In Vitro Propagation of "Salsaparrilha" [Herreria salsaparrilha Mart. (Asparagaceae)]: An Antisyphilitic Medicinal Species.

An efficient procedure for in vitro propagation of Herreria salsaparrilha Martius was established from single-node explants (fourth and fifth nodes from apex to the base) derived from donor plants maintained under shading-house conditions. After surface sterilization, explants are inoculated in test tubes containing 15 mL of Murashige and Skoog (MS) medium without growth regulators. Cultures are maintained under 35 µmol m-2 s-1 irradiance, a 16/8-h light/dark light regime, at 26 ± 2 °C. The subcultures are carried out under the same conditions, adding 6-benzyladenine 1.0 mg/L and Phytagel® 2.8 g/L. Shoots are elongated and rooted by transferring individual shoots to half-strength MS medium without growth regulators. After 25-30 days, elongated rooted shoots are transferred to plastic pots containing 25-30 mL of sterile distilled water, covered with a transparent plastic bag, and kept under the same growth room conditions for 2 days. Plants are transferred to cups containing autoclaved and washed sand and kept in a shading house (50% light interception) for acclimatization. True-to-type adult plants were successfully recovered under ex vitro conditions.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Salicylic Acid and Water Stress: Effects on Morphophysiology and Essential Oil Profile of Eryngium foetidum.

The exogenous application of bioregulators, such as salicylic acid (SA), has exhibited promising outcomes in alleviating drought stress. Nevertheless, its impact on culantro (Eryngium foetidum L.) remains unexplored. Thus, the aim of this study was to assess how SA impacts the growth, morphophysiology, and essential oil composition of culantro when subjected to drought. To achieve this, culantro plants were grown under three different watering regimes: well-watered, drought-stressed, and re-watered. Additionally, they were either treated with SA (100 µM) or left untreated, with water serving as the control. SA application did not mitigate the effects of drought in biomass production but increased biomass, leaf number, leaf area, and photosynthetic pigments under well-irrigated and re-watered conditions. After a drought period followed by re-watering, plants recovered membrane integrity independently of SA application. Water stress and the exogenous application of SA also modulated the profile of essential oils. This is the first report about SA and drought affecting growth and essential oil composition in culantro.

Chemodiversity of Dissolved Soil Organic Matter from Amazon Rainforest as Influenced by Deforestation.

Many biogeochemical processes are modulated by dissolved organic matter (DOM), but the drivers influencing the chemodiversity of DOM compounds in Amazonian soils are poorly understood. It has also been theorized whether deforestation controls the decline of DOM. In this study, we collected soil samples from thirty sites across different regions of Brazil's Legal Amazon, and we investigated the trade-offs among soil physical-chemical properties and DOM chemodiversity. We employed optical spectroscopy, Fourier transform ion cyclotron resonance, and multivariate analysis. Our results indicated that, despite variations in land use and soil physical-chemical properties, factors such as the deforested site, geometric mean diameter, weighted average diameter, and soil organic carbon were the main influencers of DOM chemodiversity variation. These findings highlight the importance of considering DOM chemodiversity as closely related to land use and its potential use in developing deforestation models for predicting soil quality decline in Brazil's Legal Amazon.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Irrigation level and substrate type on the acclimatization and development of mandacaru (Cereus jamacaru DC.): an emblematic cactus from Brazilian semiarid region.

Mandacaru is a cactus with great socioeconomic potential, but lack of information about its cultivation hinders its domestication. Here, we aimed to evaluate the acclimatization and vegetative development of mandacaru under different substrates and irrigation levels. For this, seeds inoculated in vitro were grown for 120 days, being transplanted to pots containing four types of substrate (S1-caatinga soil + gravel; S2-washed sand + organic matter + soil + charcoal; S3-washed sand + cattle manure + soil + sand; S4-commercial organic substrate). Pots were irrigated with 100% of the field capacity (FC) once-a-week, or with 50% FC twice-a-week, and kept in a greenhouse for six months. The experimental design was completely randomized, in a 4 × 2 factorial scheme, with six replications. Plant height and diameter, axial and radial growth rate, fresh and dry mass of stem and root, water content, and photosynthetic pigments were determined. Growth was affected mainly by the substrate, with S4 resulting in higher growth and pigment content, while S1 was impaired and S2 and S3 resulted in intermediate growth. The use of S4 and 100% FC once per week was the best condition for mandacaru.

Water stress and exogenous carnitine on growth and essential oil profile of Eryngium foetidum L.

Water stress influences plant growth and metabolism. Carnitine, an amino acid involved in lipid metabolism, has been related to responses of plants to abiotic stresses, also modulating their metabolites. Culantro (Eryngium foetidum L.) is a perennial herb, rich in essential oils, native to Latin America, commonly used due to its culinary and medicinal properties. Here, we investigated the effect of exogenous carnitine on morphophysiology and the essential oil profile of culantro plants under water stress. For this, plants were grown under three water conditions: well-watered, drought stress, and re-watered; and sprayed with exogenous carnitine (100 µM) or water (control). Culantro growth was impaired by drought and enhanced by re-watering. Carnitine, in turn, did not reverse drought effects on growth, and impaired the growth of re-watered plants, also improving photosynthetic pigment content. Water conditions and carnitine application changed the essential oil profile of the plants. Drought and re-watering improved the production of eryngial, which was even increased with exogenous carnitine in re-watered plants. In addition, hydroquinone was only produced with the combination of re-watering and carnitine application. The application of exogenous carnitine can be a strategy to induce the production of essential oil compounds with cosmetic and pharmaceutical importance in culantro. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03757-y.

Drought stress mitigation by foliar application of L-carnitine and its effect on radish morphophysiology.

Drought is the major abiotic stress limiting crop production worldwide, with drought events being expected to be harsher and more frequent due to the global warming. In this context, the development of strategies to mitigate the deleterious effects of drought, such as the use of biostimulants, is imperative. Radish is a globally cultivated root vegetable, with high nutritional and phytochemical value. Thus, this study aimed to evaluate the potential of exogenous carnitine application in the mitigation of drought stress on radish morphophysiology. For this, radish plants were grown for 30 days, being irrigated with 80% (well-watered) or 15% (drought stress) of water holding capacity and sprayed with carnitine (5, 50, and 500 µM) or water (0 µM-no carnitine). The experimental design was completely randomized, in a 4 × 2 factorial scheme (carnitine concentrations × water conditions) with six replicates, and each experimental unit consisted of one plant. The gas exchanges, chlorophyll a fluorescence, photosynthetic pigments, electrolyte leakage, relative water content, and biomass production and allocation were evaluated. Drought reduced the photosynthetic capacity of plants by impairing water balance and membrane integrity, decreasing biomass accumulation, mainly in globular roots. The application of low carnitine (5 µM) mitigated these negative effects caused by drought, increasing membrane integrity and water balance of plants, while higher carnitine concentration (50 and 500 µM) aggravated drought stress. This study highlights the potential of carnitine in the mitigation of drought stress on radish plants, supporting its role as a biostimulant. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01308-6.

Volatile profile and micropropagation conditions of Bauhinia forficata Link.

Bauhinia forficata Link. is a native South American plant, which possesses volatile compounds with pharmaceutical and medicinal properties such as antidiabetic and anti-inflammatory effects. However, the conservation and propagation of this plant are complicated by its recalcitrant seeds and delayed flowering transition. Hence, tissue culture is employed for the safe and efficient propagation of B. forficata. However, the optimal conditions for the in vitro cultivation of B. forficata remain unknown. Thus, this study aimed to characterize the volatile profile of adult B. forficata field plants and evaluate the effects of different light intensities (43 and 70 µmol m-2 s-1), gas exchange rates (14 and 25 µL L-1 s-1), and exogenous sucrose concentrations (0, 20, and 30 g L-1) on their in vitro development. The results showed that ß-caryophyllene is the major volatile compound produced by B. forficata. Moreover, culturing in a medium containing 30 g L-1 of sucrose and flasks with membranes that allow CO2 exchange at the rate of 25 µL L-1 s-1 produced vigorous and hardened plants with high survival rates independent of irradiance. This study is the first to report the optimal in vitro culture conditions for B. forficata as a reference for future studies on micropropagation and secondary metabolite production using this species. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03634-8.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

Salicylic acid mitigates the effects of mild drought stress on radish (Raphanus sativus) growth.

Water deficit is the most critical factor limiting plant growth and production and salicylic acid (SA) has potential for stress mitigation in plants; therefore, we evaluated the effect of SA on radish (Raphanus sativus L.) growth and ecophysiology under water deficit. Plants were sprayed with SA (100µM) or water (control), and irrigated at 80% (W80), 60% (W60), 40% (W40), and 20% (W20) of field capacity. The SA treatments and drought stress started 7days after sowing and lasted until the end of the cycle (30days after sowing). The morphophysiological analyses showed that radish plants had impaired growth at the lower water supply levels, but the treatment with SA reversed these growth restraints under moderate stress, leading to increases in shoot mass at W40 and storage root mass at W60 and W40. SA treatment also reversed the reduction of storage root volume at W60. The tendency of water deficit to increase F O and reduce F V /F M suggests possible damage to the photosystem II of drought-stressed plants. The parameters of gas exchange and photosynthetic pigments showed maintained photosynthetic efficiency, but total photosynthesis decreased due the lower shoot dry mass. Overall, exogenously applied SA reversed the growth restraints at W60 and W40, which revealed that SA was effective in mitigating the effects of moderate water deficit on biomass accumulation and partitioning in radish plants.

Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction.

Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro.

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 µmol m-2 s-1 by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 µmol m-2 s-1 than at 65 µmol m-2 s-1, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 µmol m-2 s-1 stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata. Graphical abstract.

Anatomy, Flow Cytometry, and X-Ray Tomography Reveal Tissue Organization and Ploidy Distribution in Long-Term In Vitro Cultures of Melocactus Species.

Cacti have a highly specialized stem that enables survival during extended dry periods. Despite the ornamental value of cacti and the fact that stems represent the main source of explants in tissue culture, there are no studies on their morpho-anatomical and cytological characteristics in Melocactus. The present study seeks to address the occurrence of cells with mixed ploidy level in cacti tissues. Specifically, we aim to understand how Melocactus stem tissue is organized, how mixoploidy is distributed when present, and whether detected patterns of ploidy change after long periods of in vitro culture. To analyze tissue organization, Melocactus glaucescens and Melocactus paucispinus plants that had been germinated and cultivated in vitro were analyzed for stem structure using toluidine blue, Xylidine Ponceau, Periodic Acid Schiff, ruthenium red, and acid floroglucin. To investigate patterns of ploidy, apical, medial, and basal zones of the stem, as well as, periphery, cortex, and stele (vascular tissue and pith) regions of the stem and root apexes from four- and ten-year old cultured in vitro were analyzed by flow cytometry. X-ray micro-computed tomography (XRµCT) was performed with fragments of stems from both species. The scarcity of support elements (i.e., sclereids and fibers) indicates that epidermis, hypodermis, and wide-band tracheids present in cortical vascular bundles and stele, as well as water stored in aquifer parenchyma cells along the cortex, provide mechanical support to the stem. Parenchyma cells increase in volume with a four-fold increase in ploidy. M. glaucescens and M. paucispinus exhibit the same pattern of cell ploidy irrespective of topophysical region or age, but there is a marked difference in ploidy between the stem periphery (epidermis and hypodermis), cortex, stele, and roots. Mixoploidy in Melocactus is not related to the age of the culture, but is a developmental trait, whereby endocycles promote cell differentiation to accumulate valuable water.

Leaf development stages and ontogenetic changes in passionfruit (Passiflora edulis Sims.) are detected by narrowband spectral signal.

During shoot development, leaves undergo various ontogenetic changes, including variation in size, shape, and geometry. Passiflora edulis (passionfruit) is a heteroblastic species, which means that it experiences conspicuous changes throughout development, enabling a morphological distinction between the juvenile and adult vegetative phases. Quantification of heteroblasty requires a practical, inexpensive, reliable, and non-destructive method, such as remote sensing. Moreover, relationships among ontogenetic changes and spectral signal at leaf level can be scaled up to support precision agriculture in passion fruit crops. In the present study, we used laboratory spectroscopic measurements (400-2500 nm) and narrowband vegetation indexes (or hyperspectral vegetation indexes - HVIs) to evaluate ontogenetic changes related to development and aging in P. edulis leaves. We also assessed leaf pigment concentration to further support the application of biochemical-related narrowband indexes. We report that 30-d-old leaves can be discriminated into developmental stages through their spectral signals. MSI (Moisture Stress Index) and NDVI750 (Normalized Difference Vegetation Index ρ750) contribute most to the variation of age (15 to 30-d-old leaves) and developmental stage (phytomer positions along the plant axis) in passionfruit leaves. PRI (Photochemical Reflectance Index) played an important role in detecting age and development alterations, including heteroblasty. A biochemical and spectral comparison of pigments revealed that spectroscopy offered potential for diagnosing phenology in P. edulis, as some narrowband indexes correlated strongly with chlorophylls and carotenoids content. Narrowband vegetation indexes are found to be a suitable tool for monitoring passionfruit crops.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Irradiance and light quality affect two annatto (Bixa orellana L.) cultivars with contrasting bixin production.

Light is a key factor influencing growth and development in plants. Specific irradiance and light quality can improve development and production of secondary compounds such as carotenoids during plant tissue culture. Bixin and norbixin, two apocarotenoids obtained from the seeds of Bixa orellana L. (annatto), are used as natural dyes in various industries. While annatto tissue culture has been successful, the effect of light in this species remains poorly understood. Here, we analyze for the first time the effect of irradiance regime (50, 150, 50 + 150, 200, 50 + 200 µmol m-2 s-1) and light spectral quality (fluorescent, white, blue/red LED) on in vitro development of apexes and bixin content in two contrasting bixin-producing varieties of B. orellana, namely 'Piave Vermelha' and 'UESB74'. The number of leaves per plant, stomatal density, leaf area, leaf expansion, chlorophylls and carotenoids content, malondialdehyde and bixin content were analyzed in the leaves of both cultivars. 'Piave Vermelha' produced 1.6-fold more bixin than 'UESB74'. Stomata cells of both cultivars had a paracytic arrangement with peltate trichomes along the adaxial and abaxial leaf surfaces. 'Piave Vermelha' preferred blue/red LED light; whereas fluorescent light was optimal for 'UESB74'. Under fluorescent light, an irradiance of 50 µmol m-2 s-1 is indicated for both cultivars. LED light increased bixin content only in 'Piave Vermelha', suggesting that the dye biosynthetic pathway is genotype-dependent. The present findings suggest the possibility of using light to modulate the bixin biosynthetic pathway.

Accessions of Brazilian ginseng (Pfaffia glomerata) with contrasting anthocyanin content behave differently in growth, antioxidative defense, and 20-hydroxyecdysone levels under UV-B radiation.

Ultraviolet-B (UV-B) radiation is an elicitor of secondary metabolites in plant tissue culture, but the effects on 20-hydroxyecdysone (20E) are still unclear. The 20E may show biotechnological, pharmacological, medical, and agrochemical applicability. Here, we use Pfaffia glomerata, a medically important species, to understand the impacts of UV-B radiation on their physiological performance, the expression of key genes involved in the 20E biosynthesis, and the 20E content. Two accessions (A22 and A43) of plants 20 days old grown in vitro were exposed to 0 (control), 2 (6.84 kJ m-2), and 4 (13.84 kJ m-2) h UV-B radiation for 20 consecutive days. Our data showed that UV-B reduced glucose concentration in A22 and A43 under 4 h of exposure (29 and 30%, respectively), while sucrose concentration increased (32 and 57%, respectively). UV-B also differentially impacted the accessions (A22 and A43), where the A22 under 4 h of UV-B had reduced total dry weight (8%) and electron transport rate (31%); in contrast, A43 did not change. Also, only A22 had increased POD activity under 4 h of UV-B (66%), as well as increased gene expression of the 20E pathway and the 20E content under 2 and 4 h of UV-B in leaves (28 and 21%, respectively) and roots (16 and 13%, respectively). This differential performance to UV-B can be explained by the contrasting anthocyanin contents. Notably, A43 displayed 56% more anthocyanin to the former, a possible defense against UV-B. In conclusion, UV-B radiation is a potential elicitor for increasing 20E content in P. glomerata grown in vitro.