Carrot Cells Research Articles

Effects of freeze–thaw pre‐treatment with different freezing methods on the microwave freeze drying of carrots

SummaryThree freeze–thaw (FT) pre‐treatments using different freezing methods that freezing at −80 °C (FT‐80), freezing at −25 °C (FT‐25) and vacuum freezing (VFT) were used for carrot slices, the effects and mechanisms of FT pre‐treatment on the drying characteristics and product quality of carrots during microwave freeze drying (MFD) were studied. The results showed that FT pre‐treatment under different freezing methods affected the water state of carrots, resulting in varying degrees of moisture loss and shortening drying time by 14.3% to 42.8%. VFT treatment caused the greatest water loss (35.33%) during FT and significantly increased the effective moisture diffusivity in the MFD process. FT treatments did not cause any significant degradation in the colour of the carrots. The VFT and FT‐80 treatments resulted in an elevated rehydration ratio, increased crispiness of MFD carrots and products with a porous microstructure and higher porosity. In contrast, the FT‐25 treatment led to a decrease in porosity due to destructive damage to the carrot cell structure. However, this damage favoured the extractability of carotenoids, resulting in a higher carotenoid content than the other treatments. This study provides a theoretical foundation for the practical implementation of FT pre‐treatment using various freezing methods on fresh foods prior to drying. It is recommended to employ VFT pre‐treatment before MFD due to its efficiency and effectiveness.

A Study on the Effect Mechanism of Pectin Modification on the Carrot Cell Wall’s Texture Formation under Ultrasonic and Infrared Drying

The carrot has a high water content, and dehydration is an important means to extend its edible period and reduce storage and transportation costs. In the case of infrared (IR) drying, the porosity of the product is low and the structure is compact; the textural properties of the product are improved by using combined ultrasound and infrared (US-IR) drying; however, there is a lack of reports on the mechanism of this. Pectin has an important influence on the formation of the textural properties of fruit and vegetable tissues. In order to investigate the mechanism of the change in endogenous pectin properties in the carrot cell wall under US-IR drying on the improvement of the textural properties of the product, different fractions of pectins (water-soluble pectin, chelating pectin, alkali-soluble pectin) of the carrot were extracted, separated, and analysed. The thermal stability, component and content changes, Fourier infrared (FTIR), X-ray diffraction (XRD), esterification degree, molecular weight, monosaccharide composition, Ca ion content, and atomic force microscopy (AFM) of the pectins were determined. The results showed that the changes in the contents and properties of the carrot pectins under US-IR conditions had a positive effect on the improvement of the textural properties of the carrot tissues.

Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Plant-Derived Vesicles: A New Era for Anti-Cancer Drug Delivery and Cancer Treatment.

Lipid-structured vesicles have been applied for drug delivery system for over 50 years. Based on their origin, lipid-structured vesicles are divided into two main categories, namely synthetic lipid vesicles (SLNVEs) and vesicles of mammalian origin (MDVEs). Although SLNVEs can stably transport anti-cancer drugs, their biocompatibility is poor and degradation of exogenous substances is a potential risk. Unlike SLNVEs, MDVEs have excellent biocompatibility but are limited by a lack of stability and a risk of contamination by dangerous pathogens from donor cells. Since the first discovery of plant-derived vesicles (PDVEs) in carrot cell supernatants in 1967, emerging evidence has shown that PDVEs integrate the advantages of both SLNVEs and MDVEs. Notably, 55 years of dedicated research has indicated that PDVEs are an ideal candidate vesicle for drug preparation, transport, and disease treatment. The current review systematically focuses on the role of PDVEs in cancer therapy and in particular compares the properties of PDVEs with those of conventional lipid vesicles, summarizes the preparation methods and quality control of PDVEs, and discusses the application of PDVEs in delivering anti-cancer drugs and their underlying molecular mechanisms for cancer therapy. Finally, the challenges and future perspectives of PDVEs for the development of novel therapeutic strategies against cancer are discussed.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Congener-Specific Uptake and Metabolism of Bisphenols in Carrot Cells: Dissipation Kinetics, Biotransformation, and Enzyme Responses.

Food consumption has been considered a key pathway of bisphenol compound (BP) exposure for humans. However, there is a lack of evidence concerning their congener-specific behavior and metabolism in plants. Herein, we examined the uptake and metabolism of five BPs in plants using carrot cells. Bisphenol S (BPS) and bisphenol AF (BPAF) exhibited substantially lower dissipation rates in the cells than the other BPs, indicating a strong selectivity in the uptake and metabolism among bisphenol congeners. For a total of 23 metabolites of BPs, the predominant biotransformation pathways were found to be glycosylation, methoxylation, and conjugation, while hydroxylation, methylation, and glutathionylation were only observed for some BPs. The changes in the mRNA expression of cytochrome P450 (P450) and the activities of glycosyltransferase and glutathione S-transferase were remarkably higher in cells exposed to bisphenol F, bisphenol A, and bisphenol B than in cells exposed to BPS and BPAF, indicating congener specificity in their effects on enzymes and the associated biotransformation processes. Consequently, the potential congener-specific differences in plant uptake, metabolism, and accumulation must be considered when assessing the environmental risks posed by these commonly used plasticizers.

Production of benzylglucosinolate in genetically engineered carrot suspension cultures.

Glucosinolates, a group of sulfur-containing specialized metabolites of the Brassicales, have attracted a lot of interest in nutrition, medicine and agriculture due to their positive health effects and their involvement in plant defense. Their biological activities and the extensive knowledge of their biosynthesis have inspired research into development of crops with enhanced glucosinolate contents as well as their biotechnological production in homologous and heterologous systems. Here, we provide proof-of-concept for transgenic suspension cultures of carrot (Daucus carota, Apiacae) as a scalable production platform for plant specialized metabolites using benzylglucosinolate as a model. Two T-DNAs carrying in total six genes of the benzylglucosinolate biosynthesis pathway from Arabidopsis thaliana as well as NPTII and BAR as selectable markers were transferred to carrot cells by Agrobacterium tumefaciens-mediated transformation. Putative transformants selected based on their kanamycin and BASTA resistances were subjected to HPLC-MS analysis. Of 79 putative transformants, 17 produced benzylglucosinolate. T-DNA-integration was confirmed for the five best producers. Callus from these transformants was used to establish suspension cultures for quantitative analysis. When grown in 60-ml-cultures, the best transformants produced roughly 2.5 nmol (g fw)-1 benzylglucosinolate, together with up to 10 nmol (g fw)-1 desulfobenzylglucosinolate. Only one transformant produced more benzylglucosinolate than desulfobenzylglucosinolate. The concentration of sulfate in the medium was not a major limiting factor. High production seemed to be associated with poor growth and vice versa. Therefore, future research should try to optimize medium and cultivation process and to separate growth and production phase by using an inducible promoter.

Effect of thermosonication on texture degradation of carrot tissue in relation to alterations in cell membrane and cell wall structure

Thermosonication as an alternative to hot water blanching of carrots, the effect of thermosonication on the carrot texture and cell structure required deep investigation. This study was conducted from the aspects of cell membrane, cell water, and cell wall pectin. Results showed that thermosonication at 60 °C significantly increased carrot tissue hardness by up to 10.3% compared with fresh. Rheological curves showed that the carrot tissues remained rigid after thermosonication. Cell membrane permeability was damaged to different extents during processing, inducing bound and immobilized water to migrate to free water and bring intracellular electrolytes to outflow. Thermosonication affected cell structure by altering the characteristics of pectin, including uronic acids and neutral sugar contents, degree of linearity and branching, and degree of methoxylation. The modified pectin can seize more divalent cations such as Ca2+ retained in the thermosonicated carrot cell wall, conducive to preserving the textural property of processed fruits and vegetables.

Brazzein protein production in transgenic carrot cells using air-lift bioreactor culture

Brazzein protein production in transgenic carrot cells using air-lift bioreactor culture

Carrot cells expressing the VP1 and VP2 poliovirus proteins effectively elicited mucosal immunity

Carrot cells expressing the VP1 and VP2 poliovirus proteins effectively elicited mucosal immunity

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Totipotency of Daucus carota L. Somatic Cells Microencapsulated Using Spray Drying Technology.

The carrot is considered a model system in plant cell culture. Spray drying represents a widely used technology to preserve microorganisms, such as bacteria and yeasts. In germplasm conservation, the most used methods are freeze drying and cryopreservation. Therefore, the aim of this work was to evaluate the effect of spray drying on the viability and totipotency of somatic carrot cells. Leaf, root and stem explants were evaluated to induce callus with 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Calli obtained from the stem were cultivated in a liquid medium with 1 mg/L of 2,4-D. Cell suspensions were spray dried with maltodextrin-gum Arabic and maltodextrin-xanthan gum mixtures, two outlet air temperatures (50 and 60 °C) and 120 °C inlet air temperature. Results showed that carrot cells were viable after spray drying, and this viability remained for six months at 8 °C. The totipotency of the microencapsulated cells was proven. Cells that were not spray dried regenerated 24.6 plantlets, while the spray dried cells regenerated 19 plantlets for each gram of rehydrated powder. Thus, spray drying allowed researchers to obtain viable and totipotent cells. This work is the first manuscript that reported the spray drying of plant somatic cells.

Repression of Carotenoid Accumulation by Nitrogen and NH4+ Supply in Carrot Callus Cells In Vitro.

The effect of mineral nutrition on the accumulation of the main health beneficial compounds in carrots, the carotenoid pigments, remains ambiguous; here, a model-based approach was applied to reveal which compounds are responsible for the variation in carotenoid content in carrot cells in vitro. For this purpose, carotenoid-rich callus was cultured on either BI (modified Gamborg B5) or R (modified Murashige and Skoog MS) mineral media or on modified media obtained by exchanging compounds between BI and R. Callus growing on the BI medium had abundant carotene crystals in the cells and a dark orange color in contrast to pale orange callus with sparse crystals on the R medium. The carotenoid content, determined by HPLC and spectrophotometrically after two months of culture, was 5.3 higher on the BI medium. The replacement of media components revealed that only the N concentration and the NO3:NH4 ratio affected carotenoid accumulation. Either the increase of N amount above 27 mM or decrease of NO3:NH4 ratio below 12 resulted in the repression of carotenoid accumulation. An adverse effect of the increased NH4+ level on callus growth was additionally found. Somatic embryos were formed regardless of the level of N supplied. Changes to other media components, i.e., macroelements other than N, microelements, vitamins, growth regulators, and sucrose had no effect on callus growth and carotenoid accumulation. The results obtained from this model system expand the range of factors, such as N availability, composition of N salts, and ratio of nitrate to ammonium N form, that may affect the regulation of carotenoid metabolism.

Molecular Analysis of the E2F/DP Gene Family of Daucus carota and Involvement of the DcE2F1 Factor in Cell Proliferation.

E2F transcription factors are key components of the RB/E2F pathway that, through the action of cyclin-dependent kinases, regulates cell cycle progression in both plants and animals. Moreover, plant and animal E2Fs have also been shown to regulate other cellular functions in addition to cell proliferation. Based on structural and functional features, they can be divided into different classes that have been shown to act as activators or repressors of E2F-dependent genes. Among the first plant E2F factors to be reported, we previously described DcE2F1, an activating E2F which is expressed in cycling carrot (Daucus carota) cells. In this study, we describe the identification of the additional members of the E2F/DP family of D. carota, which includes four typical E2Fs, three atypical E2F/DEL genes, and three related DP genes. Expression analyses of the carrot E2F and DP genes reveal distinctive patterns and suggest that the functions of some of them are not necessarily linked to cell proliferation. DcE2F1 was previously shown to transactivate an E2F-responsive promoter in transient assays but the functional role of this protein in planta was not defined. Sequence comparisons indicate that DcE2F1 could be an ortholog of the AtE2FA factor of Arabidopsis thaliana. Moreover, ectopic expression of the DcE2F1 cDNA in transgenic Arabidopsis plants is able to upregulate AtE2FB and promotes cell proliferation, giving rise to polycotyly with low frequency, effects that are highly similar to those observed when over-expressing AtE2FA. These results indicate that DcE2F1 is involved in the control of cell proliferation and plays important roles in the regulation of embryo and plant development.

Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation

Miraculin is a taste-modifying protein that interacts with human sweet-taste receptors and transforms a sour taste into sweet taste. Miraculin is extracted from the miracle fruit (Synsepalum dulcificum). Since mass production of miraculin is difficult because of regional and seasonal limitations, several attempts have been made to express miraculin in various cell systems. In this study, a binary vector containing the miraculin gene under the control of the SWPA2 promotor was introduced into carrot (Daucus carota) callus via Agrobacterium-mediated transformation to synthesize miraculin in carrot cell cultures. After 4 weeks of co-cultivation with Agrobacterium tumefaciens, 20 tentative transgenic callus (TC) lines were obtained on kanamycin selection medium. PCR analysis confirmed that 18 of these 20 lines (TC1–TC18) carried the miraculin gene, and 4 TC lines with high cell growth and gene expression (determined by RT-qPCR) were selected for further analysis. Protein analysis of these four TC lines by SDS-PAGE and Western blot showed that the miraculin protein was stably produced in TC lines. The cell growth showed no correlation with gene expression levels. The DNA content and G1 phase ratio were negatively correlated, whereas the S and G2/M phase ratios were positively correlated with gene expression. The ratio of cell cycle was determined by counting the number of cells in each step through flow cytometric analysis. These results indicate that gene expression was higher in TC lines with active cell division. Overall, our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems. This study expressed the miraculin gene in carrot callus via Agrobacterium-mediated transformation based on miraculin expression and cell cycle analyses. Our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems

Oxidative Stress and Antioxidative Activity in Leaves and Roots of Carrot Plants Induced by Candidatus Phytoplasma Solani.

The present study examined the effects of Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the “stolbur” group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.

Uptake and metabolism of nonylphenol in plants: Isomer selectivity involved with direct conjugation.

Nonylphenol (NP), an environmental estrogen, is actually a complicated mixture of isomers, although it is commonly considered to be a single compound. There are many routes for crops to come into contact with NP; however, little is known about the plant uptake and metabolism of NP, especially at the isomer level. This study comparatively evaluated the uptake and in-planta metabolism of 4-n-NP and its 10 isomers using both carrot cells and intact plants. The rapid metabolism of 4-n-NP was observed in the callus tissues and intact plants with half-lives of 2h and 4.72d, respectively. Six conjugates of 4-n-NP were identified in the cell extracts using high resolution mass spectrometry. The primary transformation pathway was found to be the direct conjugation (Phase II metabolism) with the parent compound at the hydroxyl. Furthermore, 4-NP isomers with short side chains and/or bulky α-substituents were more resistant to plant metabolism and showed a greater tendency for accumulation. The influence of the side chains to the isomer selectivity was verified by the molecular docking between glycosyltransferase and 4-NP isomers. This study highlighted the necessity to consider isomer-specificity in the plant accumulation of NP and the environmental and human health implications of NP conjugates.

Red Carrot Cells Cultured in vitro Are Effective, Stable, and Safe Ingredients for Skin Care, Nutraceutical, and Food Applications.

Plant biomasses growing in bioreactor could be developed as production systems for cosmetic ingredients, nutraceuticals and food additives. We previously reported that the red carrot cell line R4G accumulates high levels of anthocyanins, which are potent antioxidants with multiple health-promoting properties. To investigate the industrial potential of this cell line in detail, we tested extract for antioxidant and anti-inflammatory activity in the mouse monocyte/macrophage cell-line J774A.1 and in reconstructed skin tissue models. We also compared the R4G extract to commercial carrot extracts in terms of stability and metabolomic profiles. We found that the R4G extract have potent antioxidant and anti-inflammatory activities, protecting mammalian cells from the oxidative stress triggered by exposure to bacterial lipopolysaccharides and H2O2. The extract also inhibited the nuclear translocation of NF-κB in an epidermal skin model, and induced the expression of VEGF-A to promote the microcirculation in a dermal microtissue model. The anthocyanins extracted from R4G cells were significantly more stable than those found in natural red carrot extracts. Finally, we showed that R4G extract has similar metabolomic profile of natural extracts by using a combination of targeted and untargeted metabolomics analysis, demonstrating the safety of R4G carrot cells for applications in the nutraceutical and food/feed industries.

Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors

Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.

Increased brazzein expression by abiotic stress and bioreactor culture system for the production of sweet protein, brazzein

Plant-based recombinant protein production systems are powerful tools that can be used instead of microbial and animal cell culture systems. The aim of this study was to establish a stable brazzein production system from carrot cell suspension using bioreactor. Using Agrobacterium-mediated transformation (stress-inducible SWPA2 promoter and brazzein gene), three transgenic lines (TC1, 11, 12) were generated. During the TC12 culture period, cell proliferation increased rapidly up to 15 days, and the maximum cell division rate (log phase) was reached after 6 days of culture. Fresh weight peaked at day 18, 3.8-fold (7.6 g 100 mL−1) higher than on day 1, and gene expression was tenfold higher on day 27 than on day 0. The brazzein-encoding gene was also analyzed after treatment with various stress factors for increase of brazzein gene expression, and abscisic acid (ABA) and hydrogen peroxide (H2O2) proved most effective. In particular, the gene expression was enhanced 2.5-fold with 220 μM H2O2 and 2.8-fold with 50 μM ABA, compared with controls. Transgenic cells were then transferred to various types of air-lift bioreactor, and the column bioreactor achieved a higher biomass (238.9 g L−1) than cone and balloon bioreactors. These results demonstrate an efficient protein production system for brazzein using an air-lift bioreactor that may be suitable for the use in the food industry.