Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica

Abstract

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.

Azadirachta indica A. Juss. In Vivo Toxicity—An Updated Review

“…Mwangi et al [ 17 ] determined the safety of A. indica root bark aqueous extract using the Brine shrimp lethality test by exposing Artemia salina L. nauplii to different concentrations of the extract (10, 100, and 1000 µg/mL) for 24 h. The extract was considered moderately toxic, as LC 50 obtained was 285.8 µg/mL. Zebrafish embryo toxicity to A. indica green callus methanolic extract was carried out by Ashokhan et al [ 18 ], using six different concentrations ranging from 500–5000 µg/mL for 5 days. After treatment, LC 50 value was determined to be 4606 µg/mL and the extract was considered nontoxic.…”

“…Besides, zebrafish embryos treated with concentrations below 4000 µg/mL were able to hatch into larvae, while embryos treated with 5000 µg/mL presented a 0% hatching rate. Zebrafish larvae also presented a normal heart rate (136 beats/min) and no teratogenic defects were observed in embryos or larvae [ 18 ]. According to this study, green callus, obtained from A. indica leaf, has the potential to produce an environmentally and sustainable friendly pesticide.…”

Physicochemical properties and biological activity of extracts of dried biomass of callus and suspension cells and in vitro root cultures

“…В результате фундаментальных исследований было показано, что клетки in vitro по ряду характеристик отличаются от клеток интактного растения. Это касается интенсивности роста клеток, а также особенностей синтеза и накопления в них биологически активных веществ [3][4][5]. Клетки в культуре интенсивно делятся.…”

“…Для проведения только одного курса химиотерапии необходимо уничтожение 8-10 взрослых дерева тиса. В Германии на фирме Phyton организовано крупномасштабное производство биомассы культуры клеток тиса в биореакторах объемом 75000 литров [5][6][7]. Однако содержание пакоитаксела в культуре клеток также невысоко и стоимость конечного продукта (паклитаксела) составляет $2-3 млн.…”

“…Способность кверцетина захватывать пероксинитриты и гидроксильные радикалы, обладающие высокой реакционной способностью, является доказательством обладания протекторного свойства [7,23]. Кверцетин -это один из самых мощных антиоксидантов среди полифенолов [5,18,24]. Также были продемонстрированы его противовирусные, антибактериальные, противораковые и противовоспалительные эффекты [3,8].…”