Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Boosting Plant Photosynthesis with Carbon Dots: A Critical Review of Performance and Prospects.

Artificially augmented photosynthesis in nano-bionic plants requires tunable nano-antenna structures with physiochemical and optoelectronic properties, as well as unique light conversion capabilities. The use of nanomaterials to promote light capture across photosystems, primarily by carbon dots, has shown promising results in enhancing photosynthesis through tunable uptake, translocation, and biocompatibility. Carbon dots possess the ability to perform both down and up-light conversions, making them effective light promoters for harnessing solar energy beyond visible light wavelengths.This review presents and discusses the recent progress in fabrication, chemistry, and morphology, as well as other properties such as photoluminescence and energy conversion efficiency of nano-antennas based on carbon dots. The performance of artificially boosted photosynthesis is discussed and then correlated with the conversion properties of carbon dots and how they are applied to plant models. The challenges related to the nanomaterial delivery and the performance evaluation practices in modified photosystems, consideration of the reliability of this approach, and the potential avenues for performance improvements through other types of nano-antennas based on alternative nanomaterials are also critically evaluated. It is anticipated that this review will stimulate more high-quality research in plant nano-bionics and provide avenues to enhance photosynthesis for future agricultural applications.

Mesoporous silica nanoparticle-induced drought tolerance in Arabidopsis thaliana grown under in vitro conditions.

Nanoparticles of varying formats and functionalities have been shown to modify and enhance plant growth and development. Nanoparticles may also be used to improve crop production and performance, particularly under adverse environmental conditions such as drought. Nanoparticles composed of silicon dioxide, especially those that are mesoporous (mesoporous silica nanoparticles; MSNs), have been shown to be taken up by plants; yet their potential to improve tolerance to abiotic stress has not been thoroughly examined. In this study, a range of concentrations of MSNs (0-5000mgL-1 ) were used to determine their effects, in vitro , on Arabidopsis plants grown under polyethylene glycol (PEG)-simulated drought conditions. Treatment of seeds with MSNs during PEG-simulated drought resulted in higher seed germination and then greater primary root length. However, at the highest tested concentration of 5000mgL-1 , reduced germination was found when seeds were subjected to drought stress. At the optimal concentration of 1500mgL-1 , plants treated with MSNs under non-stressed conditions showed significant increases in root length, number of lateral roots, leaf area and shoot biomass. These findings suggest that MSNs can be used to stimulate plant growth and drought stress tolerance.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Graphene as a nano-delivery vehicle in agriculture - current knowledge and future prospects.

Graphene has triggered enormous interest in, and exploration of, its applications in diverse areas of science and technology due to its unique properties. While graphene has displayed great potential as a nano-delivery system for drugs and biomolecules in biomedicine, its application as a nanocarrier in agriculture has only begun to be explored. Conventional fertilizers and agricultural delivery systems have a number of disadvantages, such as: fast release of the active ingredient, low delivery efficiency, rapid degradation and low stability that often leads to their over-application and consequent environmental problems. Advanced nano fertilizers with high carrier efficiency and slow and controlled release are now considered the gold standard for promoting agricultural sustainability while protecting the environment. Graphene's attractive properties include large surface area, chemical stability, mechanical stability, tunable surface chemistry and low toxicity making it a promising material on which to base agricultural delivery systems. Recent research has demonstrated considerable success in the use of graphene for agricultural applications, including its utilization as a delivery vehicle for plant nutrients and crop protection agents, as well as in post-harvest management of crops. This review, therefore, presents a comprehensive overview of the current status of graphene-based nanocarriers in agriculture. Additionally, the review outlines the surface functionalization methods used for effective molecular delivery, various strategies for nano-vehicle design and the underlying features necessary for a graphene-based agro-delivery system. Finally, the review discusses directions for further research in optimization of graphene-based nanocarriers.

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Zinc- and magnesium-doped hydroxyapatite-urea nanohybrids enhance wheat growth and nitrogen uptake.

The ongoing and unrestrained application of nitrogen fertilizer to agricultural lands has been directly linked to climate change and reductions in biodiversity. The agricultural sector needs a technological upgrade to adopt sustainable methods for maintaining high yield. We report synthesis of zinc and magnesium doped and undoped hydroxyapatite nanoparticles, and their urea nanohybrids, to sustainably deliver nitrogen to wheat. The urea nanohybrids loaded with up to 42% nitrogen were used as a new source of nitrogen and compared with a conventional urea-based fertilizer for efficient and sufficient nitrogen delivery to pot-grown wheat. Doping with zinc and magnesium manipulated the hydroxyapatite crystallinity for smaller size and higher nitrogen loading capacity. Interestingly, 50% and 25% doses of urea nanohybrids significantly boosted the wheat growth and yield compared with 100% doses of urea fertilizer. In addition, the nutritional elements uptake and grain protein and phospholipid levels were significantly enhanced in wheat treated with nanohybrids. These results demonstrate the potential of the multi-nutrient complexes, the zinc and magnesium doped and undoped hydroxyapatite-urea nanoparticles, as nitrogen delivery agents that reduce nitrogen inputs by at least 50% while maintaining wheat plant growth and nitrogen uptake to the same level as full-dose urea treatments.

Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry.

INTRODUCTION: Chestnut rot caused by the fungus Gnomoniopsis smithogilvyi is a disease present in the world's major chestnut growing regions. The disease is considered a significant threat to the global production of nuts from the sweet chestnut (Castanea sativa). Conventional fungicides provide some control, but little is known about the potential of biological control agents (BCAs) as alternatives to manage the disease. OBJECTIVE: Evaluate whether formulated BCAs and their secreted metabolites inhibit the in vitro growth of G. smithogilvyi. METHODS: The antifungal potential of BCAs was assessed against the pathogen through an inverted plate assay for volatile compounds (VOCs), a diffusion assay for non-volatile compounds (nVOCs) and in dual culture. Methanolic extracts of nVOCs from the solid medium were further evaluated for their effect on conidia germination and were screened through an LC-MS-based approach for antifungal metabolites. RESULTS: Isolates of Trichoderma spp., derived from the BCAs, significantly suppressed the pathogen through the production of VOCs and nVOCs. The BCA from which Bacillus subtilis was isolated was more effective in growth inhibition through the production of nVOCs. The LC-MS based metabolomics on the nVOCs derived from the BCAs showed the presence of several antifungal compounds. CONCLUSION: The results show that G. smithogilvyi can be effectively controlled by the BCAs tested and that their use may provide a more ecological alternative for managing chestnut rot. The in vitro analysis should now be expanded to the field to assess the effectiveness of these alternatives for chestnut rot management.

Metal doped nitrogenous hydroxyapatite nanohybrids slowly release nitrogen to crops and mitigate ammonia volatilization: An impact assessment.

To supply adequate food, the ongoing and unrestrained administration of nitrogen fertilizer to agricultural fields is polluting the climate and living organisms. On the other hand, the agriculture sector urgently needs a technological upgrade to effectively confront hunger and poverty. Here, we report a rapid synthesis of zinc and magnesium-doped hydroxyapatite-urea nanohybrids for slow release and delivery of nitrogen to wheat and rice crops. Nanohybrids slowly release nitrogen for up to six weeks compared to the burst release of nitrogen from urea, and their use substantially reduces, by at least 3.8 times, ammonia emissions into the environment compared with that of urea fertilizer. A half­nitrogen dose applied as multi-nutrient complexed nanohybrids maintained crop growth, yield, and nutritional compositions in wheat and subsequent rice crops. Nanohybrids enhanced the wheat crop yield and nitrogen uptake by 22.13% and 58.30%, respectively. The synthesized nitrogen nanohybrids remained in the soil for two continuous crop cycles, reduced ammonia volatilization, and achieved nitrogen delivery to the crops. Additionally, soil dehydrogenase activity (534.55% above control) and urease activities (81.82% above control) suggest that nanohybrids exhibited no adverse impact on soil microorganisms. Our comprehensive study demonstrates the advantages of 'doping' as a method for tailoring hydroxyapatite nanoparticles properties for extended agricultural and environmental applications. The use of nanohybrids substantially reduced greenhouse gas emissions and enabled the reduction, by half, of nitrogen inputs into the agricultural fields. This study, therefore, reports a novel nano-enabled platform of engineered hydroxyapatite-urea nanohybrids as a nitrogen fertilizer for efficient nitrogen delivery that results in improved crop growth while minimizing environmental pollution.

Chitosan nanoparticles and their combination with methyl jasmonate for the elicitation of phenolics and flavonoids in plant cell suspension cultures.

Productivity enhancement approaches, such as elicitation can overcome the limitations of low metabolite(s) yield in in vitro plant cell culture platforms. Application of biotic/abiotic elicitors triggers molecular responses that lead to a concomitant enhancement in the production of metabolites. Nanoparticles have been tested as alternatives to commonly studied biotic/abiotic elicitors. However, most nanoparticles explored are of metallic origin, which raises concerns about their cytotoxicity, disposal post-elicitation, and may limit downstream applications of metabolites. Here, we report the synthesis and application of biopolymeric methyl jasmonate-loaded chitosan nanoparticles (MJ-CNPs) and empty CNPs (size <100 nm) as nano-elicitors, which were simple to synthesize, cost-effective and safe. Enzymatic and metabolic investigations revealed that MJ-CNPs and empty CNPs improve and prolong phenylalanine ammonia-lyase enzyme activity and production of phenolics and flavonoids. The data provides the first evidence of MJ-CNPs and empty CNPs as nano-elicitors that prolong the production of metabolites in plant cell suspension cultures.

Reduced Genotoxicity of Gold Nanoparticles With Protein Corona in Allium cepa.

Increased usage of gold nanoparticles (AuNPs) in biomedicine, biosensing, diagnostics and cosmetics has undoubtedly facilitated accidental and unintentional release of AuNPs into specific microenvironments. This is raising serious questions concerning adverse effects of AuNPs on off-target cells, tissues and/or organisms. Applications utilizing AuNPs will typically expose the nanoparticles to biological fluids such as cell serum and/or culture media, resulting in the formation of protein corona (PC) on the AuNPs. Evidence for PC altering the toxicological signatures of AuNPs is well studied in animal systems. In this report, we observed significant genotoxicity in Allium cepa root meristematic cells (an off-target bioindicator) treated with high concentrations (≥100 µg/ml) of green-synthesized vanillin capped gold nanoparticles (VAuNPs). In contrast, protein-coated VAuNPs (PC-VAuNPs) of similar concentrations had negligible genotoxic effects. This could be attributed to the change in physicochemical characteristics due to surface functionalization of proteins on VAuNPs and/or differential bioaccumulation of gold ions in root cells. High elemental gold accumulation was evident from µ-XRF mapping in VAuNPs-treated roots compared to treatment with PC-VAuNPs. These data infer that the toxicological signatures of AuNPs are influenced by the biological route that they follow to reach off-target organisms such as plants. Hence, the current findings highlight the genotoxic risk associated with AuNPs, which, due to the enhanced utility, are emerging as new pollutants. As conflicting observations on the toxicity of green-synthesized AuNPs are increasingly reported, we recommend that detailed studies are required to investigate the changes in the toxicological signatures of AuNPs, particularly before and after their interaction with biological media and systems.

Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells.

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14-15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due to the availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes were confirmed through a ß-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) µg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells.

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Seaweed Extract-Stimulated Priming in Arabidopsis thaliana and Solanum lycopersicum.

Plant priming is an induced physiological state where plants are protected from biotic and abiotic stresses. Whether seaweed extracts promote priming is largely unknown as is the mechanism by which priming may occur. In this study, we examined the effect of a seaweed extract (SWE) on two distinct stages of plant priming (priming phase and post-challenge primed state) by characterising (i) plant gene expression responses using qRT-PCR and (ii) signal transduction responses by evaluating reactive oxygen species (ROS) production. The SWE is made from the brown algae Ascophyllum nodosum and Durvillaea potatorum. The priming phase was examined using both Arabidopsis thaliana and Solanum lycopersicum. At this stage, the SWE up-regulated key priming-related genes, such as those related to systemic acquired resistance (SAR) and activated the production of ROS. These responses were found to be temporal (lasting 3 days). The post-challenge primed state was examined using A. thaliana challenged with a root pathogen. Similarly, defence response-related genes, such as PR1 and NPR1, were up-regulated and ROS production was activated (lasting 5 days). This study found that SWE induces plant priming-like responses by (i) up-regulating genes associated with plant defence responses and (ii) increasing production of ROS associated with signalling responses.

Designer nanoparticles for plant cell culture systems: Mechanisms of elicitation and harnessing of specialized metabolites.

Plant cell culture systems have become an attractive and sustainable approach to produce high-value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohormones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed-, stimulus-responsive-, and hormone-carrying-NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP-mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP-associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy.

Stimulation of photosynthesis and enhancement of growth and yield in Arabidopsis thaliana treated with amine-functionalized mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSNs) of 50 nm diameter particle size with a pore size of approximately 14.7 nm were functionalized with amino groups (Am-MSNs) and the effects of exposure to these positively charged Am-MSNs on each of the life cycle stages of Arabidopsis thaliana were investigated. After growth in half strength MS medium amended with Am-MSNs (0-100 µg/mL) for 7 and 14 days, seed germination rate and seedling growth were significantly increased compared with untreated controls. The seedlings were then transferred to soil and irrigated with Am-MSNs solutions every 3 days until seed harvesting. After four weeks growth in soil, Am-MSNs treated plants showed up-regulation of chlorophyll and carotenoid synthesis-related genes, an increase in the content of photosynthetic pigments and an amplification of plant photosynthetic capacity. All these changes in plants were closely correlated with greater vegetative growth and higher seed yield. In all the experiments, 20 and 50 µg/mL of Am-MSNs were found to be more effective with respect to other treatments, while Am-MSNs at the highest level of 100 µg/mL did not result in oxidative stress or cell membrane damage in the exposed plants. To the best of our knowledge, this is the first report evaluating both physiological and molecular responses following exposure to plants of these specific Am-MSNs throughout their whole life cycle. Overall, these findings indicate that following exposure Am-MSNs play a major role in the increase in seed germination, biomass, photosynthetic pigments, photosynthetic capacity and seed yield in A. thaliana.

Next-generation metabolic engineering approaches towards development of plant cell suspension cultures as specialized metabolite producing biofactories.

Plant cell suspension culture (PCSC) has emerged as a viable technology to produce plant specialized metabolites (PSM). While Taxol® and ginsenoside are two examples of successfully commercialized PCSC-derived PSM, widespread utilization of the PCSC platform has yet to be realized primarily due to a lack of understanding of the molecular genetics of PSM biosynthesis. Recent advances in computational, molecular and synthetic biology tools provide the opportunity to rapidly characterize and harness the specialized metabolic potential of plants. Here, we discuss the prospects of integrating computational modeling, artificial intelligence, and precision genome editing (CRISPR/Cas and its variants) toolboxes to discover the genetic regulators of PSM. We also explore how synthetic biology can be applied to develop metabolically optimized PSM-producing native and heterologous PCSC systems. Taken together, this review provides an interdisciplinary approach to realize and link the potential of next-generation computational and molecular tools to convert PCSC into commercially viable PSM-producing biofactories.

Nanoapplication of a Resistance Inducer to Reduce Phytophthora Disease in Pineapple (Ananas comosus L.).

Treatment of plants with a variety of abiotic and biotic inducers causes induced resistance to pathogen attack. In this study, the effect of four resistance inducers on plant diseases caused by Phytophthora cinnamomi was screened in vivo initially by using lupin, a susceptible model plant. Lupin pretreated with 0.5 mM salicylic acid (SA) showed effective resistance against P. cinnamomi with restricted lesions. Then, mesoporous silica nanoparticles (MSNs) with particle size around 20 nm and approximate pore size of 3.0 nm were synthesized and functionalized for loading and importing SA to pineapple plantlets. Decanethiol gatekeepers were introduced to the surface of MSNs via glutathione (GSH)-cleavable disulfide linkages to cover the pore entrance, which was confirmed through using Raman spectroscopy. Through free diffusion, the loading efficiency of SA in MSNs gated with gatekeepers was 11.7%, but was lower in MSNs without gatekeepers (8.0%). In addition, in vitro release profile of SA from gatekeeper-capped MSNs indicated that higher concentrations of GSH resulted in more cargo release. Moreover, the experiments in planta showed that the application of MSNs as a resistance inducer delivery system significantly improved pineapple resistance to P. cinnamomi in terms of inhibiting lesion development and improving root growth of infected plants, compared to the use of free SA and MSNs without gatekeepers. The analysis of SA, GSH, and defense-related genes, of PR1 and PR5, further confirmed that the slow and prolonged release of SA from MSNs inside the roots of pineapple plants was achieved through a redox-stimuli release mechanism. Therefore, the application of MSNs with redox-responsive gatekeepers has shown great potential as an efficient tool for delivering chemicals into plants in a controllable way.

Optimal method selection for biocompatible extraction of rosmarinic acid from mycorrhizal hairy roots of Ocimum basilicum.

Mycorrhizal hairy roots of Ocimum basilicum produce high amount of rosmarinic acid and are also valuable resource of quality mycorrhizal spores. To utilize their potential as continuous resource of biological and biochemical products, an efficient separation method is required. Solvent based extraction methods have a negative impact on mycorrhizal spore viability and vitality. Accordingly, we developed a biocompatible extraction method where spore and root viability is maintained with efficient extraction of rosmarinic acid. We screened temperature- and sonication-assisted techniques in ethanol, methanol, dimethyl sulfoxide, ionic liquid and surfactants. An inverse relationship was found between an increase in temperature and mycorrhizal and root viability. Optimum temperature for extraction was 30 °C. Most suitable solvents were 10% methanol; 0.25 M ionic liquid and dimethyl sulfoxide. Ethanol, nonane, dodecane, Triton X-100 and Tween-20 were not found suitable. Thus, our study sets a platform for optimization studies with mycorrhizal roots of other medicinal plants.