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New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

ABSTRACT: Azadirachtin is an important secondary metabolite from Azadirachta indica used as a natural biopesticide. This study is the first comprehensive report concerning the influence of plant growth regulators on callus induction, cell suspension growth, and azadirachtin accumulation and production in cell suspension cultures of A. indica. We investigated the effect of plant growth regulators including different types of auxins and cytokinins and their combinations on callus induction, cell suspension growth, and azadirachtin accumulation and production. The highest percentage of callusing (100%) obtained at different combinations of plant growth regulators on MS medium supplemented with 1 mg/L picloram and 2 mg/L kinetin and the highest fresh weight of callus (264.50 mg) was observed in MS medium containing 1.5 mg/L NAA and 3 mg/L kinetin. In cell suspension cultures, the maximum cell density, SCV, and PCV were 2.44?×?10⁶ cells per mL, 97.95%, and 81.46%, respectively, obtained in the MS medium containing 1.5 mg/L 2,4-D and 3 mg/L zeatin riboside. The highest average growth rate (0.25 days) was on MS medium containing 1.5 mg/L NAA and 3 mg/L zeatin riboside. The MS medium supplemented with 1 mg/L picloram and 2 mg/L kinetin produced the highest amount of fresh cell weight (493.02 g/L), dry cell weight (77.27 g/L), azadirachtin accumulation (3.69 mg/gDW), and azadirachtin production (285.64 mg/L). The results showed that all measured indices had positive correlation with together except FCW and DCW with azadirachtin accumulation.

SUBMITTER: Farjaminezhad R

PROVIDER: S-EPMC6661059 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

Farjaminezhad Reza R Garoosi Ghasem-Ali GA

3 Biotech 20190727 8

Azadirachtin is an important secondary metabolite from Azadirachta indica used as a natural biopesticide. This study is the first comprehensive report concerning the influence of plant growth regulators on callus induction, cell suspension growth, and azadirachtin accumulation and production in cell suspension cultures of A. indica. We investigated the effect of plant growth regulators including different types of auxins and cytokinins and their combinations on callus induction, ce ...[more]

PMID: 31355118

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Project description:For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2-0.6 mg L-1) and 2,4-D (0.2-0.6 mg L-1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L-1 of TDZ resulted in the highest fresh weight (3.38 ± 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus (214.53 ± 33.63 mg g-1 dry weight (DW)) induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 µg mL-1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

Project description:Azadirachtin-A (AzaA) from the Indian neem tree (Azadirachta indica) has insecticidal properties; however, its molecular mechanism remains elusive. The "targeted and nontargeted proteomic profiling", metabolomics, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) imaging, gene expression, and in silico analysis provided clues about its action on Helicoverpa armigera. Fourth instar H. armigera larvae fed on AzaA-based diet (AzaD) suffered from significant mortality, growth retardation, reduced larval mass, complications in molting, and prolonged development. Furthermore, death of AzaD-fed larvae was observed with various phenotypes like bursting, blackening, and half-molting. Liquid chromatography-mass spectrometry (LC-MS) data showed limited catabolic processing of ingested AzaA and dramatic alternations of primary metabolism in H. armigera. MALDI-TOF imaging indicated the presence of AzaA in midgut of H. armigera. In the gut, out of 79 proteins identified, 34 were upregulated, which were related to digestion, immunity, energy production, and apoptosis mechanism. On the other hand, 45 proteins were downregulated, including those from carbohydrate metabolism, lipid metabolism, and energy transfer. In the hemolymph, 21 upregulated proteins were reported to be involved in immunity, RNA processing, and mRNA-directed protein synthesis, while 7 downregulated proteins were implicated in energy transfer, hydrolysis, lipid metabolism, defense mechanisms, and amino acid storage-related functions. Subsequently, six target proteins were identified using labeled AzaA that interacted with whole insect proteins. In silico analysis suggests that AzaA could be efficiently accommodated in the hydrophobic pocket of juvenile hormone esterase and showed strong interaction with active site residues, indicating plausible targets of AzaA in H. armigera. Quantitative polymerase chain reaction analysis suggested differential gene expression patterns and partly corroborated the proteomic results. Overall, data suggest that AzaA generally targets more than one protein in H. armigera and hence could be a potent biopesticide.

Dataset Information

New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

Publications

New biological trends on cell and callus growth and azadirachtin production in <i>Azadirachta indica</i>.

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