Atanas Pavlov

Hairy root type plant in vitro systems as sources of bioactive substances.

Abstract“Hairy root” systems, obtained by transforming plant tissues with the “natural genetic engineer” Agrobacterium rhizogenes, have been known for more than three decades. To date, hairy root cultures have been obtained from more than 100 plant species, including several endangered medicinal plants, affording opportunities to produce important phytochemicals and proteins in eco-friendly conditions. Diverse strategies can be applied to improve the yields of desired metabolites and to produce recombinant proteins. Furthermore, recent advances in bioreactor design and construction allow hairy root-based technologies to be scaled up while maintaining their biosynthetic potential. This review highlights recent progress in the field and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.

Bioprocessing of differentiated plant in vitro systems

Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto‐pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year‐round cultivation in the bioreactor under optimal conditions with constant high‐level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.

Biosynthesis and radical scavenging activity of betalains during the cultivation of red beet (Beta vulgaris) hairy root cultures.

Betalains biosynthesis and antiradical scavenging activity were investigated during cultivation of four hairy root cultures of Beta vulgaris, obtained from different cultivars (Bordo, Egyptian, Detroit 2 and Detroit Dark Red). The best producer of betalains was a hairy root culture from Beta vulgaris cv. Detroit Dark Red (13.27 mg/g dry weight total pigment production). The ethanol extract, derived from roots of the same culture grown for 15 days under submerged conditions, showed a high antiradical activity (83% of inhibition of the stable DPPH·).

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.

Antioxidant activity of extracts from Lavandula vera MM cell cultures

Abstract The antioxidant activity of methanolic and ethyl acetate extracts from Lavandula vera MM cell culture were evaluated by the Schaal oven test in bulk sunflower oil and by the DPPH radical method. The oil oxidation was followed by measuring the quantity of primary oxidation products (peroxide value). Authentic rosmarinic acid, caffeic acid and BHT were tested in parallel for comparison. Ethyl acetate extract much better protected the oil from oxidation than methanolic extract and its antioxidant efficiency was comparable to that of pure rosmarinic and caffeic acids and much stronger than that of BHT. Both cell culture extracts and the authentic phenolic acids were much stronger scavengers of DPPH free radical than BHT on an equimolar basis.

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

Abstract The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose).

Alkaloid Spectrum in Diploid and Tetraploid Hairy Root Cultures of Datura stramonium

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots

Temporary immersion systems in plant biotechnology

Plant tissue and organ cultures in vitro usually face technological challenges. When submerged cultivation of plant cells in a controlled environment is desired, the characteristic growth morphology and physiology of differentiated organ cultures present a problem in process scale‐up. Temporary immersion systems (TIS) were developed several decades ago. These systems are providing the most natural environment for in vitro culture of plant shoots and seedlings. Over the past few years, TIS have been recognized as a perspective technology for plant micropropagation, production of plant‐derived secondary metabolites, expression of foreign proteins, and potential solutions in phytoremediation. Nowadays, several TIS, operating on similar or divergent technological principles, have been developed and successfully applied in the cultivation of various plant in vitro systems, including somatic embryos and transformed root cultures. In this article, the operational principle and technological design of the most popular TIS are reviewed. In addition, recent examples of the application of temporary immersion technology for in vitro cultivation of plant tissue and organ cultures at laboratory and pilot scales are discussed. Finally, future prospects and challenges to the industrial realization of that fast‐developing technique are outlined.

Optimization of Rosmarinic Acid Production by Lavandula vera MM Plant Cell Suspension in a Laboratory Bioreactor

The all‐round effect of dissolved oxygen concentration, agitation speed, and temperature on the rosmarinic acid production by Lavandula veraMM cell suspension was studied in a 3‐L laboratory bioreactor by means of the modified Simplex method. Polynomial regression models were elaborated for description of the process of rosmarinic acid production (Y) in the bioreactor as a consequence of the variation of the dissolved oxygen (X1) concentration between 10% and 50%; agitation (X2) between 100 and 400 rpm; and temperature (X3) between 22 and 30 °C. The optimization made it possible to establish the optimal conditions for the biosynthesis of rosmarinic acid by L. veraMM: dissolved oxygen (X1*), 50% of air saturation; agitation (X2*), 400 rpm; and temperature (X3*), 29.9 °C, where maximal yield (Ymax) of 3489.4 mg/L of rosmarinic acid was achieved (2 times higher compared with the shake‐flasks cultivation).

Nutrient Medium Optimization for Rosmarinic Acid Production by Lavandula vera MM Cell Suspension

The overall effect of NH4NO3, KNO3, and KH2PO4 on the biosynthesis of rosmarinic acid and cell biomass by Lavandula vera MM cell suspension was studied by the method of the full factor experiment. Polynomial regression models were elaborated to give a quantitative description of the processes of biosynthesis of rosmarinic acid (Y1) and cell biomass (Y2) as a result of the variation of the concentration of NH4+, 0.09 g/L ≤ X1 ≤ 0.23 g/L; NO3‐, 2.44 g/L ≤ X2 ≤ 3.02 g/L; and KH2PO4, 0.170 ≤ X3 ≤ 0.425 g/L. Optimization procedures according to the modified Simplex method allowed us to establish the optimal conditions for the biosynthesis of rosmarinic acid by Lavandula vera MM: X1* = 0.09 g/L; X2* = 3.02 g/L, and X3* = 0.170 g/L, where Y1*max = 1786.74 mg/L (27 times higher compared with the cultivation in the standard Linsmayer‐Skoog medium). As a result, modified ingredients of the Linsmayer‐Skoog nutrient medium were applied for the cultivation of Lavandula vera MM to achieve a maximum yield of rosmarinic acid.