In Vitro Culture of Lavenders (Lavandula Spp.) and the Production of Secondary Metabolites

Biotechnology Advances 31 (2013) 166–174

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Biotechnology Advances

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Research review paper In vitro culture of lavenders (Lavandula spp.) and the production of secondary metabolites

Sandra Gonçalves, Anabela Romano ⁎

Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology (IBB/CGB), Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal article info abstract

Article history: Lavenders (Lavandula spp., Lamiaceae) are aromatic ornamental plants that are used widely in the food, perfume Received 31 May 2012 and pharmaceutical industries. The large-scale production of lavenders requires efﬁcient in vitro propagation tech- Received in revised form 21 September 2012 niques to avoid the overexploitation of natural populations and to allow the application of biotechnology-based Accepted 22 September 2012 approaches for plant improvement and the production of valuable secondary metabolites. In this review we dis- Available online 27 September 2012 cuss micropropagation methods that have been developed in several lavender species, mainly based on meristem proliferation and organogenesis. Speciﬁc requirements during stages of micropropagation (establishment, shoot Keywords: Cell suspensions multiplication, root induction and acclimatization) and requisites for plant regeneration trough organogenesis, Meristem proliferation as an important step for the implementation of plant improvement programs, were revised. We also discuss differ- Organogenesis ent methods for the in vitro production of valuable secondary metabolites, focusing on the prospects for highly Secondary metabolites scalable cultures to meet the market demand for lavender-derived products. Shoot multiplication © 2012 Elsevier Inc. All rights reserved. Rooting

Contents

1. Introduction ...... 166 2. Importance and applications ...... 167 3. In vitro propagation ...... 167 3.1. Micropropagation of Lavandula spp. by meristem proliferation ...... 168 3.1.1. Establishment of in vitro cultures ...... 168 3.1.2. Shoot multiplication ...... 168 3.1.3. Root induction ...... 168 3.1.4. Acclimatization ...... 169 3.2. Plant regeneration via organogenesis ...... 169 4. Production of secondary metabolites ...... 170 5. Metabolic pathways of essential oil biosynthesis and metabolic engineering studies ...... 170 6. Concluding remarks ...... 171 Acknowledgment ...... 172 References ...... 172

1. Introduction

The lavenders (Lavandula spp.) are ﬂowering plants of the mint Abbreviations: ABA, Abscisic acid; Ads, Adenine hemisulfate; BA, 6-Benzylaminopurine family (Lamiaceae) that are endemic to the Mediterranean region, the ′ or 6-Benzyladenine; CPPU, N-(Chloro-4-pyridyl)-N -phenylurea; IAA, Indole-3-acetic acid; Arabian Peninsula, the Canary Islands and India. There are 39 species, IBA, Indole-3-butyric acid; MS, Murashige and Skoog (1962) medium; NAA, α- Naphthaleneacetic acid; PGR, Plant growth regulator; TDZ, Thidiazuron. numerous hybrids and nearly 400 registered cultivars (Upson and ⁎ Corresponding author. Andrews, 2004), many of which have aromatic and medicinal proper- E-mail address: [email protected] (A. Romano). ties that are highly valued in the fragrance, pharmaceutical, food and

flavor industries. Lavenders are also popular ornamental and decorative Table 1 plants (Touati et al., 2011). Main compounds of the essential oils from some of the most important Lavandula spp. Effective protocols for the propagation of Lavandula spp. are required Speciesa Main compounds Reference to produce large quantities of plants for the ornamentals market or for Lavandula angustifolia Linalol and linalool acetate D'Auria et al. (2005) the extraction of valuable metabolites while avoiding the exploitation Mill. of wild populations. Lavenders may be propagated sexually or asexually, Lavandula latifolia Med. Linalol, 1,8-cineole and camphor Munõz-Bertomeu et although reproduction through seeds is often slow and the plants vary al. (2007a) b significantly in terms of growth rate and essential oil composition. Vege- Lavandula×intermedia Linalol, linalool acetate, camphor Desautels et al. and 1,8-cineole (2009) tative propagation is necessary to produce genetically homogeneous in- Lavandula pedunculata Camphor and 1,8-cineole Zuzarte et al. (2010) dividuals, but multiplication via cuttings is slow and labor-intensive, and (Miller) Cav. root induction is often inefficient. Additional factors such as climate, Lavandula pinnata L. fil. α- and β-phellandrene Figueiredo et al. water availability and susceptibility to diseases may also influence the (1995) Lavandula stoechas L. Fenchon, camphor, myrtenyl Giray et al. (2008) content and composition of essential oils (Chawla, 2009; Pierik, 1987). acetate and 1,8-cineole The limitations discussed above can be overcome using in vitro Lavandula viridis L'Hér 1,8-Cineole and camphor Nogueira and propagation methods under controlled environmental conditions, fa- Romano (2002) cilitating the rapid multiplication of superior clones and the extrac- a Species names as used by the authors. tion of valuable metabolites throughout the year without seasonal b A sterile cross between L. latifolia and L. angustifolia. constraints (Zuzarte et al., 2010). Furthermore, genetic engineering is only possible in conjunction with in vitro culture techniques (Kothari et al., 2010), which could provide lavender producers with The medicinal properties of lavender essential oils were recently opportunities to improve the quality and quantity of essential oils reviewed by Woronuk et al. (2011). These oils can be absorbed into the and thus the economic potential of Lavandula spp. (Dronne et al., human body via three routes: through the respiratory system, by direct 1999a). The last review to consider the propagation of lavenders by contact with the skin and by oral ingestion (Perry and Perry, 2006). Es- tissue culture was published more than 20 years ago, prior to the de- sential oils are widely used in aromatherapy and massage, and many velopment of in vitro culture techniques with a greater emphasis on benefits are claimed for these practices (Cavanagh and Wilkinson, micropropagation (Segura and Calvo, 1991). We therefore summarize 2002). Lavender oils are thought to encourage sleep (Field et al., 2008; more recent work focusing on the application of in vitro propagation Hallschmid et al., 2004) and are widely used to reduce anxiety techniques to Lavandula spp. and the use of in vitro plant cells and tis- (Bradley et al., 2007; Dobetsberger and Buchbauer, 2011; Kritsidima et sues for the production of valuable secondary metabolites. al., 2009). Lavender essential oils have been evaluated for the treatment of dementia (Smallwood et al., 2001)andseveralLavandula spp. are thought to produce oils with antimicrobial, anticholinesterase and anti- 2. Importance and applications oxidant activities (Costa et al., 2012; Hanamanthagouda et al., 2010). In addition to volatiles, lavenders produce further active metabolites such Lavenders are widely used as ornamental and melliferous plants as phytosterols, phenolic acids and flavonoids, which act as antioxidants (Upson and Andrews, 2004) and can also help in the reforestation of (Costa et al., 2011; Georgiev et al., 2009; Nitzsche et al., 2004; Spiridon et fire-damaged areas (González-Coloma et al., 2011). However, the eco- al., 2011). Lavender extracts have been also reported with properties nomic value of Lavandula spp. predominantly relates to the properties suitable for the management of central nervous system disorders of their essential oils, which are strictly regulated by international ISO (Alnamer et al., 2012; Costa et al., 2011). standards and are used both cosmetically and therapeutically for centu- Lavender extracts are also used in the food industry because of their ries. Lavender essential oils are extensively used in the manufacturing health-promoting and nutraceutical effects. Hsu et al. (2007) found of soaps, perfumes, food flavors and other products, as pleasant fra- that aqueous extracts of L. angustifolia Vera and Lavandula stoechas grances or as antimicrobial agents (Cavanagh and Wilkinson, 2002; contained a potent tyrosinase inhibitor and were suitable as food Woronuk et al., 2011). Evidence for the therapeutic use of lavenders bleaching agents, whereas Kovatcheva-Apostolova et al. (2008) found can be traced back to the ancient Romans and Greeks, but a recent in- that the addition of Lavandula vera extract to minced chicken reduced crease in the popularity of alternative medicines has renewed interest lipid oxidation and the loss of α-tocopherol during the storage of cooked in lavenders and their essential oils as natural remedies (Woronuk et meat, confirming the antioxidant activity of the extract in a real food sys- al., 2011). tem. Lavender extracts and essential oils with phytotoxic and insecticidal Lavender essential oils are produced in specialized glandular tri- properties may also be valuable in the agrochemical industry (Haig et al., chomes found on the surface of leaves and flowers. Monoterpenes are 2009; Pavela, 2005). the main components, 50–60 of which have been identified although each species has a characteristic profile in which a small number of prominent molecules determine scent and essential oil properties 3. In vitro propagation (Demissie et al., 2011; Woronuk et al., 2011). Linalool and linalool acetate are the most abundant monoterpenes in popular lavender varieties (Lane There are two principal methods of in vitro propagation: propagation et al., 2010). Essential oil composition is determined primarily by plant from axillary or terminal buds and propagation via the formation of ad- genotype (Table 1), although environmental and propagation conditions ventitious shoots or somatic embryos (George and Debergh, 2008). The can also have a significant impact and composition may also vary be- first method requires pre-existing meristems in the explants, whereas tween different tissues (Demissie et al., 2011; Munõz-Bertomeu et al., the second involves adventitious shoot or embryo formation either 2007a). The essential oil from Lavandula angustifolia is dominated by lin- directly from explant tissues without previously-formed callus (direct or- alool and linalool acetate and the essential oil from Lavandula latifolia by ganogenesis or direct embryogenesis) or indirectly when shoots or em- linalol, 1,8-cineole and camphor (D'Auria et al., 2005; Munõz-Bertomeu bryos regenerate on previously-formed callus or in cell culture (indirect et al., 2007a). On the other hand, Lavandula×intermedia, a hybrid from organogenesis or indirect embryogenesis). In most species (including lav- L. angustifolia and L. latifolia, contains linalol, linalool acetate, camphor enders) micropropagated plants are usually produced by the first method, and 1,8-cineole as major constituents (Desautels et al., 2009). The relative because genetic stability during in vitro propagation is ensured by regen- composition of each of these monoterpenes in essential oils varies, how- eration from existing meristems and the formation of new shoots without ever, considerably between the tree species. an intervening callus phase (Table 2). 168 S. Gonçalves, A. Romano / Biotechnology Advances 31 (2013) 166–174

3.1. Micropropagation of Lavandula spp. by meristem proliferation MS basal medium (Murashige and Skoog, 1962) is widely used for in vitro studies involving Lavandula spp. but some researchers prefer During meristem proliferation, apical buds or nodal segments with less concentrated media (Nobre, 1996) or MS medium with reduced axillary buds are cultured to regenerate multiple shoots without an macronutrient levels (Dias et al., 2002). Cytokinins are required for intervening callus phase (Pati et al., 2006). Generally, micropropagation shoot multiplication, occasionally combined with low concentrations by meristem proliferation is divided into four stages (George and of auxins (Table 3). Shoot multiplication in several Lavandula spp. is Debergh, 2008): the establishment of in vitro cultures (stage 1), shoot promoted by BA (Andrade et al., 1999; Calvo and Segura, 1989; Dias multiplication (stage 2), root induction (stage 3) and acclimatization et al., 2002; Jordan et al., 1998; Sánchez-Gras and Calvo, 1996; (stage 4). Zuzarte et al., 2010). However, the use of adenine hemisulfate (Ads) combined with low levels of NAA in a basal medium containing Margara N30K macrosalts (Margara, 1978) improved shoot multipli- 3.1.1. Establishment of in vitro cultures cation in L. stoechas (Nobre, 1996). In contrast, the addition of NAA The success of micropropagation depends on the composition of the to medium supplemented with BA significantly reduced the shoot nutrient medium, the concentration and combination of plant growth number in cultures of L. vera (Andrade et al., 1999) and Lavandula regulators (PGRs), the culture environment, the genotype and physio- dentata (Echeverrigaray et al., 2005). The use of coconut milk, logical status of the donor plant, and the appropriate choice of explant. which contains vitamins, amino acids and growth regulators, promot- The selection of explants for culture initiation depends on the type of ed shoot multiplication in L. latifolia and L. dentata (Calvo and Segura, culture required, its purpose and the species. Micropropagation by mer- 1989; Jordan et al., 1998; Sánchez-Gras and Calvo, 1996). istem proliferation can be achieved using three types of explants: mer- Early culture conditions can also influence the response of shoots istem tips, shoot tips or nodes (single or multiple). Nodal explants, during multiplication. In L. latifolia and L. dentata shoot proliferation particularly those containing a single node, are the most common was affected by the type and concentration of the cytokinin used in explant type in Lavandula spp. the induction medium (Jordan et al., 1998; Sánchez-Gras and Calvo, The initiation of lavender cultures in vitro involves the surface ster- 1996). In L. pedunculata, Zuzarte et al. (2010) tested two explant ilization of the initial explants. The disinfection firstly with ethanol and types during multiplication (axillary bud explants isolated directly then with a sodium hypochlorite solution is usually effective to initiate from field-grown plants and nodal segments from the adventitious aseptic cultures of most Lavandula species. shoots of previously-established axillary buds) and found that the Culture medium supplemented with 6-benzyladenine (BA), either previously-cultured material proliferated more efficiently than the alone (Andrade et al., 1999; Dias et al., 2002; Nobre, 1996; Sánchez- fresh explants collected in the field. Gras and Calvo, 1996) or in combination with auxins (indole-3-acetic The in vitro culture of lavenders often results in hyperhydricity acid, IAA; indole-3-butyric acid, IBA; α-naphthaleneacetic acid, NAA) (Andrade et al., 1999; Echeverrigaray et al., 2005; Nobre, 1996; Zuzarte (Calvo and Segura, 1989; Jordan et al., 1998; Sánchez-Gras and Calvo, et al., 2010). This physiological malformation appears to be caused by 1996) has been used to establish in vitro cultures in several species (L. the artificial in vitro conditions, especially the humidity in the tissue con- latifolia, L. stoechas, Lavandula viridis and L. vera). Browning of the medium tainer head space, in the presence of excess cytokinins such as BA is sometimes observed, as reported by Nobre (1996) for in vitro cultures (Rojas-Martínez et al., 2010). In L. stoechas, the severity of hyperhydricity of L. stoechas and Zuzarte et al. (2010) for in vitro cultures of Lavandula is correlated with the level of BA in the culture medium and was strongly pedunculata, but this can be prevented by including ascorbic acid in the inhibited in the presence of Ads (Nobre, 1996). Hyperhydricity was also medium and exchanging it frequently. observed in L. vera (Andrade et al., 1999), L. dentata (Echeverrigaray et al., 2005)andL. pedunculata (Zuzarte et al., 2010) in the presence of 3.1.2. Shoot multiplication high levels of BA. The efficacy of shoot multiplication is determined mainly by genotype, in vitro environmental factors and medium composition, but other important physical factors include relative humidity and the ethylene and 3.1.3. Root induction carbon dioxide content of the culture vessel gas phase (Dobránszki and Successful micropropagation requires the efficient induction of roots Teixeira da Silva, 2010). on shoots growing in vitro because roots must form before plantlets can

Table 2 Methods for the in vitro propagation of Lavandula spp. and the explant type. Table 3 a Species Method Explant Reference Basal medium and PGRs used for the in vitro multiplication of lavender shoots and Lavandula latifolia Med. MP HS Calvo and Segura (1989) their impact on multiplication rate and shoot length. Lavandula stoechas L. MP AS, NE Nobre (1996) Species Basal PGR (μM) Mean Shoot Reference Lavandula latifolia Med. MP NE Sánchez-Gras and Calvo (1996) medium number length Lavandula dentata L. MP NE Jordan et al. (1998) of (mm) Lavandula vera DC MP NE Andrade et al. (1999) shoots Lavandinb OLSDronne et al. (1999a,b) Lavandula vera DC O LS Tsuro et al. (1999) L. latifolia MS BA (8.9)+IAA 15.4 >10 Calvo and Segura Lavandula vera DC O LS Tsuro et al. (2000) (0.6) (1989) Lavandula viridis L'Hér MP NE Dias et al. (2002) L. stoechas M Ads (217.2)+ 5–615–20 Nobre (1996) Lavandula dentata L. MP NE Echeverrigaray et al. (2005) NAA (0.05) Lavandula angustifolia CC LS Wang et al. (2007) L. latifolia MS BA (8.88)+IAA 6.2 16 Sánchez-Gras and ‘Munstead’ (0.57) Calvo (1996) Lavandula angustifolia OLSFalk et al. (2009) L. dentata MS BA (8.8) 22.1 Jordan et al. (1998) Mill. L. vera MS TDZ (2.25) 10.78 20.7 Andrade et al. Lavandula pedunculata MP NE Zuzarte et al. (2010) (1999) (Miller) Cav. L. viridis 1/2MS BA (0.67) 11.69 44.39 Dias et al. (2002) L. dentata MS BA (2.2)+IBA 18.60 35.3 Echeverrigaray et HS — Hypocotyl sections; AS — apical shoots; NE — nodal explants; LS — leaf segments; (2.5) al. (2005) MP — meristem proliferation; O — organogenesis; CC — cell culture. L. pedunculata MS BA (1.11) 4.07 5 Zuzarte et al. (2010) a Species names as used by the authors. b Lavandula×intermedia Emeric ex Loiseleur. M — MS with Margara N30K macrosalts (1978). S. Gonçalves, A. Romano / Biotechnology Advances 31 (2013) 166–174 169 be transferred into soil. As shown in Table 4, lavender shoots are generally the same group studied the morphology and essential oil profiles of sev- easy to root even in medium without auxins (Calvo and Segura, 1989; eral L. vera phenotypic variants obtained in the presence of BA, revealing Jordan et al., 1998; Sánchez-Gras and Calvo, 1996; Zuzarte et al., 2010). that none of the regenerated plantlets produced as much essential oil as However, the addition of NAA was necessary to achieve a high frequency the original cultivar (Tsuro et al., 2001). However, the fragrance of three of root induction in L. stoechas (Nobre, 1996), L. vera (Andrade et al., regenerated plantlets differed from the parent, probably reflecting their 1999), L. viridis (Dias et al., 2002)andL. dentata (Echeverrigaray et al., lower acetyltransferase activity. These data suggested that somaclonal 2005). In many Lavandula spp., rooting is favored in media containing variation may be useful to produce variants with different fragrances low concentrations of macronutrients, such as MS with macronutrients and also to generate taller ornamental plants. reduced to 1/2 or 1/4 (Table 4)(Andrade et al., 1999; Calvo and Segura, An efficient method for the regeneration of L. angustifolia plants from 1989; Jordan et al., 1998; Sánchez-Gras and Calvo, 1996). young leaves was described by Falk et al. (2009) using thidiazuron (TDZ) As well as the impact of macronutrients and auxins the effect of other to stimulate the development of callus on incised leaf tissues within factors in rooting was also investigated. Dias et al. (2002) found that in- 2 weeks, followed by spontaneous shoot organogenesis after 2–4addi- creasing the sucrose concentration in the medium from 58.4 to tional weeks in culture. Regenerated shoots were separated from the cal- 87.6 mM significantly increased the frequency of root induction in L. lus and allowed to grow on medium containing NAA. More than 80% of viridis shoots. Mensuali-Sodi et al. (1995) found that root induction in the shoots produced roots when they were dipped in 0.8% IBA powder lavandin (Lavandula×intermedia Emeric ex Loiseleur) was severely and then cultured on media containing 0.05 μMNAA.Theauthorsused inhibited by the endogenous ethylene precursor 1-aminocyclopropane ethyl methanesulfonate to induce mutations in order to produce new va- and by biosynthetic inhibitors of ethylene, suggesting that root induction rieties, and one of the regenerated plants produced essential oil with a and growth were strictly dependent on endogenous ethylene. The in- strikingly different composition to the wild type parent, suggesting that volvement of ethylene on axillary bud proliferation and shoot regenera- mutant lavender plants could be useful for the investigation of monoter- tion from callus of lavandin was also observed (Panizza et al., 1993, pene and sesquiterpene synthesis in plants. 1994, 1997). Ghiorghiţă et al. (2009) studied the morphogenetic behavior of L. angustifolia explants (nodes, shoot tips, internodes and leaves) and 3.1.4. Acclimatization observed that the main response in all the explants was callogenesis, The ultimate success of in vitro propagation requires regenerated and that shoot tips and nodes inoculated on PGR-free MS medium, on plants to be transferred efficiently from the culture medium to the soil MS medium with NAA, and sporadically on media with other PGRs, and acclimatized to free-living autotrophic conditions with negligible produced new plantlets. Dronne et al. (1999a) described the regener- mortality (Naik and Chand, 2011). Plants propagated in vitro are ex- ation of shoots in the lavandin cultivar Grosso 2 with the best results posed to controlled growth conditions including high concentrations achieved using four different media. Callus was generated on MS me- of organic and inorganic nutrients, PGRs, a defined carbon source, dium containing 9 μM BA and 4.5 μM NAA. After 2 weeks in culture, high humidity, low light and poor gaseous exchange, which induce callus tissue was transferred onto MS medium supplemented with structural and physiological changes that render the plants unfittosur- 18 μM BA to trigger bud regeneration, then shoot elongation was in- vive when transferred directly to the field. They must be acclimatized to duced with 1 μM gibberellic acid and roots were induced with 1 μM field or greenhouse conditions gradually. IBA. Dronne et al. (1999b) compared five lavandin cultivars in terms The acclimatization of lavender plants produced in vitro is generally of their capacity for in vitro regeneration and genetic transformation, straightforward, with few morphological changes and high survival finding that both processes were strongly cultivar-dependent. The ef- rates (e.g. 70% in L. latifolia,85–90% in L. stoechas,94%inL. vera and ficiency of bud formation from callus was 67–99% depending on the 80% in L. viridis). However, variable survival rates have been reported cultivar, and although transient expression of the Escherichia coli for L. dentata,e.g.87%byEcheverrigaray et al. (2005) but only 50–55% gusA gene (encoding the enzyme β-glucuronidase) was achieved in by Jordan et al. (1998). The quality of the root system is crucial for suc- all five cultivars, the efficiency of stable Agrobacterium-mediated cessful acclimatization so these results could be explained by differences transformation as demonstrated by the production of kanamycin- in the rooting strategy. Jordan et al. (1998) used auxin-free medium resistant callus ranged from 3% to 89%. whereas Echeverrigaray et al. (2005) used NAA-supplemented medium Wang et al. (2007) described the cultivation of L. angustifolia to significantly increase the number of roots. ‘Monstead’ cells in a bioreactor followed by the regeneration of plants from cell clusters. Callus tissue from leaf explants cultured in MS me- 3.2. Plant regeneration via organogenesis dium supplemented with 0.23 μM 2,4-dichlorophenoxyacetic and 2.22 μM BA was used to initiate suspension cell cultures in liquid me- Organogenesis is the most common in vitro regeneration pathway dium of the same composition. The cell suspensions were then and is often the most important step for successful biotechnology-based subcultured in the same medium also supplemented with 1.89 μM interventions in plants. The organogenetic process is influenced by the abcisic acid (ABA) in a 2.5 l bubble-lift bioreactor. The cell clusters cul- type of explant in addition to environmental factors and the choice of tured in the presence of 1.14 μM IAA produced a larger number of chemical additives (Duclercq et al., 2011). Cells are induced to become dedifferentiated by PGRs (particularly by controlling the ratio between cytokinins and auxins) so that they are competent to respond to new Table 4 physiological and environmental stimuli (Naik and Chand, 2011). Basal medium and PGRs used for in vitro root induction on lavender shoots and impact effect on rooting frequency. As shown in Table 2, shoot regeneration from leaf explants has been achieved in L. vera (Tsuro et al., 1999, 2000), L. angustifolia (Falk et al., Species Basal PGRs (μM) Rooting Reference 2009) and several lavandin cultivars (Dronne et al., 1999a,b). In L. vera, medium (%) Tsuro et al. (1999) observed that the urea-type cytokinin N-(chloro- L. latifolia 1/2MS – 95 Calvo and Segura (1989) 4-pyridyl)-N′-phenylurea (CPPU) was able to induce multiple prominent L. stoechas M NAA (5.4) 100 Nobre (1996) – shoots, whereas the purine-type cytokinin BA induced normal shoots. The L. latifolia 1/2MS 100 Sánchez-Gras and Calvo (1996) – μ L. dentata 1/2MS 100 Jordan et al. (1998) most effective regime for multiple shoot formation was 0.4 MCPPUbut L. vera 1/4MS NAA (1.07) 92.7 Andrade et al. (1999) the resulting shoots did not form roots. This problem was later overcome L. viridis GD NAA (10.74) 100 Dias et al. (2002) by using an ‘open culture system’ and an efficient procedure for plant L. dentata MS NAA (2.5) 100 Echeverrigaray et al. (2005) regeneration was described in which regeneration via multiple shoots L. pedunculata MS – 73.16 Zuzarte et al. (2010) was seven times more efficient than normal (Tsuro et al., 2000). Later, GD — Gresshoff and Doy medium (1972). 170 S. Gonçalves, A. Romano / Biotechnology Advances 31 (2013) 166–174 regenerated plants without an additional rooting step. This protocol 3-hydroxy-3-methlylglutaryl CoA reductase activity, and the content could therefore be useful for the micropropagation and genetic trans- and composition of the essential oil. The total quantity and secretion formation of L. angustifolia ‘Monstead’. rate of the essential oil increased 150% compared to control plantlets Nebauer et al. (2000) reported an Agrobacterium-based transfor- in the presence of BA, whereas the addition of IBA had the opposite ef- mation protocol for spike lavender (L. latifolia) which included a fect (Sudriá et al., 2001). The inhibitory effect of IBA on essential oil ac- 1-day pre-culture step for the leaf explants (on regeneration medi- cumulation was not dependent on the growth rate but was closely um) and a cocultivation period of 24 h followed by regeneration related to the number of glands and their integrity. Also, the choice of under kanamycin selection. This approach achieved a transformation PGRs in the culture medium modified the monoterpene and sesquiter- efficiency of 6% as well as transgene expression in the regenerated pene profiles in the essential oil (Sudriá et al., 1999). Results observed plants, although Mishiba et al. (2000) described another transforma- suggest that commercial-scale L. dentata cultures could be developed tion protocol using friable callus instead of leaf explants which was to produce any target oil component with industrial value. more efficient. Recently, Tsuro and Ikedo (2011) described the effect Nogueira and Romano (2002) and Gonçalves et al. (2008) com- of wild-type Agrobacterium rhizogenes strains on lavandin hairy root pared the chemical profiles of essential oils and volatiles from L. viridis formation and plant regeneration and studied the morphological phe- plants in the field, in vitro cultures and micropropagated plants, finding notypes and essential oil profiles of the regenerated plants. no significant compositional variations among the three sources. Simi- The regeneration of lavender plants by organogenesis could allow the larly, Zuzarte et al. (2010) found no chemical differences in the essential use of biotechnology-based interventions for plant improvement. oils of micropropagated and field-grown L. pedunculata plants. Somaclonal variation and genetic transformation are the major current Studies focusing on the production of secondary metabolites in lav- techniques in this genus, particularly to induce modifications in the es- ender cell cultures and the use of elicitors or biotransformation to alter sential oil profile. In the future, this approach could be used to modify a the metabolic profiles are summarized in Table 5.Theeffectofelicitors wider range of characteristics for agronomic (e.g. to confer pest and on the production of rosmarinic acid by L. vera cells is extensively docu- pathogen resistance) as well as medicinal purposes. mented and the accumulation of this metabolite can be induced by mod- ifying the nutrient supply (Georgiev et al., 2004, 2006, 2007, 2009; Ilieva 4. Production of secondary metabolites and Pavlov, 1997, 1999; Pavlov and Ilieva, 1999; Pavlov et al., 2000, 2001). The profile of volatile metabolites in L. vera cell suspension cul- The ability of cultured plant cells, tissues and organs to produce and turesisinfluenced by the cultivation mode, resulting in differences be- accumulate many of the same valuable chemical compounds as the pa- tween cells maintained in two-phase systems in the presence of resin rental plant has been recognized since the inception of in vitro technol- and in stirred tank bioreactors (Georgiev et al., 2010). ogy (Karuppusamy, 2009)andhasresultedinmanyattemptstouse Nitzsche et al. (2004) compared the metabolite profile of Lavandula plant cells and tissues for the production of valuable metabolites officinalis cell cultures growing under normal conditions, under anox- (Verpoorte et al., 2002). However, the growing demand for natural, re- ic stress and in the presence of jasmonic acid. The principal metabo- newable products has refocused attention on the use of in vitro plant ma- lite in each case was rosmarinic acid but anoxia and jasmonic acid terials for the production of such compounds (Karuppusamy, 2009). treatment induced the synthesis of caffeic acid. The use of L. officinalis Biotechnology allows cells, tissues, organs or entire plants growing in suspension cell cultures for biotransformation has also been reported, vitro to be genetically modified to promote the accumulation of particu- first for the reduction of monoterpenoid aldehydes to their corre- lar metabolites (Rao and Ravishankar, 2002). sponding primary alcohols (Shams-Ardekani et al., 2007), second The advantages of producing valuable secondary metabolites in for the deoxygenation of artemisinin (Patel et al., 2010), and most cell and tissue cultures rather than whole plants include the absence recently for the conversion of the anti-malarial compound β- of seasonal constraints, the reliability and predictability of produc- artemether into its THF-acetate derivative (Patel et al., 2011). tion, and the rapid and efficient isolation of the target compound A number of secondary metabolites have been identified in suspen- (compared to extraction from whole plants). In terms of basic sion cell cultures of L. angustifolia Mill. ssp. angustifolia. Banthorpe et al. research, products from in vitro cultures can still be used as models (1995) used callus lines that accumulate terpenoids for pulse-feeding ex- of whole plants (e.g. to test elicitation) and cell cultures can be periments that had successfully enhanced the biosynthesis of alkaloids radiolabeled so that secondary products can be traced metabolically, other species. Topçu et al. (2007) identified several fatty acids and simple e.g. in animal feeding trials (Karuppusamy, 2009). phenolics, including cis and trans p-coumaric acids and β-sitosterol. The use of differentiated plantlets or organ cultures is required when the target metabolite, such as the case of essential oils, is only 5. Metabolic pathways of essential oil biosynthesis and metabolic produced in specialized plant tissues or glands. Furthermore, the engineering studies yields of secondary metabolites are generally higher and more stable in differentiated tissues/organs than in cells (Karuppusamy, 2009; Mono- and sesquiterpenes (the C10 and C15 isoprenoids, Rao and Ravishankar, 2002). However, cell cultures can be advanta- respectively) are the major fractions of lavender essential oils. geous for the isolation of certain metabolites because the cultures Like other plant isoprenoids mono- and sesquiterpenes are synthe- can be scaled up, some metabolites can be produced at higher yields sized through condensations of the universal five-carbon precursors, in cell cultures compared to differentiated plant material and it is isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl di- also possible to alter the metabolic profiles of cells (Nitzsche et al., phosphate (DMAPP), which are derived from two independent path- 2004; Wink, 1999). ways (Fig. 1). IPP is synthesized from three molecules of acetyl-CoA The effects of different factors on the production of secondary me- by the mevalonic acid (MVA) pathway in the cytosol, while in the plas- tabolites using in vitro lavender shoots, plantlets and cell suspensions tids it is derived from pyruvate and glyceraldehyde-3-phosphate (G3P) is summarized in Table 5. Calvo and Sánchez-Gras (1993) studied the via the 2-methyl-D-erythritol-4-phosphate (MEP) pathway [also called effect of osmotic stress and ABA on monoterpene accumulation in pro- 1-deoxy-D-xylose-5-phosphate (DXP) pathway] (Dudareva et al., 2005; liferating L. latifolia cultures, and found that increasing the osmolarity of Rodriguez-Concepción and Boronat, 2002). Monoterpenes, certain the medium or supplementing it with 25–50 μM ABA resulted in a sig- sesquiterpenes and photosynthesis-related isoprenoids (carotenoids nificant increase in the monoterpene yield while the profile remained and the side chain of chlorophylls and plastoquinones) are derived qualitatively similar to the parent plants. Sudriá et al. (1999, 2001) from the MEP pathway, and certain sesquiterpenes, sterols and the reported the effect of different combinations of PGRs on L. dentata plant- side chain of mitochondrial ubiquinones are derived from the MVA lets, focusing on parameters such as growth, secretory gland activity, pathways (Lichtenthaler, 1999). Flux through MEP is controlled in S. Gonçalves, A. Romano / Biotechnology Advances 31 (2013) 166–174 171

Table 5 Production of secondary metabolites by lavender in vitro shoots, plantlets and cell cultures.

Speciesa Tissue Factor tested Secondary metabolites Reference

L. latifolia Shoots Osmotic stress and ABA Monoterpenes Calvo and Sánchez-Gras (1993) L. angustifolia Cell suspensions Mevalonate, NADPH and NADP+ Terpenoids Banthorpe et al. (1995) L. vera Cell suspensions – Phenolic acids Kovatcheva et al. (1996) L. vera Cell suspensions Sucrose Rosmarinic acid Ilieva and Pavlov (1997) L. vera Cell suspensions Nitrogen Rosmarinic acid Ilieva and Pavlov (1999) L. vera Cell suspensions Phenylalanine Rosmarinic and cafeic acids Pavlov and Ilieva (1999) L. dentata Plantlets BA and IBA Essential oils Sudriá et al. (1999, 2001) L. vera Cell suspensions Nitrogen and phosphate Rosmarinic acid Pavlov et al. (2000) L. vera Cell suspensions Culture system Rosmarinic acid Pavlov et al. (2001) L. vera Plantlets BA Essential oils Tsuro et al. (2001) L. viridis Shoots and plantlets – Essential oils Nogueira and Romano (2002) L. spica Cell suspensions Phosphate, nitrogen, iron, sucrose and inoculum size Blue pigment Trejo-Tapia et al. (2003) L. vera Cell suspensions Temperature Rosmarinic acid Georgiev et al. (2004) L. officinalis Cell suspensions Anoxic stress and jasmonic acid Ciannamic acid derivates Nitzsche et al. (2004) L. vera Cell suspensions Resistance to phenylalanine analogs Rosmarinic acid Georgiev et al. (2006) L. vera Cell suspensions Benzothiadiazole and methyljasmonate Rosmarinic acid Georgiev et al. (2007) L. angustifolia Cell suspensions – Fatty acids Topçu et al. (2007) L. viridis Shoots and plantlets – Volatiles Gonçalves et al. (2008) L. vera Cell suspensions Basal media and cultivation conditions Rosmarinic acid Georgiev et al. (2009) L. vera Cell suspensions Culture system Volatiles Georgiev et al. (2010) L. officinalis Cell suspensions Biotransformation of artemisinin Deoxyartemisinin Patel et al. (2010) L. pedunculata Shoots – Essential oils Zuzarte et al. (2010) L. officinalis Cell suspensions Biotransformation of β-artemether Tetrahydrofuran (THF)-acetate derivative Patel et al. (2011)

a Species names as used by the authors.

part by-deoxy-D-xylulose-5-phosphate synthase (DXS), the first en- without apparent detrimental effects on plant development and fitness. zyme of this pathway. The first and second steps of MVA pathway are However, this does not exclude the hypothesis that the MVA pathway catalysed by 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and could provide IPP precursors for monoterpene and sesquiterpene biosyn- 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), respectively. De- thesis. Thus, in a subsequent work, Munõz-Bertomeu et al. (2007b) inves- spite the subcellular compartmentalization of MEP and MEV pathways, tigated the possible contribution of the MVA pathway in the biosynthesis recent studies suggest the metabolic exchange between the two path- of essential oils. They generated transgenic plants of L. latifolia expressing ways (Cusidó et al., 2007; Dudareva et al., 2005). the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of There is a great interest in improving the quality and yield of lavender 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme essential oils which could be achieved through metabolic engineering. involved in the first step of the MVA pathway. Results demonstrated However, understanding secondary metabolism at the enzyme level is that up-regulation of HMGR1S increased the yield of essential oil (partic- a prerequisite for metabolic engineering of plants aiming the yield im- ularly sesquiterpenes) and sterols, supporting the involvement of the provement of important secondary products. Once the genes that control MVA pathway in the biosynthesis of these compounds. It is not clear, the biosynthesis of essential oil constituents are identified, any step lead- however, whether the increased essential oil yield resulted from the ing to the biosynthesis of these compounds can be theoretically manipu- induction of a latent MVA pathway blocked at HMGR or an up- lated in order to increase yield and/or modify the essential oil profile regulation of an existing MVA pathway. More recently, results of Lane et (Munõz-Bertomeu et al., 2008). Recently, some efforts have been al. (2010) indicate that precursor supply may represent a bottleneck in conducted towards the cloning and functional characterization of mono- the biosynthesis of sesquiterpenes justifying the very small proportion terpene (limonene synthase, linalool synthase, β-phellandrene synthase of sesquiterpenes in lavender essential oil. and 1,8-cineole synthase) and sesquiterpene synthases (bergamotene The production of transgenic plants overexpressing monoterpene synthase) in L. angustifolia (Demissie et al., 2011, 2012; Landmann et synthases is a metabolic engineering approach available to modify the al., 2007). essential oil production in aromatic species although the examples in ar- In a genomic approach towards the understanding of the regulation of omatic species are still scarce. Munõz-Bertomeu et al. (2008) transformed mono- and sesquiterpene synthesis in L. angustifolia, Lane et al. (2010) L. latifolia with the spearmint limonene synthase (MsLS)gene,whichcon- constructed two cDNA libraries from flowers and leaves and obtained verts geranyl diphosphate into limonene, under the regulation of the sequence information for around 15,000 high-quality expressed sequence CaMV 35S constitutive promoter. Authors found quantitative and qualita- tags (ESTs). Authors confirmed that the expression of essential oil-related tive alterations in terpene profiles, particularly increased amounts of biosynthetic genes is virtually restricted to glandular trichomes that limonene, in transgenic plants overexpressing the MsLS gene. Being limo- predominantly utilize the MEP pathway for the production of essential nene a minor constituent of L. latifolia essential oil, the possibility of in- oil constituents. Also, results of Munõz-Bertomeu et al. (2006) support creasing its content by metabolic engineering has obvious consequences the involvement of this pathway in essential oil biosynthesis with DXS for the commercial production of this oil (Munõz-Bertomeu et al., 2008). playing an important role. These authors used the Arabidopsis DSX gene Metabolic engineering approaches could be also applied to modify under the control of the constitutive CaMV 35S promoter and found the production of other secondary metabolites produced by lavenders, that overexpressing DSX enhanced essential oil production in transgenic namely rosmarinic acid, and not only essential oil constituents

T0 plants of L. latifolia. Because successful application of genetic engineer- (Landmann et al., 2011). Progresses in biotechnology offer promising ing depends on the transgene being expressed and inherited in a stable alternatives to improve the quality and yield of lavenders secondary and predictable way authors also studied the expression and inheritance metabolites through metabolic engineering. of the DXS gene in T1 progeny from transgenic lines. It was conﬁrmed that the enhanced monoterpene content observed in the ﬁrst generation was 6. Concluding remarks stable in the subsequent generation. Based on these results, metabolic engineering of the MEP pathway through up-regulation of DSX might be Many protocols have been developed for the in vitro propagation of considered a suitable strategy to increase the essential oil production different lavender species by meristem proliferation or organogenesis, 172 S. Gonçalves, A. Romano / Biotechnology Advances 31 (2013) 166–174 MVA pathway MEP/DXP pathway Cytosol Plastid

Acetoacetyl-CoA Glyceraldehyde-3-phosphate (GAP) + Pyruvic acid

HMGS DXS

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) 1-Deoxy-D-xylose 5-phosphate (DXP)

HMGR DXR

Mevalonate (MVA) 2-C-methyl-D-erythritol 4-phosphate (MEP)

IPPi Isopentenyl diphosphate (IPP) Dimethylallyl diphosphate (DMAPP)

GPPS

MTS Geranyl diphosphate (GPP) Monoterpenes IPP FPPS SES Farnesyl diphosphate (FPP) Sesquiterpenes

Fig. 1. The MVA and MEP pathways of isoprenoid biosynthesis in plants. HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductase; IPPi, isopentenyl diphosphate isomerase; GPPS, geranyl diphosphate synthase; FPPS, farnesyl diphosphate synthase; MTS, monoterpene synthase; SES, sesquiterpene synthase. Multiple steps are indicated by dashed lines. Adapted from Lane et al., 2010.

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