Smita Srivastava

Genetically transformed, "Hairy roots" once developed can serve as a stable parent culture for in vitro production of plant secondary metabolites. However, the major bottleneck in the commercial exploitation of hairy roots remains its successful scale-up due to oxygen transfer limitation in three-dimensionally growing hairy root mass. Mass transfer resistances near the gas-liquid and liquid-solid boundary layer affect the oxygen delivery to the growing hairy roots. In addition, the diffusional mass transfer limitation due to increasing size of the root ball (matrix) with growth also plays a limiting role in the oxygen transfer rate. In the present study, a mathematical model is developed which describes the oxygen transfer kinetics in the growing Azadirachta indica hairy root matrix as a case study for offline simulation of process control strategies ensuring non-limiting concentrations of oxygen in the medium throughout the hairy root cultivation period. The unstructured model simulates the effect of oxygen transfer limitation in terms of efficiency factor (η) on specific growth rate (μ) of the hairy root biomass. The model is able to predict effectively the onset of oxygen transfer limitation in the inner core of the growing hairy root matrix such that the bulk oxygen concentration can be increased so as to prevent the subsequent inhibition in growth of the hairy root biomass due to oxygen transfer (diffusional) limitation. © Springer Science+Business Media, LLC 2012.

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O2 in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20-100% (v/v) O2 in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O2 in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3-2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1-4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day). © Springer Science+Business Media, LLC 2012.

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg's medium vitamins, and 30 g l -1 sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g -1) and azadirachtin production (~44 mg l -1) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l -1 dry weight. To enhance the production of azadirachtin, a Plackett-Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l -1 sucrose, 0.19 g l -1 potassium dihydrogen phosphate, 3.1 g l -1 potassium nitrate, and 0.41 g l -1 magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l -1 and azadirachtin accumulation of 5.2 mg g -1, which was equivalent to an overall azadirachtin production of 73.84 mg l -1, 68% more than that obtained under non-optimized conditions. © 2011 The Society for In Vitro Biology.

Alkaloids are a diverse group of complex organic molecules found in about 20%of plant species in small quantities. Their potent biological activity has led to their exploitation as pharmaceuticals, stimulants, narcotics, and poisons. Despite their importance, inefficiency of extracting alkaloids from a myriad of other metabolites remains a significant barrier toward inexpensive bioprospecting for drug development. Furthermore, the yield is inconsistent in the natural resource due to heavy dependence on genetic and geographical diversity and also on climatic conditions. Chemical synthesis has been successful for the class of indole alkaloids. However, it is still a challenge and also impractical on industrial scale to construct other commercially important class of alkaloids. In lieu of the given limitations, plant cell/tissue cultures serve as alternative production platforms in which the biosynthesis of alkaloids has been improved through various elicitation and culture manipulation strategies. In addition, recent advances made in metabolic engineering and systems biology now have the potential to more effectively maximize the alkaloid biosynthesis in such in vitro production systems. In this respect, the chapter provides information regarding recent advancements made in bioprocessing and genetic engineering of cellular systems for large-scale alkaloid production.