer). PTX is a plant alkaloid in the taxane family of compounds obtained from bark of the Pacific Yew tree (Taxus brevifolia) [1]. Adequate market supply of PTX has remained a challenge, as paclitaxel represents only a minor proportion of the total taxoid content of the Taxus species. Over the years, research into finding an alternate to cutting down Yew trees for PTX harvesting has been on the forefront. It is estimated that up to 60 trees may need to be harvested for the treatment of one patient.

In Vitro Cultured Melissa officinalis Cells as Effective Ingredient to Protect Skin against Oxidative Stress, Blue Light, and Infrared Irradiations Damages

Skin is being increasingly exposed to artificial blue light due to the extensive use of electronic devices, which can induce cell oxidative stress, causing signs of early photo aging. The Melissa officinalis phytocomplex is a new standardized cosmetic ingredient obtained by an in vitro plant cell culture with a high content of rosmarinic acid. In this study, we examine the activity of the Melissa officinalis phytocomplex to protect skin against blue light and infrared damages, evaluating the ROS (Radical Oxygen Species) level in keratinocyte cell line from human skin (HaCaT) and Nrf2 (Nuclear factor erythroid 2-related factor 2), elastin, and MMP1 (Matrix Metalloproteinase 1) immunostaining in living human skin explants ex vivo. This phytocomplex demonstrates antioxidant activity by reducing ROS production and thus the oxidant damage of the skin caused by UV and blue light exposure. In addition, it inhibits blue light-induced Nrf2 transcriptional activity, IR-induced elastin alteration, and IR-induced MMP-1 release. This Melissa officinalis phytocomplex is a new innovative active ingredient for cosmetic products that is able to protect skin against light and screen exposure damages and oxidative stress.

Cyclodextrins: a tool in plant cell and organ culture bioprocesses for the production of secondary metabolites

Plant cell culture bioprocesses have become an alternative source for bioactive compound production to the traditional extraction technology from plants grown in nature or through agricultural techniques. Thus, environmental conditions could make the supply of these secondary metabolites intermittent and heterogeneous. Therefore, plant cell cultures assure a continuous product supply, homogeneous production, fulfilling GMP requirements. Elicitation has been an extensively used strategy to boost secondary metabolite production in plant cell cultures. Cyclodextrins, the naturally occurring cyclic oligosaccharides of glucose residues, has emerged as an elicitor that can trigger plant cell defense responses and secondary metabolite accumulation. Moreover, CDs can form complex with most of the plant secondary compounds, thus contribute to product removal, which results in the elimination of feedback inhibition of product biosynthesis and also prevents product degradation and toxicity against the plant cells. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture processes. We review different case studies, which includes the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture, which include the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. Cyclodextrins trigger secondary metabolite production in plant cell cultures and product release. Moreover, they form complexes with these bioactive compounds, reduce feedback inhibition biosynthesis and their degradation.

Plant in vitro Culture Technologies; A Promise Into Factories of Secondary Metabolites Against COVID-19

The current pandemic has caused chaos throughout the world. While there are few vaccines available now, there is the need for better treatment alternatives in line with preventive measures against COVID-19. Along with synthetic chemical compounds, phytochemicals cannot be overlooked as candidates for drugs against severe respiratory coronavirus 2 (SARS-CoV-2). The important role of secondary metabolites or phytochemical compounds against coronaviruses has been confirmed by studies that reported the anti-coronavirus role of glycyrrhizin from the roots of Glycyrrhiza glabra. The study demonstrated that glycyrrhizin is a very promising phytochemical against SARS-CoV, which caused an outbreak in 2002–2003. Similarly, many phytochemical compounds (apigenin, betulonic acid, reserpine, emodin, etc.) were isolated from different plants such as Isatis indigotica, Lindera aggregate, and Artemisia annua and were employed against SARS-CoV. However, owing to the geographical and seasonal variation, the quality of standard medicinal compounds isolated from plants varies. Furthermore, many of the important medicinal plants are either threatened or on the verge of endangerment because of overharvesting for medicinal purposes. Therefore, plant biotechnology provides a better alternative in the form of in vitro culture technology, including plant cell cultures, adventitious roots cultures, and organ and tissue cultures. In vitro cultures can serve as factories of secondary metabolites/phytochemicals that can be produced in bulk and of uniform quality in the fight against COVID-19, once tested. Similarly, environmental and molecular manipulation of these in vitro cultures could provide engineered drug candidates for testing against COVID-19. The in vitro culture-based phytochemicals have an additional benefit of consistency in terms of yield as well as quality. Nonetheless, as the traditional plant-based compounds might prove toxic in some cases, engineered production of promising phytochemicals can bypass this barrier. Our article focuses on reviewing the potential of the different in vitro plant cultures to produce medicinally important secondary metabolites that could ultimately be helpful in the fight against COVID-19.

Transient Gene Expression: an Approach for Recombinant Vaccine Production

The production of recombinant vaccines in green plants is an attractive and promising topic in genetic engineering. However, the stable transformation of green plants is a time-consuming, costly, and labor-intensive practice. Moreover, public concerns about genetically modified plants put another limitation on the development and release of transgenic plant-based recombinant vaccines. These shortcomings were addressed by developing transient gene expression systems that allow researchers to investigate candidate recombinant vaccines quickly without tedious work and high costs. A comprehensive literature review was used to gather relevant information. This approach has received much attention in various recombinant vaccine production platforms, including mammalian cell culture, insect cell culture, yeast expression systems, and, more importantly, in plant hosts. Due to their simplicity and efficiency, transient gene expression systems are now widely used to validate gene constructs and transgene expression within plant tissues. This paper describes the concept of transient gene expression and discusses the significant advantages of this approach for producing recombinant vaccines. Notably, the major types of transient gene expression viz. agroinfiltration, viral-based systems, and application of naked plasmid in plant cell culture are introduced, and some examples illustrate the pros and cons of each system. Our literature review also discusses some practical notes on the successful application of this system to provide a more comprehensive image of transient gene expression applicability in green plants. As a whole, this review contributes to the existing literature by shedding more light on various aspects of transient gene expression that have not been addressed thoroughly yet.

Back Cover: Recent applications of plant cell culture technology in cosmetics and foods

Back Cover: Recent applications of plant cell culture technology in cosmetics and foods

Application of Plant Growth Regulators Modulates the Profile of Chlorogenic Acids in Cultured Bidens pilosa Cells.

Plant cell culture offers an alternative to whole plants for the production of biologically important specialised metabolites. In cultured plant cells, manipulation by auxin and cytokinin plant growth regulators (PGRs) may lead to in vitro organogenesis and metabolome changes. In this study, six different combination ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (BAP) were investigated with the aim to induce indirect organogenesis from Bidens pilosa callus and to investigate the associated induced changes in the metabolomes of these calli. Phenotypic appearance of the calli and total phenolic contents of hydromethanolic extracts indicated underlying biochemical differences that were investigated using untargeted metabolomics, based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC–qTOF–MS), combined with multivariate data analysis. The concentration and combination ratios of PGRs were shown to induce differential metabolic responses and, thus, distinct metabolomic profiles, dominated by chlorogenic acids consisting of caffeoyl- and feruloyl-derivatives of quinic acid. Although organogenesis was not achieved, the results demonstrate that exogenous application PGRs can be used to manipulate the metabolome of B. pilosa for in vitro production of specialised metabolites with purported pharmacological properties.

Plant Tissue Culture- A New Tool for Vegetable Improvement (Indian scenario): A Review

In vitro culture of plant cells/tissues is now routine using a range of explant types from many of the important vegetable and fruit crops. Successful technologies include isolation, culture of tissues, cells, protoplasts, organs, embryos, ovules, anthers and microspores and regeneration from them of complete plantlets. The development of plant tissue culture technology represents one of the most exciting advances in plant sciences. For example, the prospect of being able to introduce, develop, produce, transfer and conserve the existing gene pool of plant sciences by using tissue culture methods opens up new opportunities for researches and entrepreneurs. The term plant tissue culture should denote in vitro cultivation of plant cells or tissues in an unorganized mass, i.e., callus culture. Plant tissue culture techniques, in combination with recombinant DNA technology, are the essential requirements for the development of transgenic plants. However, culture techniques like anther/pollen/ovule culture, meristem culture can themselves be utilized for crop improvement or may serve as an aid to conventional breeding. In recent, isolated microspore culture has developed as a breeding tool and an experimental system for various genetic manipulations. The inherent potentiality of a plant cell to give rise to a whole plant, a capacity which is often retained even after a cell has undergone final differentiation in the plant body, is described as ‘cellular totipotency’. On the other hand, production of virus-free plants via meristem culture can reduce losses caused by phyto-pathogens. Embryo culture has many potential uses ranging from overcoming seed dormancy to facilitation of inter-specific hybridization. Protoplast fusion technique can be used for the transfer of cytoplasmic male sterility from one species to another in a short period of time. In cabbage, male sterile cybrids are being utilized by seed companies to produce hybrid seeds on commercial scale and at competitive rates. Plant tissue culture and cell culture are providing useful methods for germplasm storage either by low temperature storage of organized tissue, or cryopreservation of cell or embryo culture.

Plant Cell Cultures as a Tool to Study Programmed Cell Death.

Programmed cell death (PCD) is a genetically controlled suicide process present in all living beings with the scope of eliminating cells unnecessary or detrimental for the proper development of the organism. In plants, PCD plays a pivotal role in many developmental processes such as sex determination, senescence, and aerenchyma formation and is involved in the defense responses against abiotic and biotic stresses. Thus, its study is a main goal for plant scientists. However, since PCD often occurs in a small group of inaccessible cells buried in a bulk of surrounding uninvolved cells, its study in whole plant or complex tissues is very difficult. Due to their uniformity, accessibility, and reproducibility of application of stress conditions, cultured cells appear a useful tool to investigate the different aspects of plant PCD. In this review, we summarize how plant cell cultures can be utilized to clarify the plant PCD process.

Cryopreservation of plant cell cultures – Diverse practices and protocols

Plant cell cultures can be used as biotechnological platforms for the commercial production of small-molecule active ingredients and recombinant proteins, such as biopharmaceuticals. This requires the cryopreservation of well-characterized cell lines as master cell banks from which uniform working cell banks can be derived to ensure high batch-to-batch reproducibility during production campaigns. However, the cryopreservation of plant cells is challenging due to their low viability and poor regrowth after thawing. Three approaches have been developed: slow freezing, vitrification, and encapsulation-dehydration. Typically, the protocols are iteratively adapted to accommodate the properties of different plant cell lines, taking time and resources while achieving moderate success. Since standardized processes are a prerequisite for industrial applications, this review presents an in-depth analysis of the different procedures for cryopreservation of plant suspension cell cultures, highlighting relevant parameters for effective cryopreservation and the re-establishment of vigorous plant cell cultures within weeks. The protocol variants are grouped into modules that facilitate the directed improvement of each step and allow protocol evolution by module recombination. Ultimately, such improved cryopreservation protocols will form the basis of processes that comply with good manufacturing practice and attract major biopharmaceutical companies to the benefits of plant molecular farming.

Interés de la diferenciación celular en la producción de vincristina y vinblastina en cultivos en suspensión de Catharanthus roseus (L.) G Don.

Plant cell culture technology, particularly suspension cultures, appears as a convenient tool to industrially produce molecules such as the anticancer molecules vincristine and vinblastine. Cell differentiation is needed for their in vivo synthesis and thus in vitro cultures have usually been considered as limited producing platforms. Several studies have recently detected vincristine and vinblastine in early differentiated calluses and also in suspension cultures. Nevertheless, the degree of cell differentiation has not been addressed, particularly in suspension cultures that could be used as a large-scale producing platform. Therefore, the effect of culture conditions on the production of vincristine and vinblastine, taking into account cytodifferentiation within cell aggregates, has been analyzed for the first time. Culture conditions such as light exposure and plant growth regulator regimes have been shown to affect cell differentiation. Moreover, cell differentiation was observed to be closely related to vincristine and vinblastine titers. Results provide important clues into the comprehension of in vitro culture performance for metabolites production requiring in vivo cell differentiation. They demonstrated the utility of taking into account cell differentiation for the further development of novel advanced processes of differentiated cell suspension cultures for producing valuable molecules, including biological medicines such as vincristine and vinblastine.

Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures.

This study was initiated to assess whether the supposedly axenic plant cell cultures harbored any cultivation-recalcitrant endophytic bacteria (CREB). Adopting live-cell imaging with bright-field, fluorescent and confocal microscopy and bacterial 16S-rRNA gene taxonomic profiling, we report the cytoplasmic association of abundant and diverse CREBs in long-term actively maintained callus and cell suspension cultures of different plant species. Preliminary bright-field live-cell imaging on grape cell cultures showed abundant intracellular motile micro-particles resembling bacteria, which proved uncultivable on enriched media. Bacterial probing employing DNA stains, transmission electron microscopy, and Eubacterial FISH indicated abundant and diverse cytoplasmic bacteria. Observations on long-term maintained/freshly established callus stocks of different plant species—grapevine, barley, tobacco, Arabidopsis, and medicinal species—indicated intracellular bacteria as a common phenomenon apparently originating from field shoot tissues.Cultivation-independent 16S rRNA gene V3/V3–V4 amplicon profiling on 40-year-old grape cell/callus tissues revealed a high bacterial diversity (>250 genera), predominantly Proteobacteria, succeeded by Firmicutes, Actinobacteria, Bacteriodetes, Planctomycetes, and 20 other phyla, including several candidate phyla. PICRUSt analysis revealed diverse functional roles for the bacterial microbiome, majorly metabolic pathways. Thus, we unearth the widespread association of cultivation-recalcitrant intracellular bacteria “Cytobacts” inhabiting healthy plant cells, sharing a dynamic mutualistic association with cell hosts.