Shakti Mehrotra

Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor

The study was undertaken to induce hairy roots in Glycyrrhiza glabra in leaf explants and to optimize the nutritional requirement for its growth kinetics at shake flask and bioreactor level. Pathogenecity of Agrobacterium depends upon transformation ability of strain and age, type, and physiological state of explants. Agrobacterium rhizogenes strain K599 was used to infect leaf explants of G. glabra . Explants of different age groups were obtained from 2 to 5 weeks old in vitro grown cultures. Bacterial strain K599 could induce hairy roots in 3 and 4 weeks old leaf explants cultured on B 5 , MS, NB and WP basal semi-solid medium. Leaf explants of 2 and 5 weeks old culture were not responsive to bacterial infection in terms of hairy root induction. Maximum transformation frequency (TF) of tested bacterial strain was 47% obtained in 3 weeks old explants after 25 days of incubation on MS basal semi solid medium. NB and B 5 both media composition showed 20% of transformation frequency after 28 and 38 days respectively. WP medium did not support induction of roots in cultured leaf explants infected with A. rhizogenes strain K599even after 50 days of incubation. Further, when all the four media combinations were tested for root growth it was found that though WP was not responsive for hairy root induction, yet all four basal media supported hairy root growth and a gradual increase in fresh weight biomass was observed with an increase in culture duration. However amongst all, the NB medium composition supported best growth of hairy roots followed by MS, B 5 and WP media. About 20 times increase in root biomass on fresh weight basis was recorded after 45days of culture in NB medium. Initial inoculum of roots (0.18 g. F.wt./ flask) containing 50 ml of liquid culture medium produced 3.59 g (F. wt.) biomass. A fast growing hairy root clone G6 was grown in a 5 l capacity mechanically agitated bioreactor provided with a nylon mesh septum. After 30 days of sterile run, 310 g of root biomass was harvested from the bioreactor culture vessel, recording about 20 times increase over initial inoculum (16.0 g).

Elicitor-induced cellular and molecular events are responsible for productivity enhancement in hairy root cultures: an insight study.

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.

An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first‐rate production of plant‐based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high‐value plant‐derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate‐limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by‐product formation can be accomplished either through single‐step engineering or through the multi‐step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low‐volume, but high‐value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary‐metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from ‘‐omic’ platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide‐ranging views of DNA sequence‐to‐metabolite passageway networking and their control to obtain desired results.

Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids

Hairy root cultures of Rauwolfia serpentina induced by Agrobacterium rhizogenes have been investigated extensively for the production of terpenoid indole alkaloids. Various biotechnological developments, such as scaling up in bioreactors, pathway engineering etc., have been explored to improve their metabolite production potential. These hairy roots are competent for regenerating into complete plants and show survival and unaltered biosynthetic potential during storage at low temperature. This review provides a comprehensive account of the hairy root cultures of R. serpentina, their biosynthetic potential and various biotechnological methods used to explore the production of pharmaceutically important terpenoid indole alkaloids. The review also indicates how biotechnological endeavors might improve the future progress of research for production of alkaloids using Rauwolfia hairy roots.

In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micropropagated plants, and simultaneous quantitation of reserpine, ajmaline, and ajmalicine.

Rauwolfia serpentina holds an important position in the pharmaceutical world because of its immense anti-hypertensive properties resulting from the presence of reserpine in the oleoresin fraction of the roots. Poor seed viability, low seed germination rate, and enormous genetic variability are the major constraints for the commercial cultivation of R. serpentina through conventional mode. The present optimized protocol offers an impeccable end to end method from the establishment of aseptic cultures to in-vitro plantlet production employing semisolid as well liquid nutrient culture medium and assessment of their genetic fidelity using polymerase chain reaction based rapid amplification of polymorphic DNA analysis. In vitro shoots multiplied on Murashige and Skoog basal liquid nutrients supplemented with benzo[a]pyrene (1.0 mg/L) and NAA (0.1 mg/L) and in-vitro rhizogenesis was observed in modified MS basal nutrient containing NAA (1.0 mg/L) and 2% sucrose. In-vitro raised plants exhibited 90-95% survival under glass house/field condition and 85% similarity in the plants regenerated through this protocol. Field established plants were harvested and extraction of indole alkaloid particularly reserpine, ajmaline and ajmalicine and their simultaneous quantitation was performed using monolithic reverse phase high-performance liquid chromatography (HPLC).

Dielectric Relaxation Studies of Alkyl Acrylates on Complexation with Phenols Using Time Domain Reflectometry

The dielectric relaxation measurements on binary mixtures of esters (methyl acrylate, ethyl acrylate, and butyl acrylate) with phenol derivatives (p-cresol, p-chlorophenol, and 2,4-dichlorophenol) were carried out at different concentrations at 303 K using the time domain reflectometry (TDR) over the frequency range from 10 MHz to 20 GHz. The Kirkwood correlation factor and excess inverse relaxation time were determined and discussed to yield information on the molecular interactions of the systems. The relaxation time increased with increasing concentration of phenols and increasing chain length of esters. The excess inverse relaxation time values were negative for all the systems, which indicated the solute-solvent interaction existing between esters and phenols producing a field in such a way that the effective dipole rotation was hindered.