Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Application of cell technologies for production of plant-derived bioactive substances of plant origin

Bioactive substances (BAS) of plant origin are known to play a very important role in modern medicine. Their use, however, is often limited by availability of plant resources and may jeopardize rare species of medicinal plants. Plant cell cultures can serve as a renewable source of valuable secondary metabolites. To the date, however, only few examples of their commercial use are known. The main reasons for such a situation are the insufficient production of secondary metabolites and high cultivation costs. It is possible to increase the performance of plant cell cultures by one or two orders of magnitude using traditional methods, such as selection of highly productive strains, optimization of the medium composition, elicitation, and addition of precursors of secondary metabolite biosynthesis. The progress in molecular biology methods brought about the advent of new means for increasing of the productivity of cell cultures based on the methods of metabolic engineering. Thus, overexpression of genes encoding the enzymes involved in the synthesis of the target product or, by contrast, repression of these genes significantly influences the cell biosynthetic capacity in vitro. Nevertheless, the attempts of the production of many secondary metabolites in plant cell culture were unsuccessful so far, probably due to the peculiarities of the cell culture as an artificial population of plant somatic cells. The use of plant organ culture or transformed roots (hairy root) could turn to be a considerably more efficient solution for this problem. The production of plant-derived secondary metabolites in yeast or bacteria transformed with plant genes is being studied currently. Although the attempts to use metabolic engineering methods were not particularly successful so far, new insights in biochemistry and physiology of secondary metabolism, particularly in regulation and compartmentation of secondary metabolite synthesis as well as mechanisms of their transport and storage make these approaches promising.     

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.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

TOPICAL PAPER: Utilization of Hairy Root Cultures for Production of Secondary Metabolites

The possibility of producing useful chemicals by plant cell cultures has been studied intensively for the past 30 years. However, problems associated with low product yields and culture instabilities have restricted wider industrial application of plant cell culture. The employment of hairy root culture technology, developed in the past 10 years, offers new opportunities for in vitro production of plant secondary metabolites. In contrast to cell suspension cultures, hairy root cultures are characterized by high biosynthetic capacity and genetic as well as biochemical stability. In this review, the establishment and cultivation of hairy root cultures as well as their properties and application for production of secondary metabolites are discussed.     

Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids

Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots.     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants

Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild‐type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial‐scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.     

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.     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

Long-term maintenance of a transgenic Catharanthus roseus hairy root line

Stably transformed transgenic hairy root cultures have the potential to be a valuable production platform for a variety of secondary metabolites. This study reports that a transgenic hairy root culture of Catharanthus roseus has been stably maintained for over 4.5 years. This culture carries a transgene that expresses the green fluorescent protein under the control of the glucocorticoid-inducible promoter. Genomic PCR confirmed the presence of the GFP insert within the hairy roots, and induction with dexamethasone caused a significant (p < 0.02) increase in GFP levels.     

Alkamide production from hairy root cultures of <Emphasis Type="Italic">Echinacea</Emphasis>

Hairy root cultures of Echinacea, one of the most important medicinal plants in the US, represent a valuable alternative to field cultivation for the production of bioactive secondary metabolites. In this study, the three most economically important species of Echinacea (Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia) were readily transformed with two strains of Agrobacterium that produce the hairy root phenotype. Transformed roots of all three species exhibited consistent accelerated growth and increased levels of alkamide production. Optimization of the culture of Echinacea hairy roots was implemented to enhance both growth and alkamide production concomitantly. The use of half-strength Gamborg’s B5 medium supplemented with 3.0% sucrose was twice as effective in maintaining hairy root production than any other media tested. The addition of indolebutyric acid increased the growth rate of roots by as much as 14-fold. Alkamide production increased severalfold in response to the addition of the elicitor, jasmonic acid, but did not respond to the addition of indolebutyric acid. Induced accumulation of the important bioactive compounds, alkamides 2 and 8, was observed both in transformed roots and in response to jasmonic acid treatments. The results of this study demonstrate the efficacy of hairy root cultures of Echinacea for the in vitro production of alkamides and establish guidelines for optimum yield.     

Ri质粒转化的青蒿发根培养及青蒿素的生物合成

用发根农杆菌(Agrobacterium rhizogenes)转化药用植物青蒿(Artemisia annua L．)并建立了发根体外培养系统。Southern杂交、NPT Ⅱ酶的检测证明Ri质粒的T—DNA转移并整合到植物的核基因组上。在发根培养系统中,检测了青蒿的重要次生代谢物一青蒿素的含量,检测了不同理化因子对发根生长及青蒿素含量的影响。结果表明：光照(日光灯,12h光周期,20001x)有利于次生产物青蒿素的积累。培养基的pH值为5．4。蔗糖浓度为3％不仅促进发根的生长,而且促进青蒿素的积累。低浓度萘乙酸(NAA)对发根生长具有促进作用,但抑制青蒿素的合成。赤霉素GA,对发根的生长及次生产物的合成都具有促进作用,其最适浓度为4．8mg／L。     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Elicitation is a possible aid to overcome various difficulties associated with the large‐scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator‐free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor‐specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.     

Morphological and biochemical characteristics of genetically transformed roots of Scutellaria andrachnoides

Genetically transformed roots (hairy roots) and callus tissue of skullcap (Scutellaria andrachnoides Vved.) were for the first time introduced in the in vitro culture. S. andrachnoides is the endemic plant of the Kyrgyzstan. These cultures were characterized by active and stable growth in the hormone-free liquid Gamborg nutrient medium. The growth rate of undifferentiated callus tissue was higher than that of hairy roots, which were the source of this callus. The composition of secondary metabolites in hairy roots, callus tissue, and also the roots of seedlings and adult S. andrachnoides plants was analyzed. It was found that S. andrachnoides hairy roots and callus culture retained the ability for the synthesis of flavones typical for the roots of intact plants. Substantial quantitative differences in secondary metabolites were observed between the roots of juvenile and adult plants. In the seedling roots, which like hairy roots have no secondary thickening, wogonoside, a wogonin glucuronide, predominated among flavones. In the roots of adult plants growing due to the secondary thickening, balcalin, a baicalein glucuronide, was a dominating flavon. It is proposed to use the large-scale in vitro cultivation of roots and especially the rapidly growing callus tissue of S. andrachnoides with a profitable content of only one group of flavones for the development of the biotechnological method for producing wogonin and creating on its basis a new drug — a valuable anticancer agent of plant origin with selective cytotoxic activity.     

Agrobacterium tumefaciens-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.