Plant Cell, Tissue and Organ Culture最新文献

Abstract

This study investigates the effects of artificial in vitro polyploidisation of Astragalus membranaceus, focusing specifically on gene expression and metabolic pathway associated with the biosynthesis of calycosin and calycosin-7-O-β-D-glucoside. Using oryzalin as an antimitotic agent, we have systematically investigated different genotypic lines, under both in vitro and ex vitro conditions. Amid cases of reduced gene expression in certain lines, results showed a significant upregulation in specific lines, particularly in genotypes 16, 54, and 74. Genotype 54 showed an exceptional response, manifesting a statistically significant upregulation in all investigated genes studied under in vitro conditions (i.e. AmPAL, AmC4H and AmI3′H). Interestingly, even under ex vitro conditions after two years of greenhouse cultivation, certain lines showed significant variations in gene expression. The genotype 16, although no longer tetraploid, exhibited the highest expression levels among the examined genotypes, with statistically significant upregulation of both the AmPAL and AmC4H genes. In addition, the induced autotetraploid genotype 74 showed a significant upregulation of the AmI3′H gene and a concomitant downregulation of the AmC4H gene. These results highlight the complex regulatory mechanisms affected by the polyploidisation of A. membranaceus and provide promising avenues for manipulating gene expression to enhance the production of pharmacologically significant compounds.

Key message

This study investigates the effects of artificial polyploidisation on gene expression of Astragalus membranaceus, suggesting diverse regulatory influences for potential plant bioactive compound production.

To induce polyploidy in rhododendrons, different experimental materials of four Rhododendron species (2n = 2x = 26) were treated with colchicine and oryzalin as chemical mutagens. Three materials from R. fortunei, namely, seeds, cotyledons, and adventitious buds, were treated, and the highest tetraploid induction rate (15.64%) was observed in cotyledons treated with 20 mg·L^− 1 oryzalin for 4 d. Two materials (stem tips and stem bases) were treated in R. simsii, and the highest tetraploid induction rate (4.04%) was achieved with 0.14% colchicine after soaking the stem tips for 48 h. However, when we treated the stem bases, the chimerism occurrence rate was up to 30.00%. In addition, polyploidy induction in R. ovatum was carried out at two colchicine concentrations (0.12 and 0.14%) with two materials (stem tip and stem base), which resulted in a higher chimeric rate. Three materials (stem tip, stem base, and adventitious buds) of R. molle were used, but no polyploidy was found. Polyploid identification was performed through stomatal observation and flow cytometry. Polyploid plants had larger and sparser stomata than diploid plants. Furthermore, morphological measurements revealed that the size of the stomata and leaves in tetraploid plants was larger than that in diploid plants. Stomatal density was decreased in tetraploid plants compared to diploid plants. Flow cytometry analysis identified tetraploids in the mutant plants of R. fortunei and R. simsii. Phenotypic analyses showed that tetraploid plants had smaller, rounder leaves and darker leaf colors than diploid plants. In summary, we compared the induced polyploid rhododendrons using different methods, which is of great significance for cultivating new rhododendron varieties with good ornamental properties and high resistance and for developing the rhododendron industry in China.

Plant-derived natural products, including those from bryophytes, are particularly advantageous in several aspects, such as for their antifungal, antibacterial, and anticancer properties. However, the bioactivity potential of secondary metabolites found in the liverwort species Acrolejeunea fertilis has not been extensively investigated, with its limited natural biomass posing challenges. A possible solution to address this is to propagate A. fertilis via in vitro culture. The application of plant growth regulatory substances at various concentrations is expected to enhance the growth of A. fertilis. This study aims to determine the optimum concentration of 2,4-dichlorophenoxyacetic acid and kinetin applied in the gametophyte phase of A. fertilis in vitro culture and compare the metabolite profile between A. fertilis from nature and in vitro culture conditions, using methanol and n-hexane solvent. The compounds in the extracts were identified using gas chromatography–mass spectrometry (GC-MS), and their antioxidant and antibacterial activities were also characterized. The IC₅₀ values for nature and in vitro extracts were in the range of 58.41–63.10 µg/mL and 111.73–112.10 µg/mL. The total value of phenolic compounds in A. fertilis extracts ranged from 58.51±0.02 µgGAE/g and 272.53±0.02 µgQE/g, respectively. Antibacterial activity was revealed, with inhibition on S. aureus using an n-hexane extract of A. fertilis from nature. Thus, the findings of this study highlight the diverse secondary metabolite profiles and bioactivity potential of A. fertilis extracts. These findings have important implications for the development of natural products in terms of the potential for mass propagation of the medicinal plant A. fertilis as a source of bioactive compounds.

Plant-derived hairy root cultures have the potential to be a significant biotechnological tool for producing valuable secondary metabolite compounds. This study deals with the production of broccoli’s hairy root cultures via the mediation of Agrobacterium rhizogenes and investigates the effects of different concentrations of copper sulfate (CuSO₄) and zinc sulfate (ZnSO₄) as two elicitors on the production of sulforaphane in broccoli's hairy root. The expression of the Myrosinase (MY) gene, associated with sulforaphane production, was quantified using real-time PCR. Moreover, the cytotoxic effect of different treatments of sulforaphane on the breast cancer cell line, MDA-MB-231, was estimated and the transcription levels of apoptosis-associated genes were also determined. The highest amount of sulforaphane was produced at 4 µM CuSO₄ after 16 h and 16 µM ZnSO₄ after 8 h of elicitation. Overexpression of the MY gene was found to be associated with increased production of sulforaphane. All sulforaphane treatments induced a dose-dependent reduction in the viability of cancer cells. The sulforaphane extracted from broccoli's hairy roots treated with CuSO₄ exerted a higher cytotoxic effect on the MDA-MB-231 breast cancer cell line than the sulforaphane extracted from broccoli's hairy root treated with ZnSO₄. All treatments of sulforaphane up-regulated the Apoptosis Regulator (Bax) gene, Proapoptotic Bcl2 Associated X, Caspase-3, Caspase-8, and Caspase-9, while down-regulating the B-cell lymphoma 2 (Bcl-2) gene (antiapoptotic) transcription. The overall results showed an antiapoptotic effect of sulforaphane extract derived from broccoli's hairy roots on MDAMB-231 breast cancer cells, thereby establishing the potential anticancer activity of the sulforaphane produced in broccoli's hairy root cultures.

Bamboos are perennial, arborescent, monocarpic, and industrially important non-timber grasses that are used as a pristine source of inorganic nutrients. However, conventional vegetative propagation methods demonstrated inadequate multiplication potential. This study investigates how Bambusa balcooa’s in vitro growth, photosynthetic pigment content, and antioxidant capacity were affected by citrate- and cetyltrimethylammonium bromide-coated gold nanoparticles (AuNPs). Further, to unravel the regulatory mechanism underlying gold nano-elicitation and in vitro plant behavior, we conducted RNA sequencing of non-treated control, 400 µM citrate-AuNPs-treated, and 600 µM CTAB-AuNPs-treated plantlets. Numerous morphological, physiological, and biochemical parameters were observed to be variably impacted along the citrate- and CTAB-coated AuNPs concentration gradient (200–600 µM). B. balcooa in vitro shoots supplemented with Murashige and Skoog medium enriched with 6-benzylaminopurine, naphthaleneacetic acid, and 400 µM citrate-AuNPs displayed statistically significant shoot proliferation, photosynthetic pigment accumulation, and antioxidant activities. Contrarily, a decline in growth parameters was observed in MS media supplemented with BAP, NAA, and 600 µM CTAB-AuNPs. Transcriptome profiling revealed various differentially expressed genes (DEGs) and metabolic pathways associated with nano-elicitation and plant growth. Furthermore, identifying genes (such as Glyoxalase, Expansin, and ZAT) governing in vitro proliferation and oxidative stress responses could enhance our understanding of the mechanisms underlying AuNPs’ ability to modulate various physiological and biochemical activities during micropropagation. Therefore, gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the exploration of DEGs involved in the in vitro modulations regulated by AuNPs offer novel insights into the molecular mechanisms governing nano-elicited plant organogenesis more comprehensively.

Graphical abstract

Citrus is one of the most important global fruit crops, with oranges accounting for more than half the total production. Citrus can be genetically engineered for improved traits, but the process is severely limited by shoot regeneration rates, especially in commercial varieties. Although standardizing a transformation protocol is difficult due to genotype and season dependencies, use of an optimal mineral nutrient basal culture medium increases the success of recovering transgenic Citrus cells. The purpose of this study is to improve shoot regeneration from juvenile tissue in multiple Citrus genotypes. Explants of 11 Citrus cultivars were regenerated in 21 published media to determine the optimal media for each genotype. The number of shoots from juvenile tissue were counted 90 days after the explants were first cultured. The type of basal medium strongly affected the rates of shoot regeneration from Citrus juvenile tissue, and the effectiveness was negatively correlated with the level of ammonium. For each Citrus grouping, optimized media formulations were determined, and potential improvements were predicted. In general, medium R7100 appeared to be the most effective for regeneration of the Citrus genotypes tested.

Cinchona officinalis has experienced anthropogenic pressures for nearly 400 years, such as overexploitation, habitat fragmentation, and the subsequent reduction of genetic diversity. Additionally, the challenge of regeneration in its natural environment makes it a vulnerable species. In this context, various treatments for the in vitro conservation of explants were evaluated in the present study. Conservation by slow growth, the effects of osmotic substances such as sorbitol, mannitol, and sucrose at different concentrations were assessed. Different concentrations of MS and B5 culture media were also examined for their impact on the growth, budding, mortality, and rooting of explants over 12 months without subcultures. For long-term conservation by cryopreservation, two techniques were tested: vitrification and encapsulation-dehydration. Short-term preservation of explants in sorbitol resulted in low mortality, minimal growth, and limited development of new shoots compared to preservation in sucrose or mannitol, although tissues could be recovered successfully from all storage conditions. After cryopreservation and 45 days of recovery, explants with the lowest mortality (4%) were from the control treatment (without cryoprotection) cultivated in a medium with sucrose which proved useful as a cryoprotectant. In conclusion, it is possible to conserve C. officinalis tissues in the short-term using in vitro techniques, while further assays are needed for long-term conservation.

Crocus scepusiensis (Rehm. & Woł.) Borbás ex Kulcz., a critically endangered herbaceous plant which serves as a valuable source of bioactive compounds found across Europe and Asia. The aim of this study was to produce a calli from two different plant parts (leaf and shoot tip) for the critically endangered C. scepusiensis through tissue culture techniques, characterize the resulting calli through chemical profiling, with a focus on identifying key phytoconstituents, and lay the groundwork for future research on the biological activities of these calli extracts. Leaf disc and micro shoot tip explants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA) to induce organogenic calli. The resulting calli exhibited distinct biochemical profiles. Moreover, a phytochemical analysis was conducted to compare the metabolite composition of callus 1 (derived from leaf discs) and callus 2 (derived from shoot tips). Callus 1 displayed a higher total phenolic content (30.3558 ± 1.3564 mg (GAE)/g) compared to callus 2 (29.1543 ± 0.9754 mg (GAE)/g). Similarly, callus 1 exhibited a greater total flavonoid content (26.0089 ± 1.8029 mg (RE)/g) than callus 2 (18.4464 ± 1.4797 mg (RE)/g). Liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS/MS) analysis revealed the presence of 26 and 25 constituents in callus 1 and 2, respectively. Fourteen and thirteen of these identified compounds have been previously reported in other Crocus species, with 22 constituents common to both calli. Twelve constituents were reported here in Crocus for the first time as far as we know.

This study substituted sodium molybdate dehydrate (Na₂MoO₄.2H₂O) in MS medium (Murashige and Skoog 1962) with molybdenum trioxide nanoparticles (MoO₃NPs) to evaluate their impact on the morphogenesis, growth, absorption of metal-mineral elements and the activity of antioxidant enzymes of chrysanthemum. The results indicated that 100% callus formation was observed when 1-month-old leaf explants were cultured on medium supplemented with MoO₃NPs and Na₂MoO₄.2H₂O in basic MS medium, while leaf explants cultured on MS medium without Na₂MoO₄.2H₂O did not induce callus induction. In addition, the treatment with 223.5 µL/L MoO₃NPs resulted in the highest shoot regeneration (33.33%), with 1 shoot per explant, and shoot height (1.14 cm), and fresh weight (1.21 g) compared to those in others and control treatments. Meanwhile, 1-month-old stem node (1 cm) explants cultured on medium supplemented with 149 µL/L MoO₃NPs recorded 100% shoot regeneration and the highest number of shoots larger than 2 cm (5 shoots/stem node), shoot height (3.23 cm) and fresh weight (1.87 g) of the shoot cluster after 30 days of culture. During the regeneration stage, the activity of antioxidant enzymes in 149 µg/L MoO₃NPs treatment was better than the control (+) treatment (except for SOD) and all the other treatments. Similar results were also observed during the shoot multiplication phase, where either the lack or surplus of Mo in the culture medium also caused the decline of SOD, CAT, and APX enzymes activity. Besides, at the concentration of 6.4 µg/L MoO₃NPs in the culture media, nutrients are absorbed more efficiently and rapidly by explants. These findings suggest that substituting ion salt in the culture medium with MoO₃NPs led to enhanced absorption, providing a micro-mineral source for plants to support biosynthesis and essential functions. The chrysanthemum plantlets exhibited enhanced rooting and growth when treated with 149 µg/L MoO₃NPs, particularly during the rooting stage after 15 days of culture.

Haplophyllum virgatum var. virgatum is an Iranian endemic plant. Its only natural habitat is the Geno region in the Hormozgan province of Iran. This plant’s limited distribution and special habitat conditions prompted us to investigate the possibility of its micropropagation and different biological aspects. In this study, while evaluating seed germination and seedling production of H. virgatum in two Murashige and Skoog (MS) and Gamborg (B5) culture media, shoot multiplication and rooting of single-node shoot explants of this plant were also investigated in the MS culture medium containing different concentrations of growth regulators. Flavonoids are part of the medicinal metabolites of different Haplophyllum species. Here, the flavonoid elicitation in the proliferated shoots of H. virgatum under 0, 100, and 150 µM methyl jasmonate (MeJA) treatments was done. The results demonstrated that the MS culture medium was more suitable than B5 for seed germination and natural growth of seedlings. Moreover, for H. virgatum shoot multiplication, one mg/L kinetin was more appropriate compared to other concentrations of this cytokinin (0.5, 1.5, 2, 2.5, and 3 mg/L) and the corresponding concentrations of benzyl amino purine. Rooting was achieved only in a limited number of shoots, so it needs further investigation. Meanwhile, MeJA increased total phenolic, total flavonoid, and rutin (the main flavonoid of Rutaceae) contents in the proliferated shoots. This research provided new information about the micropropagation of H. virgatum and flavonoid elicitation by MeJA in this plant.