Critical Reviews in Biotechnology ,27:29–43,2007
Copyright
c Informa Healthcare ISSN:0738-8551print /1549-7801online DOI:
w wish10.1080/07388550601173918
Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites
Smita Srivastava
and Ashok K.Srivastava Department of Biochemical Engineering and Biotechnology,Indian Institute of Technology,New Delhi,India
ABSTRACT Plant cell cultivations are being considered as an alternative to agricultural process for producing valuable phytochemicals.Since many of the products (condary metabolites)are obtained by direct extraction from plants grown in natural habitat,veral factors can alter their yield.The u of plant cell cultures has overcome veral inconveniences for the production of the condary metabolites.Organized cultures,and especially root cultures,can make a significant contribution in the production of condary metabo-lites.Most of the rearch efforts that u differentiated cultures instead of cell suspension cultures have focud on transformed (hairy)roots.Agrobacterium rhizogenes caus hairy root dia in plants.The neoplastic (cancerous)roots produced by A.rhizogenes infection are characterized by high growth rate,ge-netic stability and growth in hormone free media.The genetically transformed root cultures can produce levels of condary metabolites comparable to that of intact plants.Hairy root cultures offer promi for high production and pro-ductivity of valuable condary metabolites (ud 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 vesls (bioreactors)that permit the growth of interconnected tissues normally unevenly distributed throughout the vesl.Emphasis has focud on designing appropriate bioreactors suitable to culture the delicate and nsitive plant hairy roots.Recent reactors ud for mass production of hairy roots c
an roughly be divided as liquid-pha,gas-pha,or hybrid reactors.The prent review highlights the nature,applications,perspectives and scale up of hairy root cultures for the production of valuable condary metabolites.simplelife
KEYWORDS hairy roots,condary metabolites,bioreactors
护考120道要对多少题INTRODUCTION
The large and diver group of chemicals produced by plants,which include alkaloids,anthraquinones,anthocyanins,flavanoids,saponins and terpenes,has played an important role in the pharmaceuticals,cosmetics,perfumeries,dye-ing and flavor industry.Plants produce many of the compounds through condary metabolism.Secondary metabolites are not esntial to plant growth and hence are produced in small amounts (Kim et al.,2002b).The often ac-cumulate in specialized ichomes at distinct developmental stages,
Address correspondence to Ashok K.Srivastava,Department of Biochemical Engineering and Biotechnology,Indian Institute of Technology,Hauz Khas,New Delhi 110016,India.E-mail:ashokks@in
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making their extraction,isolation and purification dif-ficult (Kim et al.,2002b).The compounds usually have very complicated structures and/or exhibit chi-rality (Mukundan et al.,1997).Conquently,in many cas organic synthesis is not cost effective,and ex-traction from field-grown plants has been the major method ud to economically obtain the important condary metabolites (Balandrin et al.,1985;Dicosmo and Misawa,1995).Depending on the plant species,traditional agricultural methods often require months to years to obtain a crop.Furthermore,the levels of c-ondary metabolites from plants are affected by many factors,including pathogens and climate changes.In addition to factors mentioned above,decread plant resources and high labor cost involved in the extrac-tion of plant condary metabolites have accelerated the u of plant cell cultivation for their effective pro-duction.Plant cell culture is not affected by changes in environmental conditions,so improved production may be available in any place or ason.Studies on the production of uful metabolites by plant cell cultiva-tion have been carried out on an increasing scale since the 1950s.Despite considerable efforts,only a few com-merc
ial process have been achieved using plant cell cultures (Kieran et al.,1997).The biggest challenge of producing condary metabolites from plant cell sus-pension cultures is that condary metabolites are usu-ally produced by specialized cells and/or at distinct de-velopmental stages (Balandrin et al.,1985;Mukundan et al.,1997).Some compounds are not synthesized if the cells remain undifferentiated (Berlin et al.,1985).The undifferentiated plant cell cultures being genetically un-stable often lo,partially or totally,their biosynthetic ability to accumulate condary products (Rokem and Goldberg,1985;Charlwood and Charlwood,1991).A new route for enhancing condary metabolite pro-duction in tissue culture system is by transformation of desirable plant species using the natural vector sys-tem Agrobacterium rhizogenes .It is the causative agent of hairy root dia in plants (Giri and Narasu,2000;Bourgaud et al.,2001).In nature,the gram-negative soil bacterium A.rhizogenes genetically engineers dicotyle-donous plant species into chemical producers of an Agrobacterium food source (opines).This transforma-tion process leads to the emergence of “hairy roots”at the site of infection of the plant (Shanks and Morgan,1999).The genetically transformed (hairy)roots are capable of unlimited growth in culture media free of growth hormones.
Hairy root cultures of a number of dicotyle-donous/monocotyledonous plants have been estab-lished and found to produce the same condary metabolites as natural roots and hence offer a promising
system for condary metabolite production (Mukundan et al.,1997;Doran,2002;Rudrappa et al.,2005).T epfer (1990)has listed some of the plant species in which fast-growing productive hairy root lines have been established.
The greatest advantage of hairy roots is that they often exhibit about the same or greater biosynthetic capacity for condary metabolite production as com-pared to their mother plants (Kim et al.,2002a,b).Many valuable condary metabolites are synthesized in roots in vivo ,and often the synthesis is linked to root dif-ferentiation (Flores et al.,1999).Members of a num-ber of families including Balsaminaceae,Chenopodiaceae,Compositae,Juglandaceae,Labiatae,Moraceae,Ranuncu-laceae,Solanaceae,Asteraceae,Cucurbitaceae,Plumbagi-naceae,Apocynaceae,Asclepiadaceae and Umbelliferae have been reported to induce hairy root dia symptoms on getting wounded and inoculated with A.rhizogenes in a green hou (De Cleene and De Ley,1981;Dawda et al.,1997).Even in cas where a particular condary metabolite accumulates only in the aerial part of an intact plant,hairy root cultures have been shown to accumulate the same metabolite (W allaart et al.,1999).Moreover,transformed roots are often able to regener-ate whole viable plants and maintain their genetic sta-bility during continuous subculturing and plant regen-eration.Hairy root cultures are also known to produce a spectrum of condary metabolites that are not prent in the
parent plant (V eeresham,2004).Furthermore,a transgenic root system offers tremendous potential for introducing additional genes along with the Ri plas-mid,especially with modified genes,into plant cells with A.rhizogenes vector systems.Hairy root cultures have turned out to be a valuable tool to study the bio-chemical properties and the gene expression profile of metabolic pathways.Moreover,hairy root culture can be ud to elucidate the intermediates and key enzymes involved in the biosynthesis of condary metabolites (Hu and Du,2006).Various advantages of hairy root cul-ture highlighted above have led to its promising role in the mass-production of high-value condary metabo-lites.Although scale up of hairy roots is not an easy task due to its complex features,various process are cur-rently under investigation for making its commercial application feasible.Despite the few attempts reported
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in the literature (Wysokinska and Chmiel,1997;Shanks and Morgan,1999;Giri and Narasu,2000;Kim et al.,2002b),a greater insight is required to deal with the chal-lenges of commercial application of hairy root culture technology for production of plant condary metabo-lites.T o comprehend the same,the prent review deals in depth with new perspectives on the prospects and challenges in the effective production of condary metabolites from hairy root culture technology.It dis-cuss the importance of the u of various cultivation methods and the different bioreactors configurations suitable for the scale-up of hairy root culture.
HAIRY ROOT INDUCTION
礼物的英文Discovery
The name “hairy root”was first introduced in the literature by Steward et al.(1900).Riker et al.(1930)later described and named the hairy-root-causing mi-croorganism as Phytomonas rhizogenes ,which was later renamed Agrobacterium rhizogenes .This conclusion was accepted and given wide recognition by many other rearchers (White,1972).The first directed transfor-mation of higher plants using A.rhizogenes was made by Ackermann in 1973(Ackermann,1977).A large number of small,fine,hairy roots covered with root hairs originate directly from the explant in respon to A.rhizo
genes infection (T epfer and T empe,1981)and hence the term “hairy root”.Porter (1991)reported that more than 450species of many different genera and families are known to be susceptible to the infection by A.rhizogenes (Hamill and Lidgett,1997);since then many more additions have been made to the list.
Mechanism
Invasion of dicotyledonous plant tissues by A.rhizo-genes soil bacteria usually occur at a wounded site,pos-sibly caud by inct or mechanical damage.W ounded site produces phenolic compounds that attract A.rhi-zogenes by chemotaxis,which subquently infects the plant cell at the wounded site.This activity caus hairy root dia (a number of small roots protrude as fine hairs at the infection site and proliferate rapidly)(Balandrin et al.,1985).This phenotypic respon (Hairy root)results from the inrtion of T-DNA (transfer DNA)into the plant genome carried by the bacterial Ri-plasmid (root inducing plasmid),which codes for auxin synthesis (Petit et al.,1983;Ambros et al.,1986).
The integrated gment,T-DNA,also contains genes for opine biosynthesis,which are ud by A.rhizogenes as the sole carbon and nitrogen sources for further growth.Products of virulence (vir )g
enes located on non-transferred gment of the Ri plasmid (root induc-ing plasmid)are responsible for excision of the T-DNA for transfer into the plant cell,and possibly for chro-mosomal integration in the nucleus of the recipient cell (Giri and Narasu,2000).
Confirmation
The transformation of a plant cell with A.rhizogenes can be confirmed by typical transformed root morphol-ogy exhibited by hairy roots obtained after infection and their transformed regenerants.As described earlier,transformed roots have an altered phenotype,profusion of laterals,and show lack of geotropism (T epfer,1984).Also,the transformed regenerants of hairy roots inherit an aberrant phenotype in having wrinkled leaves and shortened internode compared to their normal coun-terparts (Chilton et al.,1982;Ooms et al.,1985;Guerche et al.,1987).
Since the opine synthesis in A.rhizogenes infected plant cells is encoded by T-DNA of Ri plasmid (White et al.,1982,1985),its detection rves as an effective biochemical marker in elucidating the transformed na-ture of the cultured root tissue (Petit et al.,1983;T epfer,1984).There are literature reports in which the trans-formation of hairy root culture has been confirmed by the detection of opines through paper electrophoresis (Bakkali et al.,1997;Sasaki et al.,1998;Bais et al.,2001).Althou
gh synthesis of opines is a firm indication that hairy roots are indeed transformed,the expression of opine genes in hairy root tissue may become unstable with time (Kamada et al.,1986).
T-DNA localization in the host plant genome acts as a reliable genetic marker to confirm transforma-tion (Mukundan et al.,1997).There are a number of techniques available to demonstrate and locate T-DNA incorporation in the host plant chromosomal DNA.The include localization of T-DNA by southern hy-bridization (White et al.,1982).It remains one of the earliest ud methods and is also still widely employed today.The transformation of hairy root cultures of Dacus carrota (David et al.,1988),Cinchona ledgeriana (Hamill et al.,1989),Nicotiana rustica (Rhodes et al.,1994),Artemisia annua (Chen et al.,1999),Cucurbita pepo L.(Leljak-Levani et al.,2004)are some of the examples of
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confirmation by southern hybridization.Other meth-ods include verification of the transformed nature of a tissue by screening for the prence of a foreign gene quence by DNA “blot dotting”(Draper and Scott,1988),localization of T-DNA in plant chromosome tis-sue by in situ hybridization (Ambros et al.,1986;Dong et al.,1992),and verification of transgenes as well as determination of changes in a particular gene quence resulting from tissue culture by polymera chain reac-tion (PCR)(Jaziri et al.,1994;Dong et al.,1992).Trans-formed root cultures of (hairy root)Brugmansia candida and Ginkgo biloba L have been confirmed using PCR (Giulietti et al.,1993;Ayadi and Tr´e mouillaux-Guiller,2003);however,it should be noted that it is important to verify that the plants are cured of the agrobacterium to avoid fal positives if PCR is ud.
Characteristicshuh
Hairy roots are characterized by a high degree of lat-eral branching,profusion of root hairs and abnce of geotropism (T epfer,1984).They often grow as fast as or faster than normal roots (untransformed)due to their extensive branching,resulting in the prence of many meristems (Charlwood and Charlwood,1991;Flores et al.,1999)and they do not require phytohormones in the medium (Rao and Ravishankar,2002).The increa in the number of branches is approximately logarithmic during the early stages of growth and thus the overall pattern of growth is similar to cell s
uspension cultures (Flores and Filner,1985;Flores,1986;Flores and Curtis,1992).Owing to the highly organized and small-celled region of the meristem in each lateral,cell cycle times for hairy roots average less than 10h (Gould,1982).Hairy roots do not necessarily require conditioning of the medium but physical and chemical conditioning is known to result in improved growth and metabo-lite production (Rhodes et al.,1987;V eeresham,2004).Stable integration of Ri T-DNA (root inducing transfer DNA)into host plant genome accounts for the genetic stability of transformed root cultures.The most impor-tant characteristic of transformed roots is their capa-bility of synthesizing condary metabolites specific to that plant species from which they have been devel-oped (Doran,1989;Flores,1986,1992).They exhibit biochemical stability that leads to a high growth rate with a stable and high level of production of condary metabolites (Kamada et al.,1986;Aird et al.,1988a,b).
郝彬SECONDARY METABOLITE PRODUCTION FROM HAIRY ROOTS
Various advantages of hairy root culture over cell sus-pension culture include genotypic and biochemical sta-bility,cytodifferentiation and growth in hormone free medium.The factors play a vital role during condary metabolite production.Fast growth,low doubling time,ea of maintenance of hairy roots and their ability to synthesize a large range of chemical compounds offer
an additional advantage as a continuous source for the pro-duction of valuable condary metabolites (Bourgaud et al.,2001).A number of condary metabolites have been reported to be produced from hairy root cultures (Giri and Narasu,2000).Hairy root cultures often ex-hibit about the same or greater biosynthetic capacity for condary metabolite production as compared to their normal roots (Table 1).Hairy roots have also been obrved to synthesize novel condary metabolites,which are not prent in the untransformed (control)tissue (Banerjee et al.,1995).Even in cas where c-ondary metabolites accumulate only in the aerial part of an intact plant,hairy root cultures have been shown to accumulate the metabolites.For example,lawsone nor-mally accumulates only in the aerial part of the plant,but hairy roots of Lawsonia inermis grown in half-or full-strength MS medium (Murashige and Skoog,1962)can produce lawsone under dark conditions (Bakkali et al.,1997).Similarly,artemisinin was thought to accumu-late only in the aerial part of the Artemisia annua plant (W allaart et al.,1999)but veral reports have shown that hairy roots can also produce artemisinin (W eathers et al.,1994;Jaziri et al.,1995;Liu et al.,1999;Giri et al.,2000).Hairy root culture follows a definite growth pattern;however,condary metabolite production may or may not be growth related.Secondary metabolite biosynthe-sis in transformed roots is genetically controlled (Hamill and Rhodes,1988)but it is strongly influenced by nutri-tional and environmental factors (Hilton and Rhodes,1993).Other factors like elicitors (Pitta-Alvarez et al.,2000),biotransformat
ions of precursors and genetic ma-nipulations through the Ri plasmid of A.rhizogenes also influence the yield of condary metabolites from hairy roots (Rao and Ravishankar,2002).T o obtain a high-density culture of roots,the culture conditions should be maintained at the optimum level.The composition of the culture medium affects condary metabolite pro-duction (De-Eknamkul and Ellis,1984).Variables ex-amined for their influence on growth and condary
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TABLE 1Hairy Root in which Specific Metabolites are Synthesized at Levels Higher than in Untransformed Tissue
Plant species免费少儿英语学习
Secondary metabolite
Reference
Atropa belladonna Scopolamine
Bonhomme et al.,2000A.belladonna
Atropine and hysocyamine
Jung and Tepfer,1987Cinchona ledgeriana Quinine,quinidine,and cinchonidine Hamill et al.,1989
Datura innoxia Hysocyamine and scopolamine Shimomura et al.,1991a D.quercifolia Scopolamine and hysocyamine Dupraz et al.,1994D.candida
逆拂Scopolamine and hysocyamine Christen et al.,1991Duboisia leichhardtii Scopolamine Mano et al.,1989Fagopyrun esculentum
(+)Catechin
snowwhite
Trotin et al.,1993
(−)epicatechin-3-O-gallate procyanidin B 2-3’-O-gallate Hyoscyamus niger Hysocyamine and scopol
amine Shimomura et al.,1991a Hyoscyamus niger Scopolamine Zhang et al.,2004Rubia tinctoria
Anthraquinone Saito et al.,1991
Solanum khasianum Solasodine Jacob and Malpathak,2004Tagetes patula
Thiophene Croes et al.,1989Valeriana officinalis Valpotriates
Granicher et al.,1992
metabolite production from hairy roots include differ-ent basal media (Christen et al.,1992),sucro level (Uozumi et al.,1993),exogenous supply of growth hor-mone (Bais et al.,2001),nature of the nitrogen source and their relative amounts (Norton and T owers,1986),and phosphate concentration (Taya et al.,1994).Physi-cal factors including light (Hirata et al.,1991;Y u et al.,2005),temperature (Hilton and Rhodes,1994;Y u et al.,2005),prence of chemicals inducing physical stress (Sim et al.,1994),and magnetic field induction (Kato et al.,1989)have also been reported to affect condary metabolite production from hairy roots.Betacyanin re-lea from hairy roots of Beta vulgaris was achieved by oxygen starvation (Giri and Narasu,2000).Addition of XAD-2,liquid paraffin stimulated the production of shikonin (Shimomura et al.,1991b).Permeabilization treatment using Tween-80(Pol
yoxy ethylene sorbilane monolaurate)relead a high yield of hyoscyamine from roots of Datura innoxia without any detrimental effects (Boitel et al.,1995).Treatment with 5mM hy-drogen peroxide induced a transient relea of tropane alkaloids from transformed roots without affecting vi-ability (Lee et al.,1998).The fact that individual hairy roots may have different requirements for nutrient con-ditions suggests that the culture conditions should be optimized parately for each species and for individual clones.Despite such attempts sometimes the efficiency of condary metabolite production is not as desired.Metabolic engineering offers new perspectives for im-proving the production of condary metabolites by
the over-expression of single genes in hairy root cul-ture.This approach may lead to an increa of some enzymes involved in metabolism and conquently re-sults in the accumulation of the target products (Hu and Du,2006).The hairy roots of A.belladonna transformed with the rabbit P4502E1gene displayed incread lev-els of the metabolites (Banerjee et al.,2002).Catharan-thus rous hairy roots harboring hamster 3-hydroxy-3-methylglutaryl coenzyme A (CoA)reducta (HMGR)cDNA without the membrane-binding domain were found to produce more ajmalicine and cantharanthine or rpentine and campesterol than the control (Ayora-Talavera et al.,2002).Secondary metabolite production has also been improved by over-expression of enzymes t
hat are already located in a plant (Hu and Du,2006).The tobacco putrescine N -methyltransfersa (PMT)was transformed into Datura metel L.and Hyoscyamus muticus L.(Moyano et al.,2003).The enzyme catalyzed the first committed step in the tropane alkaloid path-way and stimulated the growth of transgenic roots and the accumulation of tropane alkaloid.Oxygen defi-ciency is a usual problem in hairy root culture caud by poor mixing and mass transfer conditions.T o improve the low oxygen conditions that affect growth during fermentation,two enzymes,namely (ADH)and pyru-vate decarboxyla,were transferred into the hairy roots of Arabidopsis thaliana L.The transformant root lines maintained a similar growth rate under conditions of low oxygen to the rate achieved with full aeration (Shiao et al.,2003).
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