166 Vol.58, n.2: pp. 166-174, March-April 2015 BRAZILIAN ARCHIVES OF http://dx.doi.org/10.1590/S1516-8913201400276 ISSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL Increasing Scopolamine content in Hairy Roots of Atropa belladonna using Bioreactor Peyman Habibi1, Khosro Piri1*, Ali Deljo1, Yaser Ahmadi Moghadam1 and Taiebeh Ghiasvand1 Department of Biotechnology; Department of Agriculture; Bu-Ali Sina University; Hamedan - Iran ABSTRACT The aim of this study was to use the, hairy root system for increasing the scopolamine content in Atropa belladonna. Agrobacterium rhizogenes ATCC15834 was utilized to produce hairy roots. The culture was carried out in a 1.5-l bioreactor using the inoculum size of 0.5 g fr. wt of 10-day-old hairy roots and various parameters, including agitation, aeration, conductivity and the consumption of sucrose were evaluated. Results revealed that the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per volume per minute) and 70 rpm, respectively. Study of conductivity and the consumption of sucrose showed that the highest amount of sucrose consumption and the highest amount of minerals consumption also was at 1.25 vvm and 70 rpm. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR). Key words: Atropa belladonna, hairy root, scopolamine, bioreactor INTRODUCTION 2011). Hairy roots obtained from the transgenic plants using A. rhizogenes supply obvious Atropa belladonna L. (solonacea), generally contents of biological products and secondary referred to as the “deadly nightshade,” is a metabolites (Dechaux and Boitel-Conti 2005). perennial herbaceous plant used for commercial Genetically produced hairy roots not only increase production of tropane alkaloids in the high-value secondary metabolites, but can also pharmaceutical industry (Zhang et al. 2005). perform with greater efficiency than non- Among tropane alkaloids, scopolamine is a more transgenic roots (Kamada et al. 1986; Flores et al. valuable secondary metabolite due to its fewer 1999; Giri and Narasu 2000). The production of side-effects, usefulness in the treatment of tropane alkaloids, especially scopolamine, in Parkinson‟s disease, and relaxing and bioreactors instead of from native plants, is an hallucinogenic properties. Many efforts have been efficient way because the process is performed made to extract scopolamine from the plants but under clean and controlled circumstances, which because of the low endogenicity of scopolamine consequently controls or decreases the diversity in content, the commercial production of component quality and the alkaloid yield (Eibl et scopolamine is limited. al. 2008). Because of their fast-growing genetic and Recently, the use of plant hairy roots for the biosynthetic stability, hairy root cultures are production of various chemicals such as regarded as an effective method for biomass pharmaceuticals, pesticides, flavorings, and dyes production in plant tissue culture (Yang et al. has been taken into consideration. Plant hairy *Author for correspondence: [email protected] Braz. Arch. Biol. Technol. v.58 n.2: pp. 166-174, Mar/Apr 2015 Scopolamine Production in Hairy Roots 167 roots have proved valuable because of their great pH, etc.). Plant cell cultures are increasingly stability and productivity (Payne et al. 1992). utilized for the production of valuable natural They show indefinite and active proliferation in products such as pharmaceuticals, flavors, phytohormone-free media and are capable of fragrances, and fine chemicals. More than 20,000 producing valuable materials at similar or higher different chemicals are produced from the plants, levels compared to original plant roots (Flores and and about 1,600 new plant chemicals are Curtis 1992). Another advantage of plant hairy discovered each year (Sajc et al. 2000). On the roots is the large-scale plant micro-propagation. other hand, various problems associated with low This technique is an effective way of propagating cell productivity, slow growth, genetic instability, massive amounts of disease-free plants and is and an inability to maintain photoautotrophic genetically uniformed by “artificial” seeds, which growth have limited the application of plant cell can be achieved through direct organogenesis cultures (Bourgaud et al. 2001). from hairy roots (Honda et al. 2001). Transformed The use of bioreactors for culturing hairy roots is root cultures provide a promising alternative to complex owing to the continuous growth of hairy biotechnological exploitation and a method for roots. They must have a special configuration to constant and standard production of valuable compensate for the heterogeneous, structured, and metabolites of plant cells. entangled nature of fibrous roots (Taya et al. Several bioreactor designs for large-scale 1989). Hairy roots cultured in either shake flasks production of hairy roots have been reported or bioreactors tend to distribute unevenly (Nobuyuki and Takesh 1994; Nam 1996). throughout the vessel and to form dense and Bioreactors used for hairy root cultures are more highly tangled root clumps, which may resist flow complex owing to the continuous growth of hairy by branching the growth pattern of hairy roots. roots. A bioreactor must have a unique Conditions in the interior of the clump may be configuration to compensate for the very different from those of the bulk liquid due to heterogeneous, cohesive, structured, and mass-transfer effects. The nutrient limitation entangled nature of fibrous roots (Giri et al. 2000). caused by the formation of dense root clumps In flask and bioreactor cultures, the oxygen leads to cell death and necrosis at the core of the transferred to hairy roots and submerged in the root clumps (Yu and Doran 1994). The scale-up of medium exhibits three external mass transfer hairy root cultures becomes complicated because resistances: gas-liquid, liquid-liquid, and liquid- of the need to simultaneously provide nutrients solid resistances. In shake-flask cultures, the from both liquid- and gas- phases. To design the important parameters affecting flask properties are bioreactors for hairy root cultures many factors flask size, shaking speed, closure type, and should be considered, such as the growth medium volume (Auro et al. 1957). characteristics, nutrient requirements and Since the physical structure of hairy roots causes utilization rates, mass transfer, mechanical problems for making an efficient distribution properties, requirement of a support matrix, and during scale-up, productivity of hairy roots have the possibility of flow restriction by the root mass been reported to decrease (Lee et al. 1999). Many in certain parts of the bioreactor (Yu and Doran factors can critically affect biomass and secondary 1994). Moreover, in order to obtain an optimal metabolites production during scale-up of any biomass yield, roots should be evenly distributed kind of cell culture. These include aeration, in the bioreactor (Honda et al. 2001). agitation, pH, temperature, and inoculum density Formation of aggregates, the tendency for cells to (Humphery 1998; Kim et al. 2002). adhere to the walls of the fermenter vessel and Large-scale plant production through cell tissues sensitivity of hairy roots are significant challenges in bioreactors is a key way for industrial plant associated with the low amount of scopolamine propagation. In a biochemical context, bioreactors production. Many studies have been conducted to are usually explained as self-contained, sterile industrialize the production of plant secondary environments, which capitalize on liquid nutrient metabolites in bioreactors; however, very few or liquid/air inflow and outflow systems. They are have attained commercial success. Building and designed for intensive culture and providing handling bioreactors and controlling its conditions maximal opportunity for monitoring and have proved costly. Therefore, this study aimed to controlling micro-environmental conditions (e.g., develop the optimal conditions of aeration and agitation, aeration, temperature, dissolved oxygen, agitation required for increasing the scopolamine Braz. Arch. Biol. Technol. v.58 n.2: pp. 166-174, Mar/Apr 2015 168 Habibi, P. et al. productivity in hairy root of A. belladonna and to and 160 rpm on a shaker incubator for 24 h. The design and build a modified bioreactor to reduce cells were pelleted by centrifugation at 3,000 rpm the costs. for 15 min and re-suspended to a cell density of OD600 = 8 in liquid inoculation medium, consisting of MS salts and vitamins along with 50 mg/L MATERIALS AND METHODS sucrose. Plant materials and tissue culture Hairy root induction and culture conditions Fresh seeds of A. belladonna were washed with Different seedling parts, including roots, stems running tap water for 20 min. The seeds were then and leaves (with petioles) were isolated from in surface sterilized with 70% ethanol and vitro grown seedlings and were cut into explants. subsequently treated with 2% sodium hypochlorite The explants were randomly wounded using for 1 and 12 min, respectively. Thereafter, the sterile needle and inoculated with A. rhizogenes seeds were washed 3-4 times with sterile distilled ATCC 15834 for 10 min. The explants were dried water. Finally, these disinfected seeds were with sterile filter paper and then transferred to co- cultured on hormone-free MS/2 medium, cultivation medium, which was 1/2 MS medium. containing 30