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The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used as explants to culture the callus. The cryopreservation procedure of the callus by vitrification was initially established by using the orthogonal experimental method of four factors and three levels in the preculture, loading, and dehydration steps. Furthermore, the vitrification-based cryopreservation was optimized by changing the preculture temperature and loading solution and adding exogenous substances to the plant vitrification solution (PVS2). In this procedure, the callus was precultured at 25 °C for 2 d, and loaded in 50% PVS2 at 25 °C for 60 min. The callus was dehydrated with PVS2 containing 0.08 mM reduced glutathione (GSH) at 0 °C for 60 min. After rapid-cooling in liquid nitrogen for 1 h, it was rapid-warming in a water bath at 40 °C for 90 s and unloaded for 30 min. After 1 d of recovery, the cell relative survival rate of the cryopreserved callus was 64.60%. The results provide a valuable basic and effective method for the long-term conservation of A. andraeanum germplasm resources.

Under a water deficit, the protective proteins known as dehydrins (DHNs) prevent nonspecific interactions in protein and membrane structures and their damage, in addition to playing an antioxidant role. The DHNs of a widespread xerophytic species Scots pine (Pinus sylvestris L.) have been poorly studied, and their role in resistance to water deficits has not been revealed. In this paper, we have expanded the list of DHNs that accumulate in the cells of Scots pine under the conditions of water deficits and revealed their relationship with the effects of water deficits. In this investigation, callus cultures of branches and buds of Scots pine were used. A weak water deficit was created by adding polyethylene glycol to the culture medium. Under the conditions of a water deficit, the activity of catalase and peroxidase enzymes increased in the callus cultures. A moderate decrease in the total water content was correlated with a decrease in the growth rate of the callus cultures, as well as with an increase in the activity of lipid peroxidation. The accumulation of Mr 72, 38, and 27 kDa DHNs occurred in the callus cultures of buds, and the accumulation of Mr 72 and 27 kDa DHNs positively correlated with the lipid peroxidation activity. An increase in the content of DHNs was observed in cultures that differed in origin, growth indicators, and biochemical parameters, indicating the universality of this reaction. Thus, previously undescribed DHNs were identified, the accumulation of which is caused by water deficiency and is associated with manifestations of oxidative stress in the kidney cells of Scots pine.

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL−1 Naphthaleneacetic Acid (NAA) and 1 mgL−1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of μ = 0.059 days−1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g−1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7′,8′-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL−1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL−1) being the most sensitive to the callus culture extract.

Over recent years, extracellular vesicles (EVs), commonly termed exosomes, have gained prominence for their potential as natural nanocarriers. It has now been recognized that plants also secrete EVs. Despite this discovery, knowledge about EV biogenesis in plant cell cultures remains limited. In our study, we have isolated and meticulously characterized EVs from the callus culture of the model plant, Arabidopsis thaliana. Our findings indicate that the abundance of EVs in calli was less than that in the plant’s apoplastic fluid. This difference was associated with the transcriptional downregulation of the endosomal sorting complex required for transport (ESCRT) genes in the calli cells. While salicylic acid increased the expression of ESCRT components, it did not enhance EV production. Notably, EVs from calli contained proteins essential for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those found in intact plants. This suggests that plant cell cultures could serve as a feasible source of EVs that reflect the characteristics of the parent plant species. However, further research is essential to determine the optimal conditions for efficient EV production in these cultured cells.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound’s identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0–2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Oregano (Origanum vulgare, Lamiaceae plant family) is a well-known aromatic herb with great commercial value, thoroughly utilized by food and pharmaceutical industries. The present work regards the comparative assessment of in vitro propagated and commercially available oregano tissue natural products. This study includes their secondary metabolites’ biosynthesis, antioxidant properties, and anticancer activities. The optimization of callus induction from derived oregano leaf explants and excessive oxidative browning was performed using various plant growth regulators, light conditions, and antioxidant compounds. The determination of oregano callus volatiles against the respective molecules in maternal herbal material was performed using gas chromatography–mass spectrometry (GC/MS) analysis. In total, the presence of twenty-seven phytochemicals was revealed in both leaf and callus extracts, from which thirteen molecules were biosynthesized in both tissues studied, seven compounds were present only in callus extracts, and seven metabolites only in leaf extracts. Carvacrol and sabinene hydrate were the prevailing volatiles in all tissues exploited, along with alkanes octacosane and triacontane and the trimethylsilyl (TMS) derivative of carvacrol that were detected in significant amounts only in callus extracts. The MTT assay was employed to assess the in vitro cytotoxic properties of oregano extracts against the epithelial human breast cancer MDA-MB-231 and the human neuroblastoma SK-N-SH cell lines. The extracts displayed concentration and time-dependent responses in cell proliferation rates.

Citrus plants are important fruit tree species; however, the breeding of high-quality varieties of citrus species is a time-consuming process. Using haploid-derived plants from anther culture may reduce the time required for obtaining purebred lines. This study aimed to genetically verify whether anther culture-derived sour orange (Citrus aurantium L.) plants developed from somatic embryos or haploid tissues. Sour orange anthers were cultured in N6 and MS media to induce calli and somatic embryos. N6 liquid medium supplemented with 1 mg·L−1 gibberellic acid and 200 µM spermidine resulted in a 10% increase in callus and embryo induction rates. Regenerated plants were validated using simple sequence repeat markers. Out of the 109 regenerated plants, ploidy analysis identified 99 diploids, two haploids, and eight putative aneuploids; out of the 99 diploid plants, 33 were haploid-derived homozygous diploids. The chromosomal analysis confirmed most plants as diploids, whereas some were identified as aneuploids (19–21 chromosomes). Furthermore, phylogenetic analysis confirmed that the resultant homozygous or heterozygous plants were haploid-derived. This is the first report of haploid-derived homozygous diploid and aneuploid sour orange plants obtained through anther culture. Moreover, the anther cultivation technique described herein can be applied to other citrus varieties.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 μM 2,4-D and 2.2 μM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 μM (BA) and 1.0 μM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 μM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

Brassica juncea is a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

Cell cultures derived from strawberry fruit at different developmental stages have been obtained to evaluate their potential use to study different aspects of strawberry ripening. Callus from leaf and cortical tissue of unripe-green, white, and mature-red strawberry fruits were induced in a medium supplemented with 11.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) under darkness. The transfer of the established callus from darkness to light induced the production of anthocyanin. The replacement of 2,4-D by abscisic acid (ABA) noticeably increased anthocyanin accumulation in green-fruit callus. Cell walls were isolated from the different fruit cell lines and from fruit receptacles at equivalent developmental stages and sequentially fractionated to obtain fractions enriched in soluble pectins, ester bound pectins, xyloglucans (XG), and matrix glycans tightly associated with cellulose microfibrils. These fractions were analyzed by cell wall carbohydrate microarrays. In fruit receptacle samples, pectins were abundant in all fractions, including those enriched in matrix glycans. The amount of pectin increased from green to white stage, and later these carbohydrates were solubilized in red fruit. Apparently, XG content was similar in white and red fruit, but the proportion of galactosylated XG increased in red fruit. Cell wall fractions from callus cultures were enriched in extensin and displayed a minor amount of pectins. Stronger signals of extensin Abs were detected in sodium carbonate fraction, suggesting that these proteins could be linked to pectins. Overall, the results obtained suggest that fruit cell lines could be used to analyze hormonal regulation of color development in strawberry but that the cell wall remodeling process associated with fruit softening might be masked by the high presence of extensin in callus cultures.

Flax lignans and neolignans impart health benefits, particularly in treating different types of cancers, due to their strong phytoestrogenic and antioxidant properties. The present study enhances the comprehension on the biosynthesis of antioxidant lignans and neolignans in root-derived in vitro cultures of flax (both callus and adventitious root). The results presented here clearly showed that the adventitious root culture efficiently produced a higher amount of lignans (at day 40) and neolignans (at day 30) than callus culture of flax. High performance liquid chromatography (HPLC) analysis revealed that the accumulations of secoisolariciresinol diglucoside (SDG, 5.5 mg g−1 DW (dry weight)) and dehydrodiconiferyl alcohol glucoside (DCG, 21.6 mg/g DW) were 2-fold higher, while guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG, 4.9 mg/g DW) and lariciresinol glucoside (LDG, 11.9 mg/g DW) contents were 1.5-fold higher in adventitious root culture than in callus culture. Furthermore, the highest level of total phenolic production (119.01 mg/L), with an antioxidant free radical scavenging activity of 91.01%, was found in adventitious root culture at day 40, while the maximum level of total flavonoid production (45.51 mg/L) was observed in callus culture at day 30 of growth dynamics. These results suggest that adventitious root culture can be a good candidate for scaling up to industrial level to commercially produce these pharmacologically and nutritionally valuable metabolites.