This study explored how plant growth regulators (PGRs) impact callus induction, biomass accumulation, phytochemical synthesis, and the antioxidant and anti-aging potential of Alcea rosea L. callus cultures. Our findings revealed that the combination of 1 µM NAA and 0.1 µM TDZ leads to 100% callus induction frequency and the highest biomass accumulation, with a fresh weight of 267 g/L and a dry weight of 16.5 g/L. Maximum total phenolic (719.19 mg GAE/L) and total flavonoid (166.34 mg QE/L) production were observed in callus cultures treated with 1 µM NAA. High-performance liquid chromatography identified 9 anthocyanin compounds, with the highest total anthocyanin production (265.65 µM DW) recorded with a PGR concentration of 1 µM NAA + 0.1 µM TDZ. Antioxidant activities varied across different PGR concentrations, with notable values including DPPH-FRSA of 83.1%, ORAC value of 1089.13 µM TEAC, and maximum reduction in ROS/RNS levels by 87.56%. Anti-aging activities also varied, with tyrosinase inhibition peaking at 79.12%, vesperlysine-like AGEs’ inhibition at 68.32%, and pentosidine-like AGEs’ inhibition at 56.42%. This study introduces a novel approach that addresses traditional extraction method limitations by examining the impact of PGRs on callus culture initiation and secondary metabolite production, alongside antioxidative and anti-aging characteristics. Such methodologies potentially add to our understanding of A. rosea L.‘s therapeutic potential and contribute to sustainable techniques for producing bioactive compounds.
Patchouli (Pogostemon cablin), is an industrially important aromatic plant that produces patchouli oil. The present experiment aimed to generate polyploid plants of patchouli with increased herb yield, oil production, and higher patchouli alcohol content. In the present study, colchicine, a chemical compound known to induce polyploidy was used at concentrations of 0.2% and 0.3% for 48 h. The diploid variety CIM-Shrestha (2n = 2X = 32) was used as the starting point. Polyploidy was successfully induced in vitro conditions and confirmed through various analyses including chromosome counting, anatomical, morphological, and gene expression studies. Treating the diploid callus with 0.2% colchicine resulted in viable and stable tetraploid seedlings. These tetraploids had a chromosomal count of 2n = 4x = 64. Tetraploids exhibited distinct morphological, anatomical, and chemical characteristics, such as thicker leaves, more oil glands, higher chloroplast numbers, larger stomata size, patchouli alcohol, and α-Guaiene. In field conditions, the induced tetraploids remained stable and the majority of tetraploid lines showed higher oil content and patchouli alcohol concentrations compared to diploid plants, although herb yield was lower in tetraploids. In the gene expression study, five (PcHMGR, PcFPPS, PcTPSCF2, PcTPSB15, and PcPTS, along with the expression of the control gene Pc18S) pathway genes related to the patchouli alcohol production pathway were studied. The Real Time-qPCR results show that there was no significant change in the expression of any of the five analyzed genes.
Slash pine (Pinus elliottii Engelm.), an important economic tree species with a high resin yield, is extensively cultivated in southern China. Somatic embryogenesis (SE) technology could hasten the breeding of P. elliottii through improving seedling quality and number, but mass production of somatic embryos remains constrained by various factors. We described an efficient SE system through indirect route and investigate the effects of genotype, phytohormones and culture condition on SE. Immature zygotic embryos of three open-pollinated mother trees collected during 2020–2023 were utilized for initiation of pro-embryogenic masses (PEMs). The initiation of PEMs was significantly influenced by seed sources (families) (p < 0.05). The addition of 2,4-dichlorophenoxyacetic acid (6 mg/L), 6-benzylaminopurine (1 mg/L) and kinetin (2 mg/L) effectively improved initiation rates by up to 15.67 ± 1.89%. Furthermore, glutamine addition to the maturation medium enhanced the yield of somatic embryos during culture. The optimum concentration range of activated carbon within the germination medium spanned from 3–4.5 g/L. Inoculation with ectomycorrhizal fungus Laccaria amethystea enhanced the root length of regenerated plantlets, reaching 7.92 ± 4.06 cm. The regenerated plantlets inoculated with Pisolithus orientalis and Xerocomus chrysenteron showed improved survival during the acclimatization phase, with 100% survival of mycorrhizae-treated plantlets after 3 months of acclimatization in the greenhouse. We clarified the SE conditions suitable for P. elliottii, and the SE system and mycorrhizal method we used can be applied to mass production of plantlets.
Gamma radiation (GR) and salicylic acid (SA) enhance plant traits by regulating growth and development. However, the underlying mechanism of their combined therapies in plants is not well understood. In our study, the potential impact of 100 μM and 500 μM SA and/or 20, 40 Gy in Momordica charantia, a medicinal crop plant was assessed. We observed significant increase in plant root-shoot length and photosynthetic efficiency at 20 Gy dose, which further enhanced under SA application. Overall, simultaneous application of SA and GR also upregulated antioxidant capacity via production of secondary metabolites, such as flavonoids and phenolics. The highest antidiabetic activity with up to 81.5% inhibition of alpha-amylase enzyme was observed at 20 Gy + 500 μM SA combination. The expression of Sterol C-22 Desaturase, a key gene involved in the biosynthesis of β-sitosterol was upregulated under the treatment of GR and/or SA. β-sitosterol is an important compound with potent anti-cholesterol and anti-carcinogenic properties. Our study showed that highest content of β-sitosterol (1082.2 ± 68.2 ng/mg FW) was observed in M. charantia plants exposed to combined 20 Gy + 500 μM SA treatment. Therefore, gamma irradiation and salicylic acid can help in sustainable growth and production of various secondary metabolites in M. charantia, thereby enhancing its agronomic value.
Theobroma cacao and T. bicolor are among the most important agricultural crops of the Mexican tropics. Currently, in Mexico, the propagation of these crops is performed via seeds, which indicates that demand exceeds production. In this context, somatic embryogenesis (SE) is an alternative to this approach. Thus, we evaluated the presence of embryogenic genotypes of T. cacao and T. bicolor in the Papaloapan Basin of Mexico with the idea of implementing this technology in the region. The analysis of the phenotypic expression of the floral whorls demonstrated that, unlike T. bicolor, the combination of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid induces different morphogenetic responses in the genotypes that were evaluated in the primary callus phase of T. cacao. Staminodia presented with the highest percentage of caulogenesis in T. cacao, whereas T. bicolor presented with the highest frequency of caulogenesis in Staminodia and carpels. Some calli differentiated in the roots to a greater extent than those derived from the staminodes of T. cacao. The calli of T. bicolor did not differentiate. A parallel study using thidiazuron as an inducer demonstrated similar results for calli of both species; however, rhizogenesis from staminodes was 50% lower for the evaluated genotypes of T. cacao. Staminodes were the only structures that demonstrated primary somatic embryogenesis (PSE) in 66% of the T. cacao genotypes that were evaluated by using benzylaminopurine. Finally, secondary somatic embryogenesis (SSE) was evaluated in cotyledons and reached a 60% success rate, of which 95.48% were normal somatic embryos. Both types of embryogenesis were morphologically characterized using optical and/or scanning electron microscopy.
Determining foliar mineral status of tissue cultured shoots can be costly and time consuming, yet hyperspectral signatures might be useful for determining mineral contents of these shoots. In this study, hyperspectral signatures were acquired from tissue cultured little-leaf mockorange (Philadelphus microphillus) shoots to determine the feasibility of using this technology to predict foliar nitrogen and calcium contents. After using a spectroradiometer to take hyperspectral images for determining foliar N and Ca contents, the correlation between the hyperspectral bands, vegetation indices, and hyperspectral features were calculated from the spectra. Features with high correlations were selected to develop the models via different regression methods including linear, random forest (RF), and support vector machines. The results showed that non-linear regression models developed through machine learning techniques, including RF methods and support vector machines provided satisfactory prediction models with high R2 values (%N by RF with R2 = 0.72, and %Ca by RF with R2 = 0.99), that can estimate nitrogen and calcium content of little-leaf mockorange shoots grown in vitro. Overall, the RF regression method provided the most accurate and satisfactory models for both foliar N and Ca estimation of little-leaf mockorange shoots grown in tissue culture.
Strobilanthes cusia is an indigo-yielding plant and scarcity of both wild and cultivated plants has increased the supply pressure of the natural dye. Since hairy root cultures are suitable for the in vitro production of secondary metabolites, the hairy root culture of Strobilanthes cusia was established using two Rhizobium rhizogenes strains (ATCC 15834 and MTCC 532). Polymerase chain reaction (PCR) using rol B primer confirmed the molecular evidence of hairy root transformation. The differential transformation frequency was studied based on factors like explants types (leaves, shoot tips, and stem); infection techniques, infection period, co-cultivation period, bacterial strain types, acetosyringone concentration, and antibiotic treatment conditions. Leaf explants, infected with ATCC 15834 exhibited the highest induction rate (58.67%) among the three explants. The most efficient hairy root transformation was achieved with the immersion technique, involving 10 min of infection and 5 days of co-cultivation using 100 µM acetosyringone during infection. When the indigo content of the hairy roots and different parts of in vitro plantlets were compared, the hairy roots produced 0.314 mg/g fresh weight (FW) of indigo, slightly more than the roots (0.21 mg/g FW), but less than the leaves (1.08 mg/g FW) of in vitro plantlets. Further, the indigo content of the hairy root cultures treated with 200 µM indican and 200 µM salicylic acid were enhanced up to 0.78 mg/g FW and 0.71 mg/g FW, respectively. Thus, this study reveals the potential of hairy roots of S. cusia for indigo biosynthesis, which may serve as a potential alternative source for natural indigo.
Rhododendron is one of the top ten traditional flowers in China, with high ornamental and medicinal values. However, molecular research on this species has been impeded by the lack of efficient molecular genetic techniques. Virus-induced gene silencing (VIGS) technology is an effective tool for analyzing gene functions, which has been successfully applied to many plant species. But there have been no reports of VIGS system for Rhododendron. In this study, tobacco rattle virus (TRV) was chosen to infect Rhododendron micranthum using phytoene desaturase (PDS) as the reporter gene. After the inoculation of pTRV2-RmPDS by leaf injection, photobleaching phenomena appeared in the newly developed leaves and the RT-qPCR assay demonstrated that RmPDS was successfully silenced. Then three parameters including the developmental stage, the Agrobacterium concentration and the inoculation temperature were examined to optimize the system. The silencing efficiency was increased from 2.4 to 11.4% and the optimized conditions were as follows: the developmental stage of the two true-leaf stage, the adjustment of the inoculation solution to a final OD600 = 1.5 and the inoculation temperature of 18 ℃. To further validate the system, the most optimal combination was used to infect other six rhododendron genotypes. R. mucronulatum, R. ovatum, R. × pulchrum, R. simsii and R. yedoense displayed the silenced phenotype of PDS as expected. We successfully established TRV-mediated VIGS technology in Rhododendron which could evaluate and characterize the function of plant genes without the need for cumbersome tissue culture.
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