Callus Plant Research Articles

Hypericum Perforatum Callus Extract-Loaded Composite Hydrogel with Diverse Bioactivities for Enhanced Wound Healing and Fibrosis Prevention.

Plant Callus are a valuable source of pluripotent stem cells and bioactive phytochemicals. Meanwhile, the Hypericum perforatum callus extract (HPCE) is particularly rich in compounds such as hyperforin, hypericin, quercetin, and other phenolic and flavonoid derivatives. These phytochemicals exhibit strong antibacterial, antioxidant, anti-inflammatory, and anti-fibrotic properties, making them promising for wound healing. One of the most critical challenges following wound healing is the formation of fibrosis, which can compromise the complex structural integrity of skin. To address this issue, a poly(vinyl alcohol)/chitosan/alginate (PCA) wound dressing loaded with HPCE is developed. This hydrogel dressing features a porous structure with suitable mechanical properties and a high swelling capacity, potentially enhancing its effectiveness in promoting tissue regeneration and wound healing. In vitro studies have confirmed its biocompatibility, cell proliferation, and cell adhesion properties. Additionally, the dressing has demonstrated the ability to inhibit the proliferation of certain antibiotic-resistant bacteria. The in vivo studies revealed the anti-inflammatory properties, promotion of angiogenesis, facilitation of re-epithelialization, and stimulation of collagen deposition of the dressing under investigation. Moreover, the immunohistochemistry analysis of the two key markers, p16 and p53, has shown that the application of the dressing helps prevent fibrosis after wound healing.

Cryopreservation of Arum palaestinum plant callus as a strategy for mitigating extinction risks

Arum palaestinum is a wild perennial plant commonly known as "Al-Loof" in Jordan. Due to overharvesting, climate change, and increasing demand, its natural populations are threatened with extinction. Cryopreservation, an effective method for conserving plant material at ultra-low temperatures, is explored for A. palaestinum calli. We investigated the applicability of encapsulation-vitrification (using different plant vitrification solutions (PVS) and incubation times), encapsulation-dehydration (using sucrose or sorbitol at different concentrations and dehydration times), and the v-cryoplate (using different pre-culture times and temperatures) techniques. In the encapsulation-vitrification experiment, a notable 82.4 % regrowth rate was achieved by desiccating calli in plant vitrification solution 2 (PVS2) for 10 minutes at 25 °C. The encapsulation-dehydration technique resulted in an 82.6 % regrowth rate by incubating calli for one day in low sucrose levels (0.1 M sucrose) following one hour of air dehydration, where the moisture content of the beads was 30 %. The moisture content of the beads decreased from 81 % before chemical and air dehydration to 71 % after 0 hours of air dehydration combined with chemical dehydration using 0.1 M sucrose or sorbitol. It further dropped to 30–34 % after one day of chemical dehydration with 0.1 M sucrose and 1 hour of air dehydration. The v-cryoplate technique successfully conserved calli, showing impressive survival and regrowth percentages (96.8 %) when the callus was pre-cultured with 0.3 M sucrose for three days at 5 °C. Temperature during pre-culture significantly influenced regrowth percentages in the v-cryoplate technique. The study establishes promising cryopreservation protocols for A. palaestinum calli, offering a means to conserve germplasm and contribute to environmental and biodiversity protection by reintroducing endangered plants to their native habitats.

Influence of Primary Light Exposure on the Morphophysiological Characteristics and Phenolic Compounds Accumulation of a Tea Callus Culture (Camellia sinensis L.).

Tea plant calli (Camellia sinensis L.) are characterized by the accumulation of various phenolic compounds (PC)-substances with high antioxidant activity. However, there is still no clarity on the response of tea cells to light exposure of varying intensity. The purpose of the research was to study tea callus cultures grown under the influence of primary exposure to different light intensities (50, 75, and 100 µmol·m-2·s-1). The cultures' growth, morphology, content of malondialdehyde and photosynthetic pigments (chlorophyll a and b), accumulation of various PC, including phenylpropanoids and flavanols, and the composition of catechins were analyzed. Primary exposure to different light intensities led to the formation of chloroplasts in tea calli, which was more pronounced at 100 µmol·m-2·s-1. Significant similarity in the growth dynamics of cultures, accumulation of pigments, and content of malondialdehyde and various phenolics in tea calli grown at light intensities of 50 and 75 µmol·m-2·s-1 has been established, which is not typical for calli grown at 100 µmol·m-2·s-1. According to data collected using high-performance liquid chromatography, (+)-catechin, (-)-epicatechin, epigallocatechin, gallocatechin gallate, epicatechin gallate, and epigallocatechin gallate were the main components of the tea callus culture's phenolic complex. Its content changed under the influence of primary exposure to light, reaching the greatest accumulation in the final stages of growth, and depended on the light intensity. The data obtained indicate changes in the morphophysiological and biochemical characteristics of tea callus cultures, including the accumulation of PC and their individual representatives under primary exposure to light exposure of varying intensity, which is most pronounced at its highest values (100 µmol·m-2·s-1).

Multi-omics signatures of diverse plant callus cultures

Multi-omics signatures of diverse plant callus cultures

Potential role of ice-binding protein in mitochondria-lipid and ATP mechanisms during freezing of plant callus

Plant calli, a perpetually undifferentiated cell culture, have defects in maintaining their genetic fidelity during prolonged tissue culture. Cryopreservation using ice-binding proteins (IBP) is a potential solution. Despite a few studies on cryopreservation using IBPs in plant calli, detailed insights into the intracellular metabolism during freezing, thawing, and re-induction remain sparse. This study investigated and employed IBP from polar yeast Leucosporidium sp. (LeIBP) in the cryopreservation process across diverse taxa, including gymnosperms, monocots, dicots, and woody plants. Molecular-level analyses encompassing reactive oxygen species levels, mitochondrial function, and ATP and lipophilic compounds content were conducted. The results across nine plant species revealed the effects of LeIBP on callus competency post-thawing, along with enhanced survival rates, reactive oxygen species reduction, and restored metabolic activities to the level of those of fresh calli. Moreover, species-specific survival optimization with LeIBP treatments and morphological assessments revealed intriguing extracellular matrix structural changes post-cryopreservation, suggesting a morphological strategy for maintaining the original cellular states and paracrine signaling. This study pioneered the comprehensive application of LeIBP in plant callus cryopreservation, alleviating cellular stress and enhancing competence. Therefore, our findings provide new insights into the identification of optimal LeIBP concentrations, confirmation of genetic conformity post-thawing, and the intracellular metabolic mechanisms of cryopreservation advancements in plant research, thereby addressing the challenges associated with long-term preservation and reducing labor-intensive cultivation processes. This study urges a shift towards molecular-level assessments in cryopreservation protocols for plant calli, advocating a deeper understanding of callus re-induction mechanisms and genetic fidelity post-thawing.

Phytosynthesis of silver nanoparticles using aqueous sandalwood (Santalum album L.) leaf extract: Divergent effects of SW-AgNPs on proliferating plant and cancer cells.

The biogenic approach for the synthesis of metal nanoparticles provides an efficient eco-friendly alternative to chemical synthesis. This study presents a novel route for the biosynthesis of silver nanoparticles using aqueous sandalwood (SW) leaf extract as a source of reducing and capping agents under mild, room temperature synthesis conditions. The bioreduction of Ag+ to Ago nanoparticles (SW-AgNPs) was accompanied by the appearance of brown color, with surface plasmon resonance peak at 340-360 nm. SEM, TEM and AFM imaging confirm SW-AgNP's spherical shape with size range of 10-32 nm. DLS indicates a hydrodynamic size of 49.53 nm with predominant negative Zeta potential, which can contribute to the stability of the nanoparticles. FTIR analysis indicates involvement of sandalwood leaf derived polyphenols, proteins and lipids in the reduction and capping of SW-AgNPs. XRD determines the face-centered-cubic crystalline structure of SW-AgNPs, which is a key factor affecting biological functions of nanoparticles. This study is novel in using cell culture methodologies to evaluate effects of SW-AgNPs on proliferating cells originating from plants and human cancer. Exposure of groundnut calli cells to SW-AgNPs, resulted in enhanced proliferation leading to over 70% higher calli biomass over control, enhanced defense enzyme activities, and secretion of metabolites implicated in biotic stress resistance (Crotonyl isothiocyanate, Butyrolactone, 2-Hydroxy-gamma-butyrolactone, Maltol) and plant cell proliferation (dl-Threitol). MTT and NRU were performed to determine the cytotoxicity of nanoparticles on human cervical cancer cells. SW-AgNPs specifically inhibited cervical cell lines SiHa (IC50-2.65 ppm) and CaSki (IC50-9.49 ppm), indicating potential use in cancer treatment. The opposing effect of SW-AgNPs on cell proliferation of plant calli (enhanced cell proliferation) and human cancer cell lines (inhibition) are both beneficial and point to potential safe application of SW-AgNPs in plant cell culture, agriculture and in cancer treatment.

Nutritional supplementation of an apple callus extract to target epidermal aging

The global increase in life expectancy has led to a growing elderly population, which places a significant burden on healthcare systems. As a result, there is a growing interest to improve healthspan and promote healthy aging with accessible means, such as nutrition and lifestyle interventions. The skin, the body's largest organ and first line of defense against environmental stressors, is particularly vulnerable to aging due to both intrinsic and extrinsic factors. Various molecular and biochemical changes can drive skin aging, including the depletion of stem cells, which can lead to impaired skin properties. Nutrition plays a crucial role in skin aging, and dietary supplements could be a viable intervention to support a healthy skin. In this study, a plant callus extract from a specific apple variety (Malus Domestica Borkh), the Uttwiler Spätlauber, was evaluated as a dietary supplement to combat epidermal aging. The extract was found to maintain the function of keratinocyte progenitor cells during aging in vitro. Furthermore, an open-label pilot clinical study involving 31 female volunteers showed that the intake of apple callus extract led to improvements in skin elasticity, density, and firmness after 56 days of supplementation. This study highlights the potential of plant callus extracts as natural dietary supplements to support healthy skin aging. The use of plant callus cultures provides a sustainable and reproducible source of bioactive molecules, which is gaining acceptance in the nutraceutical market. These findings suggest that dietary interventions can play a role in promoting healthy skin aging and contribute to a holistic approach to improving healthspan during the aging process.

Влияние липополисахаридов грамотрицательных бактерий различных таксономических групп на морфогенетическую активность каллусов мягкой пшеницы

In vitro tissue culture is actively used in modern plant breeding for genetic transformation, effective selection, rapid production and propagation of genetically homogeneous lines. For many crops, the low yield of regenerated plants is limiting for the use of calluses in the breeding process. Some bacterial lipopolysaccharides are able to increase the morphogenetic activity of plant calluses, increasing the efficiency of in vitro technology. In this work, we investigated the effect of lipopolysaccharides (LPS) from the rhizobacterial strains Ensifer adhaerens T1Ks14, Ochrobactrum quorumnocens T1Kr02, and Pseudomonas chlororaphis K3 on the processes of callusogenesis, the formation of morphogenic calluses and regenerant plants of common wheat (Triticum aestivum L.) of a low-morphogenic line with the Rht-B1a dwarfing gene, obtained on the basis of the cultivar Saratovskaya 29. It was found that LPS preparations at a concentration of 10 μg/ml had different effects on wheat tissue cultures. The yield of calluses from explants obtained from immature embryos significantly increased by 2.5% when exposed to LPS of the O. quorumnocens strain T1Kr02 and decreased by 4% in the variant with LPS from P. chlororaphis K3. The yield of morphogenic calluses increased under the influence of LPS from E. adhaerens T1Ks14 (+57%) and P. chlororaphis K3 (+41%). LPS strain O. quorumnocens T1Kr02 had a negative effect on the yield of regenerated plants, while the other two LPS preparations did not differ from the control in this parameter. As a result, the effectiveness of using the LPS preparations from explants to regenerants was significantly higher only in the variant of the E. adhaerens strain T1Ks14 (+64%). The data obtained indicate significant differences in the response of plant tissue cultures to LPS of various rhizosphere bacteria.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

In vitro production of steroidal saponin, total phenols and antioxidant activity in callus suspension culture of Paris polyphylla Smith: an important Himalayan medicinal plant.

Paris polyphylla Smith (Melanthiaceae) family, which is native to the Himalayan region, has received a lot of attention recently due to its extensive history of usage in traditional medicine. The production of steroidal saponin from callus suspension cultures of P. polyphylla was observed in the current study. The current study attempted to develop a P. polyphylla plant callus suspension culture through optimization of cultivation technique for callus suspension, quantification of total phenolic components and estimation of the extract's antioxidant activity. A light-yellow callus was formed within six weeks of cultivating rhizomes on Murashige and Skoog (MS) media supplemented with Thidiazuron (TDZ). Furthermore, the effect of TDZ, Methyl Jasmonate (MeJA), and Yeast Extract (YE) on callus growth, steroidal saponin (dioscin and diosgenin), total phenolic content, total flavonoids, total tannin, and total antioxidant activity was also measured. The medium containing 0.5 μM TDZ depicted the maximum callus biomass (2.98 g fresh weight). Significantly high phenolic and tannin content was observed in the MS medium containing 50 μM MeJA, whereas, no significant increase was observed in total tannin production in any treatment. Three in vitro assays, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid)) and FRAP (ferric ion reducing antioxidant potential) and FC (Folin-Ciocalteu), were used to assess antioxidant potential of callus. Maximum antioxidant analysis reported in 1.0 μM TDZ (6.89 mM AAE/100 g) containing medium followed by 50 μM MeJA (6.44 mM AAE/100g). The HPLC analysis showed a high presence of dioscin and diosgenin (5.43% and 21.09%, respectively) compared to the wild sample (2.56% and 15.05%, respectively). According to the results, callus produced on media supplemented with 50 μM MeJA have significant phenolic contents and elevated antioxidant activity; nevertheless, callus growth was greater in the presence of 0.5 μM TDZ. The findings of the current study have commercial implications since greater biomass production will result in active phytochemicals that the pharmaceutical and nutraceutical sectors are in need desperately.

The Influence of Phytohormones on Antioxidative and Antibacterial Activities in Callus Cultures of Hypericum perforatum L.

Natural extracts of Hypericum perforatum L. are widely used in the pharmaceutical industry due to their antiviral, antioxidant, antibacterial, antidiabetic, and antidepressant activities. Plant biotechnological methods are gaining more attention as efficient and biosustainable approaches for the production of high value compounds by using plant callus cultures in vitro. The aim of this research was to evaluate the influence of phytohormones on the formation of Hypericum perforatum L. callus culture, to explore antioxidant and antibacterial activities, and to determine the amounts of phytochemicals in the analyzed extracts. Callus cultures were induced on Murashige and Skoog basal medium supplemented with phytohormones (auxins, cytokinins, and salicylic acid), and containing newly synthesized compound N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide. The antioxidant activities of callus cultures were evaluated using DPPH and FRAP assays and the enzyme antioxidants (superoxide dismutase, catalase, and ascorbate peroxidase) were evaluated. The antibacterial activity of plant extracts was screened against Escherichia coli and Bacillus subtilis bacteria by the diffusion agar method. There was a significant positive correlation between total phenolic content and high antioxidant efficiency in Hypericum perforatum L. callus cultures, which were formed on Murashige and Skoog medium supplemented with auxin, cytokinin, and salicylic acid (100 µM). Our results have demonstrated that callus cultures generated on MS medium supplemented with N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide and cytokinin BAP (0.5 mg L−1) exhibited improved antioxidant activities for the first time.

Estimate of some Effective Compounds of the Four O’clock Rose Plant Callus Mirabilis jalapa L. Outside in vivo

Seeds of the plant Mirabilis jalapa were collected from private nurseries in the Salah al-Din governorate and analyzed at the laboratory of plant cell and tissue transplantation in the department of horticulture engineering in the Faculty, of Agriculture at the University of Tikrit, and after the success of the process of sterilization of the seeds of the plant of Mirabilis jalapa and the induction of callus, some of the active compounds within the callus of the plant internode were estimated and compared with the use of growth regulators of different concentrations, When using a combination of Bracinolide at a concentration of 0.01 mg L-1 and sodium chloride salt at a concentration of 100 mmol liters, the highest concentration of P-Coumaric was given at 128.9 μg/gm.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Elucidating the Metabolism of 2,4-Dibromophenol in Plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized inthe plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

Effects of Hydrogen Peroxide on In Vitro Cultures of Tea (Camellia sinensis L.) Grown in the Dark and in the Light: Morphology, Content of Malondialdehyde, and Accumulation of Various Polyphenols.

Tea plants (Camellia sinensis L.) are phenol-accumulating crops that are widely used for public health. The healing effect of tea leaf products is due to the biosynthesis of such phenolic compounds (PCs) as flavans, which have P-vitamin capillary-strengthening activity. Due to their limited habitat and the value of their specialized metabolites of a phenolic nature, a promising approach is to establish in vitro cultures from them that retain the ability to form PCs, which is characteristic of ex vivo tea plants. The aim of this study was to investigate the effect of exogenic H2O2 (0.01 mM; 0.1 mM; 1 mM) on the growth, morphology, degree of stress response, and accumulation of various phenolic compounds in tea plant callus cultures of different ages (24 or 36 days) grown under different cultivation conditions (darkness or light). According to the results obtained, the H2O2 effect on tea callus cultures of different ages did not cause changes in their morphophysiological characteristics, both after 2 h of exposure (rapid response of callus culture, RRCC) and after 48 h (delayed response of callus culture, DRCC). The determination of the malondialdehyde (MDA) content, which serves as an indicator of changes in the level of lipid peroxidation (LPO) and the presence of stress responses in plant cells, indicated either its maintenance at the control level, a decrease, or an increase. All these effects depended on the growth conditions of the tea callus cultures (darkness or light), their age, the duration of exposure (rapid or delayed response), and the H2O2 concentration. Similar trends were noted for the total content of PCs as well as the amount of flavans, proanthocyanidins (soluble and insoluble forms), and lignin. The plant cell responses reflected changes in its adaptation programs, when specialized metabolites act as a target for the action of H2O2, thereby contributing to an increase in their resistance.

A simple mid-term preservation method (SMPM) of plant callus under low temperature conditions

A simple mid-term preservation method (SMPM) of plant callus under low temperature conditions

Risk-based Approach in Obtaining and Analysing the Characteristics of Baikal Skullcap Suspension Cultures

Introduction . The use of biologically active compounds of Baikal skullcap ( Scutellaria baicalensis Georgi) becomes especially important under pandemic conditions. The plant's value becomes the cause of overexploitation of the skullcap subterranean parts, which leads to the reduction of the population distribution range and inappropriate conditions of collection and harvesting. To prevent the extinction hazard of the species, in vitro cultivation of the plant's cells becomes a promising technology. This opens up the prospect of studying the physiology and biochemistry of plant cells, regulating the direction of valuable secondary metabolites synthesis and creating medicines without interfering with the natural habitat. Modern technologies provide opportunities to automate cell cultivation in order to minimize human involvement in the process. However, cultivation of plant cells is a probabilistic process that is influenced by a range of factors. The use of risk management tools will reduce the number of unintended consequences leading to dangerous and critical cell culture conditions. Aim . Risk analysis resulting from in vitro cultivation of Baikal skullcap ( Scutellaria baicalensis Georgi). Materials and methods . The object of the study was suspension cultures of Scutellaria baicalensis obtained from loose plant callus. Suspensions of Scutellaria baicalensis cells were grown in 250 ml flat-bottomed conical flasks under conditions of constant mixing on a shaker (rotation speed 100 rpm). The subculture cycle was 21 days. For microscopy of cell biomass, we used 0.1 % neutral red (NK) and 0,025 % Evans blue (EG) solution. Process risks were assessed using a cause-and-effect diagram (Ishikawa diagram). Results and discussion . It was found that the cells in the suspension cultures of skullcap were characterized by an elongated oval shape. Primary suspensions were distinguished by the presence of predominantly parenchymal cells (70 ± 2,0 %). A large aggregation was observed in the primary suspensions. The diameter of the aggregates reached about 8–15 mm. The growth properties of Baikal skullcap suspension depended on the density of the inoculum. As the amount of transplanted biomass decreased, cells showed a tendency to destruction. The results indicated the need for risk analysis of the laboratory technology of Baikal skullcap suspension cultivation. Five most important factors in plant cell cultivation were identified: callus quality, quality of prepared liquid nutrient medium, equipment serviceability and suitability, operator actions, and suitability of cell culture technology and biomass viability index sampling. Conclusion . An algorithm for analyzing the security of a Baikal skullcap suspension culture is proposed. The risk assessment can be used to make a conclusion about the probability of undesirable effects and their impact on the process. Risk management tools provide the basis for modernization of the available technology and automatic outfitting of the process.

Carbon-nanotube for Transient Expression in Rice Calli

Transient gene expression is an important technique in gene functional analysis, protein production and in plants. However, traditional transient expression methods using Agrobacterium are time-consuming with low efficiency. In this study, we demonstrated the use of a single-walled carbon nanotube (SWCNT) to deliver 35S:mCherry:pCXSN plasmid into rice calli. This transient expression protocol used a plastic medical syringe to create the physical pressure to help the delivery of plasmid DNA into plant cells. This protocol is relatively easy to perform and low cost. The transient expression was observed under fluorescence microscopy, and the mCherry fluorescence signal was quantified. The plasmid DNA was delivered into the rice cell using a 3:1 ratio (plasmid: carbon nanotube). The result showed that the mCherry signal of carbon nanotube + plasmid DNA treatment was the highest signal at 3 days post-transformation and decreased to a lower signal at 6 days post-transformation. Moreover, the quantitative analysis of mCherry mean intensity revealed that the signal intensity of carbon nanotube + plasmid DNA treatment was the highest level, and significantly higher than the control treatments at 3 days post-transformation. Plasmid DNA can be transported easily into plant calli using this carbon nanotube transient expression protocol.

Genome-Wide Identification and Comparative Analysis of WOX Genes in Four Euphorbiaceae Species and Their Expression Patterns in Jatropha curcas.

The WUSCHEL-related homeobox (WOX) proteins are widely distributed in plants and play important regulatory roles in growth and development processes such as embryonic development and organ development. Here, series of bioinformatics methods were utilized to unravel the structural basis and genetic hierarchy of WOX genes, followed by regulation of the WOX genes in four Euphorbiaceae species. A genome-wide survey identified 59 WOX genes in Hevea brasiliensis (H. brasiliensis: 20 genes), Jatropha curcas (J. curcas: 10 genes), Manihot esculenta (M. esculenta: 18 genes), and Ricinus communis (R. communis: 11 genes). The phylogenetic analysis revealed that these WOX members could be clustered into three close proximal clades, such as namely ancient, intermediate and modern/WUS clades. In addition, gene structures and conserved motif analyses further validated that the WOX genes were conserved within each phylogenetic clade. These results suggested the relationships among WOX members in the four Euphorbiaceae species. We found that WOX genes in H. brasiliensis and M. esculenta exhibit close genetic relationship with J. curcas and R. communis. Additionally, the presence of various cis-acting regulatory elements in the promoter of J. curcas WOX genes (JcWOXs) reflected distinct functions. These speculations were further validated with the differential expression profiles of various JcWOXs in seeds, reflecting the importance of two JcWOX genes (JcWOX6 and JcWOX13) during plant growth and development. Our quantitative real-time PCR (qRT-PCR) analysis demonstrated that the JcWOX11 gene plays an indispensable role in regulating plant callus. Taken together, the present study reports the comprehensive characteristics and relationships of WOX genes in four Euphorbiaceae species, providing new insights into their characterization.

Highly Efficient Bioconversion of trans-Resveratrol to δ-Viniferin Using Conditioned Medium of Grapevine Callus Suspension Cultures.

δ-Viniferin is a resveratrol dimer that possesses potent antioxidant properties and has attracted attention as an ingredient for cosmetic and nutraceutical products. Enzymatic bioconversion and plant callus and cell suspension cultures can be used to produce stilbenes such as resveratrol and viniferin. Here, δ-viniferin was produced by bioconversion from trans-resveratrol using conditioned medium (CM) of grapevine (Vitis labruscana) callus suspension cultures. The CM converted trans-resveratrol to δ-viniferin immediately after addition of hydrogen peroxide (H2O2). Peroxidase activity and bioconversion efficiency in CM increased with increasing culture time. Optimized δ-viniferin production conditions were determined regarding H2O2 concentration, incubation time, temperature, and pH. Maximum bioconversion efficiency reached 64% under the optimized conditions (pH 6.0, 60 °C, 30 min incubation time, 6.8 mM H2O2). In addition, in vitro bioconversion of trans-resveratrol was investigated using CM of different callus suspension cultures, showing that addition of trans-resveratrol and H2O2 to the CM led to production of δ-viniferin via extracellular peroxidase-mediated oxidative coupling of two molecules of trans-resveratrol. We thus propose a simple and low-cost method of δ-viniferin production from trans-resveratrol using CM of plant callus suspension cultures, which may constitute an alternative approach for in vitro bioconversion of valuable molecules.