Abstract
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.
Highlights
Production of secondary metabolites from plant tissue culture has emerged as a promising and feasible option attracting the attention of scientists worldwide
Embryos at early–late dicotyledonary stages were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxy acetic acid (2,4-D), N6-benzylamino purine (BAP) and thidiazuron (TDZ) either alone or in combination with a-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), N6-furfuryladenine, abscisic acid (ABA), gibberellic acid (GA3) and casein hydrolysate (CH), for callus induction and morphogenesis
The results of this study revealed that organized in vitro callus cultures supported higher azadirachtin biosynthesis, while unorganized callus cultures supported the least
Summary
Production of secondary metabolites from plant tissue culture has emerged as a promising and feasible option attracting the attention of scientists worldwide. The entire exercise becomes obligatory if we aim at metabolites from an out-breeding tree species, like neem, due to the variability inflicted upon by heterozygosity in the genus. The tree has been claimed to possess several biological activities, such as immune stimulation, blood purification, anti-inflammation, antitumour activity, insect repulsion, bactericidal activity and growth-disrupting properties (Biswas et al 2002; Haque et al 2006). These properties are attributed to several secondary metabolites present in the genus, most of which chemically belong to the class of terpenoids like azadirachtin, nimbin, salannin, margosane and meliacin. Azadirachtin is one of the most complex and important compounds, which has been the focus of research since its isolation and characterization by Butterworth and Morgan (1968)
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