In this context, plant cell cultures have proven useful for the production of high-value secondary metabolites due to the consistency in quality and quantity of the desired product. These properties can be attributed to several secondary metabolites present in the genus, most of which, chemically, belong to the terpenoid class. Tables and graphs are included in the text, while all figures are compiled at the end of the thesis, in the form of plates.
3A.4: Effect of three induction media on shoot-bud differentiation from ovary calli in the regeneration medium. 3B.3: Effect of different sucrose concentrations on in vitro roots of the excised shoots grown on ½ MS basal medium. It is a native of the Indian subcontinent (Koul et al. 1990) and has spread to many parts of the world.
These properties can be attributed to the many secondary metabolites present in the genus, most of which chemically belong to the terpenoid class, such as azadirachtin, nimbin, salanin, margosan and meliacin. In addition, seed propagation is also undesirable due to the highly heterozygous nature of the plant due to protandry, which prevents self-pollination (Reddy et al. 2004).
IN VITRO STUDIES
In this case, multiple small shoots formed on ½ MS medium (major inorganic salts reduced to half strength) supplemented with BAP (1.0 µM) and Gibberellic acid (GA3; 0.5 µM). 2001) observed shoot differentiation of neem root explants on MS medium supplemented with BAP (8.9 µM) and IAA (0.6 µM). On B5 (Gamborg et al. 1968) medium containing BAP (4.4 µM), the explants formed nodular structures, which appeared bipolar.
1993) reported very high incidence of embryogenesis in the cultures of cotyledons on MS medium supplemented with NAA (8.0 µM). Indirect somatic embryogenesis via callusing was achieved from leaf explants grown on MS medium supplemented with a combination of TDZ (2.3–4.5 µM) and 2,4-D (0.5 µM). Calli induced on MS medium supplemented with Kinetin (6.9 µM) and IAA (8.6 µM) were embryogenic, subsequently giving rise to somatic embryos.
They reported multiple shoot propagation from hypocotyl explants of 1-week-old seedlings on MS medium supplemented with BAP (2.2 μM) and NAA (0.54 μM). Aseptic axillary buds formed multiple shoots within five weeks when cultured on MS medium supplemented with BAP (8.8 μM) and NAA (0.54 μM).
PHARMACOLOGICAL APPLICATIONS OF SECONDARY METABOLITES
In addition to its action against insects, bacteria, fungi, viruses and protozoa, which are the main enemies of crops, are also reported to be sensitive to azadirachtin (Mordue et al. 1993). Apart from this, antiviral activity of aqueous neem leaf extracts against vaccinia, chikungunya, group B Coxsackie and measles virus has also been reported (Rao et al.). Induction of apoptosis in rat oocytes was observed upon treatment with neem leaf extracts (Chaube et al.). 2006).
Ethanolic extract of neem leaves also induced cell death of prostate cancer cells (PC-3) by inducing apoptosis (Kumar et al. 2006). As for Spilanthes, it contains a wide range of compounds with a diverse range of bioactivity such as alkylamides (spilanthol), phenolics (ferulic acid and vanillic acid), coumarin (scopoletin) and triterpenoids such as β-sitostenone and stigmasterol (Scheme 2) ( Prachayasittikul et al. 2009). In addition, Spilanthes is known for its larvicidal and insecticidal properties (Ramsewak et al. 1999; Saraf and Dixit 2002; Pandey et al. 2007).
The analgesic activity of spilanthol has been attributed to an increased gamma-aminobutyric acid (GABA) release in the temporal cerebral cortex (Rios et al. 2007). The scopoletin (coumarin) and ferulic acid (phenols) found in this plant are reported to be of immense pharmacological interest (Prachayasittikul et al. 2009).
OBJECTIVES
The main obstacles in neem, regarding the availability of metabolites such as azadirachtin, from tissue culture lie in its variable and low productivity. In the present study, we made an effort towards the systematic selection and screening of elite cell lines in vitro for continuous and improved azadirachtin production. Experiments regarding the bioactivity of neem were not performed because the plant is known for its biological properties and there are sufficient reports on this aspect.
All biochemical studies conducted to date have been conducted on plants growing in the wild. Recognizing that environmental currents cause changes in the type and amount of metabolites produced, establishing in vitro cultures will help utilize biomass and negate the effect of seasonal variation on secondary metabolite levels.
MATERIALS AND METHODS ………………………..……………………...33-54
The remainder of the stem was cut back to single nodes and used for propagation. All aseptic manipulations of plant material, including inoculations, were done inside a laminar airflow cabinet. The ploidy level of the regenerants, derived from leaf, ovary and zygotic embryo cultures, was determined by flow cytometry.
The stock solution (1000 µg/ml) of azadirachtin (Sigma, USA) was prepared by dissolving 0.5 mg of the standard compound in 0.5 ml of HPLC grade methanol (Merck, India). The standard equation obtained from curve was used for quantification of the compound in unknown samples. The experimental design and statistical analysis of the data were developed using Minitab 15.5 statistical software package.
Calluses maintained in semi-solid responsive medium were harvested at the end of the 5-week growth period, washed with distilled water and vacuum filtered. Stock solutions (1000 μg/ml) of scopoletin and dodeca-2(E), 4(E)-dienoic acid isobutylamide (a reference standard for spilanthol) were prepared by dissolving 5 mg of the compound in 5 ml of HPLC-grade methanol. The standard equation obtained from the curve was used for the quantification of scopoletin and spilantol in the unknown samples.
Correlation coefficients (R2) were also generated in Microsoft Ofiice (Excel) Professional Edition 2003 by fitting linear trend lines to standard curves obtained for each of the two compounds. The pH and residual nutrients (phosphate, nitrate and sugar) of the suspension cultures were monitored every third day. Spilanthes cells were inoculated so that each 250 ml Erlenmeyer flask containing 50 ml of medium contained 0.1 g of cells.
Callus cells weighing approximately 0.1 g were collected at the end of the growth period and re-inoculated into 50 ml of fresh medium of the same composition. The free radical scavenging activity of the plant extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The standard deviation of the mean is calculated and shown as bars on the graph.
Interestingly, excessive adventitious root proliferation was observed at all lower concentrations of BAP (1.0-3.0 µM). Effect of different concentrations of sucrose on in vitro rooting of excised shoots grown on ½ MS basal medium. The ion at m/z 244 was due to the formation of the potassium ion adduct [M+K]+. The characteristic fragment at m/z 149 resulted from the dissociation of the C-N bond.
In this study, various in vitro strategies were extensively investigated in two important medicinal plants, Azadirachta indica A. In this study, in vitro cultures of neem were established using different explants viz. However, shoots exposed to high concentration of BAP (7.0-15.0 µM) were short and stunted.
Another interesting finding of this study was the production of spilanthol from in vitro cultures of Spilanthes. Ed.), The Neem tree: source of unique natural products for integrated pest management, medicine, industry and other purposes. Antifeedant effect of in vitro culture extracts of the neem tree, Azadirachta indica, against the desert locust (Schistocera gregaria (Forskal).
Production of callus-associated limonoids and azadirachtin by in vitro culture of neem (Azadirachta indica A. Juss). In vitro organogenesis and plant regeneration from an unpollinated ovary: a new explant of neem (Azadirachta indica A. Juss.). In vitro organogenesis and plant regeneration from an unpollinated ovary: a new explant of neem (Azadirachta indica A. Juss.).
