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BOLETÍN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES Y AROMÁTICAS 17 (5): 453 - 491 (2018) © / ISSN 0717 7917 / www.blacpma.usach.cl

Revisión | Review Traditional uses, conservation status and biotechnological advances for a group of aromatic / medicinal native plants from America

[Usos tradicionales, estado de conservación y avances biotecnológicos para un grupo de plantas aromáticas / medicinales nativas de América]

Jesica Iannicelli1,4, Julián Guariniello1, Sandra Pitta Álvarez2,3 & Alejandro Escandón1

1Instituto de Genética “Ewald A. Favret”, Instituto Nacional de Tecnología Agropecuaria, Hurlingham, Buenos Aires, Argentina 2Laboratorio de Cultivo Experimental de Plantas y Microalgas, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina 3Instituto de Micología y Botánica (INMIBO), Universidad de Buenos Aires-CONICET, Ciudad Autónoma de Buenos Aires, Argentina 4Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) Contactos | Contacts: Jesica IANNICELLI - E-mail address: [email protected]

Abstract: Medicinal and aromatic plants are biologically and economically valuable species because of their intrinsic value as plants, ability to produce secondary metabolites, possible use in the pharmaceutical and food industries, germplasm availability and applications in traditional medicine. In addition, they hold social and economic importance due to the ancestral knowledge they represent and because they are part of the livelihood of many families. Most of them are collected from the wild and are in serious danger of extinction. Through biotechnological tools it is possible to develop their germplasm and obtain new and improved varieties from wild material, while advocating the alternative of production by cultivation instead of extracting it from nature. The objective of this review is to provide an updated perspective on the traditional uses, conservation status and biotechnological advances in a group of 30 plant species native to the American continent.

Keywords: Biotechniques; Aromatic-medicinal plants; Exploitation; Conservation

Resumen: Las plantas medicinales y aromáticas deben ser valoradas tanto por su valor intrínseco como tales, por su capacidad de producir metabolitos secundarios, su posible uso en las industrias farmacéutica y alimentaria y por sus aplicaciones en medicina tradicional. Además, tienen importancia social y económica debido al conocimiento ancestral que representan y porque son parte del sustento de muchas familias. La mayoría de estas especies son recolectadas de la naturaleza y están en grave peligro de extinción. A través de herramientas biotecnológicas es posible desarrollar su germoplasma y obtener variedades nuevas y mejoradas a partir de material silvestre; esta estrategia propicia la alternativa de producción por cultivo en lugar de extraerla de la naturaleza. El objetivo de esta revisión es proporcionar una perspectiva actualizada de los usos tradicionales, el estado de conservación y los avances biotecnológicos en un grupo de 30 especies de plantas nativas del continente americano.

Palabras clave: Biotecnicas; Plantas medicinales aromáticas; Explotación; Conservación

Recibido | Received: May 14, 2018 Aceptado | Accepted: July 7, 2018 Aceptado en versión corregida | Accepted in revised form: August 25, 2018 Publicado en línea | Published online: September 30, 2018 Declaración de intereses | Declaration of interests: This work was conducted with funds from PNHFA-1106094, Instituto Nacional de Tecnología Agropecuaria (INTA). The authors are also thankful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) for fellowship financial support. Este artículo puede ser citado como / This article must be cited as: J Iannicelli, J Guariniello, S Pitta Álvarez, A Escandón. 2018. Traditional uses, conservation status and biotechnological advances for a group of aromatic / medicinal native plants from America. Bol Latinoam Caribe Plant Med Aromat 17 (5): 453 – 491.

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INTRODUCTION wild state. However, they have not yet been properly Biodiversity, defined as the variability of all of and exhaustively explored. nature's living beings in a particular ecosystem or in One of the main problems facing wild plants the entire world (Pathak & Abido, 2014), exists in is that, in recent years, the increased demand resulted three different levels: genes, species and ecosystems, in indiscriminate harvesting (Barata et al., 2016). For each with its own composition and structure. example, in the past decades in Argentina, the Biodiversity sustains every ecosystem, enabling its productive chain for MAPs was very simple and possible exploitation (Cardinale et al., 2012) and, as limited. It consisted of collectors from different such, it is in the origins of agriculture and is the regions that harvested the wild “yuyos” (‘weeds’) to source of all the cultivars and domesticated livestock obtain an additional income, selling them to tourists from the beginning of human civilization. A relevant and to an incipient herbal industry. part of biodiversity is constituted by medicinal and This type of collection, carried out by expert aromatic plants (MAPs), which are species that hands, was performed in harmony with nature, synthesize pharmacologically active secondary favoring development while avoiding predation metabolites (active principles). Strictly speaking, (Elechosa, 2009). This is part of the "good practices aromatic plants are those whose active principles, of collection" and it consists in respecting the plant´s responsible for their characteristic aroma, are volatile cycles, harvesting only the aerial part and, if there are compounds (essential oils). seeds, shaking the branches to distribute them, From the beginning of history, mankind has promoting their germination. These practices allow turned to the Plant Kingdom for food and medicine. sustainable harvests because they exploit the part of In fact, the history of civilization began with the the population that would otherwise die from natural domestication of some plant species and the ensuing causes. Therefore, if off-take does not exceed natural emergence of agriculture (which merits the question: mortality rates, and simply replaces it, harvesting is who domesticated whom?). MAPs rapidly became sustainable (Tarciso et al., 2017). As can be part both of mankind´s kitchen and medicine cabinet. observed, determining sustainable harvesting They made food more attractive by providing new strategies requires basic ecological information and, aromas, tastes and colors. Furthermore, they also although this detailed information is extremely cured or alleviated illnesses and, in some species, useful, a considerable amount of local knowledge has expanded states of consciousness. not been documented or critically examined (Shanley The importance of MAPs can be analyzed & Luz, 2003). from different perspectives. First, they can be Over the years, other protagonists with higher examined as living beings: their ecological niche, commercial influence, such as the liquor, herbal and their intrinsic value as plants, their genes, and the food industries, came into play and, consequently, the nutrients and metabolites codified by those genes. demand increased significantly. Furthermore, the This biological assessment of MAPs intermingles middleman, who sets prices by buying from the small with their economic importance, since they have producers/collectors and supplying the large-scale multiple industrial applications as pharmaceuticals, wholesalers, became increasingly important. The food or perfume (also determined by their genes). In original saddlebag on a donkey became a cart, then a addition, their social importance is manifold, because truck and, with time, dozens of trucks. The collection they not only embody ancestral cultural and religious started to be performed in an unsustainable manner, knowledge, but also represent the livelihood of many using a metal roll that pulled out the material from its families. roots and left a razed mount. The future of MAPs It is estimated that approximately 400,000 worldwide was further endangered by the burning of plant species exist and around 60,000 have medicinal lands that were ultimately assigned to cattle raising, properties (UN COMTRADE, 2013; Barata et al., tourism and real estate business. It is estimated that 2016), but only a few hundred species are cultivated. 15,000 of the global medicinal species are now Although these are estimates, the data presents an endangered because of overharvesting and habitat alarming view concerning the situation of the vast loss (Schippmann et al., 2006; Barata et al., 2016). In majority of MAPs used by man. Our native species Argentina, an outstanding example can be found in are an important source of genetic variability and an the province of Cordoba, where natural vegetation invaluable gene pool, since most of them are in a was lost in the past decades due, mainly, to

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants unstainable collection practices, indiscriminate facilitates the preservation of phytogenetic resources deforestation, urbanization, the advance of the through in vitro culture methods that, through agricultural frontier and periodic fires, among other adequate changes in the culture environment (Scocchi factors (Menseguez et al., 2007; Arias Toledo et al., & Rey, 2010), can decelerate tissue growth and 2010). This situation demands that we become aware extend the time span between subcultures. The of the threats surrounding these genetic resources and ultimate long-term technique for this strategy is address them in order to reverse the present state of cryopreservation, which consists in storage at liquid affairs. nitrogen temperature (-196º C) to suppress growth Modern biotechnology has several tools that until a state of “suspended animation” is reached are being increasingly applied to characterize the (Scocchi & Rey, 2010). genetic diversity of plant germplasm. In this respect, Informatics and technology have also it has an essential role in supporting plant experienced significant advances, commensurable conservation programs and the complementary plant with Biotechnology. The progress in these fields has breeding programs. The incorporation of revolutionized the research paradigms of plant biotechnology in conservation programs requires the breeders and conservationists. Currently, it is possible interaction of multiple and diverse areas, and, in the to access DNA databases and available genes easily near future, professionals from different disciplines and rapidly, thus supplying a genomic map that and with diverse expertise should be included in this allows a more efficient management of genetic effort. resources. However, this is not available for the In modern biotechnology, three areas should majority of the species mentioned in this review. be included as tools in conservation programs: i) Secondary metabolites are organic molecules molecular markers, ii) tissue culture, and iii) synthesized in some plant species and stored in cryopreservation. relatively low concentrations. They don´t play a role Considering that germplasm is a source of an in the growth or development of the plant, but they indeterminate number of genes, molecular markers are significantly important in defense mechanisms are an essential tool to recognize and measure the and as pollinator attractants. These secondary extant and available genetic variability, and to study metabolites bestow high commercial value to natural the structure of different populations and the products due to their enormous potential as drugs and distribution of genes within the ecosystem. This other uses for a wide array of industries. Among would contribute towards the sustainability of in situ these active principles, we can mention pigments, conservation programs and would also secure the essential oils, steroids, phenolic compounds progress of genomic studies in these species (Pathak (coumarins, flavonoids, tannins and lignin), saponins, & Abido, 2014). cardiotonic and cyanogenic glucosides and alkaloids. Tissue culture is perhaps one of the most In the biotechnological productive paradigm, popular tools of modern biotechnology and, in this all these compounds could be produced in vitro, by context, it is highly relevant for the ex situ way of cell, shoot or root cultures, avoiding multiplication of genetic resources, the sanitary status exploitation of the wild resource. There are already of materials and the recovery of plants in risk of several examples of substances that are being extinction. It is also the groundwork for the obtained in vitro, such as paclitaxel, ajmalicine, application of biotechniques, such as transgenesis or atropine, codeine, dopamine, digitoxin and morphine polyploidization, in programs that aim to improve the (Pathak & Abido, 2014). In the context of this paper, production of interesting active principles. In the Taxus and paclitaxel example is revealing: ten addition, tissue culture is a fundamental tool to trees are needed to produce one dose of paclitaxel to sustain germplasm banks through micropropagation treat only one patient, which turns the extraction of techniques. These, in turn, enable establishing paclitaxel from the Taxus bark completely cultivars in the short to medium term, producing unsustainable. Consequently, biotechnology allows synthetic seeds by way of somatic embryogenesis the implementation of strategies for economic and, due to their axenic nature, facilitating the exploitation without threatening the survival of the transportation and exchange of germplasm between species (Tabata, 2006). Furthermore, it produces a nations (Escandón et al., 2010). positive social impact generating new sources of As previously mentioned, tissue culture work and specialized workforce. This would be an

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants alternative for regions rich in biodiversity whose literature and further corroborated that information population must exploit genetic resources to promote with the following bibliography: Martínez (2005); economic development, while preserving biodiversity Elechosa (2009) and The IUCN Red List (2017). for future generations. The objective of this review is to provide an RESULTS updated picture on the traditional uses, conservation 1) Achyrocline satureioides (Lam.) DC. Asteraceae status and biotechnological advances for a group of (“jate'i ka'a”, “marcela”, “marcela hembra”, “vira- 30 MAPs native to the American continent. The vira”). The aerial parts and the inflorescences have species under study were chosen because most of antispasmodic, hepatoprotective, antioxidant, them are employed by the pharmaceutical, cosmetic colagogue, choleretic, smooth muscle relaxant, and food industries, but are exploited in an febrifuge and antitussive properties (Kadanian et al., unsustainible manner, placing them in a vulnerable 2002; Barboza et al., 2009; Retta, 2014). Cosmetic situation. products containing its active principles (mainly Regardless of the success achieved with those quercetin derivates) are commercialized because of techniques, our aim was to show what has been its antioxidant and antiinflammatory properties. It is accomplished and encourage the integration of also used in the manufacture of food products, such biotechnology for the conservation and sustainable as tonic beverages. It was officially incorporated in exploitation of MAP biodiversity. the Brazilian National Pharmacopeia, 4th edition (Juliani et al., 2007; Retta et al., 2010). MATERIALS AND METHODS The current demand is met by collecting Criteria for the selection of plant species material in the wild. This not only affects its ecology, We selected a group of medicinals and aromatic but it is also frequently found mixed with similar plants native to the American continent, used in species that partially share the distribution area, like traditional medicine and exploited by different Achyrocline flaccida (Weinm.) DC. and industries. The botanical, taxonomical and Pseudognaphalium gaudichaudianum (DC.) Anderb. geographic description of each plant can be found in synonymous Gnaphalium gaudichaudianum DC. the following literature: CONABIO (2009); Randall (Retta et al., 2010). A. satureioides is one of the few (2012); Instituto de Botánica Darwinion (2017); The native plants that has been subjected to domestication International Plant Names Index (2017); Missouri and cultivation processes, mainly in Uruguay and Botanical Garden (2017); Flora do Brasil 2020 Brazil (Davies, 2011). However, the traditional (2017); The Plant List (2017). The scientific names propagation method of this species is by seeds or by and current synonyms for each species were cuttings. With the first method, there is no uniformity corroborated in The Plant List (2017). in the population obtained; and with the second one it is possible to obtain few individuals for commercial Search strategy plantations (Severin et al., 2008). In this sense, the We carried out an extensive review of the available use of biotechnology through in vitro culture literature to establish if the species involved were techniques has been proposed in order to obtain a threatened with extinction and if any biotechnological high number of plants with controlled quality. tools had been applied in each case. We used the In the last two decades there have been following databases: Google Scholar, Scopus and the important advances concerning the in vitro MINCyT (Argentine National Ministry for Science introduction and multiplication of “marcela” (Gattuso and Technology) database. The keywords used were: et al., 2007; Severin et al., 2008; Kotik et al., 2014; “biotechnology”, “tissue culture”, “molecular Rosso et al., 2015). All these authors have been markers”, “cultivation” and “crops”. We estimate that successful regenerating shoots and producing for every reference mentioned in this review, five plantlets from nodal segments, cotyledons, references were examined. In many cases, only one hypocotyls and leaf cultures. Among these reports, or two references were found and all of them were Kotik et al. (2014) informed the most promising used. results, with interesting multiplication rates and ex vitro plants transferred and tested in the field. Criteria to evaluate the risk of extinction The National Institute of Agricultural To assess the risk of extinction, we checked the Research (INIA) in Uruguay has amassed an

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants extensive and outstanding experience in the closely related species could be mistakenly included management of this and other related species, both in in or substituted for “black cohosh” products on the traditional cultivation and breeding (Davies, 2011), market, we found only one citation pertaining to the and in different aspects of tissue culture. Recently, a use of the “fingerprint” technique, Amplified new variety was developed by the State University of fragment length polymorphism (AFLP), to identify Campinas (Brasil), which was obtained through the species among other related sympatric species to classic breeding. This variety presents less variability ensure quality control (Zerega et al., 2002). in different phenotypic aspects (Garcia, 2017). However, no improved varieties were yet obtained 3) Adesmia emarginata Clos. Fabaceae, through biotechniques. (“paramela”). The plant is used in traditional medicine, mainly because of its analgesic and diuretic 2) Actaea racemosa L., synonymous: Cimicifuga properties (Fischer et al., 2011). Also, infusions or racemosa (L.) Nutt. Ranunculaceae (“cimicifuga”, decoctions of the dry leaves are used as aphrodisiacs “hierba de cascabel”, “hierba de San Cristóbal”, (Montes & Wilkomirsky, 1985). “hierba rica”, “raíz de cascabel”, “raíces de cohosh Wild harvesting and the increase in the use of negro”, “raíz de culebra negra”, “raíz de serpiente de land for crop and forest cultivation (mainly in Chile) cascabel”). This plant has been employed in the have resulted in the decrease of plant population treatment of menopause symptoms (nocturnal hot density. However, only a few attempts have been flashes and vaginal dryness, among others) as well as made to domesticate and cultivate this species to in irregular menstrual cycles, premenstrual syndrome prevent its extinction. Fischer et al. (2011) reported a and vaginal discharge. In fact, the “cimicifuga” preliminary study to characterize the environment rhizome is used mainly as a dietary supplement and to rescue, propagate and characterize plants both aimed at treating pre-menstrual syndrome, in situ and ex situ. We found no literature regarding menopause and related gynecological problems the use of biotechniques. (Laboratorios Bagó, 2012). In addition, it is used to treat rheumatism, arthritis, dizziness, sleep disorders, 4) Aloysia citriodora Palau., synonyms: Aloysia pain and muscular spasm. Its use as triphylla (L'Hér.) Britton, Lippia citriodora (Palau) antiinflammatory, tonic, diuretic, aperitif, antitussive Kunth, Lippia triphylla (L'Hér.) Kuntze, Verbena and antidiarrheal (Vademécum Colombiano de citriodora (Palau) Cav. Verbenaceae (“cedrón”, Plantas Medicinales, 2008) has also been reported. “cidrinha”, “hierba luisa”, “salvia limão”, “verbena The plant is extensively used as an ornamental. de Indias”). This plant is characterized by its lemon- Notwithstanding the important like aroma. commercialization of this species, cultivated plants In traditional medicine, the leaves are used in represent a minimum part of the total harvest. It was infusions and decoctions as stomachic, digestive, included in the list of rare species (Robbins, 1999) tonic, carminative, eupeptic, in the treatment of and it may become an endangered species since wild dyspepsias, indigestion, nauseas and vomits (Shanley populations have declined because of collection & Luz, 2003), as well as in the treatment of coughs pressure (Lata et al., 2002; Hyung et al., 2005). and colds (Barboza et al., 2009). Tissue culture techniques have been reported As a result of its digestive properties, for conservation and propagation of this endangered “cedrón” is in high demand worldwide by the herbal medicinal plant, but only one report on in vitro industry, and is used as an infusion, either alone or culture for plantlet regeneration through leaf-derived mixed with other herbs (Juliani et al., 2007). calli was found (Lata et al., 2002), and its purpose It is assumed that its essential oil and was plant conservation to increase the number of phenolic compounds (flavonoids, and mainly propagules for cultivation or to replace of natural verbascoside) are responsible for the digestive populations. However, this report did not indicate if properties (Pascual et al., 2001). Concerning the the process included the transfer of ex vitro plants to chemical composition of the essential oil, Elechosa soil. Also, there is only one communication on (2009) reported a large variety of aromas in the same hydroponic culture (Hyung et al., 2005) and the population, and determined the presence of citral, purpose of this study was to determine the potential citronellal, carvone-dihydrocarvone, thujones and for “black cohosh” production in perlite. Although cineol-carvone. The citral type is the most abundant

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants and the most accepted in the market. In Argentina, up 5) Aloysia polystachya (Griseb.) Moldenke, to ten chemotypes have been reported in studies of synonymous: Lippia polystachya Griseb., populations in different provinces, being citral-neral Verbenaceae (“burrito”). The infusion of leaves and the preferred one. However, several chemotypes are flowers is used to treat stomach aches, hepatic also very interesting due to their composition and disorders, slow digestions, indigestions possible application in the aromatic industry, such as (“empachos”), acid reflux, nausea and vomiting the ones rich with limonene-citronellal, β-thujone, (Menseguez et al., 2007; Barboza et al., 2009). It is linalool, carvone, and citronellal (Di Leo Lira et al., demanded by the herbal (“yerbatera”) industries for 2008; Juárez et al., 2012). In turn, in samples the formulation of compounded herbs (Juliani et al., collected in Chile, the main compounds were 2007). Also, the plant has a promising potential for sabinene and 1,8-cineol (Di Leo Lira et al., 2008). the pharmaceutical industry as eupeptic, carminative Progress in several aspects of the propagation and sedative (Mora et al., 2005; Hellión-Ibarrola et of “cedrón” has been made. The specifications for its al., 2008). culture are described in Alonso & Desmarchelier In Argentina, only two chemotypes were (2015). described (Elechosa, 2009): carvone-limonene, with The plant has been subjected to different in digestive and carminative properties attributed to the vitro biotechniques, such as direct propagation presence of carvone (Burdyn et al., 2006), and α- (Severin et al., 2005; Severin et al., 2006; Moradi et thujone-β-thujone, with possible toxic effects since α- al., 2014) and, very recently, propagation through a thujone is mentioned as an animal neurotoxin (Millet, previous callus stage (El-Hawary et al., 2012; 1981). Boustani et al., 2016). All these reports evaluated the There are references regarding experimental production of active principles in the cultures, cultivation of “burrito” in Argentina and Paraguay. highlighting the differences found between in vitro- This species is usually cultivated in home gardens derived materials and wild-types. Both Severin et al. (Alonso & Desmarchelier, 2015), and recent studies (2006) and Moradi et al. (2014) reported high have suggested that these may be important essential oil content in micropropagated plants; reservoirs of unique genetic diversity (Watson & whereas El-Hawary et al. (2012) suggested that Eyzaguirre, 2002). Despite these reports, there are further studies are still required to increase the references of harvesting from the wild (Martínez, flavonoid content in the callus obtained. Besides, a 2005) but no reliable ones concerning the cultivation protocol for the obtention of hairy roots was of “burrito”. described (Figueroa et al., 2013), but no references of In vitro propagation is mentioned only in one the effects on the production of metabolites were paper (Burdyn et al., 2006). In one report, the authors found. described the use of these “burrito” micropropagated Although the taxonomical identification was plantlets for the manufacture of tablets by direct confirmed as Aloysia citriodora (The Plant List, compression of the extract (Aguado et al., 2006). The 2017), there are reports of synonymia for this species, in vitro cultivation of plantlets provided uniform so it can be found with the names Lippia citriodora, materials for the formulation of this product. Also, L. triphylla, A. triphylla (Boustani et al., 2016). the authors highlight the importance of starting from However, very little is known about genetic diversity uniform plant material (in this case micropropagated levels and genetic structure within and between plants) when a pharmaceutical product is developed. populations, not only in this species but between genera (Suárez González et al., 2007). We found only 6) Aniba rosaeodora Ducke. Lauraceae (“palo de a pilot study concerning chloroplast and nuclear rosa”). It is a species with great economic importance DNA polymorphisms cpDNA, Internal transcribed in the international market due to its linalol-rich spacer (ITS) and Inter Simple Sequence Repeats essential oil, used as a fixative in the perfumery (ISSR) with the aim to document the level of genetic industry (Sampaio, 1999). The oil is extracted almost structure with contrasting distributions (natural entirely from the wood, and the extraction method environments versus home gardens) and contrasting results in the destruction of the tree (May & Barata, modes of propagation (natural versus human-induced 2004). The high demand for this product has resulted clonal propagation) (Suárez González et al., 2007). in the overexploitation of the species through the indiscriminate extraction of complete adult

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants individuals. Besides, there are factors related to the considered a weed in various parts of the world physiology and ecology of the species, such as the (Randall, 2012). Different parts of this plant are used intensive predation of the seeds by birds and insects, in chronic skin diseases, and also as emetic, and the extreme recalcitrance and low germination expectorant, demulcent and diuretic; the seeds and rate of the seeds (Yepes et al., 2010). seed oil are employed as a remedy for dysentery, This has led to the decline of many natural ulcers, asthma and other intestinal affections populations and the loss of important variability, to (Brahmachari et al., 2013). This plant produces the point that it has been included as a species alkaloids (protopine, berberine, sanguinarine, inter threatened with risk of extinction in countries such as alia) in all its organs and it is reputed to have Colombia, Brazil, and Surinam (IBAMA, 1992; sedative, sleep-inducing, antiinflammatory and Calderón, 1997; Oldfield et al., 1998; May & Barata, antibacterial effects (Harborne & Williams, 1983; 2004; The IUCN Red List, 2017). Due to this Brahmachari et al., 2013). situation, the export of essential oil has drastically An interesting work reports the use of leaf fallen in the last 30 years. extract of A. mexicana for the synthesis of To reverse this situation, multiplication nanoparticles in the search of value-added products procedures for this species have been developed for biomedical and nanotechnology based industries (Viera, 1970; Sampaio, 1987; dos Santos Britto & (Singh et al., 2010). Sampaio, 2003; Yepes et al., 2010). However, the The plant that is used for medicinal purposes results have not been satisfactory in terms of is harvested in the wild. However, some blog spots increasing survival through the production of indicate that in the United States and Europe it is seedlings, due to the slow growth of the species cultivated as an ornamental. (Valencia et al., 2010). In vitro cell cultures of this species have been Concerning biotechnology, in vitro culture established, and they produce and accumulate techniques to promote the propagation of the species sanguinarine at levels comparable to those in plant have been established. Only two reports dealing with tissues (Ziegler & Facchini, 2008; Trujillo- micropropagation were found (Handa et al., 2005; Villanueva, 2010). However, we did not find further Jardim et al., 2010). These works analyzed aspects of references concerning the use of these cell cultures as the in vitro culture, such as the use of embryos´seeds a source of metabolite production at industrial scales. and bud explants (Handa et al., 2005) or the effects of In addition, there is a protocol that describes transient different growth regulators (Jardim et al., 2010). transformation via A. tumefaciens (Godoy-Hernández Although these reports showed successful results, et al., 2008), but there are no recent reports there were no references concerning further uses of concerning transgenic A. mexicana. the developed materials in conservation programs. Molecular markers were developed for this 8) Baccharis articulata (Lam.) Pers. and 9) B. species to estimate genetic diversity in the trimera (Less.) DC. Asteraceae, (“carquejas”). populations which remained in their natural habitat. Several species of the genus Baccharis are known as To contribute to effective strategies for the “carquejas” because they are very similar conservation and management of this threatened morphologically and cannot be easily distinguished Amazonian forest resource, Angrizani et al. (2013) (Simões-Pires et al., 2005). Also, their uses in evaluated SSR to estimate gene flow and characterize traditional medicine are similar. The aerial non- population genetic diversity, structure and mating woody parts of both species are mainly used in the system of the species. Santos et al. (2008) used manufacture of non-alcoholic bitter beverages Random Amplified Polymorphic DNA (RAPD) and because of their hepatoprotective, digestive and they informed that populations retain high diversity, antiinflammatory properties. Furthermore, infusions mainly in a population that had been protected for and decoctions are traditionally used as antidiabetics more than forty years in a Reserve. They suggest the due totheir antihyperglycemic properties (Juliani et creation of a new reserve as a strategy for the al., 2007; Barboza et al., 2009). Hepatoprotective and protection of the variability of this threatened species. antihyperglycemic properties in both species are mainly attributed to the presence of luteolin, 7) Argemone mexicana L. Papaveraceae (“cardo quercetin and 4'-O-beta-D-glucopyranosyl-3',5'- amarillo”, “cardo santo”, “chicalote”). It is dimethoxybenzyl caffeate (BaII) (Soiche & Leng

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Peschlow, 1987; Oliveira et al., 2005; Kappel et al., value increase, greater attention will be paid to tissue 2012). In addition, B. articulata has been ascribed culture. Currently, reliable protocols exist for the aphrodisiac and sexual stimulant properties (Barboza micropropagation of several Bauhinia species. In this et al., 2009). With respect to its important volatile context, it is important to highlight that there are a fraction, whereas the main principles of the essential few reports on the use of cell cultures (Mello et al., oil of B. articulata are β-pinene, spathulenol, (E)- 2000; Appezzato-da-Glória & Machado, 2004; Lima, nerolidol, β-caryophyllene and bicyclogermacrene 2009). Mello et al. (2000) proposed the establishment (Zunino et al., 2004), in the case of B. trimera, of a protocol for in vitro plant regeneration from cells carquejyl acetate is the most important constituent, of B. forficata that could be used in plant propagation not only for its antispasmodic activity (Torres et al., and transformation assays. However, no further 2000) but also because it could be a chemical marker reports concerning transformation were found. Lima for this species (Simões-Pires et al., 2005; (2009) established a cell culture protocol to produce Minteguiaga et al., 2018). phenolic compounds and proteins with hypoglycemic The exploitation of these species is carried activities, but we could find no current advances for out by wild harvesting and, due to its high demand this work. (Martínez, 2005), the wild populations in Argentina Despite its medicinal importance, are subjected to immense pressure. In the case of B. commercial cultivation is not feasible since its trimera, many of its populations are exposed to the propagation is negatively affected by the long period risk of extinction (Céspedes de Zárate et al., 2009). (10-12 months) between flowering and seed However, we could not find data regarding its production (Lorenzi, 1992; Mello et al., 2000). cultivation, except for staking assays (De Bona et al., However, in Brazil it is included in the National 2005) and small-scale gardening. Policy of Medicinal Plants and Phytotherapics and Regarding biotechniques, in the reviewed the National Program of Medicinal and literature only in B. trimera there are reports on Phytotherapeutic Plants within the Unified Health studies of inter-population genetic variability through System (SUS), with common objectives aimed at isoenzymes (Cúneo, 2012) and molecular markers of guaranteeing safe access and rational use of the alu ISSRs type in studies of genetic diversity medicinal plants and phytotherapics (Brasil, 2006). between species (Vaco, 2011); no references were found concerning B. articulata. The propagation 11) Carapichea ipecacuanha (Brot.) L. Andersson, under in vitro conditions is not documented, neither synonyms: Callicocca ipecacuanha Brot., Psychotria for B. articulata nor B. trimera. ipecacuanha (Brot.) Standl., Cephaelis ipecacuanha (Brot.) A. Rich. Rubiaceae, (“ipecacuana”). It is well 10) Bauhinia forficata Link. subsp. pruinosa known for its expectorant and emetic properties, and (Vogel) Fortunato & Wunderlin. Fabaceae it is also effective in the treatment of amoebiasis. Its (“bauhinia”, “pata de chivo”, “pata de vaca”, “pezuña main active principles are the isoquinolic alkaloids de vaca”). This species has been claimed to possess cephaeline, emetine and dehydroemetine (Teshima et antidiabetic activity and strong hypoglycemic effects, al., 1988). making it a therapeutic alternative for the treatment For approximately 300 years, “ipecacuana” of diabetes mellitus (Marques et al., 2013). The has been subjected to over-extraction practices in active principle responsible for this pharmacological Tropical America, mainly in Brazil. At that time, effect is trigonelline, which is extracted from the harvesting was uncontrolled and there was no leaves (Toloza-Zambrano et al., 2015). Currently, in replanting after the uprooting of native populations Uruguay, a product (drops) developed from B. (Oliveira & Martins, 1998). Around the mid-20th forficata is commercialized for the treatment of century, plantations were initiated under the arboreal diabetes mellitus Type 2 (Diabenat, 2011). cover of the tropical rainforest, first in Nicaragua and Concerning the development of later in Costa Rica. Hence, the cultivation of high biotechnological tools, micropropagation of the genus quality roots, aimed exclusively at satisfying the Bauhinia has been extensively reviewed by Teixeira international market demand, was established. In da Silva (2013). According to this author, the studies contrast, the root´s supply in Brazil is procured on the in vitro culture of Bauhinia species are still at through wild harvesting of the populations in the an early stage. As their medicinal and ornamental Mato Grosso state. As a result, the roots present

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants higher variability in their secondary metabolite reported. Rossi et al. (2009) found high genetic content and the plant is at high risk of extinction differentiation between Amazonian and Atlantic (Ocampo, 2007). A similar situation has been “ipecacuanas”. De Oliveira et al. (2010) applied these described in Colombia (Yoshimatsu et al., 2003; molecular markers to assess the implications of Lucía Aterhotua, personal comunication). clonality for the in situ genetic conservation of Biotechnological tools for this species have “ipecacuana” to establish efficient strategies for the been extensively developed, beginning with reports conservation of this species. on micropropagation (Jha & Jha, 1989; Yoshimatsu & Shimomura, 1994), indirect regeneration (Rout et 12) Cecropia pachystachya Trécul, synonymous: al., 1992) and somatic embryogenesis (Rout et al., Cecropia adenopus Mart. ex Miq. Cecropiaceae 2000). In relation with the work carried out in tissue (“ambaí”, “ambay”, “amba-hu”, “palo lija”). The culture, long term cultures were achieved. This in leaves of “ambay” have a long tradition of medicinal vitro germplasm, maintained through reduced growth use in the treatment of respiratory diseases, mainly as conditions for more than a decade, turned out to be a an antitussive and expectorant (Argentina-Paraguay- successful mode of preservation when it was used as Brazil) (Arias Toledo, 2009; Alonso & source material for micropropagation, obtaining Desmarchelier, 2015). It is accepted in herbal complete plant regeneration with phenotypic and medicine for treating coughs and asthma, and as chromosomal stability (Chaudhuri & Jha, 2008). cardiotonic and diuretic, but it also used as a dietary However, Yoshimatsu & Shimomura (1994) pointed supplement (Gupta, 1995). out an important limitation to this clonal propagation. The pharmaceutical industry combines it with They found that field-cultivation of “ipecacuana” guayacol to prepare tablets (Laboratorios Weltrap). plants is restricted to a tropical or subtropical region Recently, “ambay” has been introduced in the because the plants were significantly damaged when Primary Health Care System in the province of the monthly average of minimum temperatures was Misiones, Argentina (Alonso & Desmarchelier, below 10° C. 2015). As an alternative source of active principles, “Ambay” is harvested from the wild (Alonso production of in vitro alkaloids via root culture & Desmarchelier, 2015). These authors also indicate (Teshima et al., 1988; Yoshimatsu & Shimomura, that this species can be found as cultivated, but the 1991; Karuppusamy, 2009) and via callus culture provided information is very scarce and vague and, in (Jha et al., 1988; Teshima et al., 1988; Tripathi & fact, there are no references about the location of Tripathi, 2003) has been reported. Roots cultured in these cultivations. Besides, they include requirements solid and liquid medium yielded similar content of of culture and forms of reproduction although no emetine and cephaeline as the roots from a mother reference is reported. Despite the constant demand plant grown in greenhouse conditions (Teshima et al., for its active principles H (ambain and ambainin) and 1988). C (cecropine and cecropinine), we did not find Furthermore, transformed cultures via reports in the literature concerning the use of any Agrobacterium rhizogenes have been established biotechnique, such as propagation, breeding or (Yoshimatsu et al., 2003). The yield of cephaeline genetic diversity studies. reported by these authors in transformed roots was over twice that in the non-transformed roots of 13) Equisetum giganteum L. and 14) E. bogotense similar age reported previously by Yoshimatsu & Kunth. Equisetaceae (“cavalinho gigante”, “cola de Shimomura (1991). However, when Yoshimatsu et caballo”). Although they are different, they share the al. (2003) regenerated plants from transformed roots, same vernacular names and applications in traditional they found that the alkaloid contents in the medicine. The infusions prepared with the aerial parts transformed plants, both in the leaves and roots, were are used as diuretic (Menseguez et al., 2007). In lower than those of non-transformed plants. Thus, addition, this species was found as a constituent of these authors proposed the transformed root cultures aphrodisiac mixtures (van Andel et al., 2012). as a promising tool for an alternative source of ipecac Both species possess interesting potential for alkaloids and for biosynthetic and transport studies of the pharmaceutical industry, and E. giganteum, ipecac alkaloids. whose diuretic activity is known (Pérez Gutiérrez et Finally, the use of ISSRs in this species was al., 1985; Cáceres et al., 1987), is frequently

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants employed to replace E. arvense, being extensively 2011). commercialized as a dietary supplement (Francescato Efforts to multiply and preserve this valuable et al., 2013). In addition, E. giganteum was proposed species through the application of biotechniques, as a promising alternative for the such as micropropagation, have been made (Rosal et treatment/prevention of dental stomatitis and oral al., 2007; Prudente et al., 2016). However, both candidiasis (Alavarce et al., 2015), and as references only describe an established protocol to antiinflammatory in rheumatoid arthritis and other multiply this species, since ex vitro germination rate inflammatory afflictions (Farinon et al., 2013). of the seeds is considered low. In this context, the No bibliographic references were found objective was to establish a protocol for in vitro concerning domestication and/or cultivation of either germination and multiplication, aiming at the rapid species. There are indications of over-harvesting of multiplication of the species. We could not find any E. giganteum in Argentina, which may endanger the reports concerning the use of in vitro culture to species (Martínez, 2005; Alonso & Desmarchelier, produce the active principles of interest. Concerning 2015). However, Alonso & Desmarchelier (2015) the use of molecular markers, RAPDs have been also indicate that E. giganteum is cultivated as applied in order to study its genetic diversity (Moura, ornamental around lakes and lagoons. In the case of 2005; Estopa et al., 2006; Freitas et al., 2008). E. bogotense, Rodriguez et al. (1994) have reported that since very little has been done on cultivation in 16) Fabiana imbricata Ruiz & Pav. Solanaceae Chile, the plant is usually gathered in nature. (“palo pichi”, “pichi”, “pichi romero”). The infusion Only one report on the micropropagation of obtained from the bark and stems of this shrub is used E. bogotense was found (Rodríguez et al., 1994), but in traditional medicine as a digestive and diuretic no further techniques were used for breeding. For E. agent (Muñoz et al., 2001; Navas, 2001; Schmeda- giganteum, no references were found. However, there Hirschmann et al., 2004). In Chilean folk medicine, have been studies regarding its secondary metabolism this infusion is also used to treat Fasciola hepatica and its products (Alavarce et al., 2015) and also on infections in goats and sheep (Navas, 2001). This the taxonomical relations of the genus Equisetum species is registered in the Homeopathic German through the sequencing of chloroplast DNA and by Pharmacopoeia. In other countries, this shrub is used “fingerprints” with ISSRs (Des Marais et al., 2003; as an ornamental plant in gardens (Grüneberg, 1995). Brune et al., 2008, respectively). The health-promoting properties reported for the infusions of F. imbricata can be associated with 15) Eremanthus erythropappus (DC.) MacLeisch, the presence of rutin, scopoletin, 3-O-caffeoylquinic synonymous: Vanillosmopsis erythropappa (DC.) acid, quercetin together with its derivatives (Reyes et Sch.Bip., Albertinia erythropappa DC. Asteraceae al., 2005; Quispe et al., 2012). (“candeia”). This species is used in folk medicine as a This species is collected from the wild and, in scarring agent and in the treatment of infections and this context, a few attempts have been made to stomach ulcers (Silvério, 2004). Currently, the main domesticate and cultivate this species (Schmeda- product of interest is the oil, rich in α-bisabolol, Hirschmann et al., 2004; Fischer et al., 2011). extracted from the whole plant. This active principle Concerning the application of biotechniques, is widely used in the pharmaceutical and cosmetic an in vitro propagation system leading to the industries (Rosal et al., 2007). formation of shoots, calli, roots, cell suspensions and The tree is under threat because of intensive plantlets has been developed (Razmilic et al., 1994; exploitation (Souza et al., 2007) and because its Schmeda-Hirschmann et al., 2004). Schmeda- natural environment is constantly shrinking, mainly Hirschmann et al. (2004) evaluated conditions due to the accelerated anthropic action (Vieira & (temporary immersion system, morphogenesis and Martins, 2000; Pereira & Gama, 2010). maintenance of cell suspensions) for the regeneration Since sustainable supply of “candeia” oil can of different organs of F. imbricata grown under in no longer be guaranteed, the Symrise Company has vitro conditions as an alternative for active compound decided to stop harvesting natural bisabolol from the production and regeneration of selected plants. Their “candeia” tree. This company focuses now on the results showed an important variation in the production of highly pure nature-identical bisabolol: production of the metabolites, but there is no Dragosantol® and Dragosantol® 100 (Symrise, information on the secondary metabolite production

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants of in vitro cultures and its variation according to dominated by “jarillas” indicates that the area was explant-type and growth regulators. subjected to intense grazing for long periods of time or suffered periodic fires (Femenia, 2014). 17) Justicia pectoralis Jacq. Acanthaceae Several preparations are obtained from the L. (“amansatoros”, “anador”, “curia”, “té criollo”, divaricata plant, especially from its leaves and stems. “tilo”). The decoction of the fresh or dry leaves is Traditionally, the leaves´ infusion is recommended used generally as a neurosedative (Fernández et al., for the treatment of stomach disorders, arthritis, 1989; Rodríguez et al., 1989). The plant is also rheumatism, fever, bronchitis and other respiratory employed as an expectorant, antiasthmatic and ailments (Barboza et al., 2009; Palacio et al., 2012). analgesic (Lino et al., 1997). In addition, it is The species is reputed to have traditionally used in menopause and premenstrual antiinflammatory, antitumoral, antiviral and syndrome due to its estrogenic and progestogenic antimicrobial properties. These properties have been activity (Locklear et al., 2010). attributed to its most frequent biologically active One of the most representative compounds of compound: nor-dihydroguaiaretic acid (NDGA) this species is justicidine B. This is a potent cytotoxic (Anesini et al., 1998; Anesini et al., 1999; Konigheim substance on several cell lines, especially chronic et al., 2005; Svetaz et al., 2013). In addition, its myeloid and chronic lymphoid leukemia. This abundant flavonoids, mainly quercetin, kaempferol, property makes justicidin B a potential ferulic acid and coumaric acid, are considered onchopharmacological agent (Hemmati & Seradj, anticancer agents, and there is a possible synergism 2016). among the compounds (Palacio et al., 2012). The properties of J. pectoralis make it a Concerning its essential oil, α-thujene, promising commercial species, and the Cuban myrcene, limonene, (E)-ocimenone, thymyl acetate, pharmaceutical industry is actively developing γ-elemene, spathulenol, β-oplopenone, α-cadinol and technological processes to obtain pharmaceutical the eudesmol isomers are the main constituents quality products from the natural compounds of this (Barboza et al., 2009). However, it yielded very low species. In this sense, medicinal syrups (Martín et al., amounts of essential oils. In contrast, high yields of 2011) and tablets (Rodríguez et al., 2015) have been alcoholic resinoids were obtained, which suggest that obtained. this species could be used as a natural fixative in Despite all the pharmacological progress with perfumery (Juárez et al., 2016). this species and the pharmaceutically valuable Commercially, two types of shampoo can be characteristics of justicidin B, we could only find one found: antidandruff and to treat alopecia. Both were recent report on micropropagation (Oliveira Freitas et developed by CONICET researchers from extracts of al., 2016). Although this work is very relevant since “jarilla” and “café” (EcoHair, 2013). it is the first one related to J. pectoralis tissue culture, No references were found concerning the it refers only to the evaluation of different basal cultivation of this species, nor its conservation status. media and growth regulators, and it does not show Nevertheless, it has been propagated through in vitro results in terms of the acclimation phase and transfer culture techniques such as micropropagation, indirect to soil. Furthermore, it does not evaluate the regeneration through calli and cell cultures (Palacio production of neither justicidin nor other principles. et al., 2008; Palacio et al., 2012). This is disturbing because in the bibliography Despite the growing potential of clinical uses regarding the production of justicidin B using for NDGA and its derivatives, its production in L. biotechnology, this principle is not currently being divaricata plant cell cultures has only been carried obtained from this species. J. pectoralis is also out in the last six years (Palacio et al., 2011; Palacio cultivated as an ornamental for urban woodland. et al., 2012). In this sense, studies have evaluated the optimal conditions for the in vitro growth of calli and 18) Larrea divaricata Cav. Zygophyllaceae (“jarilla cell suspensions, as well as cell growth kinetics and del cerro”, “jarilla hembra”). Together with another the production of active principles. Calli, both with species of this genus, L. cuneifolia Cav., they and without organogenesis, produced NDGA and constitute the “jarillales”, which are frequently quercetin. NDGA, together with p-coumaric acid, considered desertification indicators. Both are ferulic acid and sinapyl alcohol, was also produced regarded as anthropic species, since a terrain by the cell suspension cultures (Palacio et al., 2012).

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These authors demonstrated that the capacity of hyperhydricity. This system was able to produce a undifferentiated tissues to form phenolic compounds large number of uniform plants. These reports (NDGA and quercetin) is very limited, but when the showed an auspicious scenario for “maca” calli underwent organogenesis, developing mainly propagation and for obtaining clonal plants from adventitious shoots, the phenolic compound selected material. Besides, the use of microsatellites production significantly increased. This data on chloroplastid DNA was reported (Hasan et al., indicated that the content of NDGA and quercetin 2010). Furthermore, “maca” has been used for the increased with differentiation. However, plantlets creation of somatic hybrids with Brassica oleracea L. regenerated from adventitious shoots of L. divaricata (Ryschka et al., 2003). calli did not show the same phenolic pattern as wild plants, with lower levels of NDGA and quercetin. 20) Lippia alba (Mill.) N. E. Br. ex Britton & P.Wilson. Verbenaceae (“herva cidreira”, “pronto 19) Lepidium meyenii Walp. Brassicaceae (“ayak alivio”, “salvia de campo”, “salvia de monte”, “salvia chichira”, “ayak willku”, “maca”, “maca-maca”, morada”). It is used extensively in traditional “maino”). This species has been domesticated in the medicine, mainly to treat digestive and respiratory Puna agro-ecological zone of central Peruvian Andes, disorders, but it also has sedative, analgesic, where it is cultivated for its edible underground antipyretic and antiinflammatory properties (Pascual organs (called "roots") (Wang et al., 2007). et al., 2001; Hennebelle et al., 2008a). This species is “Maca” has been consumed for many years characterized by the variability in the chemical and it is considered as an adaptogen because of its composition of the essential oils. There are several special properties (nutritional and energizing), since chemotypes – at least seven described – which differ it provides the energy and endurance needed for daily in the main components of the essential oils. These activities (there is a large bibliography about it). are: chemotype I (citral, linalool and β-caryophyllene, Recently, “maca” has been treated as a plant with as the main constituents), chemotype II (tagetenone), added value and many people in the world are using chemotype III (limonene and carvone or related it as a tonifier and powerful revitalizing agent monoterpenic ketones), chemotype IV (myrcene), (Polzerová et al., 2011; Sifuentes-Penagos et al., chemotype V (γ-terpinene), chemotype VI (camphor- 2015). In addition, it is highly demanded as a dietary 1,8-cineole) and chemotype VII (estragole) supplement, because of its nutrient content (Ronceros (Hennebelle et al., 2008a; Ricciardi et al., 2009; et al., 2005). Also “maca” is employed traditionally López et al., 2011). to improve sexuality and fertility (Sifuentes-Penagos The essential oils, either extracts or their et al., 2015), properties that have been demonstrated constituents, have been claimed to possess in studies not only in male rats and mice but also in antispasmodic, antiinflammatory, antiviral, men (Gonzales et al., 2001; Gonzales et al., 2002). antibacterial, antifungal and antiparasitic activities Due to its aphrodisiac effects, its (Ruffa et al., 2004; Ara et al., 2009; Ocazionez et al., consumption is gradually increasing. However, the 2010; Blanco et al., 2013; Gómez et al., 2013). source of available “maca” seeds is restricted, Among the non-volatiles, flavonoids and iridoids because the growth conditions for its cultivation are have been detected, and these are apparently not favorable in certain environments (Wang et al., responsible for the sedative effects of the species 2007; Polzerová et al., 2011). In this context, “maca” (Hennebelle et al., 2008b). has been subjected to the application of tissue and Although in Argentina there have been organ culture techniques with the aim of producing attempts to domesticate some of the chemotypes an adequate number of plantlets for cultivation. There described above (Ringuelet & Cerimele, 2010), the are reports on its in vitro propagation through preferred choice of exploitation is the collection of micropropagation by shoots derived from calli wild specimens (Alonso & Desmarchelier, 2015). (Cheng et al., 2004; Wang et al., 2007), but These last authorts also indicate data on cultivation, hyperhydricity was frequent. On the other hand, although there is no reference report. Cultivated Polzerová et al., (2011) established an effective specimens can be found mainly for ornamental and multiplication system through direct organogenesis culinary uses, particularly in Central America, from nodal segments (axillary buds) with good Mexico, Colombia and Brazil (Urdaneta & Kanter, regeneration rates, high rooting efficiency and no 1996). Asexual propagation methods are preferred for

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants this species (Pimenta et al., 2007; Seaforth et al., obtained by in vitro polyploidization (Iannicelli et al., 2008; Herrera-Moreno et al., 2013) due to the low 2016a), with a higher yield in essential oils. In germination of seeds of L. alba (Gupta et al., 2001). autotetraploids there was an increase in the Concerning the use of biotechnology, for this production of essential oils and also quantitative ethnobotanical useful species, only two reports on its differences in the proportions of some of the in vitro propagation have been found (Gupta et al., compounds. This was the first variety of “incayuyo” 2001; JibinaBai et al., 2014). Based on the registered in Argentina. In addition, the evaluation of conclusion drawn by Gupta et al. (2001), who plants regenerated through in vitro culture in natural observed that the phenotype and oil profile of the conditions has been reported, and this paves the way micropropagated plants were identical to the normal to future assays related to re-population (Iannicelli et vegetatively propagated ones, the micropropagation al., 2016a). Furthermore, the ISSR technique to study protocol adjusted for L. alba ensures the cloning of the in vitro genetic variability has been described selected genotypes. On the other hand, the (Iannicelli et al., 2016b), and in vitro regenerated interrelation between different accessions and plants showed polymorphisms with respect to the chemotypes of L. alba have been studied through field mother plant. Also, plants derived from in vitro ISSRs and RAPDs (Manica-Cattani et al., 2009; culture showed quantitative differences in some Pierre et al., 2011). Also, cpDNA, ITSs and ISSRs compounds of their essential oils, but no differences were used in this species within a molecular study of in their yields were detected (Iannicelli et al., 2016a). the genus Lippia (Suárez González et al., 2007). From an ecological standpoint, some of the populations are in risk because the species is not 21) Lippia integrifolia (Griseb.) Hieron. cultivated and, at the same time, it is in high demand Verbenaceae (“incahierba”, “incayuyo”, “poleo”, from the liquor and herbal (“yerbatera”) industries to “pulco”, “té del inca”, “yerba del inca”). Infusions or prepare bitters and compounded herbs for infusions decoctions prepared with the aerial parts (leaves and (Elechosa, 2009). Besides, it represents a high portion flowers) of “incayuyo” are used traditionally for the of the income of families that have been harvesting it treatment of gastrointestinal disorders or ailments. Its from the wild and commercializing it for more than use as diuretic, emmenagogue and for the treatment 20 years. In view of this situation, the Instituto of coughs and colds has also been documented Nacional de Tecnología Agropecuaria (INTA) in (Iannicelli et al., 2016a). In Argentina, “incayuyo” is Argentina has started to promote sustainable harvest used in the preparation of bitters (“amargos techniques to help “maintain the populations and plan serranos”) and compounded herbs (Juliani et al., the harvests´ collection” (INTA Informa, 2015). 2007), and it is included in the Argentine Food Codex for preparation of beverages intended for infusions or 22) Lippia turbinata Griseb. Verbenaceae (“poleo”, macerations (Alonso & Desmarchelier, 2015). “té del país”, “manzanillo”). In traditional medicine, Because of the variable composition of the essential the stems and leaves are used to prepare infusions (té) oil of L. integrifolia, five different chemotypes were that have digestive, diuretic and stimulant properties described in Argentina. These are the chemotypes: (Pascual et al., 2001; Arias Toledo, 2009; Elechosa, trans-davanone, lippifolienone, β-davanone-2-ol, 2009). “Poleo” is part of bitters and compounded spathulenol/bicyclogermacrene/β-caryophyllene and herbs (Juliani et al., 2007), and it is usually trans-nerolidol (Marcial et al., 2016). The essential consumed, together with other herbs, in “mate” oils not only confer it its digestive properties, but (Menseguez et al., 2007). they also have antioxidant (Barbieri et al., 2015) and Analysis of the essential oils revealed the mosquito repellent (against Aedes aegypti) activities presence of limonene, piperitone oxide, bornyl (Gleiser et al., 2011). Among the non-volatiles, the acetate and 1,8-cineol although some authors report main compound is verbascoside (Marcial et al., as the main constituents α-thujone, carvone, 2014), which is apparently responsible for limonene, β-caryophyllene oxide, bornyl acetate, antiinflammatory effects on stomach cells and spathulenol, germacrene-D and bicyclogermacrene antiadhesive properties against Helicobacter pylori (Pascual et al., 2001; Barbieri et al., 2015). (Marcial et al., 2014). Recently, his species was There is a high demand for this species and it macropropagated, and also introduced and multiplied is under pressure because the plants are gathered by in vitro (Iannicelli et al., 2016b). A new variety was unsustainable wild harvesting (Martínez, 2005;

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Elechosa, 2009). Alonso & Desmarchelier (2015) Edition of the Argentine National Pharmacopoeia describe its cultivation requirements and the forms of (Alonso & Desmarchelier, 2015). reproduction; however, no studies are reported Besides its uses as digestive, the essential oils concerning its real cultivation. of “peperina” have great potential for the Only one report on its micropropagation has development of products such as toothpastes and been found (Ortiz et al., 2007), and it describes a mouthwashes (Huari Guerrero, 2014) due to its protocol for indirect organogenesis through calli proven antibacterial and antimycotic activities culture. (Carhuapoma et al., 2009; Zapata et al., 2009). Regarding the active principles, the 23) Maytenus laevis Reissek. Celastraceae composition of its essential oils is well known, and (“chuchuguaza”, “chuchuguasi capinuri”, the diverse aromas detected in the populations “chuchuhuasha”, “chuchuhuasi”, “chuchuhuazo”). analyzed are due to a large chemical variety of There is a certain confusion regarding this species compounds. For example, in Argentina, 13 different because, in the extensive ethnobotanical literature chemotypes were detected and these were available, many different plants; e.g.: M. krukovii differentiated according to regions. The mentone- A.C. Sm. and M. macrocarpa (Ruiz & Pav.) Briq. pulegone chemotype is the one that confers the have received this vernacular name. The bark, roots, typical aroma required by the market for this species. and leaves of these different plants also share The rest of the chemotypes have linalool, carvacryl pharmacological properties, such as antiinflammatory acetate, carvacrol, dihydrocarvone, carvone, activity for the treatment of rheumatism and arthritis. limonene and menthyl acetate, as the main The “chuchuhuasi” has also been used to treat constituents (Elechosa, 2009). tuberculosis, bronchitis, stomach aches and fevers. In Traditionally, this species has been exploited traditional medicine, chewing the bark of through unsustainable, wild harvesting practices, “chuchuhuasi” is recommended to treat diarrhea, particularly in the central region of Argentina (Ojeda arthritis and menstrual problems (González et al., et al., 2001; Bustos & Bonino, 2005). Although there 1982). have been initiatives to domesticate and cultivate this We found numerous references regarding the species, these have encountered difficulties because pharmacological properties of this species and the “peperina” is highly demanded by the herbal industry development of phytopharmaceuticals (Lucero & and the market. The situation is further complicated Dehesa, 2011), and there are even websites that offer because many “peperina” populations, at least in products containing the active principles of this plant, Argentina and according to some authors, are in but we could not find any reports on its ecological serious risk of extinction (Martínez, 2005; Origlia, status, nor on its cultivation or the application of 2015). Concerning its cultivation, the most interesting biotechniques. This situation would place this species study is that of Ojeda et al. (2004) who tested the best at serious risk of extinction. form of cultivation. These authors found significant differences in plant mortality, productivity and 24) Minthostachys mollis (Benth.) Griseb. essential oil yield between the regimes, but also that Lamiaceae (“muña”, “peperina”, “toronjil”). The some populations were better adapted to cropping main use of “peperina” is as a digestive, particularly than others. Furthermore, we found references in infusions or compounded digestive herbs (Juliani pertaining to the development of cultivation in the et al., 2007). However, ethnobotanists have work of Alonso & Desmarchelier (2015). documented a surprisingly wide array of traditional Concerning the use of biotechniques, this uses for this species. These include uses as a species has been successfully cultivated in vitro. hypotensive, for antimycotic and antiparasitic Chebel et al. (1998) micropropagated plantlets which purposes, against diarrhea, colics, and respiratory were then successfully transferred to soil. When their illnesses (bronchitis, asthma), headaches, and to essential oils were analyzed, little differences were induce menstruation. Besides, it is used in the found in the composition compared to the plant formulation of “yerba compuesta”, for the production grown in the wild. Continuing the work of Ojeda et of liqueur and as a condiment (Cortella & Pochettino, al. (2004), shoots of a cultured clone were used to 1999; Schmidt-Lebuhn, 2008; Arias Toledo, 2009). optimize experimental conditions for rapid in vitro The leaves of “peperina” are registered in the 5th multiplication and transfer to soil of selected

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“peperina” clones. These results will allow the different organs of the plant, we can mention a multiplication of selected clones to use them for mixture of steroidal saponins known as charantins, breeding programs or for homogeneous commercial insulin-like peptides and alkaloids that are productions, with verified quality (Bima et al., 2006). responsible for the hypoglycemic activity of this In addition, we found a reference mentioning the use species (Ananya & Sarmistha, 2010). of expressed sequence tag - Inter-Simple Sequence In the field of the biotechnology, several Repeat (EST-ISSR) to study genetic diversity in protocols have been established with different populations (Bonafede et al., 2014), in which the strategies and explants in tissue culture and authors report that, despite the excessive exploitation micropropagation. For example, direct and indirect of these populations, there are still diversity hubs that regeneration from leaves, apices, nodal segments, could be used for conservation or breeding. The use cotyledonary nodes and embryogenic suspension of AFLP in the genus Minthostachys has been culture have been reported. Agarwal (2015) has reported by Schmidt-Lebuhn (2008) in a work that reviewed these large applications of in vitro culture supplements field and morphological studies within techniques, describing methodologies and results. the framework of a taxonomic and systematic However, in this review there is no information revision. Besides, this work suggests that M. mollis, concerning the effects on the production of secondary considered as the most complicated species, is metabolites. In fact, despite the existence of a large genetically very heterogeneous and, according to number of references, only a few have as their their geographical origin, exhibits a high discrepancy objective the production and evaluation of the active with the pattern of morphological variation. principles. In this sense, Agarwal & Kamal (2007) Despite the fragile ecological status of this reported the use of callus cultures and different in species, it is important to highlight the work carried vitro developmental stages for the production of out by the group of the National University of flavonoids. Agrobacterium rhizogenes–mediated Córdoba (Argentina) in terms of the domestication hairy root induction to produce secondary metabolites and improvement of “peperina” (Ojeda et al., 2004; was reported. Swarna & Ravindhran (2012) obtained Ordóñez et al., 2006; Rodríguez Pardina et al., 2013). transformed hairy roots to produce charantin. The One of the most important result obtained by the authors informed the detection of this compound but group of Dr. Marta Ojeda from the above mentioned there were no references about the amounts obtained. University is the development of a variety of Also, Thiruvengadam et al. (2014) established a “peperina” registered in the Argentine National protocol for the production of phenolic compounds Institute for Seeds (INASE) in order to facilitate its from hairy root cultures. The flavonoids (catechin, industrial cultivation (Carrizo, 2017; Tolchinsky, myricetin and quercetin) and the phenolic acids 2017). Currently, this new variety is being cultivated (gallic acid, chlorogenic acid, gentisic acid and in fields in the province of Cordoba, Argentina. salicylic acid) levels were highly increased in hairy However, we could not find information about its roots compared to untransformed control roots; commercialization. Finally, it is important to mention consequently, there was an increase in both the a website that compiles most of the existing antioxidant and antimicrobial activities in the hairy references on “peperina” (Phylobotanist, 2012). roots. Furthermore, different types of molecular markers have been used. Ananya & Sarmistha (2010) 25) Momordica charantia L. Cucurbitaceae reviewed the use of RAPD and Sequence (“balsamina”, “cundeamor”, “melón amargo”, Characterized Amplified Region (SCAR) to study the “pepinillo”, “pepino cimarrón”, “tomaco”). In genetic diversity of the species. The authors suggest traditional medicine, fruits and seeds of M. charantia that both molecular markers could be used to explore are considered as a powerful antidiabetic, but they the genes associated with medicinal properties of M. also have numerous applications, such as anti-HIV, charantia. Besides, the efficiency of RAPD, ISSR antiparasitic, emmenagogue, antihelmintic, and AFLP in detecting genetic diversity was antimycotic, antirheumatic, antihipertensive, evaluated (Singh et al., 2007; Behera et al., 2008a; antimalarial, carminative, choleretic, antipyretic and Behera et al., 2008b) and internal transcribed spacer abortive (Chauhan et al., 2010; Ananya & Sarmistha, 2 (ITS2) was used as an identifying tool (Michel et 2010; van Andel et al., 2014). al., 2016). Among the compounds isolated from This species is commonly cultivated in South

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America and the Caribbean, Asia and Africa (Basch conservation. Regarding the use of tissue culture, P. et al., 2003; Portillo, 2009). In Argentina it is caerulea has been extensively studied and there are considered as an edible weed (Rapoport et al., 2009). numerous papers dealing with its in vitro culture Also, in Mexico, the National Commission for the through different methodologies, including Knowledge and Use of Biodiversity (CONABIO) micropropagation and regeneration from calli considers it a weed (CONABIO, 2009). (Carnier Dornelas & Carneiro Vieira, 1994; Severin There are products already in the market such et al., 2011; Ozarowski & Thiem, 2013; Rathod et as of the immune system strengthening capsules in al., 2014). Using leaf, nodal or internodal segments, Spain (Thaivita, 2018) and capsules and extracts with regeneration of shoots was obtained with and without hypoglycemic activity in Mexico (Tecnobotanica de callus development and the plantlets were México, 2018). successfully transferred to ex vitro conditions. In this sense and considering the problems with 26) Passiflora caerulea L. Passifloraceae conventional propagation, Rathod et al. (2014) (“burucuyá”, “maracuyá”, “mburucuyá”: names reported that a complete plant along with shoots and derived from the guaraní language; “flor de la roots can be developed in around 54 days, which may pasión", “pasionaria”, “pasionaria azul”). It is widely take about two seasons or sometimes a year for a seed employed in traditional medicine, where all the parts to germinate. of the plant are used. The infusion prepared with the On the other hand, there is a lack of leaves and flowers is traditionally consumed for its systematic phytochemical evaluation in in vitro sedative and anxiolytic properties, in the treatment of clones for the detection of flavonoids, phenolic acids insomnia. In ethnomedicine, other uses are also and alkaloids in regenerated plantlets. Only a few reported for its aerial parts, as diuretic, antitussive reports have confirmed the occurrence of secondary and antiasthmatic and spasmolytic, as eupeptic (by metabolites in plantlets, showing the presence of decoction of its fruits), antiscurvy, urinary apigenin, luteolin, vitexin, isovitexin, rutin, antiinfectant, and its root is used as anthelmintic, hyperoside, chlorogenic and rosmarinic acids menstrual cycle regulator and contraceptive (Alonso, (Busilacchi et al., 2008; Ozarowski & Thiem, 2013). 2004; Menseguez et al., 2007; Arias Toledo, 2009). It is important to highlight a specific reference in Furthermore, the ornamental value of the flowers is which micropropagated plants of P. caerulea were highly appreciated. evaluated in the field, and it was observed that there Its use as a sedative and anxiolytic is mainly were no qualitative differences in the secondary due to the presence of chrysin, which is similar to metabolites found in the plant from where the diazepan, although its effect is ten times lower and it explants were originally obtained (mother plant) has no myorelaxant properties (Medina et al., 1990; compared to the ones obtained by in vitro culture Wolfman et al., 1994). (Busilacchi et al., 2008). Although in other species of In Argentina, P. caerulea is highly demanded the genus Passiflora the use of transformation by consumers and it is obtained by extractive wild methods and polyploidization were reported, in P. harvesting, causing a marked decrease in its relative caerulea no references were found regarding the abundance decreased in the last years (Martínez, application of these biotechniques. Genetic marker 2005; Mendiondo & Garcia, 2006). Conventional techniques, such as RAPDs, to study genetic diversity propagation displays certain problems concerning the among different species have also been used (Fajardo percentage of seed germination and growth rates. et al., 1998; Crochemore et al., 2003). Crochemore et Considering the economic importance and the al. (2003) generated fingerprints that would allow the conservation problems of P. caerulea, Mendiondo & identification of improved varieties/populations. Garcia (2006) examined the seed emergence pattern There has been remarkable progress in the from the standpoint of either in situ or ex situ plant breeding, cultivation and production species of this preservation and propagation. These authors genus with great importance as food, such as P. informed low levels of seed emergence and slow edulis Sims or P. alata Curtis (Cerqueira-Silva et al., growth rates. 2014). However, the species with medicinal uses, Since conventional propagation of this such as P. caerulea, are still exploited through species seems to be a difficult task, the application of collection of wild specimens. This not only results in biotechniques has been considered for its conservation problems, as in the case of P. caerulea,

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants but also produces batches of raw material with high cultures, works dealing with the search for alternative variability and low quality (Severin et al., 2011). production methods are scarce. In this sense, the most recent one refers to the generation of calli and 27) Phyla scaberrima (Juss. ex Pers.) Moldenke, cellular suspensions (Urrea et al., 2009) as synonyms: Lippia dulcis Trevir., Phyla dulcis preliminary stages for the study of hernandulcine (Trevir.) Moldenke. Verbenaceae (“hierba buena", production and upscaling. “hierba dulce”, “orozús”). It is one of the species with the most number of ethnobotanical references, 28) Quillaja saponaria Molina. Quillajaceae and there are bibliographic data dating back to the (“quillay”). The saponins have great economic 15th century (Compadre et al., 1986). Although the importance and they are used with pharmaceutical, US Pharmacopoeia has mentioned it as an industrial and agronomic purposes (Hoffmann, 1995; expectorant since the late 19th century, in traditional Vega & Prehn, 2005). medicine it is used as a sweetener, for the treatment Due to the high demand for quillay bark (the of respiratory diseases (bronchitis, coughs and colds) main source of saponins) and changes in the use of and as abortive (Compadre et al., 1986; Pascual et soils where it grows, the existence of adult trees was al., 2001; Gornemann et al., 2008). reduced (Prehn et al., 2003). Its sweetening properties are due to This situation prompted the development of a hernandulcine (500 sweeter than sucrose, Compadre forest management program for this species, et al., 1986). However, the production of supported by a propagation program (Copaja et al., hernandulcine from leaves and flowers has low yields 2003; Prehn et al., 2003). The objective of this (Urrea et al., 2009). Furthermore, the presence of program is to obtain a massive, efficient and significant quantities of camphor in its essential oil, economical production of “quillay” trees with a high which is responsible for the medicinal use in the content of good quality and low toxicity saponins. treatment of respiratory ailments (Gornemann et al., For this purpose, in vitro culture techniques for the 2008), confers an unpleasant taste and restricts its use multiplication of this species (micropropagation and in the food industry (Urrea et al., 2009). embryogenesis) were implemented (Prehn et al., Commercial cultivation is very recent and at 2003; Vega & Prehn, 2005). Besides, there have been a small scale and it is sold falsely as “Stevia”. attempts to use in vitro-grown shoots as a source of Information about its cultivation, at least in Mexico, extracts with biological activity (Ribera et al., 2008), is nonexistent or very scarce (Fierro et al., 2015). The focusing on the value of developing in vitro plant species is also used in gardens and family orchards as tissue cultures in order to obtain desirable an ornamental. compounds. The vegetative propagation of the species has been reported (Fierro et al., 2015) and diverse 29) Stevia rebaudiana (Bertoni) Bertoni. Asteraceae biotechnological tools have been used to optimize the (“stevia”, “estevia”, “ka'a he'ẽ”). The main use is as a production of hernandulcine, such as natural sweetener (Durán et al., 2012), and there are micropropagation, callus culture and cell suspensions many brands in the market that use this species. (Urrea et al., 2009), and in vitro culture of apices in Besides, “stevia” has been suggested as solid and liquid media (Sauerwein et al., 1991a). hypoglycemic, antioxidant, antimicrobial, antifungal There are also reports on the use of hairy root and anticancer agent (Gupta et al., 2013). cultures to obtain hernandulcine in vitro (Sauerwein The main metabolites are stevioside and et al., 1991b), indicating that the transformed roots rebaudioside A, which are 250 to 300 times sweeter can produce secondary metabolites present in the than sucrose. Both compounds are thermo and pH- mother plant, even if those compounds are not found stable and non-fermentable. For these reasons, in the root system of the plant. Molecular markers to “stevia” is one of the main sucrose substitutes and it analyze its genetic diversity (together with other also possesses potential as a health promoter (Gupta species of the genus Lippia) have also been used, et al., 2013). through cpDNA ITSs and ISSRs (Suárez González et Due to the real and potential economic value al., 2007). of “stevia”, there is a large quantity of publications Despite the high sweetening and medicinal dealing with biotechnological applications, power of P. scaberrima and the high costs of in vivo particularly tissue culture and breeding of the species.

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There are several reports on its micropropagation, polymorphisms between cultivated plants, which with different conditions and techniques (Oviedo- could be helpful for the identification of cultivars Pereira et al., 2015). The highest multiplication rates designated to the production of steviosides. Khan et were obtained in semisolid mediums by Mohamed al. (2014) reported the generation of somaclones in in (2011), Preethi et al. (2011), Verma et al. (2011), and vitro-derived plants with lower contents of steviol Thiyagarajan & Venkatachalam (2012). Temporary than the mother plant. immersion systems (TIS) were also developed for Finally, this species is cultivated since early this species, but the multiplication rates were lower 1970s and as a result of the introduction of “stevia” in (Jiménez-Quesada, 2011; Noordin et al., 2012; Japan and their research to evaluate the potential of Alvarenga-Venutolo & Salazar-Aguilar, 2015). the species, nowadays commercial cultivation is However, the quality of the obtained plantlets was extended/attempted in Japan, southeast Asia, and different. The plantlets from TIS showed higher United States and there are guidelines available to length and biomass, and this facilitated their develop its cultivation (Ramesh et al., 2006; acclimatization (Alvarenga-Venutolo & Salazar- Carrascal, 2017). Aguilar, 2015). Another successful methodology used in this species is direct organogenesis using thin 30) Valeriana carnosa Sm. Valerianaceae (ñancú cell layer (TCL) (Ramírez-Mosqueda & Iglesias- lahuén, yerba del aguilucho blanco). Its underground Andreu, 2016). With this technique the authors have organs (rhizomes) have similar properties as obtained high multiplication rates and vigorous Valeriana officinalis L., and the mapuche ethnic, shoots. which considers it a magical plant, has used it from Several types of cultures were developed for time immemorial as a sedative, antispasmodic, the production of steviosides, such as callus, shoots, hepatoprotector, and to treat rheumatism (Estomba et roots, cell suspensions (Rajasekaran et al., 2007; al., 2005). It has also been cited as diuretic and Dheeranapattana et al., 2008; Karuppusamy, 2009; digestive (Bach et al., 2011). Among its active Mathur & Shekhawat, 2013). Rebaudioside-A is of principles, the major constituents are β- particular interest among the glycosides produced in sesquiphellandrene, α-pinene, bornyl acetate and the leaves of “stevia” because the most desirable mainly isovaleranic acid, a compound found in the flavour profile is the one with the most rebaudioside- widely used euro-asian commercial species V. A (Yadav et al., 2011). In this sense, Rajasekaran et officinalis, and associated with the sedative properties al. (2007) compared the stevioside production in both of “valerianas” (Bach et al., 2013). in vitro and ex vitro grown plants and in its different Contrary to V. officinalis, there are no organs. They found the highest contents of important commercial cultivars of this species. rebaudioside-A in in vitro leaves. Currently, the laboratories and herbalists´ providers One of the strategies used to increase the gather their material from wild populations, with the production of active principles is the generation of expected loss of the biological resource. The species autotetraploids (Li et al., 2012; Yadav et al., 2013). grows by forming isolated and low-number Yadav et al. (2013) reported the obtention of populations and the root and/or rhizome are used to tetraploids with higher content of rebaudioside-A induce sleep or as sedative. As a result, the whole than diploids. plant is lost when used, placing this and other species Genetic transformation protocols via A. of the genus Valeriana in risk of extinction. tumefaciens have also been reported (Khan et al., Recently, attempts to introduce this species 2014). Transformed plants showed a different into cultivation have been reported. It is expected in glycoside profile, where the content of steviol was the medium term that the results obtained will allow higher in the transformed plants than in mother plant, to start a rational productive economic management whereas the content of stevioside was higher in the with social inclusion (original groups and small mother plant. The level of rebaudioside-A was producers) that contemplate its conservation similar in both types of plants. (Nagahama et al., 2016). Concerning the application Genetic variability studies with ISSRs have of biotechnology, no references were found for this also been performed (Garro-Monge et al., 2014; species, which is disturbing since this species is Khan et al., 2014). Garro-Monge et al. (2014) found under risk.

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DISCUSSION developing world is limited by the resilience of Understanding that society at large depends to some nature and the ability of societies to manage it. extent on the extractive use of wild species is Indefinite harvesting, under growing human paramount (Hutton & Leader-Williams, 2003), but pressures, could be the harbinger of the extinction of the fate of many of the natural resources in the many species (Tarciso et al., 2017).

Figure No. 1 Ecological situation of the MAPs of the American continent involved in this review

In this paper, we reviewed the ecological the development of alternatives for their situation of 30 species, native to America, widely conservation. employed in the pharmaceutical, cosmetic and food On the other hand, nine of the selected industries. Figure No. 1 shows that, despite their species are domesticated and cultivated. However, as extensive use as raw materials for these industries, we mentioned before, in four of them the extractive more than half the species analysed (n=21) are regimes are still in use. We found that, from the list exploited directly from the environment by wild of species analysed, the only five that are cultivated harvesting. Only four of them are extracted from the for their exploitation in the market are Aloysia wild but also cultivated in certain countries: citriodora (Elechosa, 2009; Alonso & Desmarchelier, Achyrocline satureioides (Retta et al., 2010; Davies, 2015), Lepidium meyenii (Wang et al., 2007), Phyla 2011), Aloysia polystachya (Alonso & scaberrima (Fierro et al., 2015), Momordica Desmarchelier, 2015; Martínez, 2005), Carapichea charantia (Portillo, 2009) and Stevia rebaudiana ipecacuanha (Oliveira & Martins, 1998; Ocampo, (Ramesh et al., 2006). Furthermore, another five 2007) and Lippia alba (Ringuelet & Cerimele, 2010; species are also cultivated mainly for their Alonso & Desmarchelier, 2015). ornamental purposes, and secondarily for their Harvesting wild species is frequently medicinal properties. We could find no references unsustainable and a major source of biodiversity loss concerning extraction or production management for (Rice et al., 1997; Struhsaker, 1998). Thus, 17 two species. species are in some risk of extinction, with varying Likewise, we found that the state-of-the-art degrees. Among the studied species, Aniba concerning the use of biotechnology in the different rosaedora, Eremanthus erythropappus, species was also very variable. It is well known that Minthostachys mollis and Quillaja saponaria are the in vitro propagation methods play a vital role not most affected ones. Their critical situation has led to only in searching for alternatives to produce desirable

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants medicinal compounds from plants, but also in the in order to establish the conditions to propagate the conservation and preservation of plant biodiversity species. Although this is necessary for conservation (Rao & Ravishankar, 2002; Karuppusamy, 2009; and/or subsequent improvement, in several of the Cruz-Cruz et al., 2013). In this respect, we found that works the continuity of the results obtained was not 77% of the selected species (23 species, Figure No. 2, found. These are the cases of Achyrocline Table No. 1) had been propagated through some satureioides (Gatusso et al., 2007; Severin et al., tissue culture technique (micropropagation, cell 2008; Kotik et al., 2014), Actaea racemosa (Lata et culture, indirect regeneration, somatic al., 2002), Aniba rosaedora (Handa et al., 2005; embryogenesis, protoplast culture, thin cell layer Jardim et al., 2010), Equisetum bogotense (Rodriguez and/or culture in bioreactors). Nevertheless, only in et al., 1994), Eremanthus erythropappus (Rosal et al., 50% of these propagated plants, tissue culture 2007; Prudente et al., 2016), Justicia pectoralis techniques were also implemented as a source for (Oliveira Freitas et al., 2016), Lepidium meyenii alternative production of secondary metabolites (Polzerová et al., 2011) and Lippia turbinata (Ortiz et (Figure No. 2a). This indicates that, despite the many al., 2007). Furthermore, in 23% of the species promising results reported, biotechniques are rarely studied, we could not find any report concerning their used to improve the rational profit of this germplasm. propagation despite the fact that they are in high For instance, there has been research dealing with demand and the possibilities that it could be used in production of the following compounds: essential oils the conservation of this species. These are the cases in micropropagated plants of Aloysia citriodora and of Adesmia emarginata, Baccharis articulata and B. Lippia integrifolia (Severin et al., 2006; Moradi et trimera, Cecropia pachystachya, Equisetum al., 2014; Iannicelli et al., 2016b), phenolic giganteum, Maytenus laevis and Valeriana carnosa. compounds in cell culture of Bauhinia forficata Biotechnological tools such as in-vitro (Lima, 2009), alkaloids (emetine and cephaeline) in regeneration together with genetic transformation or root cultures of Carapichea ipecacuanha (Teshima et polyploidization are important to facilitate efforts to al., 1988; Yoshimatsu & Shimomura, 1991), NDGA engineer secondary product metabolic pathways and flavonoids in organogenic calli of Larrea (Tripathi & Tripathi, 2003; Gómez-Galera et al., divaricata (Palacio et al., 2012), flavonoids in callus 2007). Genetic transformation may provide increased culture of Momordica charantia (Agarwal & Kamal, and efficient systems for in-vitro production of 2007), flavonoids in plantlets of Passiflora caerulea secondary metabolites (Tripathi & Tripathi, 2003). (Busilacchi et al., 2008), steviosides in cell Polyploids are usually more valuable because they suspension and plantlets in Stevia rebaudiana exhibit increased biomass and content of effective (Rajasekaran et al., 2007; Mathur & Shekhawat, compounds (Dhooghe et al., 2011). 2013). However, some of these reports are not In this sense, Table No. 1 details that 15 followed through, as far as scaling-up and/or species of the list were not only propagated in vitro production of the metabolites of interest, and we did but also subjected to some type of biotechnique. not find current applications of these reports as According to Figure No. 2b, this represents 50% of sources of active principles. This is disturbing, the studied species. In seven of these species (23%, because there exist examples of the integration Figure No. 2b), in vitro culture and propagation between biotechnological techniques, such as in vitro techniques were combined with transgenesis or cultures, and sustainable exploitation for the polyploidization strategies aimed at studying and/or formulation of a drug. This is the case of Aloysia improving the production of secondary metabolites. polystachya (Aguado et al., 2006). Also, molecular markers were applied to them. From Likewise, Figure No. 2a shows that, although a biotechnological perspective, Carapichea micropropagation protocols have been established ipecacuanha is a very good example to follow. In this and applied to half of the species, improved plants case, biotechnology was clearly used not only to have not been obtained from all of them. For preserve the germplasm, but also to develop this instance, in 27% of the species studied, the valuable medicinal species. Stevia rebaudiana is the implementation of micropropagation techniques has only species developed employing all the tools not been developed further to introduce an analysed in this review and its development improvement in the species. In many of the reports corresponds to the importance this species has gained analysed, the use of in vitro culture is only considered for the industries. In the remaining eight species

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(27%, Figure No. 2b), genetic variability was studied cuttings (Magalhães, 2000), we could not find any through a combination of in vitro culture and the use studies with molecular markers to analyze this of molecular markers. However, in some species the variability at the genetic level. Lippia turbinata is application of molecular markers still remains another case in which no genetic variability studies unexploited. This is the case of Achyrocline were detected, even though the technique used for its satureioides, in which despite the high variability propagation could compromise the genetic stability of expressed in important characters such as biomass the propagated material. Furthermore, although productivity, morphology and architecture of the Elechosa (2009) mentioned the possible existence of leaves and crowns, time of flowering, active different chemotypes, the extant genetic diversity is principles contents and rooting percentage of the completely unknown.

b

Only Propagation Propagation through through tissue tissue culture and culture others

Molecular Transgenesis or Markers Polyploidization and Only Molecular Molecular Markers markers

No Biotechnology

Figure No. 2 a) Application of plant propagation and active principles (AP) production studies on the MAPs of the American continent mentioned in this review. b) State-of-the-art concerning the combination of biotechnological tools in the MAPs of the American continent mentioned in this review Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/473

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Species PTC T P MM Biotec. Ref. 1) Achyrocline M - - - Gattuso et al., 2007; Severin et al., 2008; Kotik et satureioides (Lam.) DC. al., 2014; Rosso et al., 2015 2) Actaea racemosa L. IR - - AFLP Lata et al., 2002; Zerega et al., 2002; Hyung et H al., 2005 3) Adesmia emarginata - - - - - Clos 4) Aloysia citriodora M Ar - cpDNA Severin et al., 2005; Severin et al., 2006; Suárez Palau IR ITS González et al., 2007; El-Hawary et al., 2012; ISSR Figueroa et al., 2013; Moradi et al., 2014; Boustani et al., 2016; 5) Aloysia polystachya M - - - Aguado et al., 2006; (Griseb.) Moldenke Burdyn et al., 2006 6) Aniba rosaeodora M - - SSR Handa et al., 2005; Santos et al., 2008; Angrizani Ducke RAPD et al., 2013; Jardim et al., 2010 7) Argemone mexicana M At - RAPD Godoy-Hernández et al., 2008; Ziegler & L. CC Facchini, 2008; Trujillo-Villanueva, 2010 8) Baccharis articulata - - - Isoenzymes Vaco, 2011; Cúneo, 2012 (Lam.) Pers. and 9) B. ISSR (in 9) trimera (Less.) DC. 10) Bauhinia forficata M - - - Mello et al., 2000; Appezzato-da-Glória & Link subsp. pruinosa CC Machado, 2004; Lima, 2009; Teixera da Silva, (Vogel) Fortunato & 2013 Wunderlin 11) Carapichea M Ar - ISSR Jha et al., 1988; Teshima et al., 1988; Jha & Jha, ipecacuanha (Brot.) L. IR 1989; Yoshimatsu & Shimomura, 1991; Rout et Andersson SE al., 1992; Yoshimatsu & Shimomura, 1994; Rout et al., 2000; Yoshimatsu et al., 2003; Tripathi & Tripathi, 2003; Chaudhuri & Jha, 2008; Rossi et al., 2009; De Oliveira et al., 2010 12) Cecropia - - - - - pachystachya Trécul 13) Equisetum M (in - - Sequencing Rodriguez et al., 1994; Des Marais et al., 2003; giganteum L. and 14) CpDNA Brune et al., 2008 14) E. bogotense Kunth ISSR 15) Eremanthus M - - RAPD Moura, 2005; Estopa et al., 2006; Rosal et al., erythropappus (DC.) 2007; Freitas et al., 2008; Prudente et al., 2016 MacLeisch 16) Fabiana imbricata M - - - Razmilic et al., 1994; Schmeda-Hirschmann et al., Ruiz & Pav. IR 2004 CC TIS 17) Justicia pectoralis M - - - Oliveira Freitas et al., 2016 Jacq. 18) Larrea divaricata M - - - Palacio et al., 2008; Palacio et al., 2011; Palacio Cav. IR et al., 2012 CC 19) Lepidium meyenii M - - SSR Cheng et al., 2004; Wang et al., 2007; Hasan et Walp. IR cpDNA al., 2010; Polzerová et al., 2011 20) Lippia alba (Mill.) M - - ISSR Gupta & Khanuja, 2001; Suárez González et al., Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/474

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N. E. Br. ex Britton & IR RAPD 2007; Manica-Cattani et al., 2009; Pierre et al., P.Wilson CpDNA 2011; JibinaBai et al., 2014 ITS 21) Lippia integrifolia IR - P ISSR Iannicelli et al., 2016a; Iannicelli et al., 2016b (Griseb.) Hieron. 22) Lippia turbinata IR - - - Ortiz et al., 2007 Griseb. 23) Maytenus laevis - - - - - Reissek. 24) Minthostachys mollis M - - EST-ISSR Chebel et al., 1998; Bima et al., 2006; Schmidt- (Benth.) Griseb. AFLP Lebuhn, 2008; Bonafede et al., 2014 25) Momordica M Ar RAPD Agarwal & Kamal, 2007; charantia L. IR AFLP Singh et al., 2007; Behera et al., 2008a; Behera et CC ISSR al., 2008b; Ananya & Sarmistha, 2010; Swarna & SE SCAR Ravindhran, 2012; Thiruvengadam et al., 2014; ITS2 Agarwal, 2015; Michel, 2016 26) Passiflora caerulea M - - RAPD Carnier Dornelas & Carneiro Vieira, 1994; L. IR Fajardo et al., 1998; Crochemore et al., 2003; Busilacchi et al., 2008; Severin et al., 2011; Ozarowski & Thiema, 2013; Rathod et al., 2014 27) Phyla scaberrima M Ar - ISSR/ Fierro et al., 2015; Sauerwein et al., 1991a; (Juss. ex Pers.) CC cpDNA/IT Sauerwein et al., 1991b; Suárez González et al., Moldenke S 2007; Urrea et al., 2009 28) Quillaja saponaria M - - - Prehn et al., 2003; Vega & Prehn, 2005; Ribera et Molina SE al., 2008 29) Stevia rebaudiana M At P ISSR Rajasekaran et al., 2007; Dheeranapattana et al., Bertoni (Bertoni) IR 2008; Karuppusamy, 2009; Jiménez-Quesada, CC 2011; Mohamed, 2011; Preethi et al., 2011; TIS Verma et al., 2011; Li et al., 2012; Noordin et al., TCL 2012; Thiyagarajan & Venkatachalam 2012; Mathur & Shekhawat, 2013; Yadav et al., 2013; Garro-Monge et al., 2014; Khan et al., 2014; Alvarenga-Venutolo & Salazar-Aguilar, 2015; Oviedo-Pereira et al., 2015; Ramírez-Mosqueda & Iglesias-Andreu, 2016 30) Valeriana carnosa - - - - - Sm.

Table No. 1 Biotechnological techniques applied to MAPs of the American continent mentioned in this review. References: PTC: Propagation through tissue culture; T: Transgenesis; P: Polyploidization; MM: Molecular Markers; M: microprogation; CC: cell culture; SE: somatic embryogenesis; P: protoplasts; IR: indirect regeneration; H: hydroponics; TIS: temporary immersion systems; TCL: thin cell layer; Ar: A. rhizogenes; At: A. tumefaciens.

The use of bioreactors and the TIS are two of method of plantation, or even plant cell culture, since the most powerful tools in biotechnology. Bioreactors scaled-up and automated systems help overcome are the key step towards commercial production of and/or minimize production costs, increase secondary metabolites by plant biotechnology multiplication rates and reduce the amount of (Tripathi & Tripathi, 2003). Large-scale plant tissue handling during the steps required for culture is an attractive alternative to the traditional micropropagation (Watt, 2012; Paek et al., 2005). Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/475

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However, TIS are not widely applied. In fact, from charantia, but there are many native species – weeds the list of species analysed in this review, we found – with important value as medicinals such as that this technology was used only in two species: Alternanthera pungens Kunth (“yerba del pollo”), Stevia rebaudiana (Jiménez-Quesada, 2011; Noordin Dysphania ambrosioides (L.) Mosyakin & Clemants et al., 2012; Alvarenga-Venutolo & Salazar-Aguilar, (“paico”) or Commelina erecta L. (“Santa Lucía”) 2015) and Fabiana imbricata (Schemeda- among others. Hirschmann et al., 2004). Safeguarding biodiversity is a global chore Finally, we found that in five species (17%, that should engage all of us. Through our systematic Figure No. 2b) no biotechnology was applied. These literature search and interviews with our colleagues, species are Adesmia emarginata, Baccharis we thought that the perilous situation of the MAPs articulata, Cecropia pachystachya, Maytenus laevis germplasm is similar in all the American continent and Valeriana carnosa. This is an alarming and and, perhaps, worldwide. disturbing fact, because the conservation and Stevia rebaudiana is a clear and paradigmatic breeding of these species is not being properly example of the mismanagement and exploitation of undertaken, although they are under pressure and the natural resources in our region, since Paraguay, even in risk. and particularly the Guaraní population, lost the Concerning the state-of-the-art in germplasm in the 70´s to the Japanese sweetening biotechnology, we must highlight the differences industry (Delgado Jiménez, 2016). Today, Japan is a found among the species analysed in this review. As major grower and marketer for the sweetener and has an example, we can compare Carapichea approved it for use in many food products, including ipecacuanha or Stevia rebaudiana versus Cecropia cereals, teas, and soft drinks (Ramesh et al., 2006). pachystachya. For “ipecacuana” many biotechniques The case of Larrea divaricata is another example of have been developed, including different forms of in the appropriation of genetic resources, without vitro propagation (relevant for its conservation) and recognizing the origin of the species and the ancestral the use of hairy roots, obtained by genetic knowledge that allowed the use of this species in folk transformation with Agrobacterium rhizogenes, as a medicine. In fact, the extraction process of NDGA source of active principles. Meanwhile, in “stevia” from L. divaricata was developed and patented by the practically all in vitro propagation methods have been University of Minnesota and produced in a explored. Even temporary immersion systems were commercial scale (Pardo, 1965). However, this patent developed with successful results. Concerning has expired. biotechniques, not only genetic transformation has The sustainable use and exploitation of been applied but also the obtaining of autopolyploids aromatic/medicinal native species is a multi- was described. Both techniques resulted in materials disciplinary task that requires the collaboration of with higher content of active principles. These cases ethnobotanists, taxonomists, ecophysiologists, are the opposite of “ambay”, which has not been chemists, pharmacologists, agronomists, propagated and its conservation status is not known, biotechnologists, together with the social work of although it is widely used and exploited by the local extensionists, economists and politicians. In this pharmaceutical industry. scenario, the countries in our region, through a It would be interesting to use the data network of laboratories, should collaborate in presented in this review, and further additional ones, studying, developing and proposing a rational and to promote programs to study native plants and their sustainable exploitation of the germplasm at risk. potential as cultivars for the future. Brazil developed This coordinated and concerted action will such a program during the past decade, and they enable the reversal of the status of many of these published a book where they detailed the possibilities species. In this context, biotechnology can play a of using many of their native plants as food, even relevant role, ranging from propagation methods to including cookbook recipes for each one (Ferreira the analysis of the genetic variability available in the Kinupp & Lorenzi, 2014). In Argentina, Rapoport et germplasm to be employed in breeding programs. al. (2009) have published a book on edible weeds, In addition, it has been demonstrated that where many of them, native to the Southern Cone biotechnological methods are reliable and safe and region, are medicinal as well as being classified as contribute to the conservation of biodiversity. They weeds. We mentioned the case of Momordica also provide high value products, such as

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Iannicelli et al. Conservation and biotechnology for some aromatic/medicinal plants pharmaceuticals, cosmetics, dietary supplements and with other more “profitable” ones, precluding the other industrial products. necessary concentration on species that need to be The strategy proposed by our team, which is studied urgently. constituted by pharmacists, agronomists, chemists, biologists and biochemists, is to obtain improved ACKNOWLEDGEMENTS genotypes of the wild plants and offer them to the This work was conducted with funds from PNHFA- harvesters/producers. Since we can use INTA´s 1106094, Instituto Nacional de Tecnología structure, we can work together with extensionists Agropecuaria (INTA). The authors are also thankful and take advantage of the social programs carried out to Consejo Nacional de Investigaciones Científicas y by INTA and the National Universities involved, Técnicas (CONICET, Argentina) for fellowship turning an extractivist model into a productive one. financial support. This is impossible without a State initiative to standarize and regulate the extraction from the wild, REFERENCES according to good extractive practices. Agarwal M. 2015. Tissue culture of Momordica With respect to the technological aspects, in charantia L.: A review. J Plant Sci 3: 24 - our research group we proposed the use of 32. polyploidization-nature´s own way of accelerating Agarwal M, Kamal R. 2007. Studies on flavonoid evolutionary processes-to develop MAPs´ germplasm production using in vitro cultures of rapidly. In agreement with the literature, in previous Momordica charantia L. Ind J Biotechnol 6: studies with ornamental/medicinal species, we 277 - 279. observed an increase in the size of organs when we Aguado MI, Núñez MB, Dudik HN, Bela A, Raisman applied this strategy (Escandón et al., 2005, 2006; JS, Sansberro P. 2006. Diseño de González Roca et al., 2015; Iannicelli et al., 2016a). comprimidos de extracto de Aloysia Since there were also reports of polyploidization on a polystachya por compresión directa. Acta number of MAPs, we adopted this strategy for Farm Bonaerense 25: 225 - 230. species at risk, starting with Lippia integrifolia, and, Alavarce RAS, Saldanha LL, Almeida NLM, Porto after 4 years, we registered a new variety of VC, Dokkedal AL, Lara VS. 2015. The “incayuyo”, Tawa INTA (Register number: 15679. beneficial effect of Equisetum giganteum L. Date: 2015/07/06), at the National Institute of Seed against Candida biofilm formation: new (INASE) of the Argentine Republic. This approaches to denture stomatitis. Evid-based development was declared of interest by Argentina´s Compl Altern Med 2015: Article ID Honorable National House of Representatives in 939625, 9 pages. 2015. Alonso JR. 2004. Tratado de Fitofarmacia y Nutracéuticos. Corpus Libros, Rosario, CONCLUSIONS Argentina. As a starting point, it is essential to understand our Alonso J, Desmarchelier C. 2015. Plantas knowledge with respect to the biology and the medicinales autóctonas de la Argentina. propagation of a certain species. From a Bases científicas para su aplicación en biotechnological viewpoint, and taking into account a atención primaria de la salud. Corpus few MAPs, we can observe that there is still a great Libros, Buenos Aires, Argentina. deal to do in the field and that rational and systematic Alvarenga-Venutolo S, Salazar-Aguilar T. 2015. work has been undertaken only with a few species. Micropropagación masiva de Stevia This is probably due to the economically valuable rebaudiana Bertoni en sistemas de inmersión active principles of those species and the publicity temporal. Cultivos Tropicales 36: 50 - 57. surrounding them. Ananya P, Sarmistha SR. 2010. Medicinal uses and This is a major hindrance, since this type of molecular identification of two Momordica research requires abundant funding and MAPs are not charantia varieties – a review. Elect J Biol 6: a priority in Western society, except when their illicit 43 - 51. exploitation yields good dividends. Consequently, in Anesini C, Genaro A, Boccio J, Cremaschi G, an effort to continue working with the native species Zubillaga M, Sterin Borda L, Borda E. 1998. in risk of extinction, we combine this line of work In vivo antitumour activity of Larrea

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