Culture condition optimization and FT-IR analysis of Polygonum multiflorum Thunb. adventitious root cultures grown in an air-lift bioreactor system

Plant Cell, Tissue and Organ Culture (PCTOC) - Bioreactor cultures have been used for biomass production and bioactive compounds accumulation in adventitious root cultures of medicinal plants. In...     

Influence of mist intervals and aeration rate on growth and second metabolite production of Pseudostellaria heterophylla adventitious roots in a siphon-mist bioreactor

Plant adventitious root culture in bioreactors is a promising alternative for the efficient production of medicinal herbs. Adventitious roots of Pseudostellaria heterophylla were induced from callus and then cultivated in a siphon-mist bioreactor. An orthogonal test established that the optimal medium for adventitious root induction was MS medium supplemented with 1.0 mg/L naphthaleneacetic acid and 2.0 mg/L 3-indolybutyric acid. Under these conditions, the average root number was more than 14 on each 1.0 cm diameter callus and the rooting rate reached 100%. The bioreactor was equipped with an integral siphon-spraying device designed to automatically supply the liquid medium. The operation parameters of the bioreactor were assessed by varying the mist interval and the aeration velocity. The mist interval was negatively related to average growth rate of the adventitious roots and positively related to saponin and polysaccharide content. A relatively high aeration rate was necessary to achieve the maximum biomass production, but the secondary metabolite production was not enhanced by increasing the aeration velocity.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Tools for biotechnological production of useful phytochemicals from adventitious root cultures

Adventitious roots are natural roots which develop from the differentiated cells of any organ such as leaf, root or stem. These roots are attractive source of phytochemicals due to their genetic and biosynthetic stability. Their stability offers new scope for the in vitro production of valuable secondary metabolites. In the past two decades, adventitious roots have been induced from different plant systems and the suspension cultures have been established. However, the special morphology of adventitious roots hampers the scale-up processes, which needs modifications with the existing bioreactors to facilitate proper mixing, aeration and which can overcome shear stress. Various modifications have been done with the stirred tank and air-lift bioreactors and successfully used for the cultivation of adventitious roots. The progress made in this area is tremendous, in few cases adventitious root cultures are succeeded up to commercial scale with step-wise viz. small scale to large-scale by establishing the standards with various parameters which affect the biomass accumulation and metabolite productivity. This review addresses the recent progresses made in this area and discusses the potential applications and limitations.     

Ginsenosides: prospective for sustainable biotechnological production

Panax ginseng C.A. Meyer (ginseng) is a well-known medicinal plant that has been traditionally used in the oriental countries for centuries. Wild ginseng is a scarce and rare commodity. Field cultivation of the ginseng plant is a time-consuming and labor-intensive process. Ginsenosides, a group of glycosylated triterpenes, also known as saponins, are the principal bioactive constituents of ginseng. The use of cell and organ culture processes has been sought as a potential alternative for the efficient mass production of ginseng raw material. Various bioprocessing strategies have been developed to date. Cells and adventitious roots have been cultured in large-scale bioreactors and various strategies have been developed accordingly for the enhancement of biomass and ginsenoside accumulation. This review highlights the recent progress in the cultivation of ginseng cell and organ cultures for the production of ginsenosides from bioreactor cultures. In addition, the metabolism and biochemistry of ginsenoside biosynthesis, genomic and proteomic studies in ginseng, metabolic engineering, biosafety, toxicological evaluation, and efficacy assessment of ginseng raw material are also summarized and thoroughly discussed.     

Adventitious root culture of <Emphasis Type="Italic">Polygonum multiflorum</Emphasis> for phenolic compounds and its pilot-scale production in 500 L-tank

Polygonum multiflorum Thunb. is an important medicinal plant that synthesizes an array of phenolic compounds. Its roots are used in a variety of pharmacological and cosmetic formulations, notably as hair dye. In the present study, the inoculum density (3–15 g/L) and culture period (1–7 weeks) were optimized in a 3 L bioreactor. High root biomass (14.18 g/L dry weight (DW)) was recorded with an inoculum of 7 g/L (p?≤?0.05), which is consistent with the results for 5 and 10 g/L. However, significantly higher yield of bioactive compounds (53.87 mg/g DW total phenolics and 27.96 mg/g DW total flavonoids) with high free radical scavenging activity was obtained in root samples from 5 g/L inoculum density. A 4 week culture period was sufficient for optimum root growth and metabolite production. The optimized conditions were used for large-scale (5 and 20 L) and pilot-scale (500 L) studies. Considering that the continuous aeration of root cultures may lead to oxidative stress, antioxidant enzyme activity and lipid peroxidation also were studied. The results revealed high catalase (CAT) and guaiacol peroxidase (G-POD) activities, and low malondialdehyde (MDA) production, with increasing culture scale (20 and 500 L), which may indicate low-level oxidative damage to the cultures. An optimal yield of 4.01 kg dry root biomass with 287.12 mg/L of total phenolic productivity was achieved in a 500 L pilot-scale bioreactor. This work can pave the way for commercial production of biomass and secondary metabolites at the industrial level, and meet the rising demand for natural ingredients, especially in the pharmaceutical and cosmetic industries.     

Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives

In an attempt to scale-up of adventitious root cultures of Echinacea angustifolia for the production of biomass and caffeic acid derivatives, i.e. echinacoside, chlorogenic acid, cichoric acid, caftaric acid, and cynarin, the effects of Murashige and Skoog (MS) medium dilutions, and initial sucrose concentrations were investigated in a 5-L airlift bioreactor. In addition, the kinetics of adventitious root growth and accumulation of secondary metabolites were also studied. The greatest root dry weight (6.50 g L^?l) and accumulation of total phenolics [22.06 mg g^?1 DW (dry weight)], total flavonoids (5.77 mg g^?1 DW) and total caffeic acid derivatives (10.63 mg g^?1 DW) were obtained at quarter-strength MS medium. Of the various gradients of sucrose tested, 5 % sucrose supplementation was regarded as an optimal concentration for enhancing productivity of biomass and bioactive compounds. Neither higher salt strength (3/4–2 MS) nor sucrose concentrations (7 and 9 %) showed promotive effect on root growth and metabolite production. The kinetic studies revealed that 4 weeks of culture period is the optimal time to achieve highest productivity of metabolites. Based on these results, a large-scale (20 L) and a pilot-scale (500 L) adventitious root culture system was established. In the pilot-scale bioreactor, adventitious roots were elicitor-treated with 100 μM methyl jasmonate (MJ) on day 28. After 1 week of elicitation, 1.75 kg dry root biomass was harvested containing 60.41 mg g^?1 DW of total phenolics, 16.45 mg g^?1 DW of total flavonoids, and 33.44 mg g^?1 DW of total caffeic acid derivatives. Among the caffeic acid derivatives, the accumulation of echinacoside (the major bioactive compound) in MJ-treated adventitious roots grown in the 500-L bioreactor was the highest (12.3 mg g^?1 DW), which is approximately threefold more than the non-MJ-treated roots cultured in 5- and 20-L bioreactors.     

Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Biosafe Ginseng: A Novel Source for Human Well‐Being

Given the enormous biodiversity in field cultivation, bioengineers were only to look within nature to find new promising phytomolecules or biochemical pathways from plant biofactories. The present review discusses pilot‐scale bioreactor cultivation of adventitious roots of ginseng and the enhanced production of pesticide‐free bioactive phytomolecules. This biotechnological method is described together with a discussion on economic and biosafety aspects. Besides the “classical” tissue culture as a subject of research on ginseng, new developments and efforts in pilot‐scale bioreactor tissue modeling and in the generation of artificial adventitious roots are considered. Additionally, recent progress in the field of medicinal and nutritional roles of adventitious roots produced in a pilot‐scale bioreactor are discussed.     

Gradually scale-up culture in a bioreactor promotes radical scavenging activity of <Emphasis Type="Italic">Panax ginseng</Emphasis> (C. A. Meyer) adventitious roots on 1,1-diphenyl-2-picrylhydrazyl

A scale-up culture of adventitious roots of ginseng was established using a balloon-type bubble bioreactor (BTBB). Maximum growth rates of ~52-fold and ~50-fold in 3 and 5 L BTBBs were obtained, respectively after 40 days of inoculation, which was significantly higher than that in 0.5 L conical flask (~15-fold). Gradually scale-up culture of adventitious roots increased the root biomass, while the contents of ginsenoside and polysaccharides were not affected. This study also revealed that radical scavenging activity of dried adventitious roots on 1,1-diphenyl-2-picrylhydrazyl was higher than that of native roots at 20–100 mg L⁻¹ methanolic extract.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.     

Biotechnological production of plant-based insecticides

The demand for natural and nonpersistent insecticides is increasing day by day. Plant cell cultures could be an alternative to conventional methods of production of insecticides from field-grown plants. In vitro cultured plant cells produce a wide array of insecticides as a part of their secondary metabolism. Their ability to synthesize key enzymes and the manipulation of these could lead to the enhanced production of many insecticides of industrial importance. The development of a high-yielding hairy root culture system for thiophenes, nicotine, and phytoecdysones is of considerable interest. In this article, the current literature on various factors that influence the growth, production, and secretion of six insecticidal compounds, namely, pyrethrins, azadirachtin, thiophenes, nicotine, rotenoids, and phytoecdysones which have been prospects for the scale-up of cell cultures, genetic engineering to obtain transgenic plants, and metabolically engineered plants for increased production of bio-molecules, has been discussed. Environmental safety clearance and the future prospects of application of biomolecules for plant-derived insecticides are presented.     

Effects of cultivation years on effective constituent content of Fritillaria pallidiflora Schernk

Fritillaria pallidiflora Schrenk has been treasured in traditional classic medicine as an antitussive, antiasthmatic and expectorant for hundreds of years. With gradually decreasing wild F. pallidiflora resources, the herb can no longer satisfy the demand. Artificial cultivation is one of the most effective ways to solve the contradiction between supply and demand in the medicinal material market. During the growth of Rhizomes medicinal plants, root biomass and active ingredient content showed dynamic accumulated variation with increasing cultivation years. Up to now, hardly any attempts have been made to investigate the relationship between quality and cultivation years of F. pallidiflora. Therefore, in this paper, we determined the optimum harvesting time by comparing biomass and biological characteristics of F. pallidiflora at different cultivation times. High-performance liquid chromatography with evaporative light scattering detection and phenol-sulfuric acid visible spectrophotometry was performed to determine imperialine and polysaccharide content of F. pallidiflora bulbs. From year 1 to 6 of cultivation, we observed an upward trend in plant height, diameter and dry weight of F. pallidiflora, while water content decreased. Plant height and dry weight increased remarkably during the fourth year of cultivation. The content of imperialine and polysaccharide of F. pallidiflora bulbs, on the other hand, showed an upward trend from year 1 to 3, after which it decreased from year 3 to 6. By comparing plant growth, biomass development and the accumulation of imperialine and polysaccharide, the best harvesting time of F. pallidiflora was determined to be after 4 years of cultivation. Our results showed that it is possible to establish a “safe, effective, stable and controllable” production process, which could play an important role in achieving sustainable utilization of F. pallidiflora resources.     

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Crescentia cujete L. is a widely distributed medicinal tree with a diverse range of phytochemicals used as medicinal compounds. Seedlings of wild-harvested C. cujete were established in vitro and used as the starting material for the establishment of axenic cultures. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 1.0 μmol l⁻¹ kinetin. De novo regeneration was induced on petiole sections cultured onto a medium containing thidiazuron in combination with 2,4-dichlorophenoxyacetic acid. Axenic cultures were also used to test the efficiency of three different cultivation systems for production of biomass of C. cujete. Growth of plantlets in a temporary immersion bioreactor resulted in significant increases in biomass, leaf number, shoot height and transplant efficiency. Plantlets grown in the bioreactors were acclimatized under greenhouse conditions. Together, these experiments have established optimized parameters for propagation and growth of C. cujete plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products.     

Submerged cultivation of Stephania glabra (Roxb.) Miers cells in different systems: Specific features of growth and accumulation of alkaloid stepharine

Strains of a Stephania glabra suspension culture grown in flasks and two types of bioreactors (laboratory-scale bubble and pilot-scale stirred reactors) have been compared according to their growth characteristics and accumulation of the alkaloid stepharine. The best characteristics have been recorded for strains 113 and 261. In the case of batch cultivation in flasks, the maximal accumulation of dry biomass by these strains reaches 19–21 g/l; that of the alkaloid stepharine, 0.30–0.35% of dry biomass. The used strains differ in their response to cultivation scale-up from flasks to bioreactors, strain 254 displaying the lowest adaptation to such changes. A bubble reactor is the most beneficial system for submerged cultivation of S. glabra. The absence of detectable stepharine synthesis on the background of a considerable decrease in all growth characteristics of the cultures has been observed when using a pilot stirred bioreactor. The batch cultures of strains 113 and 261 in a bubble bioreactor accumulate 11–16 g/l of dry biomass containing 0.05–0.16% of the alkaloid. It has been shown that strains 113 and 261 retain satisfactory physiological characteristics in a semi-flow regime of a bubble bioreactor. This scale-up scheme can be used for further industrial cultivation.     

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.     

Adventitious root production and plastic resource allocation to biomass determine burial tolerance in woody plants from central Canadian coastal dunes

BACKGROUND AND AIMS: Burial is a recurrent stress imposed upon plants of coastal dunes. Woody plants are buried on open coastal dunes and in forested areas behind active blowouts; however, little is known about the burial responses and adaptive traits of these species. The objectives of this study were: (a) to determine the growth and morphological responses to burial in sand of seven woody plant species native to central Canadian coastal dunes; and (b) to identify traits that determine burial tolerance in these species. METHODS: Field experiments were conducted to determine the responses of each species to burial. Saplings were exposed to burial treatments of 0, 10, 25, 50 and 75 % of their height. Burial responses were evaluated based on regressions of total biomass, height, adventitious root production and percentage allocation to shoot, root and adventitious root biomass on percentage burial. KEY RESULTS: Pinus strobus and Picea glauca lacked burial tolerance. In response to the burial gradient, these species showed a strong linear decline in total biomass, minimal adventitious root production that peaked at moderate levels (25-50 % burial) and no change in allocation to shoots vs. roots. The tolerant species Juniperus virginiana, Thuja occidentalis and Picea mariana showed a quadratic response to burial, with little change in biomass up to 50 % burial, but a large decline at 75 %. These species produced abundant adventitious roots up to 50 % burial, but did not alter allocation patterns over the range of burial levels. Populus balsamifera and Salix cordata were stimulated by burial. These species showed linear increases in biomass with increasing burial, produced copious adventitious roots across the gradient and showed a clear shift in allocation to vertical shoot growth and adventitious root production at the expense of the original roots under high burial conditions. CONCLUSIONS: Adventitious root production and plastic resource allocation to biomass are adaptive traits of coastal dune woody plants in central Canada, and provide a basis for assessing burial tolerance in woody plants on coastal dunes throughout the world.     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.     

Hairy root research: recent scenario and exciting prospects

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.