Production Of Azadirachtin Research Articles

Enhanced azadirachtin production in neem (Azadirachta indica) callus through NaCl elicitation: Insights into differential protein regulation via shotgun proteomics

With their remarkable bioactivity and evolving commercial importance, plant secondary metabolites (PSMs) have gained significant research interest in recent years. Plant tissue culture serves as a credible tool to examine how abiotic stresses modulate the production of PSMs, enabling clear insights into plant stress responses and the prospects for controlled synthesis of bioactive compounds. Azadirachta indica, or neem has been recognized as a repository of secondary metabolites for centuries, particularly for the compound named azadirachtin, due to its bio-pesticidal and high antioxidant properties. Introducing salt stress as an elicitor makes it possible to enhance the synthesis of secondary metabolites, specifically azadirachtin. Thus, in this research, in vitro callus cultures of neem were micro-propagated and induced with salinity stress to explore their effects on the production of azadirachtin and identify potential proteins associated with salinity stress through comparative shotgun proteomics (LCMS/MS). To induce salinity stress, 2-month-old calli were subjected to various concentrations of NaCl (0.05–1.5%) for 4 weeks. The results showed that the callus cultures were able to adapt and survive in the salinity treatments, but displayed a reduction in fresh weight as the NaCl concentration increased. Notably, azadirachtin production was significantly enhanced in the salinity treatment compared to control, where 1.5% NaCl-treated calli produced the highest azadirachtin amount (10.847 ± 0.037 mg/g DW). The proteomics analysis showed that key proteins related to primary metabolism, such as defence, energy, cell structure, redox, transcriptional and photosynthesis, were predominantly differentially regulated (36 upregulated and 93 downregulated). While a few proteins were identified as being regulated in secondary metabolism, they were not directly involved in the synthesis of azadirachtin. In conjunction with azadirachtin elicitation, salinity stress treatment could therefore be successfully applied in commercial settings for the controlled synthesis of azadirachtin and other plant-based compounds. Further complementary omics approaches can be employed to enhance molecular-level modifications, to facilitate large-scale production of bioactive compounds in the future.

Mediators of arrested development as attractive malaria vector control tools: The Azadirachta indica and azadirachtin routes

Insecticides are utilized as key components in agriculture and public health tool-kits. Mosquitoes are targeted using out/indoor insecticide sprays and treated bed nets to reduce malaria. These practices are associated with increasing resistance in various species of mosquitoes, multiple insecticide use, environmental pollution, high cost and spread of malaria into regions where they did not exist before. This paper explores the possibility of developing a safer route to mosquito control through the use of plant secondary metabolites. Peer-reviewed literature on safe substances from biological sources with attractive potentials to mediate arrested development on water-based stages of mosquitoes were appraised. Google Scholar search engine was used to locate published works from reputable journals and institutions by feeding its dialogue search box with relevant key words. Insecticide use is bedeviled with lots of impediments which embolden malaria vectors to acquire resistance, destabilize the ecosystem besides causing public health problems. Plants such as Citrullus colocynthi, Azadirachta indica, etc. contain secondary metabolites that are effective in arresting eggs, larva and pupa in water. Azadirachta indica extract is reputed to have the highest activity against insect vectors. Its most active ingredient against vectors is azadirachtin. Its use in mosquito control will be sustainable as it is well endowed with both reduced cost, accessibility and effectiveness in small water bodies around human surroundings. Small- and large-scale production of azadirachtin can be done through plant tissue culture which is boosted with new editing tools in genetic engineering. Use of A. Indica’s azadirachtin is a safe malaria vector control that can be accomplished through imposition of arrested development on immature mosquitoes. Its role in deceiving egg laden mosquitoes deserve further investigations while funding agencies such as World Health Organization (WHO) and governments of Sub Sahara Africa should take advantage of this harmless route to eliminate malaria.

Construction of yeast cell factories for production of azadirachtin precursor tirucalla-7,24-dien-3β-ol

Azadirachtin, as a botanical insecticide, is a highly oxidized limonoid triterpenoid existing in the seeds of Azadirachta indica. However, due to the low content in the seeds, the production of azadirachtin by seed extraction has low yield. Chemical synthesis of azadirachtin is characterized by complex process and low yield. Synthetic biology provides an alternative for the supply of azadirach-tin. In this study, two oxidosqualene cyclases AiOSC1 and MaOSC1 respectively derived from A. indica and Melia azedarach were identified in yeast. A yeast strain producing tirucalla-7,24-dien-3β-ol was constructed by integration of AiOSC1, Arabidopsis thaliana-derived squalene synthase gene(AtAQS2), and Saccharomyces cerevisiae-derived truncated 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene(PgtHMGR) into the delta site of yeast. Then, the function of MaCYP71BQ5 was successfully verified in yeast after this gene was introduced into the constructed yeast strain. This study not only laid a foundation for the biosynthesis of tirucalla-7,24-dien-3β-ol, but also provided a chassis cell for the functional identification of cytochrome oxidases(CYP450 s) in azadirachtin biosynthesis pathway.

Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (azadirachta indica)

BackgroundAzadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study.ResultsWe attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning.ConclusionsBy integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

Influence of fungal elicitor on bio production of Azadirachtin in cell cultures of Azadirachta indica

The present investigation involves the influence of elicitor feeding on bioproduction of important secondary metabolites in cell suspension cultures of Azadirachta indica. The content of azadirachtin in MS media supplemented with NAA (1 mg/l) + Kinetin (0.5 mg/l) and Dicamba (2 mg/l) + Kinetin (1 mg/l) hormonal combinations was found to be (0.42±0.04 mg/l) and (0.30 mg/l±0.07) respectively. Out of these two combinations, NAA (1 mg/l) + Kinetin (0.5 mg/l) combination was found optimum for bioproduction of azadirachtin. The addition of Fusarium solani significantly enhanced the amount of azadirachtin over control cultures. The cell extracts (CE) and culture filtrates (CF) of Fusarium solani were added in different concentrations (5% v/v and 10% v/v) to the cell cultures of A. indica separately, while maintaining suitable control. There is no significant change in the amount of azadirachtin between control and cell extract (CE) treated cultures. The addition of culture filtrate (5% v/v) of Fusarium solani showed significant improvement in bioproduction of azadirachtin (0.87±0.12 mg/l) over to control cultures (0.38±0.07 mg/l). It showed approximately 2-fold improvement in the bioproduction of azadirachtin. Present study constitute an important milestone in realizing commercial production of these metabolites.

Production of biopesticide azadirachtin using plant cell and hairy root cultures.

The extensive use of nondegradable chemical pesticides for pest management has developed serious environmental hazards. This has necessitated the urgent need to switch over to an alternative mode of biopesticide development for mass agriculture and field crop protection. Azadirachta indica A. Juss (commonly known as neem) houses a plethora of bioactive secondary metabolites with azadirachtin being the most active constituent explored in the sector of ecofriendly and biodegradable biopesticides characterized by low toxicity toward nontarget organisms. It has been reported that the highest content of azadirachtin and related limonoids is present in the seeds, available once in a year. Moreover, the inconsistent content and purity of the metabolites in whole plant makes it imperative to tap the potential of in vitro plant tissue culture applications, which would allow for several controlled manipulations for better yield and productivities. This review gives a summarized literature of the applied research and achievements in plant cell/hairy cultures of A. indica A. Juss mainly in context with the biopesticide azadirachtin and applications thereof.

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2gl(-1)dry weight and azadirachtin yield of 3.2mgg(-1) leading to a volumetric productivity of azadirachtin as 1.14mgl(-1)day(-1). The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97% at a concentration of 2% w/v and 83% at a concentration of 0.05% (w/v), respectively, in ethanol).

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

Azadirachtin has high industrial demand due to its immediate application as an ecofriendly, biodegrad- able biopesticide and also due to its various other significant bioactivities. To date, the only commercially feasible way to produce azadirachtin is extraction from seeds, but their availability is very limited as the tree flowers only once a year and only one-third of the fruits are collected due to operational problems. Further, due to the strict out- breeding nature of the plant, the seeds are highly heterozygous, resulting in inconsistent metabolite production. There- fore, in the present study, to achieve sustainable production of azadirachtin, dedifferentiated and redifferentiated calli derived from various explants of neem—zygotic embryo, leaf and ovary—were investigated for their potential to bio- synthesize azadirachtin. High-performance liquid chromatography analysis of theinvitro cell lines showed the presence of azadirachtin in all the samples tested, the content of which in cultured cells varied with explant source and cell dif- ferentiation response. The presence of azadirachtin in samples was further confirmed by positive electrospray ionization mass spectroscopy. The zygotic embryo cultures of neem accumulated much higher amounts of azadirachtin than leaf and ovary cultures. Furthermore, organizedinvitro callus cultures (redifferentiated) supported higher azadirachtin bio- synthesis, while unorganized callus cultures (dedifferentiated) supported the least. The maximum azadirachtin content of 2.33 mg g 21 dry weight was obtained from redifferentiated immature zygotic embryo cultures.

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l−1 sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g−1) and azadirachtin production (∼44 mg l−1) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l−1 dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l−1 sucrose, 0.19 g l−1 potassium dihydrogen phosphate, 3.1 g l−1 potassium nitrate, and 0.41 g l−1 magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l−1 and azadirachtin accumulation of 5.2 mg g−1, which was equivalent to an overall azadirachtin production of 73.84 mg l−1, 68% more than that obtained under non-optimized conditions.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss.

This report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on 1/4 MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n=x=12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

Integrated yield and productivity enhancement strategy for biotechnological production of Azadirachtin by suspension culture of Azadirachta indica

AbstractAzadirachtin is considered as the best pesticide among the biopesticides available in market for crop protection due to its broad spectrum activity and varying mode of action. To meet the increasing demand and primarily due to geographical and variability limitations, its biotechnological production from plant cell culture has gained momentum in last two decades. The major requirement for economic production is to increase the yield as well as the productivity of Azadirachtin production in the bioreactor. In general, different strategies used in the plant cell culture result in either yield or productivity enhancement. The simultaneous increase of both the parameters has remained a challenging task, particularly in plant cell cultivations. In this work, an integrated approach has been adopted by combining the various yield as well as productivity enhancement strategies, i.e. coupling the mathematical model–derived nutrient‐feeding strategy for continuous cultivation with elicitation, precursor addition, and permeabilization, etc. With this integrated approach, a yield of 0.0122 g/g and a productivity of 7.25 × 10−4 g/l/h Azadirachtin were obtained, which is so far highest in plant cell suspension culture of Azadirachta indica to the best of our knowledge. Copyright © 2010 Curtin University of Technology and John Wiley & Sons, Ltd.

In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate: ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate: ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.

Statistical media optimization for cell growth and azadirachtin production in Azadirachta indica (A. Juss) suspension cultures

Azadirachtin is one of the most widely used biopesticide originating from a plant source. Its production from plant cell cultivation was viewed to overcome constraints associated with its regular supply from the seed kernels. In order to select the effective carbon and nitrogen source, different concentrations of carbon (sucrose/glucose) and nitrogen (NO3−/NH4+ ratio) were studied in A. indica suspension culture. Glucose turned out to be a better carbon source over sucrose yielding high biomass (6.32g/L) and azadirachtin (11.12mg/L) content. Nitrate alone as nitrogen source was favorable for both biomass and azadirachtin accumulation. Plackett–Burman design was adopted to select the most important nutrients influencing the growth and azadirachtin accumulation in suspension culture. After identifying effective nutrients, Central Composite Design (CCD) was used to develop mathematical model equations, study responses and establish the optimum concentrations of the key nutrients for higher growth and azadirachtin production. A maximum of 15.02g/L biomass and 2.98mg/g azadirachtin was produced using optimum nutrient concentrations representing 99 and 96% validity of the model prediction with respect to biomass and azadirachtin, respectively. This optimized media can be used for cultivation of A. indica cells in bioreactor for mass production of azadirachtin.

Induction of hairy root cultures of Azadirachta indica A. Juss. and their production of azadirachtin and other important insect bioactive metabolites

Hairy root cultures were established from stem and leaf explants of Azadirachta indica A. Juss (neem) following infection with Agrobacterium rhizogenes. Transformation was confirmed using polymerase chain reaction analysis. The cultures had a relatively fast growth rate (0.134 g dry weight day–1) and showed a 100-fold increase in biomass over a 4-week culture period. Both the roots and cultivation medium were antifeedant as determined by a no-choice bioassay using the desert locust, Schistocerca gregaria. Azadirachtin, nimbin, salannin, 3-acetyl-1-tigloylazadirachtinin and 3-tigloylazadirachtol were detected in the hairy roots by supercritical fluid chromatography. The cultivation medium contained 3-acetyl-1-tigloylazadirachtinin, 3-tigloylazadirachtol and desacetylsalannin.

Production of azadirachtin from plant tissue culture: State of the art and future prospects

With increasing awareness towards environment-friendly and non-toxic pesticide azadirachtin obtained from neem tree (Azadirachta indica) is gaining more and more importance. Its broad-spectrum activity, peculiar mode of action. eco-friendly and non-toxic action towards beneficial organisms has offered many advantages over chemical pesticides. All currently use commercial formulations based on azadirachtin contains azadirachtin extracted from seeds of naturally grown whole plants which is labour intensive process depending upon many uncontrollable geographical and climatic factors. Plant tissue culture can be a potential process for the production, offering consistent, stable and controlled supply of this bioactive compound, However the research on tissue culture aspects of production are in preliminary stage and requires culture and process optimization for the development of a commercially viable process. This review states the present status and future challenges of plant tissue culture for azadirachtin production.

Callus cultures ofAzadirachta indica and their potential for the production of Azadirachtin

The content of azadirachtin, an insecticidally active ingredient in neem tree [Azadirachta indica (A. Juss.)] seeds, varies considerably due to environmental factors and for genetic reasons. Furthermore, the tree does not grow in moderate climates. It seems appropriate, therefore, to employ tissue culture techniques for the production of azadirachtin in order to obtain constant amounts of standardized quality. We investigated if and how azadirachtin contents of callus cultures derived from various cell lines are affected by different nutrient media and carbohydrate concentrations. The azadirachtin contents of the calli were analyzed by TLC and HPLC. Azadirachtin contents of callus cultures varied depending on the cell line, the nutrient medium and the carbohydrate source employed.

The production of azadirachtin by in‐vitro tissue cultures of neem, Azadirachta indica

AbstractCallus produced from leaves of a Ghanaian strain of the neem tree, Azadirachta indica A. Juss has been shown to produce the natural insecticide azadirachtin when grown in a defined medium. The azadirachtin was isolated by standard procedures of solvent partition and column chromatography monitored by supercritical fluid chromatography. Biological activity was monitored with antifeedant tests using the desert locust (Schistocerca gregaria Forsk.). The azadirachtin was identified by chromatography on three independent chromatographic systems (SFC, HPLC & TLC) and two thin‐layer colour tests. It has 100% antifeedant activity at < 0.04mg litre−1. The yield of azadirachtin was 0.0007% based on dry weight of callus.