Hairy roots, obtained after genetic transformation of higher plant cells by Agrobacterium rhizogenes, are genetically stable and express faster growth rate than the normal roots. Above all, such roots are amenable for scale-up in large bioreactors and hence offer better advantages compared to cell cultures. For these reasons, hairy roots are widely preferred for the production of various chemical substances that are akin to root systems. Peroxidase (E.C.1.11.1.7) (POD) is an enzyme known to play a very crucial role in scavenging free radicals (peroxides) within the plant system in addition to its involvement in various metabolic activities. Outside the plant system this enzyme has several commercial applications; the major ones being its use as an important component in chemical diagnostics and laboratory experiments. Because of broader catalytic activity, a wide range of chemicals can be modified using POD and hence has varied applications such as waste water treatment to remove phenolics, synthesis of various aromatic compounds and removal of peroxides from foodstuffs, beverages and industrial wastes. Presently, horseradish has been the source of high quality POD for biochemical / clinical applications whereas certain agricultural wastes / by products have been suggested for the production of commercial-grade POD. However, the latter sources of POD are limited by difficulties in purification. Certain cultured plant cells and organs are known to produce POD enzyme where the productivity was invariably hampered due to slow growth rate of cell cultures, inconsistent product yield and genetic instability rendering such systems with limited applications.

This communication reports, for the first time, that the genetically transformed root cultures (hairy roots) of red beet can be used for the production of high levels of POD enzymes. The study focuses on the establishment of different clones of red beet hairy root cultures using different A. rhizogenes strains and follow their growth patterns as well as the levels of POD production to select the most suitable hairy root clone for scale up in a bioreactor. The present study also considers other aspects that might influence the enzyme productivities such as the effect of different metal ions on the production and secretion of POD into the medium so that the enzyme is amenable for in situ recovery (Uozumi et al. 1992; Thimmaraju et al. 2004).

Establishment of hairy roots

The cotyledonary leaf explants were aseptically infected, by following standard protocols, with different strains of A. rhizogenes such as- LMG-150, A 2/83, A4, A 20/83 (Doran, 2002; Thimmaraju et al. 2003). The hairy root clone arising independently from each transformed cell was made bacteria-free using cefotaxime (200 mg L^-1) and the bacteria-free clones were maintained in 50 ml conical flask containing 15 ml of Murashige and Skoog's (Murashige and Skoog, 1962) liquid medium with 30 g sucrose (MS).

Growth of hairy roots

For testing growth performance, about 50 mgs of root tips of 10 hairy root clones were subcultured in 50 ml conical flasks as mentioned above and the biomass accumulation was monitored at an interval of 5 days for a total period of 25 days. Fresh weight increase was recorded after surface drying the roots by keeping between folds of blotter sheets.

POD production

The POD activity in each clone was monitored at an interval of 5 days for a total period of 30 days following the method of Wititsuwannakul et al. (1997). Intracellular and extra-cellular POD i.e., the enzyme within the biomass and in the medium respectively was also monitored periodically. Effect of various growth regulators and elicitors on the biomass production as well as elicitation of POD in all the ten clones were also recorded arriving at the best performing clone in terms of biomass and maximum POD production. While the activity of POD at different pH was checked, the stability at different temperatures was also recorded.

Production of POD in bioreactor

A best performing clone was selected and grown in a 3L bubble column reactor with a working medium volume of 1.75 L. Hairy root inoculum was prepared by sub-culturing about 100 mg of hairy roots of clone LMG-150 in 40 ml medium in conical flasks for 10 days under standard conditions mentioned earlier. Totally 10 g fresh weight of actively growing hairy root inoculum was transferred aseptically through inoculation port into the anchorage basket of the growth chamber. The bioreactor was maintained in dark at 23 ± 2ºC with air supply through a sparger at a rate of 33.4 cm³ s^-1. After the running period of 10 days, the biomass and the spent medium were analyzed for the units of POD produced.

Of the ten clones established using different strains of A. rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods, (Hamill and Lidgett, 1997). Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L^-1) as well as POD (ranging from 8000-9000 U g^-1 FW and 1.18 x 10⁶ U L^-1 with a specific activity of 600 U mg^-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10⁶ U L^-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of R_m 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at R_m 0.06, 0.16, 0.25 and one extra band of R_m 0.575 in the spent medium where isozymes of R_m 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.

The present study has clearly established that certain clones of red beet hairy roots produce copious levels of POD having good thermal stability. Since POD is also involved in lignin biosynthesis via phenyl propanoid pathway and the latter gets elicited to various biotic and abiotic elicitors, there is a high possibility for further enhancing the levels of POD by means of elicitation. The additional fact that the clone LMG -150 produced similar amounts of POD in a bubble column reactor as of shake flask cultures, indicate that the red beet hairy roots can be exploited for scaled up production of POD and the method appears to form a better alternative for horseradish POD. The observation that nearly half of the synthesized POD is secreted into the medium indicates the possibility of permeabilizing the roots for enhanced efflux of the enzyme even in bioreactor, as reported for the pigments from the same system (Thimmaraju et al. 2003). Several other unit operations for scale-up of hairy root biomass have also been worked out and, therefore, the red beet hairy root system appears very promising for the production of this expensive enzyme.

The authors thank Dr. V. Prakash, Director, CFTRI for his encouragement in the research activities.

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