Red Beet Hairy Roots Research Articles

Gamma rays as an effective tool for removing undesirable color without adverse changes in biological activities of red beet extracts

The ethanolic extracts of red beet (Beta vulgaris L.) hairy root were used to investigate the removal of color and improvement of biological activity for enhanced industrial applications. The extracts were exposed to gamma rays ranging from 2.5 to 30kGy. The red beet hairy root is composed of two major red-colorants, betanin and isobetanin. Gamma ray radiation at 5kGy remarkably reduced the levels of the major colorants by 94% and the reddish color was eliminated by doses greater than 10kGy. Color removal was likely due to the gamma ray radiolysis of ethanol. Although details on the mechanism responsible for the decay of the chromophore have not been entirely determined, our results suggest that the free radicals that are produced during this process are capable of destroying the chromophore group in isobetanin, thus bleaching the substrate solution. In spite of the degradation of the major colorants, the biological activities of constituents of the extract such as DPPH radical scavenging and tyrosinase inhibition were negligibly affected by the gamma ray radiation up to 20kGy. The antioxidant activity was 92.7% in control samples and 90.0–92.0% in irradiated samples (2.5–20kGy), and a slight decrease to 87.5% was observed for gamma ray radiation at 30kGy. In addition, tyrosinase inhibition activity has also the same pattern; the activity is slightly increased from 50.7% of control to 49.1–52.8% of irradiated samples (2.5–20kGy) with a 46.8% at 30kGy.

Bioreactor for cultivation of red beet hairy roots and in situ recovery of primary and secondary metabolites

AbstractTo arrive at an appropriate bioreactor design and in situ recovery of the products, red beet hairy roots were used as a model system where the levels of betalain pigments (betacyanins and betaxanthins) were followed as secondary metabolite and the peroxidase enzyme as primary metabolite. Medium volume and other kinetic parameters were found to play significant roles by way of directly affecting the biomass yield rather than a specific metabolite. The hydrodynamic stress created on the roots by large culture volume could be minimized by pulse‐feeding of medium in shake‐flasks; and by separating the biomass chamber from the aerated medium reservoir in circulatory fed‐batch bioreactor. Accordingly the bioreactor was modified to provide anchorage and air‐enrichment chamber which resulted in higher formation of both the metabolites than in shake‐flasks. Various down‐stream processing aspects such as in situ release of pigments by non‐destructive methods, followed by adsorption through a column and recovery by desorption were optimized for betalains. A strategy for simultaneous recovery of pigment and peroxidase was worked out using aqueous two phase extraction (ATPE).

Improved procedure for nucleus extraction for DNA measurements by flow cytometry of red beet (Beta vulgaris L.) hairy roots

Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Münster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully.

Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations

It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.

Batch and Fed-Batch Production of Betalains by Red Beet (Beta vulgaris) Hairy Roots in a Bubble Column Reactor

Hairy root cultures from red beet (Beta vulgaris L.), which could be used for the commercial production of biologically active betalain pigments, were cultivated in a 3 L bubble column bioreactor in batch mode with various rates of air supply. Both the growth of the roots and betalain volumetric yields were highest (12.7 g accumulated dry biomass/L and 330.5 mg/ L, respectively) with a 10 L/h (0.083 vvm) air supply. The air flow rate also influenced the betacyanins/betaxanthins ratios in the cultures. Growth and betalains production were then examined in two fed-batch regimes (with a 10 L/h air supply), in which nutrient medium was fed just once or on five occasions, designated FBI and FBII, respectively. The root mass accumulation was increased in the FBI feeding regime (to 13.3 g accumulated dry biomass/ L), while in FBII the betalains content was ca. 11% higher (15.1 mg betacyanins/g dry weight and 14.0 mg betaxanthins/g dry weight) than in the most productive batch regime. Data on the time course of the utilization of major components in the medium during both operational modes were also collected. The implications of the information acquired are discussed, and the performance of the hairy roots (in terms of both growth and betalains production) in the bubble column reactor and previously investigated cultivation systems is compared.

Purification and characterization of an intracellular peroxidase from genetically transformed roots of red beet ( Beta vulgaris L.)

An intracellular peroxidase (POD) produced by genetically transformed root cultures of red beet ( Beta vulgaris L.) was purified using a combination of (NH 4) 2SO 4 fractionation and ion exchange chromatography resulting in 15-fold enhancement of activity. This enzyme exhibited highest activity (10,500 U mg −1 protein) and stability at pH 5.0 and retained over 70% of the activity for 20 min at 70 °C where horseradish peroxidase (HRP) – a vastly used commercial source, had lost its activity after 11 min. The purified enzyme showed highest preference for H 2O 2 as the substrate ( K m value of 0.1). Among the H donors, the enzyme appeared to have affinity in the order of orthodianisidine > 2,2′-azino-bis(3-ethylbenz-thiazoline)-6-sulfonic acid > guaiacol. The purified POD was completely and competitively inhibited by periodate ( HIO 6 - , K i = 0.2 mM) whereas sodium azide (NaN 3) was a non-competitive inhibitor. The purified POD had a molecular mass of 45 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and activity staining. This is the first report describing purification and characterization of POD from red beet hairy roots showing its better efficacy than commercial HRP.

Elicitation of peroxidase activity in genetically transformed root cultures of Beta vulgaris L.

Genetically transformed roots of red beet produce copious levels of peroxidase (POD) - a multifunctional enzyme with a number of commercial applications. In an effort to elicit the POD activity, the cultures were treated with biotic elicitors such as dry cell powders of microbial cultures (0.1-0.5% w/v) and the respective culture filtrates (1-5% v/v). Similarly, abiotic elicitors, particularly metal ions (2-8 folds of that present in the nutrient medium), the plant hormone Thidiazuron (at 0.25-1 ppm) and other bio-molecules such as Glutathione (at 0.5-10 mM) and Methyl jasmonate (at 20-100 µM) were used. It was observed that dry cell powder of Candida versatilis significantly elicited the enzyme activity (3.52-fold higher than the control) followed by glutathione (3.44-fold) and Rhizophus oligosporus (3.09-fold) . Among abiotic elicitors, thidiazuron, Mg and Ca salts elicited 2.49, 3.03 and 2.8 fold activities respectively. While most of the biotic elicitors were effective when added on 15 th day of culture, the abiotic elicitors were effective when added on 20 th day. Combination of highly effective elicitors indicated that glutathione (1 mM) and dry cell powder of R. oligosporus caused a 4-fold enhancement in enzyme activity, accounting for 10.9 x 10 6 U L -1 . The present study is the first report on red beet hairy roots where a large number of elicitors have been systematically screened and their probable involvements in eliciting POD activities have been discussed.

Betalain biosynthesis by red beet ( Beta vulgaris L.) hairy root culture

The time course of growth and betalains biosynthesis by Beta vulgaris L. (cv. Detroit Dark Red) hairy root culture were investigated. It was established that hairy root culture produced 42.2 mg/flasks betalains (26.2 mg/flasks betaxanthins and 16.0 betacyanins) between 6th and 15th days of cultivation. The uptake of sucrose, nitrate and phosphate ions were followed during the cultivation and data on the physiology of the B. vulgaris hairy root culture are presented.

Various Hexoses and di-hexoses Differently Influence Growth, Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.

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.).

Evaluation of growth property of red beet hairy roots depending on condition of inocula and its application to culture control with fuzzy logic theory

The influence of period of preculture on growth was examined for the development of control system suitable to the culture of red beet hairy roots. The prolongation of preculture caused the decrease in viability of root tips, resulting in the extension of lag phase during the following culture. The number of lateral roots emerging from the inoculated hairy roots could be used as a parameter for the identification of the culture phases. A bioreactor system including a solid-culture unit to monitor the variation in the number of growing points (GPs) was constructed, and the fuzzy inferencing was employed for the control of shear stress in the culture, based on the membership functions with state variables concerning the growth indexes in terms of the number of GPs and concentration of cell mass. The successful performances were achieved by means of appropriate recognition of culture phases in the cultures of hairy roots with different levels of tip viability. It was verified that this control system could be available for the culture with variation of inoculum states by employing the state variable based on the number of elongating tip meristems of the hairy roots.

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.

Culture of red beet hairy roots by considering variation in sensitivity of tip meristems to hydraulic stress

In the culture of red beet hairy roots in shaking flasks, a period for acclimation without lateral root generation existed at the early stage, and the root tip meristems containing growing points (GPs) were found to be damaged under an elevated shear stress condition. The loading experiments of shear stress to the hairy roots revealed that the GPs subjected to the acclimation acquired tolerance to shear stress, retaining relatively high viability of GPs up to 0.6 N/m 2 of loaded shear stress. Next, the hairy roots after culture for 50 h at 0.05 N/m 2 of shear stress were exposed to conditions at various levels of shear stress in a single column reactor, and a relatively high growth rate was obtained in the vicinity of 1.0 N/m 2 of shear stress. According to these results, two-stage cultures of hairy roots were then performed, which was comprised of a first stage for 50 h at 0.05 N/m 2 of shear stress for the prevention of decay of the GPs caused by hydraulic stress and a second stage for 110 h at 1.0 N/m 2 of shear stress for active elongation of the GPs with sufficient nutrient supply by regulation of the medium flow rate. The cell concentration ultimately reached 7.6 kg dry cells/m 3, although no growth was observed in the case where the hairy roots did not undergo the first stage.

High-density culture of red beet hairy roots by considering medium flow condition in a bioreactor

The effect of liquid flow condition on the growth of red beet hairy roots was investigated with a single-column reactor (SCR) at superficial velocities of medium ranging from 2 to 28 m/h, and the optimal growth was obtained when the superficial velocity was kept at 15 m/h in the culture for 190 h. The increase in medium flow caused the enhancement of elongation rate and decline of viability at tip meristems of the hairy roots, resulting from the enhancement of both the dissolved oxygen (DO) concentration and shear stress at hairy root surfaces, respectively. Specific cross-sectional area of bioreactor was defined as an evaluation parameter and a variation in the specific cross-sectional area was found to affect DO concentration at hairy root surfaces. Based on evaluated value of the specific cross-sectional area, a radial flow reactor (RFR) was constructed to perform the high-density culture of red beet hairy roots. Under the conditions of void fraction of hairy roots of 0.3–0.7, superficial velocity of 15 m/h and specific cross-sectional area of 40 m -1, it was estimated that the DO concentration at hairy root surfaces increased with increasing cell mass concentration. During the culture in the RFR at superficial velocity of 15 m/h, the serious declination of DO concentration in bulk liquid did not occur owing to improvement of oxygen supply. The final density of hairy roots in a growth unit of the RFR reached 51 kg dry weight/m 3 at culture time of 280 h.

Effect of Liquid Flow on Pigment Formation of Red Beet Hairy Roots.

Red beet hairy roots are cultivated in a single column reactor to investigate the influence of superficial velocity of medium on formation of betanin (a major pigment component in red beet hairy roots). The betanin content in the cells increases with increasing superficial velocity in the range of 13 to 40 m/h. In the cultures at superficial velocities of 15 and 40 m/h for 166 h, the betanin content increases with elapsed time of cultures. The growth of the hairy roots is responsible for an increase in shear stress on the root surfaces by means of lowered void fraction of hairy root bed, and the estimated values of shear stress range from 0.22 to 188 N/m2 during both the cultures. The activity of monophenol monooxygenase in the cells, which is a key enzyme involved in the biosynthesis of betanin, can be correlated with the value of shear stress in such a manner as to approach plateaus gradually with increasing shear stress.

Effect of liquid flow on culture of red beet hairy roots in single column reactor.

The influence of liquid flow on the growth of red beet hairy roots is investigated by using a single column reactor operated at 25 °C under a fixed value of superficial velocity of medium. In the cultures of the hairy roots for 172–190 h, average growth rates of 2.8 × 10–2, 1.6 × 10–2 and 0.98 × 10–2 kg/(m3·h) are obtained at superficial velocities of 4.2 × 10–3, 7.8 × 10–3 and 11.1 × 10–3 m/s, respectively. Using the packed column with the hairy roots at void fractions of 0.49 to 0.71, it is found that pressure drop through the root bed increases with an increase in superficial velocity of medium elevated up to about 14 × 10–3 m/s. The effective diameter of the hairy roots is estimated at 1.5 × 10–4 m, considering the root hairs found on the surfaces of the main roots. Pressure drop through the root bed in the column can be correlated with superficial velocity of the medium by using an extended Ergun’s equation expressed in terms of effective diameter of the hairy roots and void fraction in the bed. When the hairy roots are kept for 50 h in the column under pressure drop values of 8.4 × 103 to 3.1 × 104 Pa/m, the reduction in viability of growing points at the hairy root tips is observed with increasing pressure drop in the range.

Extracellular production of pigment from red beet hairy roots accompanied by oxygen starvation.

Extracellular production of pigment was conducted in a culture of red beet hairy roots accompanied by pigment release under oxygen starvation. The phenomenon of pigment release into medium by oxygen starvation was found that a part of hairy root cells associated with a declination of cell viability decayed and the pigment release arose from the cells. The kinetics of pigment release was expressed by the declination of cell viability during oxygen starvation as the time response of first-order lag plus dead time. Moreover, the variation in cell viability affected the growth rate after oxygen starvation and the growth rate was evaluated by the extent of the decay of growing points in hairy roots. According to the kinetics, the culture accompanied by repeated pigment release was performed under oxygen starvation to achieve maximum extracellular production. From experimental data, the extracellular production rate was found to attain to 2.70x10 -4 kg/(m 3 .h) when oxygen starvation of 16 h duration was repeated three times.

Kinetic Expression for Pigment Production in Culture of Red beet Hairy Roots.

A kinetic model was proposed to represent the profile of pigment production associated with the growth of red beet hairy roots. This model was constructed on the basis of a concept of cellular age distribution arising from a linear growth mode of the roots. The kinetics of root proliferation were formulated using a Monod-type rate equation of root elongation with intracellular phosphorus as a limiting substrate. A positional dependence of pigment content was observed along with the hairy roots; namely, content increased with increasing distance from the root tips and gradually approached a saturated value correlated with intracellular phosphorus content. The kinetics of pigment formation of hairy roots were presented by considering the variation in pigment content along the roots and the intracellular phosphorus effect. It was demonstrated that the model made it possible to describe the kinetic behaviors of growth and pigmentation during hairy root culture in a fermentor.

Culture of Red Beet Hairy Roots in a Column-type Reactor Associated with Pigment Release.

Culture of Red Beet Hairy Roots in a Column-type Reactor Associated with Pigment Release.

Influence of medium constituents on enhancement of pigment production by batch culture of red beet hairy roots

For the enhancement of pigment production by red beet hairy roots, the effects of medium constituents (Murashige-Skoog (MS) medium) on hairy root cultures were investigated in flasks. In a series of cultures using media with diluted medium components, it was found that phosphate was a key nutrient involved in pigment accumulation in the hairy roots, and that higher pigment contents in the roots were obtained at lower phosphate concentrations (range of 0–2.5 mol/m 3). In an 18 d batch culture using phosphate-free medium, the total amount of pigment production was 4.8 times that obtained in a control culture using normal MS medium with 1.25 mol/m 3 phosphate.