Abstract
Guayusa (Ilex guayusa) is an endemic plant from the Amazon with potential medicinal applications. Indigenous people are familiar with such applications and use guayusa based on ancestral knowledge. There is a growing interest in guayusa-based products in urban areas of Ecuador and internationally. The supply cannot meet the demand. Currently, traditional practices are used for guayusa growth and the potential use of the protected forest is foreseen. This work describes a protocol for the in vitro propagation of guayusa, a sustainable solution to generate high quality plants in reduced space. Stakes obtained from stems were used as explants. Chemical sterilization with ethanol and sodium hypochlorite resulted in 100% surface-sterilized stakes. The growth medium mWPM resulted in favorable outcomes regarding shoot development and elongation, as well as rooting. Supplementation with activated charcoal resulted in reduced browning, only 10% of the shoots presented necrosis during the elongation phase. More than two thirds of shoots were able to develop roots spontaneously. Medium supplementation with the auxin indole-3-butyric acid, IBA, may be considered when rooting does not occur spontaneously. Acclimatization was performed in soil. The protocol was tested under different light spectra, revealing that guayusa growth is affected by light quality. The photobiology of this shade tolerant plant requires further characterization, but the data uncovered a potential role for green and far-red light in root development.
Key message
Guayusa was propagated on mWPM medium supplemented with activated charcoal. Spontaneous root development occurred in most shoots. Light quality affected plant development, green and far-red light could influence root growth.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- AC:
-
Activated charcoal
- B:
-
Blue light
- FR:
-
Far-red light
- G:
-
Green light
- IBA:
-
Indole-3-butyric acid
- LED:
-
Light-emitting diode
- MS:
-
Murashige & Skoog
- mWPM:
-
Modified Woody Plant Medium
- NAA:
-
1-Naphthaleneacetic acid
- R:
-
Red light
- STN:
-
Shoot tip necrosis
- W:
-
White light
- WPM:
-
Woody Plant Medium
References
Ascrizzi R, Fraternale D, Flamini G (2018) Photochemical response of parsley (Petroselinum crispum (Mill.) Fuss) grown under red light: The effect on the essential oil composition and yield. J Photochem Photobiol B Biol 185:185–191. https://doi.org/10.1016/j.jphotobiol.2018.06.006
Azad MAK, Yokota S, Ohkubo T et al (2005) In vitro regeneration of the medicinal woody plant Phellodendron amurense Rupr. through excised leaves. Plant Cell Tissue Organ Cult 80:43–50. https://doi.org/10.1007/s11240-004-8809-5
Bairu MW, Stirk WA, Van Staden J (2009) Factors contributing to in vitro shoot-tip necrosis and their physiological interactions. Plant Cell Tissue Organ Cult 98:239–248. https://doi.org/10.1007/s11240-009-9560-8
Ballaré CL, Pierik R (2017) The shade-avoidance syndrome: multiple signals and ecological consequences. Plant Cell Environ 40:2530–2543. https://doi.org/10.1111/pce.12914
Bonga JM, Aderkas P, von Aderkas P (1992) In vitro culture of trees. Springer Science & Business Media, Berlin
Bussmann RW (2013) The Globalization of traditional medicine in Northern Peru: from Shamanism to molecules. Evid Based Complement Altern Med. https://doi.org/10.1155/2013/291903
Carvalho SD, Folta KM (2014) Environmentally modified organisms–expanding genetic potential with light. Crit Rev Plant Sci 33:486–508
Carvalho SD, Schwieterman ML, Abrahan CE et al (2016) Light quality dependent changes in morphology, antioxidant capacity, and volatile production in sweet basil (Ocimum basilicum). Front Plant Sci 7:1328
Chianese G, Golin-Pacheco SD, Taglialatela-Scafati O et al (2019) Bioactive triterpenoids from the caffeine-rich plants guayusa and maté. Food Res Int 115:504–510. https://doi.org/10.1016/j.foodres.2018.10.005
Dang JC, Kumaria S, Kumar S, Tandon P (2011) Micropropagation of Ilex khasiana, a critically endangered and endemic holly of Northeast India. AoB PLANTS. https://doi.org/10.1093/aobpla/plr012
Darko E, Heydarizadeh P, Schoefs B, Sabzalian MR (2014) Photosynthesis under artificial light: the shift in primary and secondary metabolism. Philos Trans R Soc Lond B Biol Sci 369:20130243. https://doi.org/10.1098/rstb.2013.0243
de Cássia TJ, Degenhardt-Goldbach J, Lucia Grunennvaldt R et al (2019) In vitro establishment of shoot meristems of Ilex paraguariensis and identification of endophytic bacteria. J for Res 30:1765–1777. https://doi.org/10.1007/s11676-018-0763-x
Dennis Thomas T, Yoichiro H (2010) In vitro propagation for the conservation of a rare medicinal plant Justicia gendarussa Burm. f. by nodal explants and shoot regeneration from callus. Acta Physiol Plant 32:943–950. https://doi.org/10.1007/s11738-010-0482-1
Dolce NR, Mroginski LA, Rey HY (2011) Enhanced seed germination of Ilex dumosa R. (Aquifoliaceae) through in vitro culture of cut pyrenes. HortScience 46:278–281. https://doi.org/10.21273/HORTSCI.46.2.278
Dueñas JF, Jarrett C, Cummins I, Logan-Hines E (2016) Amazonian Guayusa (Ilex guayusa Loes.): a Historical and Ethnobotanical Overview. Econ Bot 70:85–91. https://doi.org/10.1007/s12231-016-9334-2
El Comercio (2018) Napo exporta 120 toneladas de la hoja sagrada de guayusa a tres países. https://www.elcomercio.com/actualidad/napo-exportacion-guayusa-ecuador-bebida.html. Accessed 14 Jul 2020
El-Sherif NA (2019) Impact of Plant Tissue Culture on Agricultural Sustainability. In: Negm AM, Abu-hashim M (eds) Sustainability of agricultural environment in Egypt: Part II: soil-water-plant nexus. Springer International Publishing, Cham, pp 93–107
Fankhauser C, Christie JM (2015) Plant phototropic growth. Curr Biol 25:R384-389. https://doi.org/10.1016/j.cub.2015.03.020
FAO (2008) Erosion of plant genetic diversity. http://www.fao.org/newsroom/en/focus/2004/51102/article_51107en.html. Accessed 14 Jul 2020
Fu X, Chen Y, Mei X et al (2015) Regulation of formation of volatile compounds of tea (Camellia sinensis) leaves by single light wavelength. Sci Rep. https://doi.org/10.1038/srep16858
Fundación Futuro Latinoamericano and Grupo FARO (2020) ODS Territorio Ecuador. https://odsterritorioecuador.ec/. Accessed 14 Jul 2020
Galvão VC, Fankhauser C (2015) Sensing the light environment in plants: photoreceptors and early signaling steps. Curr Opin Neurobiol 34:46–53. https://doi.org/10.1016/j.conb.2015.01.013
Gamboa F, Muñoz C-C, Numpaque G et al (2018) Antimicrobial activity of Piper marginatum jacq and Ilex guayusa loes on microorganisms associated with periodontal disease. Int J Microbiol 2018:4147383. https://doi.org/10.1155/2018/4147383
Gan R-Y, Zhang D, Wang M, Corke H (2018) Health benefits of bioactive compounds from the Genus Ilex, a source of traditional caffeinated beverages. Nutrients. https://doi.org/10.3390/nu10111682
García-Ruiz A, Baenas N, Benítez-González AM et al (2017) Guayusa (Ilex guayusa L.) new tea: phenolic and carotenoid composition and antioxidant capacity. J Sci Food Agric 97:3929–3936. https://doi.org/10.1002/jsfa.8255
Giovannini P (2015) Medicinal plants of the Achuar (Jivaro) of Amazonian Ecuador: ethnobotanical survey and comparison with other Amazonian pharmacopoeias. J Ethnopharmacol 164:78–88. https://doi.org/10.1016/j.jep.2015.01.038
Gu R, Wang Y, Long B et al (2014) Prospecting for bioactive constituents from traditional medicinal plants through ethnobotanical approaches. Biol Pharm Bull 37:903–915. https://doi.org/10.1248/bpb.b14-00084
Haissig BE (1986) Metabolic processes in adventitious rooting of cuttings. In: Jackson MB (ed) New root formation in plants and cuttings. Springer, Netherlands, Dordrecht, pp 141–189
Huang D, Dai W (2011) Direct regeneration from in vitro leaf and petiole tissues of Populus tremula ‘Erecta.’ Plant Cell Tissue Organ Cult 107:169–174. https://doi.org/10.1007/s11240-011-9955-1
Innerhofer S, Bernhardt K-G (2011) Ethnobotanic garden design in the Ecuadorian Amazon. Biodivers Conserv 20:429–439. https://doi.org/10.1007/s10531-010-9984-9
Isbell F, Adler PR, Eisenhauer N et al (2017) Benefits of increasing plant diversity in sustainable agroecosystems. J Ecol 105:871–879. https://doi.org/10.1111/1365-2745.12789
Jain SM, Häggman H (eds) (2007) Protocols for micropropagation of woody trees and fruits. Springer, Netherlands
Jou J-H, Lin C-C, Li T-H et al (2015) Plant growth absorption spectrum mimicking light sources. Materials 8:5265–5275. https://doi.org/10.3390/ma8085240
Kapp RW, Mendes O, Roy S et al (2016) General and Genetic toxicology of guayusa concentrate (Ilex guayusa). Int J Toxicol 35:222–242. https://doi.org/10.1177/1091581815625594
Klem K, Gargallo-Garriga A, Rattanapichai W et al (2019) Distinct morphological, physiological, and biochemical responses to light quality in barley leaves and roots. Front Plant Sci 10:1026. https://doi.org/10.3389/fpls.2019.01026
Krause T, Ness B (2017) Energizing agroforestry: Ilex guayusa as an additional commodity to diversify Amazonian agroforestry systems. Int J Biodivers Sci Ecosyst Serv Manag 13:191–203. https://doi.org/10.1080/21513732.2017.1303646
Landi M, Zivcak M, Sytar O et al (2020) Plasticity of photosynthetic processes and the accumulation of secondary metabolites in plants in response to monochromatic light environments: a review. Biochim Biophys Acta Bioenerg 1861:148131. https://doi.org/10.1016/j.bbabio.2019.148131
Legris M, Ince YÇ, Fankhauser C (2019) Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nat Commun 10:5219. https://doi.org/10.1038/s41467-019-13045-0
Leone A, Grillo S, Monti L, Cardi T (2007) Molecular tailoring and boosting of bioactive secondary metabolites in medicinal plants. In: Ranalli P (ed) Improvement of crop plants for industrial end uses. Springer, Netherlands, Dordrecht, pp 471–507
Luna CV, Gonzalez AM, Mroginski LA, Sansberro PA (2017) Anatomical and histological features of Ilex paraguariensis leaves under different in vitro shoot culture systems. Plant Cell Tissue Organ Cult 129:457–467. https://doi.org/10.1007/s11240-017-1191-x
Martínez MT, Corredoira E, Vieitez AM et al (2017) Micropropagation of mature Quercus ilex L. trees by axillary budding. Plant Cell Tissue Organ Cult 131:499–512. https://doi.org/10.1007/s11240-017-1300-x
McCown BH (2000) Special symposium: in vitro plant recalcitrance recalcitrance of woody and herbaceous perennial plants: dealing with genetic predeterminism. In Vitro Cell Dev Biol Plant 36:149–154. https://doi.org/10.1007/s11627-000-0030-6
McCown BH, Lloyd G (1981) Woody plant medium (WPM)—a mineral nutrient formulation for microculture of woody plant species. HortScience 16:453–453
Mccown BH, Sellmer JC (1987) General media and vessels suitable for woody plant culture. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry: general principles and biotechnology. Springer, Netherlands, Dordrecht, pp 4–16
Mølmann JA, Hansen E, Johansen TJ (2020) Effects of supplemental LED light quality and reduced growth temperature on swede (Brassica napus L. ssp. rapifera Metzg.) root vegetable development and contents of glucosinolates and sugars. J Sci Food Agric. https://doi.org/10.1002/jsfa.10866
Morelli G, Ruberti I (2000) Shade avoidance responses. driving auxin along lateral routes. Plant Physiol 122:621–626. https://doi.org/10.1104/pp.122.3.621
Mroginski LA, Rouvier SM, Fabisik JC et al (1999) Effect of medium composition and light supply on in vitro shoot proliferation in Ilex paraguariensis (Aquifoloaceae). J Plant Nutr 22:359–368. https://doi.org/10.1080/01904169909365633
Nazari M, Zarinkamar F (2020) Ultraviolet-B induced changes in Mentha aquatica (a medicinal plant) at early and late vegetative growth stages: investigations at molecular and genetic levels. Ind Crops Prod 154:112618. https://doi.org/10.1016/j.indcrop.2020.112618
Opabode JT (2017) Sustainable mass production, improvement, and conservation of African indigenous vegetables: the role of plant tissue culture, a review. Int J of Veg Sci 23:438–455. https://doi.org/10.1080/19315260.2017.1319006
Paik I, Huq E (2019) Plant photoreceptors: multi-functional sensory proteins and their signaling networks. Semin Cell Dev Biol 92:114–121. https://doi.org/10.1016/j.semcdb.2019.03.007
Pardau MD, Pereira ASP, Apostolides Z et al (2017) Antioxidant and anti-inflammatory properties of Ilex guayusa tea preparations: a comparison to Camellia sinensis teas. Food Funct 8:4601–4610. https://doi.org/10.1039/c7fo01067b
Pennisi G, Blasioli S, Cellini A et al (2019) Unraveling the role of red: blue LED lights on resource use efficiency and nutritional properties of indoor grown sweet basil. Front Plant Sci. https://doi.org/10.3389/fpls.2019.00305
Perreault T (2005) Why Chacras (swidden gardens) persist: agrobiodiversity, food security, and cultural identity in the Ecuadorian Amazon. Hum Organ 64:327–339. https://doi.org/10.17730/humo.64.4.e6tymmka388rmybt
Pierik R, de Wit M (2014) Shade avoidance: phytochrome signalling and other aboveground neighbour detection cues. J Exp Bot 65:2815–2824. https://doi.org/10.1093/jxb/ert389
Piovene C, Orsini F, Bosi S et al (2015) Optimal red:blue ratio in led lighting for nutraceutical indoor horticulture. Sci Hortic 193:202–208. https://doi.org/10.1016/j.scienta.2015.07.015
Prefectura Napo, Ministerio del Ambiente, FAO, Gef (2017) Plan de manejo integral de la guayusa. http://info.napo.gob.ec/assets/bio_comercio_descargas/PMI_Guayusa%20Ruku%20Kawsay%20PKR_Portada.pdf. Accessed 14 Jul 2021
Radice M, Scalvenzi L, Sablón Cossio N (2017) Ilex guayusa: A systematic review of its Traditional Uses, Chemical Constituents, Biological Activities and Biotrade Opportunities, in Proceedings of the MOL2NET 2016, International Conference on Multidisciplinary Sciences, 2nd edition, 15 January–15 December 2016, MDPI: Basel, Switzerland. https://doi.org/10.3390/mol2net-02-03868
Rathore JS, Rathore V, Shekhawat NS et al (2005) Micropropagation of woody plants. In: Srivastava PS, Narula A, Srivastava S (eds) Plant biotechnology and molecular markers. Springer, Netherlands, Dordrecht, pp 195–205
Robles Arias DM, Cevallos D, Gaoue OG et al (2020) Non-random medicinal plants selection in the Kichwa community of the Ecuadorian Amazon. J Ethnopharmacol 246:112220. https://doi.org/10.1016/j.jep.2019.112220
Sabzalian MR, Heydarizadeh P, Zahedi M et al (2014) High performance of vegetables, flowers, and medicinal plants in a red-blue LED incubator for indoor plant production. Agron Sustain Dev 34:879–886. https://doi.org/10.1007/s13593-014-0209-6
Sansberro P, Rey H, Mroginski L, Collavino M (1999) In vitro plant regeneration of Ilex paraguariensis (aquifoliaceae). In Vitro Cell Dev Biol Plant 35:401–402. https://doi.org/10.1007/s11627-999-0054-5
Schuchovski CS, Biasi LA (2019) In vitro establishment of ‘Delite’ rabbiteye blueberry microshoots. Horticulturae 5:24. https://doi.org/10.3390/horticulturae5010024
Schultes RE (1994) Amazonian ethnobotany and the search for new drugs. Ciba Found Symp 185:106–112 (discussion 112–115). https://doi.org/10.1002/9780470514634.ch8
Sellaro R, Crepy M, Trupkin SA et al (2010) Cryptochrome as a sensor of the blue/green ratio of natural radiation in Arabidopsis. Plant Physiol 154:401–409. https://doi.org/10.1104/pp.110.160820
Skirycz A, Kierszniowska S, Méret M et al (2016) Medicinal bioprospecting of the amazon rainforest: a modern eldorado? Trends Biotechnol 34:781–790. https://doi.org/10.1016/j.tibtech.2016.03.006
Tarragó J, Filip R, Mroginski L, Sansberro P (2012) Influence of the irradiance on phenols content and rooting of Ilex paraguariensis cuttings collected from adult plants. Acta Physiol Plant 34:2419–2424. https://doi.org/10.1007/s11738-012-1009-8
Teixeira da Silva JA, Nezami-Alanagh E, Barreal ME et al (2020) Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions. Planta 252:47. https://doi.org/10.1007/s00425-020-03449-4
Thomas E, Semo L, Morales M et al (2011) Ethnomedicinal practices and medicinal plant knowledge of the Yuracarés and Trinitarios from Indigenous Territory and National Park Isiboro-Sécure, Bolivian Amazon. J Ethnopharmacol 133:153–163. https://doi.org/10.1016/j.jep.2010.09.017
Trigiano RN, Gray DJ (1999) Plant tissue culture concepts and laboratory exercises, 2nd edn. CRC Press, Boca Raton
Tripathi S, Hoang QTN, Han Y-J, Kim J-I (2019) Regulation of Photomorphogenic Development by Plant Phytochromes. Int J Mol Sci. https://doi.org/10.3390/ijms20246165
van Gelderen K, Kang C, Paalman R et al (2018a) Far-red light detection in the shoot regulates lateral root development through the HY5 transcription factor. Plant Cell 30:101–116. https://doi.org/10.1105/tpc.17.00771
van Gelderen K, Kang C, Pierik R (2018b) Light signaling, root development, and plasticity. Plant Physiol 176:1049–1060. https://doi.org/10.1104/pp.17.01079
Wang W, Chen Q, Botella JR, Guo S (2019) Beyond light: insights into the role of constitutively photomorphogenic1 in plant hormonal signaling. Front Plant Sci 10:557. https://doi.org/10.3389/fpls.2019.00557
Wang P, Chen S, Gu M et al (2020) Exploration of the effects of different blue LED light intensities on flavonoid and lipid metabolism in tea plants via transcriptomics and metabolomics. Int J Mol Sci. https://doi.org/10.3390/ijms21134606
Webb D (1981) Effects of light quality on root elongation and nodulation of Zamia floridana DC. Seedlings in sterile culture. Z Pflanzenphysiol 104:253–258. https://doi.org/10.1016/S0044-328X(81)80119-5
Wiñak Association (2020) Small Kichwa Farmers working together for the progress of their families and communities. https://www.winak.org/. Accessed 14 Jul 2020
Winkelmann T (2013) Recent advances in propagation of woody plants. Acta Hortic. https://doi.org/10.17660/ActaHortic.2013.990.47
Wise G, Negrin A (2020) A critical review of the composition and history of safe use of guayusa: a stimulant and antioxidant novel food. Crit Rev Food Sci Nutr 60:2393–2404. https://doi.org/10.1080/10408398.2019.1643286
World Health Organization (2002) WHO traditional medicine strategy 2002–2005. http://digicollection.org/hss/en/d/Js2297e/. Accessed 14 Jul 2021
Wu S-H (2014) Gene expression regulation in photomorphogenesis from the perspective of the central dogma. Annu Rev Plant Biol 65:311–333. https://doi.org/10.1146/annurev-arplant-050213-040337
Xu Y, Yang M, Cheng F et al (2020) Effects of LED photoperiods and light qualities on in vitro growth and chlorophyll fluorescence of Cunninghamia lanceolata. BMC Plant Biol 20:269. https://doi.org/10.1186/s12870-020-02480-7
Yadav A, Singh D, Lingwan M et al (2020) Light signaling and UV-B-mediated plant growth regulation. J Integr Plant Biol. https://doi.org/10.1111/jipb.12932
Yokoya NS, Yoneshigue-Valentin Y (2011) Micropropagation as a tool for sustainable utilization and conservation of populations of Rhodophyta. Rev Bras Farmacogn 21:334–339. https://doi.org/10.1590/S0102-695X2011005000077
Zhang T, Maruhnich SA, Folta KM (2011) Green light induces shade avoidance symptoms. Plant Physiol 157:1528–1536. https://doi.org/10.1104/pp.111.180661
Zhang T-J, Zheng J, Yu Z-C et al (2018a) Functional characteristics of phenolic compounds accumulated in young leaves of two subtropical forest tree species of different successional stages. Tree Physiol 38:1486–1501. https://doi.org/10.1093/treephys/tpy030
Zhang T-J, Zheng J, Yu Z-C et al (2018b) Variations in photoprotective potential along gradients of leaf development and plant succession in subtropical forests under contrasting irradiances. Environ Exp Bot 154:23–32. https://doi.org/10.1016/j.envexpbot.2017.07.016
Zheng C, Ma J-Q, Ma C-L et al (2019) Regulation of Growth and flavonoid formation of tea plants (Camellia sinensis) by blue and green light. J Agric Food Chem 67:2408–2419. https://doi.org/10.1021/acs.jafc.8b07050
Acknowledgements
The LED sources were installed in the plant tissue culture room with the dedicated contribution from Nelson Herrera and the Physical Plant Team of USFQ. We thank Runa Foundation for sharing the guayusa plants used as source material, the Laboratory of Plant Biotechnology at USFQ for daily and continuous support during the development of this project, and Wa and David Garcia for sharing information on methods used for guayusa growth and details on production systems. We thank Venancio Arahana, Andrea Montero and María Mercedes Cobo for assisting in experimental design of preliminary propagation trials. We thank Raquel Carvalho for helpful discussion and reviewing of the manuscript.
Funding
This work was supported by Fundación Runa and by Universidad San Francisco de Quito USFQ, Colegio de Ciencias Biológicas y Ambientales COCIBA Grants – Hubi 12431.
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MLT supervised the project. MLT, SC conceptualized the project. SC, MaO, and MLT designed the experiments, examined results. MaO, MiO, and MR performed the experiments, compiled and analyzed the data. KF made available the light sources and provided insight in result interpretations. SC and MaO prepared and wrote the manuscript. MLT provided feedback and editions during manuscript preparation. SC and MLT obtained funding for the study, oversaw experiments. All authors approved the manuscript.
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Carvalho, S.D., Ortega, M., Orellana, M. et al. In vitro propagation of the Amazonian medicinal plant guayusa (Ilex guayusa) and effects of light in the growth and development of this shade tolerant plant. Plant Cell Tiss Organ Cult 147, 503–517 (2021). https://doi.org/10.1007/s11240-021-02142-y
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DOI: https://doi.org/10.1007/s11240-021-02142-y