Plant Cell, Tissue and Organ Culture最新文献

The sex-dependent differences in the response to salt stress of the dioecious Rumex thyrsiflorus and the influence of different concentrations of sodium chloride (NaCl) on the biosynthesis of phenolic compounds in callus suspension cultures were evaluated. The cultures originated from callus obtained on hypocotyls isolated from male and female seedlings were cultured on Murashige and Skoog medium supplemented with 0.4 mg/L 6-benzylaminopurine and 1 mg/L 2,4-dichlorophenoxyacetic acid. The results showed that the response to salt stress depends on both the sex of the plant and the NaCl concentration in the culture medium. The analysis of the main morphometric parameters showed that callus tissue derived from the hypocotyls of female seedlings was less sensitive to salt stress than that from male seedlings, which was correlated with higher concentrations of phenolic compounds. The optimal NaCl concentration to increase the production of phenolic compounds was 129 mM for females and 43 mM for males. In the methanolic tissue extracts 22 compounds were determined using the HPLC–DAD method. In general, higher amounts of compounds were detected in the extracts from the female tissue. The following dominated: catechin (max. 213.31), cryptochlorogenic acid (max. 76.35) and epicatechin (max. 54.84) (mg/100 g DW). This comprehensive phytochemical analysis of the sex-related aspects of the response to salt stress was performed for the first time in this dioecious model species. The results revealed potential application of NaCl as the ecological friendly and inexpensive elicitor to increase the production of pharmaceutically valuable compounds and highlight the importance of dioecy in phytochemistry.

This study established in vitro root cultures of Tarenaya atropurpurea from root segments of seedlings and from in vitro propagated plants. Moreover, culture conditions were manipulated aiming to optimize root biomass accumulation and shoot regeneration from newly formed roots was determined. A phytochemical assessment was performed using two extraction methods — dynamic maceration (DM) and ultrasonic assisted extraction (UAE) — and two chromatographic methods for extract analysis (TLC and HPLC). MS medium supplemented with 3.0 mg.L^− 1 of indole-3-butyric acid (IBA) induced the highest root multiplication. Root cultures initiated from seedling explants achieved higher biomass accumulation. However, improved root multiplication was achieved using explants from in vitro propagated plants in an optimized culture formulation called Optimum Root Culture Medium (ORCM), which combines MS medium with 1/4 concentration of mineral salts + 3.0 mg.L^− 1 IBA + 70 g.L^− 1 sucrose, pH 6.5, stirring speed at 130 r.p.m., and 16 h/light. Shoot regeneration from newly formed roots was successfully obtained on MS containing 6-benzylaminopurine (BA). Analysis by TLC suggests the presence of saponins, mainly in root extracts, with the most intense bands acquired by UAE, while HPLC analysis suggests the presence of flavonoids in extracts from aerial parts, with intense signals in extracts obtained by DM. This study was able to establish in vitro root cultures of T. atropurpurea and optimize root biomass accumulation through the manipulation of culture conditions. Phytochemical assessment indicated the presence of saponins and flavonoids, demonstrating potential commercial use of in vitro cultures to produce secondary metabolites in T. atropurpurea.

Graphical Abstract

Sphaeralcea angustifolia is a plant with confirmed anti-inflammatory, immunomodulatory and gastroprotective effects. These properties can be attributed to scopoletin, tomentin, sphaeralcic acid, iso-sphaeralcic acid and 8-methyl-iso-sphaeralcic acid compounds isolated from cells in suspension and hairy root cultures. Genetic transformation with Agrobacterium rhizogenes were used in S. angustifolia cell cultures to increase the production of active secondary metabolites and stimulate the production of other compounds. We observed that non-transformed cells in a suspension of S. angustifolia had a higher growth index after two and three weeks of culture (9.29 and 11.84, respectively) compared with cells in suspension transformed with A. rhizogenes. Both cultures produced sphaeralcic acid, and enhanced production was detected in the transformed cells (0.19 and 0.16 mg/g, respectively). In addition, transformed and non-transformed cells produced a new compound identified as sphaeralgin (dicumarine); a higher yield of sphaeralgin was detected in the transformed cells (2.21 mg/g dry biomass). The anti-inflammatory effects of sphaeralgin in edema models induced with 12-O-tetradecanoylphorbol-13-acetate and ʎ-carrageenan inhibited edema formation in a dose-dependent manner, with a mean effective dose (ED₅₀) of 0.25 mg/ear and 64.56 mg/kg, respectively. During the late phase of the formaline test, sphaeralgin had an antinociceptive effect, with an ED₅₀ of 1.35 mg/kg.

Hyptis suaveolens (L.) Poit is known for containing podophyllotoxin (PTOX), a natural lignan used as the lead compound in antitumor agent preparations, in its roots. The use of phloroglucinol (PHL) and indoleamines, such as melatonin (MEL) and serotonin (SER), in plant tissue culture is a promising alternative for eliciting rare and high-value secondary metabolites of medical importance. Aimed to evaluate the impact of different concentrations of PHL, either alone or in combination with MEL or SER, on the growth and accumulation of PTOX in H. suaveolens. Apical segments were vertically inoculated in MS medium containing different concentrations of MEL or SER (0; 0.25; 0.50; 1.00; 5.00 and 10.00 µM) and PHL (0 and 50 mg L^-1). After 40 days, growth parameters, chlorophyll content and PTOX content were assessed. Plantlet rooting and root dry weight notably increased with the use of 0.5 µM MEL alongside PHL. Overall, treatments containing PHL showed superior results, except for chlorophyll content. SER supplementation was effective in increasing root growth, root number and leaf area. The combination of 1.0 µM MEL or SER and 50 mg L^-1 PHL increased the production of PTOX in the roots of H. suaveolens by 2.0 and 1.6 times, respectively. In conclusion, the application of PHL and indoleamines demonstrated an eliciting effect on both growth and PTOX production in H. suaveolens roots, presenting a promising avenue for further research and application in eliciting targeted secondary metabolites.

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

Cerium oxide nanoparticles (Ce₂O₃-NPs) are widely used for their catalytic and oxidative properties, such as in diesel additives and potential oncology treatments. However, limited data exists on their impact on callus formation and plant regeneration in wheat. This study investigates the effects of different genotypes and Ce₂O₃-NPs concentrations on callus formation and plant regeneration in wheat (Triticum aestivum L.) using mature embryo culture. Our hypothesis was that Ce₂O₃-NPs would enhance callus and plant regeneration rates in a genotype-dependent manner. We used Murashige and Skoog (MS) medium with varying concentrations of Ce₂O₃-NPs (1–7 mg/L) to assess their effects. The Kırik genotype showed the highest callus formation (7 mg/L Ce₂O₃-NPs), while the Rumeli genotype showed the lowest (1 mg/L Ce₂O₃-NPs). Embryogenic callus formation was the highest in Kırik without Ce₂O₃-NPs and the lowest in Rumeli with 3 mg/L Ce₂O₃-NPs. Root and shoot formation rates in Rumeli were 16.03% and 51.40%, respectively, compared to 3.94% and 40.49% in Kırik. The main outcomes demonstrated that 1 mg/L Ce₂O₃-NPs promoted root formation, while 7 mg/L was optimal for shoot formation. Genotypic effects on callus, root, and shoot formation, as well as plant regeneration, were significant. MS media with Ce₂O₃-NPs enhanced callus formation and regeneration in wheat tissue culture, highlighting the importance of genotype in these processes. Future research should explore the underlying mechanisms of Ce₂O₃-NPs’ effects on plant tissue culture and extend the study to other plant species to validate these findings.

Rapid climate change affects the supply of goods and services needed for a growing world population, reducing crop yields and threatening biodiversity, with potentially catastrophic effects on ecosystems. Particularly at risk are plant species with long life cycles, such as woody species. To address this crisis, there is an urgent need to accelerate breeding pipelines for these species to withstand new environmental conditions and mitigate the effects of climate change. In this article, we highlight examples of genetic engineering in woody species that have the potential to contribute not only to economic development, but also to the conservation and sustainable use of genetic biodiversity, including: the introgression of traits into ecologically important threatened species for large-scale forest restoration, the reduction of air pollution from foliar emissions from bioenergy crops, the acceleration of biomass production to increase CO₂ sequestration, the improvement of bioenergy crops to reduce dependence on fossil fuels, and the induction of flowering to reduce the time and resources needed to develop fruit and forest trees varieties with greater resistance to biotic and abiotic stresses. These examples are examined not only for their economic potential but also for their environmental and social impacts. This compilation of biotechnological applications in woody species, aims to enrich the debate on the adoption of genetic engineering in these species.

Saussurea laniceps is renowned for its extraordinary medicinal ingredients diversity, which is an important Tibetan medicinal plant in China. Flavonoids play an important role in the medicinal efficacy of S. laniceps. The species, however, has become increasingly scarce and endangered. Therefore, the use of callus to propagate medicinal resources is of great significance. We found that cold acclimation increased flavonoids content in S. laniceps callus. RNA-seq revealed a total of 18,414 and 21,215 differentially expressed genes at 6 d and 9 d cold (4 °C) periods, respectively. Besides, 456 differential expressed transcriptional factor genes were identified, among which AP2-EREBP, WRKY, NAC, MYB, bHLH and WD40 had the ability to induce the transcription of genes associated with flavonoid synthesis. A total of 34 unigenes were identified and linked to the flavonoids and related derivative biosynthesis pathway. The high expression levels of genes in regulating flavonoids biosynthesis (e.g., PAL, 4CL, CHS, CHI and F3’H) were identified. Besides, 66 up-regulated transporter genes were also detected, including the ATP-binding cassette (ABC) family (23), GST (13), MATE (12), vacuolar-associated proteins (8) and H⁺-ATPase (10). Consequently, these findings suggest that cold-acclimated callus could be an effective alternative resource for flavonoids in S. laniceps, which is valuable for the conservation and usage of this wild and endangered plant.

Reynoutria japonica Houtt. (Japanese knotweed) is an invasive plant belonging to the Polygonaceae family. However, being native to East Asia, it has been used in natural medicine for ages because of its broad range of biological activity. Although R. japonica is known as a rich source of phenolic compounds, plant biomass collected from the field may be contaminated with toxic elements like heavy metals, and the level of metabolite accumulation depends on environmental conditions. Therefore, the aim of this study was to derive Japanese knotweed tissue cultures and investigate biomass production and phenolic compound synthesis in in vitro conditions. Plants were cultivated in a traditional agar-solidified medium, in a liquid medium with rotary shaking (agitated culture), and in a temporary immersion bioreactors Plantform™, as well as in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted to determine the sum of phenolic compounds, as well as the quantity of selected phenolic acids, catechins, and other flavonoids. Results have shown that agitated cultures and temporary immersion bioreactors increased biomass accumulation compared to solid medium cultures. Tissue cultures of R. japonica had increased synthesis of phenolic compounds compared to plants from ex vitro conditions. Shoots and roots from agitated cultures were 2.8- and 3.3-fold richer in catechins, respectively, compared to plants cultivated in soil. Based on the obtained results it can be concluded that agitated and bioreactor cultures are the best source of Japanese knotweed biomass rich in valuable secondary metabolites.

Salvia aristata Aucher ex Benth. is considered a valuable endemic medicinal plant in Iran due to its two main bioactive metabolites: phenolic acids and tanshinones. This study established hairy root (HR) cultures from 14-day-old seedlings inoculated with Rhizobium rhizogenes strain ATCC 15834. In addition, the effects of elicitation with Ag⁺ ions (15 and 25 µM) on growth indices, phenolic acid and tanshinone contents in the HRs, as well as changes in the expression patterns of key genes involved in their biosynthetic pathways were investigated in a time-course experiment. The results showed that exposure of HRs to both Ag⁺ ions concentrations significantly increased rosmarinic acid (1.34- to 1.43-fold) and salvianolic acid B (1.71- to 1.82-fold) content. Notably, exposure to 25 µM Ag⁺ ions for seven days resulted in increases of 7.25-, 7.78-, 6.47-, and 3.9-fold in total tanshinones, tanshinone I, tanshinone IIA, and cryptotanshinone levels, respectively, compared to the control groups. The study also revealed a significant increase in the release of tanshinones, especially tanshinone IIA (31.49 ± 0.65 mg L⁻¹) by HRs into the culture medium following Ag⁺ ions elicitation. Changes in the transcription levels of key genes related to the biosynthetic pathways of phenolic acids (PAL, TAT, and RAS) and tanshinones (CPS and CYP76AH1) were also associated with their contents in the elicited HRs. The findings confirm the effectiveness of elicitation as a strategy to enhance metabolite production in HR cultures of S. aristata, a potent natural source of phenolic acids and tanshinones.