Introduction Agrobacterium Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience-bio:

2 A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22

1. Introduction

Plants remain a major source of pharmaceuticals and fine chemicals. Despite considerable efforts, only a few commercial processes have been achieved using cell cultures e.g. shiko- nin, berberine. The major constraint with cell cultures is that they are genetically unstable and cultured cells tend to produce low yields of secondary metabolites. A new route for en- hancing secondary metabolite production is by transformation using the natural vector system Agrobacterium rhizogenes , the causative agent of hairy root disease in plants. Genetically transformed hairy roots obtained by infection of plants with A. rhizogenes , a gram-negative soil bacterium, offers a promising system for secondary metabolite production [1]. The fast growing hairy roots are unique in their genetic and biosynthetic stability and their fast growth offers an additional advantage. These fast growing hairy roots can be used as a continuous source for the production of valuable secondary metabolites. Moreover, transformed roots are able to regenerate whole viable plants and maintain their genetic stability during further subculturing and plant regeneration.

2. Agrobacterium

and Ri T-DNA genes Agrobacterium recognizes some signal molecules exuded by susceptible wounded plant cells and becomes attached to it chemotactic response. Infection of plants with A. rhizo- genes causes development of hairy roots at the site of infection. The rhizogenic strains con- tain a single copy of a large Ri plasmid. In the Agropine Ri plasmid T-DNA is referred to as left T-DNA T L -DNA and right T-DNA T R -DNA. T R T-DNA contains genes homologous to Ti plasmid tumor inducing genes. Genes involved in agropine synthesis are also located in the T R DNA region. T-DNA is transferred to wounded plant cells and it gets stably integrated into the host genome [2]. Genes encoded in T-DNA are of bacterial origin but have eukary- otic regulatory sequences enabling their expression in infected plant cells. Synthesis of aux- ins can be ascribed to the T R -DNA. However, even in the absence of T R -DNA directed auxin synthesis as in the mannopine type which lacks tms loci, root induction occurs. Genes of Ri T L -DNA direct the synthesis of a substance that recruits the cells to differentiate into roots under the influence of endogenous auxin synthesis [3,4]. With the exception of border sequences, none of the other T-DNA sequences are required for the transfer. Virulence genes that form the vir region of the Ri plasmid, and chv genes found on bacterial chromosomes mediate transfer of T-DNA. Transcription of the vir region is in- duced by various phenolic compounds released by wounded plant cells such as acetosyringone and a -hydroxy-actosyringone. Recalcitrant plant species for transformation can be transformed by inducing the vir genes of the bacteria by signal molecules or it can be achieved in vitro by co-cultivating Agrobacterium with wounded tissues or in media that contains signal molecules [5]. Acetosyringone or related compounds have been reported to increase Agrobacterium medi- ated transformation frequencies in a number of plant species [6]. Various sugars also act syner- gistically with acetosyringone to induce high level of vir gene expression. Different strains of Agrobacterium rhizogenes vary in their transforming ability [7,8]. Hairy roots obtained by in- fection with different bacterial strains exhibit different morphologies. The differences in viru- lence and morphology can be explained by the different plasmids harbored by the strains [9]. A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 3 The growth medium has a significant effect on hairy root induction. High salt media such as LS [10] or MS [11] favors hairy root formation in some plants. Low salt media such as B 5 [12] favor excessive bacterial multiplication in the medium and therefore the explant needs to be transferred several times to fresh antibiotic containing medium before incubation. The bacterial concentration also plays an important role for the production of transformed roots, suboptimal concentrations may result in lower availability of bacteria for transforming the plant cells while high concentrations may decrease it by competitive inhibition [7]. Hairy roots are fast growing and plagiotropic and require no external supply of growth hormones; the plagiotropic characteristic is advantageous as it increases the aeration in liquid medium and roots grown in air have an elevated accumulation of biomass. 2.1. Secondary metabolite production Hairy root cultures are characterized by a high growth rate and are able to synthesize root de- rived secondary metabolites. Normally, root cultures need an exogenous phytohormone supply and grow very slowly, resulting in poor or negligible secondary metabolite synthesis. However, the use of hairy root cultures has revolutionized the role of plant tissue culture for secondary metabolite synthesis. These hairy roots are unique in their genetic and biosynthetic stability. Their fast growth, low doubling time, ease of maintenance, and ability to synthesize a range of chemical compounds offers an additional advantage as a continuous source for the production of valuable secondary metabolites. To obtain a high-density culture of roots, the culture condi- tions should be maintained at the optimum level. Hairy root cultures follow a definite growth pattern, however, the metabolite production may not be growth related. Hairy roots also offer a valuable source of root derived phytochemicals that are useful as pharmaceuticals, cosmetics, and food additives. These roots can also synthesize more than a single metabolite and therefore prove economical for commercial production purposes. Transformed roots of many plant spe- cies have been widely studied for the in vitro production of secondary metabolites [13–17] Ta- ble 1. Transformed root lines can be a promising source for the constant and standardized pro- duction of secondary metabolites. Hairy root cultures produce secondary metabolites over successive generations without losing genetic or biosynthetic stability. This property can be uti- lized by genetic manipulations to increase biosynthetic capacity. Sevon et al. [18] characterized transgenic plants derived from hairy root cultures of Hyoscyamus muticus and concluded that a single hairy root that arises from the explant tissue is a clone. Secondary metabolite biosynthesis in transformed roots is genetically controlled but it is influenced by nutritional and environmental factors. The composition of the culture medium affects growth and secondary metabolite production [8,19]. The sucrose level, exogenous growth hormone, the nature of the nitrogen source and their relative amounts, light, tempera- ture and the presence of chemicals can all affect growth, total biomass yield, and secondary metabolite production [20]. Sucrose is the best source of carbon and is hydrolyzed into glu- cose and fructose by plant cells during assimilation; its rate of uptake varies in different plant cells [21]. In hairy roots the source of new cells are in the tips so proliferation occurs only at the apical meristem and laterals form behind the elongation zone. Such a defined growth pat- tern leads to steady accumulation of biomass in root cultures. To obtain a high density root culture, the culture conditions should be maintained at the optimum level. Hairy root cultures 4 A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 Table 1 Secondary metabolite production from hairy root cultures Plant Secondary metabolite References Aconitum heterophyllum Aconites [8] Ajuga replans var. atropurpurea Phytoecdysteroids [81] Ambrosia sps. Polyacetylenes and thiophenes [82] Amsonia elliptica Indole alkaloids [39] Anisodus luridus Tropane alkaloids [83] Armoracia laphthifolia Peroxidase, Isoperoxidase, Fusicoccin [84,85] Artemisia absinthum Essential oils [86] Artemisia annua Artemisinin [87–90] Astragalus mongholicus Cycloartane saponin [91] Atropa belladonna Atropine [24,92] Azadirachta indica A. Juss. Azadirachtin [93] Beta vulgaris Betalain pigments [13,94] Bidens sps. Polyacetylenes and thiophenes [82] Brugmansia candida Tropane alkaloids [95] Calystegia sepium Cuscohygrine [96,97] Campanula medium Polyacetylenes [98] Carthamus Thiophenes [82] Cassia obtusifolia Anthraquinone [99,100] Polypeptide pigments Catharanthus roseus Indole alkaloids, Ajmalicine [101–103] Catharanthus tricophyllus Indole alkaloids [104] Centranthus ruber Valepotriates [92,105] Chaenatis douglasis Thiarubrins [106] Cinchona ledgeriana Quinine [107] Coleus forskohlii Forskolin [108] Coreopsis Polyacetylene [109] Datura candida Scopolamine, Hyoscyamine [110] Datura stramonium Hyoscyamine, Sesquiterpene [111–113] Daucus carota Flavonoids, Anthocyanin [114,115] Digitalis purpurea Cardioactive glycosides [116] Duboisia myoporoides Scopolamine [117] Duboisia leichhardtii Scopolamine [118] Echinacea purpurea Alkamides [119,120] Fagra zanthoxyloids Lam. Benzophenanthridine [121] Furoquinoline alanine Fagopyrum Flavanol [122] Fragaria Polyphenol [123] Geranium thubergee Tannins [124] Glycyrrhiza glabra Flavonoids [125] Gynostemma pentaphyllum Saponin [126] Hyoscyamus albus Tropane alkaloids, Phytoalexins [27,127] Hyoscyamus muticus Tropane alkaloids [18,128] Hyoscyamine, Proline [129] Hyoscyamus niger Hyoscyamine [130] Lactuca virosa Sesquiterpene lactones [131] Leontopodium alpinum Anthocyanins Essential oil [132] A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 5 are able to synthesize stable amounts of phytochemicals but the desired compounds are poorly released into the medium and their accumulation in the roots can be limited by feed- back inhibition. Media manipulations have been reported to aid in the release of metabolites. Betacyanin release from hairy roots of Beta vulgaris was achieved by oxygen starvation. Per- meabilization treatment using Tween-20 Polyoxy ethylene sorbilane monolaurate released high yield of hyoscyamine from roots of Datura innoxia without any detrimental effects [22]. Addition of XAD-2, liquid paraffin stimulated production of shikonin [23]. Lee et al. [24] reported that treatment with 5 mM H 2 O 2 induced a transient release of tropane alkaloids from transformed roots without affecting its viability. Table 1 Continued Plant Secondary metabolite References Linum flavum Lignans 5-methoxy podophyllotoxins [133] Lippia dulcis Sesquiterpenes, hernandulcin [39] Lithospermum erythrorhizon Shikonin, Benzoquinone [23,134] Lobelia cardinalis Polyacetylene glucosides [135] Lobelia inflata Lobeline, Polyacetylene [136] Lotus corniculatus Condensed tannins [137] Nicotiana hesperis Nicotine, Anatabine [138] Nicotiana rustica Nicotine, Anatabine [13] Nicotiana tabacum Nicotine, Anatabine [139] Panax ginseng Saponins [38,78] Panax Hybrid P. ginseng X P. quinqifolium Ginsenosides [140] Papaver somniferum Codeine [141,142] Perezia cuernavcana Sesquiterpene quinone [143] Pimpinella anisum Essential oils [144] Platycodon grandiflorum Polyacetylene glkucosides [145,146] Rauwolfia serpentina Reserpine [16,147] Rubia peregrina Anthraquinones [71] Rubia tinctorum Anthroquinone [147] Rudbeckia sps. Polyacetylenes and thiophenes [82] Salvia miltiorhiza Diterpenoid [6] Scopolia japanica Hyoscyamine [14] Scutellaria baicalensis Flavonoids and phenylethnoids [148] Serratula tinctoria Ecdysteroid [149] Sesamum indicum Naphthoquinone [150] Solanum aculeatissi Steroidal saponins [151] Solanum lacinialum Steroidal alkaloids [1] Solanum aviculare Steroidal alkaloids [40] Swainsona galegifolia Swainsonine [152] Swertia japonica Xanthons [153] Tagetus patula Thiophenes [82,154] Tanacetum parthenium Sesquiterpene coumarin ether [155] Tricosanthes kirilowii maxim var japonicum Defense related proteins [156] Trigonella foenum graecum Diosgenin [157] Valeriana officinalis L. Valepotriates [184] Vinca minor Indole alkaloids vincamine [158] Withania somnifera Withanoloides [159] 6 A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 Production of certain secondary metabolites requires participation of roots and leaves. Metabolic precursors produced by organ-specific enzymes in roots are presumed to be trans- located to aerial parts of the plant for conversion to another product by the leaves. If the ex- pression and activity of enzymes retain the organ specificity in vitro then the end product synthesis will be difficult. A solution to this problem is the root-shoot co-culture using hairy roots and their genetically transformed shoot counterparts shooty teratomas [25,26]. Intergeneric co-culture of genetically transformed hairy roots and shooty teratomas is effec- tive for improving tissue specific secondary metabolites. It resembles the whole plant in local- ized metabolite synthesis and translocation of compounds between organs for further biocon- version. Developments in transgenic organs make co-culture feasible by sharing common medium requirement without any hormone supplement. Besides this, transformed green roots have been obtained in a few species belonging to Asteraceae, Solanaceae, and Cucurbitaceae [27]. Green hairy roots are known to produce certain metabolites that are normally synthesized in green parts of the plant [28]. Chloroplast-dependent reactions are a vital part of certain meta- bolic pathways and could result in a novel pattern of compounds produced by roots. This aspect has been studied recently using soybean hairy roots by functional analysis of the tobacco Rubisco large subunit AN-methyltransferase promoter and its light controlled regulation [29]. 2.2. Scaling up of hairy roots and bioreactors Hairy roots once established can be grown in a medium with low inoculum with a high growth rate. The main constraint for commercial exploitation of hairy root cultures is the scaling up at industrial level. Hairy roots are complicated biocatalysts when it comes to scal- ing up and pose unique challenges. Mechanical agitation causes wounding of hairy roots and leads to callus formation. With a product of sufficiently high value it is feasible to use batch fermentation, harvest the roots, and extract the product. For less valuable products it may be desirable to establish a packed bed of roots to operate the reactor in a continuous process for extended periods collecting the product from the effluent stream. Scale up becomes difficult in providing nutrients from both liquid and gas phases simultaneously. Meristem dependent growth of root cultures in liquid medium results in a root ball with young growing roots on the periphery and a core of older tissue inside. Restriction of nutrient oxygen delivery to the central mass of tissue gives rise to a pocket of senescent tissues. Due to branching, the roots form an interlocked matrix that exhibits a resistance to flow. The main problem with hairy roots is supply of oxygen. The ability to exploit hairy root culture as a source of bioactive chemicals depends on development of suitable bioreactor system where several physical and chemical parameters must be taken into consideration. 2.3. Chemical parameters Nutrient availability is the major chemical factor involved in scaling up. For large-scale cultivation in a bioreactor several aspects play an important role. Periodic estimations of spe- cific nutrients at different periods provide information regarding nutrient uptake, biomass, and metabolite production in bioreactors. Carbon, nitrogen, oxygen, and hydrogen depletion in the medium along with the biomass increase and alkaloid production has been studied in Atropa belladonna by Kwok and Doran [30]. These types of studies can be extended to other A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 7 plant species, where product leaches into the medium and can be recovered by adsorbents. The medium can be rejuvenated to maintain the supply of nutrients. By leaching of second- ary metabolite synthesized by hairy roots, the uptake of nutrients gets altered so leachate needs to be removed regularly. Leaching of phenolics by hairy roots and their oxidation leads to inhibition of uptake of other nutrients which can be avoided by passing the spent medium through adsorbents or metabolite traps. Mass transfer is also an important factor that influences the uptake of nutrients by hairy root cultures. The availability of water and nutrients to any region of a hairy root network in a bioreactor at different periods is known as mass transfer. Hairy root bioreactor chambers become more heterogeneous owing to continuous growth of culture. Oxygen is the most im- portant chemical that needs to be supplied continuously to a bioreactor, judicious mixing leads to efficient oxygen transfer. At initial stages in a bioreactor oxygen transfer is not diffi- cult as the medium contains enough dissolved oxygen to support the growth of the inoculum. Mixing is a very important factor because it serves the dual purpose of supplying dissolved oxygen and driving away the carbon dioxide. The rate of uptake of oxygen by a unit of bio- mass in a unit of time is known as the oxygen transfer coefficient. Other dissolved gaseous metabolites namely carbon dioxide and ethylene also affect the overall productivity. A high biomass transfer resistance by hairy roots will result in development of stagnant zones and non-uniform gaseous metabolite concentrations. The sampling of the inlet and exit gases by passing through rotameter and then to a mass spectrophotometer interphased to a computer is an important factor in analysis of bioreactor functioning. Few attempts have been made for scaling up hairy root cultures for secondary metabolite production. Several bioreactor de- signs have been reported for hairy roots taking into consideration their complicated morphol- ogy and shear sensitivity. These features call for a specially designed bioreactor that permits the growth of interconnected tissue unevenly distributed throughout the culture vessel. The design of bioreactors for hairy root cultures should take into consideration factors such as the requirement for a support matrix and the possibility of flow restriction by the root mass in certain parts of the bioreactor. Moreover, for optimal biomass yields, an even distribution of roots is needed within the bioreactor. For a continuous mode of operation in a bioreactor, the product must be in part released from the roots, and it should be possible to maintain a high density of packed root cultures without loss of viability. Several bioreactor designs have been formulated for hairy root cultures Table 2. 2.3.1. Stirred tank reactor STR This type of bioreactor includes impeller or turbine blades which facilitate mass transfer, and is not usually suitable for hairy root cultures because of the wound response and callus formation that results from the shear stress caused by the impeller rotation [31,32]. However, recently some modified stirred tank bioreactors have been developed. These modified STRs have large impellers and baffles that are agitated at a very low speed; alternatively, hairy roots can be grown in a steel cage inside the STR. 2.3.2. Airlift or submerged bioreactors These are similar to STRs but lack an impeller. Plants cells have large vacuoles and slow growth so hairy roots require comparatively low oxygen supply of about 0.05–0.4 vol of air 8 A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 vol of liquidmin. Humidified air is passed through glass grid that functions as aerators. These have been found to be successful for hairy roots [31,33]. 2.3.3. Bubble column reactor Like an airlift bioreactor, in a bubble column the bubbles create less shear stress, so that it is useful for organized structures such as hairy roots. In this case, the bubbling rate needs to Table 2 Bioreactor types used for the growth and secondary metabolite production from hairy roots Bioreactor Volume Plant species Secondary metabolite References Air-sparged vessel 880 mL Nicotiana rustica Nicotine [160] Stirred tank 330 mL Armoracia rusticana [84] 1.0 L Atropa belladonna Tropane alkaloids [96] 1.0 L Calystegia sepium Tropane alkaloids [96] Stirred tank with impeller isolated 1.0 L Atropa belladonna The impeller is separated by a mesh from the roots Tropane alkaloids [96] 1.0 L Calystegia sepium Tropane alkaloids [96] 12.0 L Datura stramonium Tropane alkaloids [32] 1.0 L Duboisia leichhardtii Scopolamine [118] Fermenter with mechanical stirring Catharanthus tricophyllus Indole alkaloids [104] Air lift 300 mL Armoracia rusticana Roots immobilized in reticulated polyurethane foam [84] 9.0 L Trigonella foenum- graceum Draft tube Diosgenin [161] 9.0 L Trigonella foenum- graceum Nylon mesh replacing draft tube Diosgenin [161] Panax ginseng Saponins [38] Lippia dulcis Hernandulcin [39] Concentrically arranged three sparged set-up used to provide air bubbles 2.0 L Lithospermum erythrorhizon Reactor connected to column containing polymeric adsorbent for continuous production of shikonin [23] 15 L Solanum tuberosum [33] Bubble column 2.5 L Atropa belladonna Tropane alkaloids [162] 1.0 L Catharanthus roseus Indole alkaloids [102] 6.0 L Tagetes patula Thiophene [34] 2.5 L Atropa belladonna Atropine [30] Trickle bed nutrient mist rotating drum 2.0 L Hyoscyamus muticus Tropane alkaloids [163] 1.4 L Beta vulgaris Betacyanins [36] 1.0 L Daucus carota Anthocyanins [35] A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 9 be gradually increased with the growth of hairy roots. Moreover, the division of a bubble col- umn into segments, and installation of multiple spargers increases the mass transfer [34]. 2.3.4. Gas sparged bioreactor Here humidified air is introduced from the bottom of the reactor through a sintered glass sparger. This is useful for mixing and oxygenation. 2.3.5. Turbine blade reactor This is a combination of airliftstirred tank reactor. Here cultivation space is separated from agitation space by stainless steel mesh, so that hairy roots do not come in contact with impeller and the air is introduced from the bottom and dispersed by an eight-blade impeller that stirs the medium. This is efficient for hairy roots [35]. 2.3.6. Mist bioreactor trickle bed reactor Here the medium trickles over a Whatman filter paper containing the biomass, then spent medium is drained from the bottom of the bioreactor to a reservoir and is recirculated at a specific rate. The degree of distribution of liquid varies according to the mechanism of liquid delivery at the top of the reactor chamber. For better dispersion spraying is done by mixing humidified air with medium that creates the mist [36,37]. 2.3.7. Rotating drum bioreactor This consists of a drum-shaped container mounted on rollers for support and rotation. The drum is rotated at only 2–6 rpm to minimize the shear pressure on the hairy roots. Kondo et al. [35] used this system for hairy roots from carrot. Hairy roots adhere to the walls of the re- actor and as the drum rotates the roots tend to break up. To overcome this problem, a poly- urethane foam sheet was fixed on to the surface of the drum, to which the hairy roots get at- tached. This resulted in higher growth without any detachment. In a gas sparged reactor the oxygen is delivered by local transfer from gas bubbles that rise through the reactor and the inoculum gets distributed evenly in the vessel and circulates. Be- sides the cultivation of free roots in a stirred tank reactor and an airlift column, the growth of hairy roots was also tested after immobilization in polyurethane foam. Buitelaar et al. [34] tested growth and thiophene production by Tagetes patula hairy roots in three different types of fermenters and found the best productivity with a bubble column bioreactor. Shimomura et al. [23] used an airlift reactor connected to a column containing a polymorphic adsorbent for continuous production of shikonin by hairy root cultures of Lithospermum erythrorhizon. Yoshikawa and Furuya [38] successfully used an airlift reactor with Panax ginseng hairy roots and for hernandulin production from Lippia dulcis [39]. 2.3.8. Spin filter bioreactor In this bioreactor the rotating filter mixes the cultures and simultaneously allows for spent medium removal and fresh medium addition. 2.4. Parameters that affect productivity Although roots do not require additional illumination, certain hairy roots produce higher levels of metabolites in the presence of light. Bioreactors can be illuminated externally or in- 10 A. Giri, M.L. Narasu Biotechnology Advances 18 2000 1–22 ternally. Temperature also plays an important role. Yu et al. [40] studied the effects of tem- perature on Solanum aviculare hairy roots and found 258C to be optimal. Root morphology is an important parameter for scale-up. The shear sensitivity of hairy root systems is of special interest because their rheology changes continuously because of their indefinite proliferation. Their cell walls are relatively weak and rupture easily which makes them more sensitive to- ward shear stress. Asepsis is another parameter that plays an important role; it can be achieved through effective system design, operating procedures, scheduled checks, and maintenance [36]. All the above-mentioned parameters and variables result in highly complicated opera- tional procedures for successfully running a bioreactor. Computer-aided models can help in planning for efficient product formation and recovery. Kim et al. [41] developed hairy root models based on a branching pattern that helps to monitor shear stress and stoichiometry. Al- biol et al. [42] used an artificial neural network model for plant cell cultures and adapted it for hairy roots. Wyslouzyl et al. [43] found good agreement between experimental models and predicted values. Padmanabhan et al. [183] have done computer vision analysis of so- matic embryos for assessing their ability to be converted to plants; the same type of analysis for hairy roots may be beneficial for assessing their growth, genetic and biosynthetic stabil- ity. For complex hairy root cultures modeling involves multiple factors as rheology, oxygen consumption, and product excretion.

3. Plant regeneration