It is certified that the work described in this thesis is entitled "Plant tissue culture and production of triterpenoids from medicinal plants: Azadirachta indica A. and Lantana camaraL." by Priyanka Srivastava for the award of the degree of Doctor of Philosophy is an authentic record of the results obtained from the research work carried out under my supervision in the Department of Biotechnology, Indian Institute of Technology Guwahati, India, and this work is not not submitted. elsewhere for a degree. Their constant help and encouragement, especially during the final stages of the work, is greatly appreciated. The tables and graphs are included in the text while all the figures are compiled in the form of plates at the end of the thesis.
Chapter 5 sheds light on the main points of the current work and its future purpose. 3B.4: Results of paired t-tests (p=0.05a and 0.01b) between treatment groups (organic extract) (HeLa and BHK-21) at different time intervals. 3B.2: Effect of different carbon sources on cell growth and the production profile of two triterpenoids, OA and UA, in cell suspension cultures of L.
3B.3: Effect of different stirring speeds on the fresh and dry weight of cells in suspension cultures.
INTRODUCTION AND LITERATURE REVIEW
TISSUE CULTURE TECHNIQUES
Micropropagation by induction of shoots from preexisting meristems is a most popular approach for clonal propagation of plants because the cells in the shoot tip are uniformly diploid and least susceptible to genotype changes under culture conditions. Thus, it guarantees that the characteristics of the source plant are preserved (Rao and Venkateswara 1985). It offers many advantages over the conventional methods of vegetative propagation: (1) The rate of propagation is extremely fast and can continue throughout the year, regardless of the season.
Adventitious shoot proliferation in cell and tissue cultures, in response to hormonal manipulation of the culture medium, requires de novo differentiation of meristematic region, randomly, throughout the tissue other than the preexisting meristem. In addition, since the development of microspores is independent of the sporophytic tissues, the media components and culture treatments have direct access to the microspores. Pelletier and Ilami (1972) introduced the concept of "Wall Factor", according to which the somatic tissues of the anther play an important role in the induction of sporophytic divisions in pollen.
Gynogenic development of plants from unfertilized female gametophyte (embryo-sac) cells in egg/ovule cultures is one of the alternatives available for haploid production.
IN VITRO STUDIES
The standard equation obtained from the curve was used for quantification of the three triterpenes in the unknown samples. However, the presence of extraneous tissues (e.g. the anther wall) makes it a messy system for cell biology and other precise studies (Forster et al. 2007). Most susceptible microorganism in their study was C. The range of minimum inhibitory concentration of tested extracts was mg/ml while minimum bactericidal/fungicidal concentration ranged from mg/ml. 2009) reported the biochemical composition and antibacterial activities of the leaves and flowers of four Lantana camara (Verbanaceae) plants with yellow, lavender, red and white flowers.
Ed.), The Neem Tree: A Source of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. Antifeedant effects of extracts from an in vitro culture of the neem tree, Azadirachta indica against the desert locust (Schistocera gregaria (Forskal). Haploid plants from an in vitro anther culture of the legume tree, Peltophorum pterocarpum (DC) K. Plant cell organ culture.
Ed.), The Neem Tree Azadirachta indica A. VCH Publications, Weinheim, Germany, pp. p53 induces apoptosis through caspase activation through mitochondrial cytochrome c release.
SECONDARY METABOLITES AND THERAPEUTIC APPLICATIONS
OBJECTIVES
The bottlenecks in Neem, in terms of extraction and availability of metabolites like azadirachtin, lie in its out-breeding nature and long reproductive cycle that makes one wait for long periods to get the seeds. Our goal and interest in growing haploids in Azadirachta therefore arose from the fact that they bring us one step closer to obtaining genetically pure, homozygous diploid lines that produce uniform quality and quantity of compounds. No tissue culture reports exist to date and all the biochemical studies carried out so far have been carried out on plants growing in nature.
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. Also, secondary metabolite studies require an in-depth understanding of biosynthetic pathways, which is often difficult to perform in whole plants because the biosynthetic activities may only be expressed in certain cell types within a specific plant organ or at a certain time of the season. Cell cultures have a higher metabolic rate than intact differentiated plants because the initiation of cell growth in culture leads to rapid proliferation of cell mass and to a condensed biosynthetic cycle.
These cell cultures can further aid in scale-up operations, for isolating desired compounds in bulk.
MATERIALS AND METHODS
In this study, the extract derived from in vitro elevated androgenic callus lines showed no activity with most of the strains tested, except some strains of Staphylococcus aureus. Qualitative analysis of the organic extract by TLC revealed the presence of three anti-cancer triterpenoids: BA, OA and UA. Quantitative estimation by HPLC of the cell culture-derived organic extract further showed that all three triterpenoids BA, OA and UA were produced and accumulated simultaneously in the same cultures.
The results obtained by DNA fragmentation analysis conclusively demonstrated the apoptotic activity of the extract. A very mild inhibitory activity of the extract against certain pathogenic bacterial strains was observed in the leaf extract of Lantana. The beneficial effect of sucrose was also seen in the present study through the experiments.
The type and concentration of plant growth regulators in the environment have been recognized as one of the critical factors in plant development in anther cultures (Reynolds 1987; Later, in 2001, they identified a multivariate calibration technique for the estimation of azadirachtin related limonoids as well as simple terpenoids in different parts of the Neem tree. As mentioned in the introduction, the different activities can be attributed to a spectrum of compounds localized in different parts of the plant.
In the present study, extracts derived from in vitro established anther culture cell lines were evaluated for antimicrobial activity. Detection of the presence of azadirachtin as the main constituent of the extract by chemical analysis. Establishment of in vitro cultures of woody plants is strongly affected by browning of the medium and necrosis of explants.
The importance of in vitro cultures lies in the uniformity and stability of the metabolites produced by them, which is independent of plant availability and seasonal variations. Based on ethnopharmacological evidence, the in vitro cytotoxic activity of cell culture-derived ethyl acetate and Lantana aqueous extracts were evaluated against HeLa cancer cell lines, in the present work. The production of three pentacyclic triterpenoids was observed to be growth related and increased with an increase in fresh cell weight.
Investigation of locust feeding inhibition of neem tree seeds, Azadirachta indica.
RESULTS
DISCUSSION 88-109
The natural occurrence of sporophytic haploids was first described by Dorothy Bergner in the weed species Datura stramonium in 1922 (Blakeslee et al. 1922). However, above a certain level, sucrose above 12%, as in the current study, showed a decrease in the percentage of responsive anthers. In the present study, the anthers were cultured at the early to late non-nuclear stage of microspores.
In the previous report by Chaturvedi et al. 2003b), the regeneration medium consisted only of a cytokinin (BAP), while in the present case, it had to be mixed with an axin (NAA). In the present study, stem elongation required a GA3 (3 μM) pretreatment before transferring to MS + BAP (0.5 μM). In the present study, the reported azadirachtin values are from cells containing the haploid (n) genome.
In this study, a very simple ultrasound-based extraction protocol for azadirachtin was adopted. The haploid plants obtained in this study can be a continuous source of homogeneous quality and quantity of an important metabolite such as azadirachtin. In this study, much higher amount of azadirachtin was obtained by different haploid callus lines of Neem.
In this study, extracts obtained from different anther culture lines were used to study the antibacterial activity against certain clinical isolates and reference strains. In this study, it was found that complete utilization of phosphate from the culture medium results in the initiation of the stationary phase. This shortened growth period, however, negatively affected the production of BA, OA and UA in cell suspension cultures in this study.
Such effects on biomass production were observed in cell suspension cultures of Psoralea corylifolia (Shinde et al. 2009). In addition to cytotoxic activity, antibacterial activity of Lantana was also evaluated in the present study.
CONCLUSIONS AND FUTURE PROSPECTS ………………. 110-112
In vitro clonal propagation of a mature tree of neem (Azadirachta indica A. Juss.) by forced axillary branching. Callus and azadirachtin related limonoids production by in vitro culture of neem (Azadirachta indica A. Juss). In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Rapid clonal propagation of Plumbago zeylanica Linn.
Effect of glycine on in vitro biosynthesis of nimbin and β-sitosterol in tissues of Azadirachta indica. In vitro organogenesis and plant regeneration from unpollinated ovary: a novel explant of neem (Azadirachta indica A. Juss.). Accumulation of betulinic acid, oleanolic acid and ursolic acid in in vitro cultures of Lantana camara L.
