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

“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.     

Production of biopesticide azadirachtin using plant cell and hairy root cultures

The extensive use of nondegradable chemical pesticides for pest management has developed serious environmental hazards. This has necessitated the urgent need to switch over to an alternative mode of biopesticide development for mass agriculture and field crop protection. Azadirachta indica A. Juss (commonly known as neem) houses a plethora of bioactive secondary metabolites with azadirachtin being the most active constituent explored in the sector of ecofriendly and biodegradable biopesticides characterized by low toxicity toward nontarget organisms. It has been reported that the highest content of azadirachtin and related limonoids is present in the seeds, available once in a year. Moreover, the inconsistent content and purity of the metabolites in whole plant makes it imperative to tap the potential of in vitro plant tissue culture applications, which would allow for several controlled manipulations for better yield and productivities. This review gives a summarized literature of the applied research and achievements in plant cell/hairy cultures of A. indica A. Juss mainly in context with the biopesticide azadirachtin and applications thereof.     

An efficient in vitro shoot regeneration from leaf petiolar explants and ex vitro rooting of Bixa orellana L.- A dye yielding plant

Physiology and Molecular Biology of Plants - Bixa orellana L. (Bixaceae) is a multipurpose tree grown for the production of commercially important dyes. In the present study, an efficient,...     

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.     

Rhizosecretion of recombinant proteins from plant hairy roots

Rhizosecretion of a target protein in the hydroponic medium provides an alternative manufacturing platform that simplifies the downstream purification procedure and increases protein yield. In order to increase the production rates of rhizosecreted proteins, we have exploited the ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP). The secretion of SEAP from hairy roots induced on the stems of transgenic tobacco plants was 5–7 times higher than that from adventitious transgenic roots.Abbreviations mRNA Messenger RNA - Ri Root-inducing - SEAP Secreted alkaline phosphataseCommunicated by W. Harwood     

Psoralen production in hairy roots and adventitious roots cultures of Psoralea coryfolia

Psoralea corylifolia is an endangered plant producing various compounds of medical importance. Adventitious roots and hairy roots were induced in cultures prepared from hypocotyl explants. Psoralen content was evaluated in both root types grown either in suspension cultures or on agar solidified medium. Psoralen content was ~3 mg g⁻¹ DW in suspension grown hairy roots being higher than in solid grown hairy roots and in solid and suspension-grown adventitious roots.     

Automatic image recognition to determine morphological development and secondary metabolite accumulation in hairy root networks

This study focuses on the morphological development and secondary metabolite production of the red pigments from the group of betacyanins in hairy roots of Beta vulgaris. We demonstrate a working, medium throughput, customized, automatic image recognition solution for hairy roots on agar plates including the evaluation of 12 experimental samples. Image acquisition is conducted under comparable para‐meters using a tripod with light emitting diode background lighting and a digital single lens reflex camera. The server‐based image recognition system developed together with Wimasis GmbH, Munich, Germany helps to obtain not only quantitative values for morphological parameters, such as segment lengths and widths or metabolite concentrations, but also global parameters of root growth, such as total root length or the number of branching points. Using timed diagrams the development of the total root length, the total number of branching points, and the mean pigment concentration during the cultivation period were determined. The generated data present the basis for detailed mathematical modeling in order to achieve a structured growth model for hairy roots. A mathematical model for growth of hairy roots can be used to decrease experimental efforts as well as to optimize cultivation conditions and the bioreactor design.     

荞麦高频离体再生及发根农杆菌转化体系的建立

荞麦无菌苗下胚轴切段在不同激素配比的MS培养基上诱导愈伤组织,出愈率均为100％。在2．0mg/L2,4-D和1．5mg/L 6-BA组合下诱导产生的愈伤组织;转入2．0mg/L 6-BA和1．0mg/L KT的MS培养基,再生苗分化率在80％以上。根尖色体分析表明再生植株具一定的遗传稳定性。发根农杆菌A4转化荞麦下胚轴和子叶获得发状根,纸电泳检测所有随机取样测定的发状根均有相应冠瘿碱的存在。     

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l^–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l^–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed. Received: 24 February 1997 / Revision received: 10 July 1997 / Accepted: 28 July 1997     

不定根发生机理的研究进展

综述了近年来试管苗不定根发生机理的研究进展,在不定根发生过程中,专一基因的表达,内源激素水平的动态变化,酶活性的变化,多胺和钙水平以及碳水化合物水平的变化与不定根诱导,根原基形成和根发育生长有密切关系。自发性生根和诱导生根系统的植物或组织在不定根发生过程中存在较大的生理生化差异。通过了解不定根发生机理的研究动态,有助于更好地研究试管苗生根和试管苗商品化生产。     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

Design of bioreactors suitable for plant cell and tissue cultures

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10 years. Plants for human health in the post-genome era, PSE congress 26.8.2007–29.8.2007, Helsinki.     

Application of plant tissue cultures in phytoremediation research: Incentives and limitations

The aim of this review is to critically assess the benefits and limitations associated with the use of in vitro plant cell and organ cultures as research tools in phytoremediation studies. Plant tissue cultures such as callus, cell suspensions, and hairy roots are applied frequently in phytoremediation research as model plant systems. In vitro cultures offer a range of experimental advantages in studies aimed at examining the intrinsic metabolic capabilities of plant cells and their capacity for toxicity tolerance. The ability to identify the contributions of plant cells to pollutant uptake and detoxification without interference from microorganisms is of particular significance in the search for fundamental knowledge about plants. However, if the ultimate goal of plant tissue culture experiments is the development of practical phytoremediation technology, the limitations inherent in the use of in vitro cultures as a representative of whole plants in the field must be recognized. The bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution are likely to be substantially different in the two systems; this can lead to qualitative as well as quantitative differences in metabolic profiles and tolerance characteristics. Yet, many studies have demonstrated that plant tissue cultures are an extremely valuable tool in phytoremediation research. The results derived from tissue cultures can be used to predict the responses of plants to environmental contaminants, and to improve the design and thus reduce the cost of subsequent conventional whole plant experiments. Biotechnol. Bioeng. 2009;103: 60–76.     

Influence of inoculum density and aeration volume on biomass and bioactive compound production in bulb-type bubble bioreactor cultures of Eleutherococcus koreanum Nakai

This study deals with the effects of initial inoculum density and aeration volume on biomass and bioactive compound production in adventitious roots of Eleutherococcus koreanum Nakai in bulb-type bubble bioreactors (3-L capacity). While the fresh and dry weights of the roots increased with increasing inoculum density, the highest percentage dry weight and accumulation of total target compounds (eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids) were noted at an inoculum density of 5.0 g L⁻¹. Poor aeration volume (0.05 vvm) stunted root growth, and high aeration volume (0.4 vvm) caused physiological disorders. Moreover, an inoculum density of 5.0 g L⁻¹ and an aeration volume of 0.1 vvm resulted in the highest concentration of total target compounds and least root death. Such optimization of culture conditions will be beneficial for the large-scale production of E. koreanum biomass and bioactive compounds.     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor

The effects of inoculum density, aeration volume and culture period on accumulation of biomass and secondary metabolites in adventitious roots of Hypericum perforatum in balloon type airlift bioreactors (3 l capacity) were investigated. The greatest increment of biomass as well as metabolite content occurred at an inoculum density of 3 g l⁻¹ and an aeration volume of 0.1 vvm. After 6 weeks of culture, an approximately 50-fold increase in biomass was recorded containing 60.11 mg g⁻¹ dry weight (DW) of phenolics, 42.7 mg g⁻¹ DW of flavonoids and 0.80 mg g⁻¹ DW of chlorogenic acid. Liquid chromatography–mass spectroscopy/mass spectroscopy demonstrated that the presence of quercetin and hyperoside in adventitious roots at a level of 1.33 and 14.01 μg g⁻¹ DW, respectively after 6 weeks of culture. The results suggest scale-up of adventitious root culture of H. perforatum for the production of chlorogenic acid, quercetin and hyperoside is feasible.     

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.     

Cell fate switch during in vitro plant organogenesis

Plant mature cells have the capability to reverse their state of differenUation and produce new organs under cultured conditions. Two phases, dedifferentiation and redifferentiation, are commonly characterized during in vitro organogenesis.In these processes, cells undergo fate switch several times regulated by both extrinsic and intrinsic factors, which are associated with reentry to the cell cycle, the balance between euchromatin and heterochromatin, reprogramming of gene expression, and so forth. This short article reviews the advances in the mechanism of organ regeneration from plant somatic cells in molecular, genomic and epigenetic aspects, aiming to provide important information on the mechanism underlying cell fate switch during in vitro plant organogenesis.     

Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro

Although use of embryonic or seedling tissues for mass clonal micropropagation in vitro in conifer reforestation programmes is questionable, there is a potential application in the regeneration of plants from scarce and costly seed derived from controlled pollination. In addition, in vitro culture shortens considerably the lag phase in numbers during the initial stages of vegetative propagation via rooted cuttings. Successive steps of the present technique are described whereby cotyledonary needles (secondary explants) were subcultured on a hormone-free medium after administration of cytokinin or auxin to 14-day-old seedling (primary) explants of Picea abies. For bud induction, N⁶-benzyladenine (BA) was applied either as a short-duration (3 h), high-concentration (125 μM) pulse or by vacuum infiltration and incubation in a BA-containmg (5 μM) infusion medium. Induced adventitious shoots were elongated with the aid of far-red light and rooted in vivo after a long-duration (12 h), high-concentration (625 μM) application of indolebutyric acid. Pulse and infusion treatments resulted in the induction of greater numbers of adventitious buds (average of 12 per needle) over a three to four week shorter culture period than was the case with the conventional inclusion of growth regulators in the agarified medium. No exogenous auxin was required in the bud-induction programme; its inclusion even at nanomolar levels promoted histo- rather than morphogenesis. In cotyledonary needles, to the primary explants of which BA was applied as a pulse or by infusion, the cell divisions which gave rise to the meristemoids from which adventitious buds were produced, appeared to commence mainly in undifferentiated hypodermal layer cells but also in the mesophyll immediately below. By contrast, where BA was incorporated in the agarified medium the first divisions occurred mainly in cells of the epidermal layer. A number of factors affected plantlet regeneration, for instance seed variability, age of seedlings, and mode of application of growth substances. It should also be accepted that the xeromorphic nature of the conifer leaf might impose physiological and morphological constraints on its culture in vitro that could militate against easy morphogenic manipulation. It is deemed essential that the current mean ratio of regenerated plants to cotyledonary needles of 1:1 be increased 10 to 20 fold in order to approach commercial feasibility.