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2.16.2 Culture Conditions

      To achieve maximum production of azadirachtin, organized and unorganized calli derived from various explants (zygotic embryo, leaf, and ovary) were investigated. The zygotic embryo-derived calli were more suitable for higher accumulation of azadirachtin than leaf and ovary-derived callus cultures. Similarly, higher biosynthesis of azadirachtin was observed in organized calli, while unorganized callus cultures contain the least amount of azadirachtin (Singh and Chaturvedi 2013). A. indica has its origin linked to Indian history and is considered as a symbol of health. Several phytochemicals have been isolated from this plant species (Biswas et al. 2002). The isolated phytochemicals (azadirachtin A, nimbin, and salannin) demonstrated several biological activities (Subapriya and Nagini 2005). The production of azadirachtin was optimized by manipulating in vitro cell cultures (Veerasham et al. 1998; Wewetzer 1998; Jarvis and Morgan 2000) and cell suspension cultures (Kuruvilla et al. 1999; Balaji et al. 2003; Raval et al. 2003). Nimbin accumulation was observed in both callus cultures (Sanyal et al. 1981) and in in vitro regenerated shoots and roots of neem (Srividya and Devi 1998), but salannin accumulation was not reported in tissue cultures (Babu and Nair 2004). The callus regenerated by intermodal segments contains azadirachtin A, nimbin, and salannin (Babu et al. 2006).

The organized and unorganized callus culture of A. indica showed the presence of azadirachtin, the content was found in variable concentration due to diverse nature of explants (Govindachari et al. 1992). The accumulation of azadirachtin was higher (threefold) in the zygotic embryo cultures than in leaf and ovary cultures. The organized callus was richer in azadirachtin than the unorganized. Similarly, BAP (individually) or in combination with IAA promoted the induction of azadirachtin. The ratio of nitrogen and ammonium (4 : 1) increased production of azadirachtin 1.5-fold higher. The levels of phosphate in culture medium also affected the accumulation of azadirachtin (Sujanya et al. 2008). The lower concentration of sucrose (1.5%) reduced the proliferation of cells along with accumulation of azadirachtin (Anuradha et al. 2010; Nagella and Murthy 2010).

      Gradual enhancement of biomass and azadirachtin production was assessed in neem cell suspension cultures. By increasing the nitrate levels in cell suspension cultures, the maximum enhancement of azadirachtin accumulation was reported. It was proposed that the higher nitrate concentration increased the cell permeability, which also enhances the leaching of azadirachtin into culture medium (Prakash and Srivastava 2005, 2006). The cell cultures also promoted higher accumulation of azadirachtin in wild tree species of A. indica in Sri Lanka (Allan et al. 1994; Eeswara et al. 1997). For further enhancement of azadirachtin accumulation in cell cultures, the culture medium was supplemented with picloram and BAP with different concentrations (Kaii-a-Kamb et al. 1992; Fulzele and Satdive 2001; Satdive et al. 2011).

      Due to biopesticide property of azadirachtin, it has attracted the attention of researchers to enhance the production. The calli were regenerated on Woody Plant Medium (liquid medium) supplemented with glucose, casein hydrolysate, and methyl jasmonate. The maximum accumulation was achieved in methyl jasmonate elicited cells. Methyl jasmonate as signal transducer enhanced the secondary metabolism in plant cells that might have induced the production of azadirachtin (Rodrigues et al. 2014). Normally azadirachtin B was not detectable by normal characterization methods in somatic embryos, but the extract of somatic embryos affected the larval development of locusts (Schaaf et al. 2000; Prakash et al. 2002; Festucci-Buselli et al. 2008; Nakabayashi et al. 2010).

      To find the effect of azadirachtin A on callus induction, the seedlings used as explant selected from those seeds, which were having high azadirachtin concentration. The seedlings were inoculated on MS culture medium supplemented with NAA. The observed results revealed that the callus induction was rapid and more proliferative in this explant. In addition, the leaf explant was also used and exposed to clod treatment. The biochemical studies revealed that azadirachtin concentration was higher in those calli that were induced from the cotyledons of higher azadirachtin containing seeds (Kota et al. 2006; Rafiq and Dahot 2010).

      Azadirachtin, an important biopesticide, is one of the active compounds obtained from seeds of neem. To reduce the effectivity of barriers of variation in its source, availability, genetic diversity, and purity maintenance, the tissue culture studies with elicitation are considered as an important technique (Savitha et al. 2006). With the incorporation of fungal elicitors in cell cultures, fourfold production of azadirachtin was enhanced. Similarly, cyanobacterial elicitors were also used for the enhancement of azadirachtin production (Poornasri Devi et al. 2008).

Neem is a multipurpose plant species that possesses medicinal and agrochemical properties. Besides these properties, the secondary metabolites from neem demonstrated anti-inflammatory, antitumor, insect repellent, and bactericidal properties. The traditional methods of tree improvement have been hampered due to prolonged juvenile period, enormous heterozygosity, and recalcitrant nature. The production of plant seedlings was increased by developing somaclonal variants. The somatic embryogenesis is the best method of tree improvement (Salvi et al. 2001; Rout 2005). Agrobacterium rhizogenes strains were used for the development of hairy roots. The leaves were used as explants. In hairy roots, 100-fold enhancement of biomass was obtained. The presence of azadirachtin, nimbin, salannin, 3-acetyl-1-tigloylazadirachtinin, and 3-tigloylazadirachtinol were isolated by supercritical fluid chromatography (Allan et al. 2002; Akula et al. 2003).

      Azadirachtin is considered as potent biopesticide. Effects of Nitsch, Gamborg, and MS medium were investigated. Nitsch medium was found more suitable than other types of media in increasing the production of azadirachtin. The biotic elicitors (jasmonic acid and salicylic acid) enhanced the fivefold production of azadirachtin in neem cell cultures (Satdive et al. 2007).

References

      1 Akhila, A. and Rani, K. (1999). Chemistry of the neem tree (Azadirachta indica A. Juss.). In: Progress in the Chemistry of Organic Natural Products (eds. W. Herz, H. Falk, G.W. Kirby, et al.), 47–149. Vienna: Springer.

      2 Akula, C., Akula, A., and Drew, R. (2003). Somatic embryogenesis in clonal neem, Azadirachta indica A. Juss. and analysis for in vitro azadirachtin production. In Vitro Cell. Dev. Biol. Plant 39: 304–310.

      3 Allan, E.J., Eeswara, J.P., Johnson, H. et al. (1994). The production of azadirachtin by in vitro tissue cultures of neem, Azadirachta indica. Pestic. Sci. 42: 147–152.

      4 Allan, E., Eeswara, J., Jarvis, A., and Mordue, A. (2002). Induction of hairy root cultures of Azadirachta indica A. Juss. and their production of azadirachtin and other important insect bioactive metabolites. Plant Cell Rep. 21: 374–379.

      5 Anuradha, V.E., Jaleel, C.A., Salem, M.A. et al. (2010). Plant growth regulators induced changes in antioxidant potential and andrographolide content in Andrographis paniculata Wall. ex Nees. Pestic. Biochem. Physiol. 98: 312–316.

      6 Babu, V.S. and Nair, G.M. (2004). Bioproduction of azadirachtin from callus cultures of neem (Azadirachta indica A. Juss.). In: Proceedings of the IUPAC International Conference on Biodiversity and Natural Products: Chemistry and Medicinal Applications, New Delhi, 52.

      7 Babu, V.S., Narasimhan, S., and Nair, G.M. (2006). Bioproduction of azadirachtin-A,

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