Abstract
The present study is to assess the morphophysiological development in vitro of Lippia grata Schauer, an endemic “Caatinga” species that shows pharmacological properties. Young cuttings containing nodal segments were divided into three explant types, namely E1 (apical bud), E2 (nodal segment below the apical bud) and E3 (nodal segment below E2); the explants were inoculated in MS medium supplemented with antioxidant compound (polyvinylpyrrolidone) or adsorbing agent (activated charcoal). The survival index, the occurrence of oxidation, contamination and the number of leaves and sprouts were assessed. The explants were cultivated in MS medium supplemented with polyethylene glycol 6000 (0, 0.5 and 1 g L−1) and closed with aluminum foil (total sealing) or with aluminum foil containing one hole (partial sealing), in the multiplication stage, due to hyperhydricity in vitro. Histological and biometric analyses were performed at the 45th cultivation day, as well as the analyses of antioxidative enzyme, and hydrogen peroxide and malondialdehyde levels. Normal leaves have presented greater organization and cellular development than hyperhydric leaves. The use of antioxidants was not efficient to control the oxidation of inoculated apical buds (E1), only. The lowest hyperhydricity rates in the multiplication stage were found in the partial sealing treatments; however, the total sealing of tubes, in combination with the PEG addition, has led to increased lipid peroxidation. In view of the results, it is concluded that the explant types, as well as the suitability of a functional gas exchange system in vitro, have favored the growth and development of cells and tissues, thus attenuating the hyperhydricity symptoms. In addition, this study demonstrates the suitability of the culture system through the facilitation of gas exchange allows the use of the protocol for in vitro propagation without compromising the leaf morphoanatomy.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anjum S, Xie X, Wang L (2011) Morphological, physiological and biochemical responses of plants to drought stress. African J Agric Res 6:2026–2032. https://doi.org/10.5897/AJAR10.027
Araujo MCR, Chagas EA, Garcia MIR, Pinto STS, Chagas PC, Vendrame W, Mota Filho AB, Souza OM (2016) Micropropagation of caçari under different nutritive culture media, antioxidants, and levels of agar and pH. Afr J Biotechnol 15:1771–1780
Argyropoulou C, Akoumianaki-Ioannidou A, Christodoulakis NS, Fasseas C (2010) Leaf anatomy and histochemistry of Lippia citriodora (Verbenaceae). Aust J Bot 58:398–409. https://doi.org/10.1071/BT10072
Azevedo RA, Alas RM, Smith RJ, Lea PJ (1998) Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation, in the leaves and roots of wild-type and a catalase-deficient mutant of barley. Physiol Plant 104:280–292. https://doi.org/10.1034/j.1399-3054.1998.1040217.x
Barbosa LMP, de Paiva Neto VB, Dias LLC, Festucci-Buselli RA, Alexandre RS, Iarema L, Finger FL, Otoni WC (2013) Biochemical and morpho-anatomical analyses of strawberry vitro plants hyperhydric tissues affected by BA and gelling agents. Rev Ceres 60:152–160. https://doi.org/10.1590/S0034-737X2013000200002
Barbosa MR, de Silva MMA, Willadino L, Ulisses C, Camara TR (2014) Geração e desintoxicação enzimática de espécies reativas de oxigênio em plantas. Ciênc Rural 44:453–460. https://doi.org/10.1590/S0103-84782014000300011
Bezerra RMDF, Aloufa MAI, Aurélio F, Freire DM (2014) Efeito de 6-benzilaminopurina sobre a propagação in vitro de Mimosa caesalpiniifolia Benth. (Fabaceae). Rev Árvore 38:771–778. https://doi.org/10.1590/S0100-67622014000500001
Bitu VCN, Martins da Costa JG, Rogrigues FFG et al (2015) Effect of collection time on composition of essential oil of Lippia gracilis Schauer (Verbenaceae) growing in Northeast Brazil. J Essent Oil Bear Plants 18:647–653. https://doi.org/10.1080/0972060X.2014.935043
Bukatsch F (1972) Bermerkungen zur Doppelfärbung: Astrablau-Safranin. Mikrokosmos 61:255
Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2006) Hyperhydricity in apple: ultrastuctural and physiological aspects. Tree Physiol 26:377–388
Costa AS, Arrigoni-Blank MF, Blank AF, Mendonça AB, Amancio VF, Ledo AS (2007) Estabelecimento de alecrim-pimenta in vitro. Hortic Bras 25:68–72. https://doi.org/10.1590/S0102-05362007000100013
Cruz EMDO, Pinto JAO, Fontes SS et al (2014) Water deficit and seasonality study on essential oil constituents of Lippia gracilis schauer germplasm. Sci World 314626:1–9. https://doi.org/10.1155/2014/314626
de Cruz EMO, Costa-Junior LM, Pinto JAO et al (2013) Acaricidal activity of Lippia gracilis essential oil and its major constituents on the tick Rhipicephalus (Boophilus) microplus. Vet Parasitol 195:198–202. https://doi.org/10.1016/j.vetpar.2012.12.046
Dewir YH, El-Mahrouk MES, Hafez YM et al (2015) Hyperhydricity in African violet (Saintpaulia ionantha H. Wendl)—biochemical aspects of normal versus hyperhydric shoots regenerated via direct adventitious shoots formation. Propag Ornam Plants 15:53–62. https://doi.org/10.4172/2329-955X.1000139.D
dos Santos CP, Pinto JAO, dos Santos CA et al (2016) Harvest time and geographical origin affect the essential oil of Lippia gracilis Schauer. Ind Crops Prod 79:205–210. https://doi.org/10.1016/j.indcrop.2015.11.015
Esyanti RR (2016) Efficiency evaluation of vanda tricolor growth in temporary immerse system bioreactor and thin layer culture system. J Adv Agric Technol 3:63–66. https://doi.org/10.18178/joaat.3.1.63-66
Fernandes LCB, de Albuquerque CC, Sales Júnior R et al (2015) Fungitoxicidade dos extratos vegetais e do óleo essencial de Lippia gracilis Schauer sobre o fungo Monosporascus cannonballus Pollack e Uecker. Summa Phytopathol. https://doi.org/10.1590/0100-5405/1978
Gao H, Xu P, Li J, Ji H, An L, Xia X (2017) AgNO3 prevents the occurrence of hyperhydricity in Dianthus chinensis L. by enhancing water loss and antioxidant capacity. In Vitro Cell Dev Biol Plant 53:561–570. https://doi.org/10.1007/s11627-017-9871-0
Giannopolitis CN, Ries SK (1977) Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol 59:309–314
Gomes SVF, Nogueira PCL, Moraes VRS (2011) Aspectos químicos e biológicos do gênero Lippia enfatizando Lippia gracilis Schauer. Eclet Quim 36:64–77. https://doi.org/10.1590/S0100-46702011000100005
Gribble K, Sarafis V, Conroy J (2003) Vitrified plants: towards an understanding of their nature. Phytomorphol Int J Plant Morphol 53:1–10
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. Arch Biochem Biophys 125:189–198. https://doi.org/10.1016/0003-9861(68)90654-1
Ivanova M, van Staden J (2009) Nitrogen source, concentration, and NH4 +:NO3 − ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla. Plant Cell Tissue Organ Cult 99:167–174. https://doi.org/10.1007/s11240-009-9589-8
Jausoro V, Llorente BE, Apóstolo NM (2010) Structural differences between hyperhydric and normal in vitro shoots of Handroanthus impetiginosus (Mart. ex DC) Mattos (Bignoniaceae). Plant Cell Tissue Organ Cult 101:183–191. https://doi.org/10.1007/s11240-010-9675-y
Johansen DA (1940) Plant microtechnique. McGraw Hill, New York
Karuppanapandian T, Moon JC, Kim C et al (2011) Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Aust J Crop Sci 5:709–725
Loreto F, Velikova V, Nazionale C et al (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127:1781–1787. https://doi.org/10.1104/pp.010497.Sharkey
Luz JMQ, Santos VA, Rodrigues TM et al (2014) Estabelecimento in vitro e aclimatização de Lippia alba (Mill.) NE Brown. Rev Bras Plantas Med 16:444–449. https://doi.org/10.1590/1983-084X/12_140
Marinho MJM, Albuquerque CC, Morais MB et al (2011) Estabelecimento de protocolo para micropropagação de Lippia gracilis Schauer. Rev Bras Plantas Med 13:246–252. https://doi.org/10.1590/S1516-05722011000200019
Martínez-Natarén DA, Parra-Tabla V, Dzib G, Calvo-Irabién LM (2011) Morphology and density of glandular trichomes in populations of Mexican oregano (Lippia graveolens H.B.K., Verbenaceae), and the relationship between trichome density and climate. J Torrey Bot Soc 138:134–144. https://doi.org/10.3159/TORREY-D-10-00007.1
Mohammad MA, Abbas Y, Mehdi HM (2014) Somayeh Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock. J Genet Eng Biotechnol 12:103–110. https://doi.org/10.1016/j.jgeb.2014.10.002
Morais MB, Camara TR, Ulisses C, Carvalho Filho JLS, Willadino L (2018) Multiple stresses on the oxidative metabolism of sugarcane varieties. Cienc Rural 48:1–8. https://doi.org/10.1590/0103-8478cr20141487
Murashige T, Skoog F (1962) A revised medium for rapid growth and Bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
O’Leary N, Denham SS, Salimena F, Múlgura ME (2012) Species delimitation in Lippia section Goniostachyium (Verbenaceae) using the phylogenetic species concept. Bot J Linn Soc 170:197–219. https://doi.org/10.1111/j.1095-8339.2012.01291.x
Palma D, Schuelter AR, Stefanello S, Fortes AMT (2011) Aspectos morfofisiológicos e controle da hiperhidricidade na cultura de tecidos vegetais. Rev Bras Agrociênc 17:174–184
Pinheiro MVM, Martins FB, Xavier A, Otoni WC (2013) Trocas gasosas influenciam na morfogênese in vitro de duas cultivares de oliveira (Oleaeuropaea L.). Rev Árvore 37:19–29. https://doi.org/10.1590/S0100-67622013000100003
Rodrigues M, Costa THF (2017) Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachtaindica A. Juss.). In Vitro Cell Dev Biol Plant 48:1–6. https://doi.org/10.1007/s11627-011-9398-8
Saher S, Piqueras A, Hellin E, Olmos E (2004) Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Physiol Plant 120:152–161. https://doi.org/10.1111/j.0031-9317.2004.0219.x
Sass JE (1951) Botanical microtechnique, 2nd edn. State College Press, Iowa
Sato Yoshiko A, Dias Teixeira HC, de Andrade Alves L, Souza Camello V (2001) Micropropagação de Celtis sp: controle da contaminação e oxidação. CERNE 7:117–123
Sen A, Alikamanoglu S (2013) Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture. In Vitro Cell Dev Biol Plant 49:396–404. https://doi.org/10.1007/s11627-013-9511-2
Silva VN, Souto LS, Dutra Filho JA, Souza TMA, Borges CHA (2015) Deposição de serapilheira em uma área de caatinga preservada no semiárido da Paraíba, Brasil Litter deposition in a savanna area preserved in the semiarid region of Paraíba, Brazil. Rev Verde Agroecol Desenvolv Sustent 10:21–25. https://doi.org/10.18378/rvads.v10i2.3409
Silveira DG, Vidal ÁM, Alberto C et al (2013) Aspectos morfofisiológicos na pré-aclimatização in vitro e aclimatização de plantas de caroá. Rev Ciênc Agron 44:544–553
Souza AVV, Souza DD, Silva FP, Santos US, Santos MC, Oliveira FJV (2012) Cultivo do alecrim do mato no Vale do São Francisco em função da adubação organo-mineral. Hortic Bras 30:S6010–S6015
Tabart J, Franck T, Kevers C, Dommes J (2015) Effect of polyamines and polyamine precursors on hyperhydricity in micropropagated apple shoots. Plant Cell Tissue Organ Cult 120:11–18. https://doi.org/10.1007/s11240-014-0568-3
Tozin LRS, Carvalho SF, Machado SR, Rodrigues TM (2015) Glandular trichome diversity on leaves of Lippia origanoides and Lippia stachyoides (Verbenaceae): morphology, histochemistry, and ultrastructure. Botany 93:297–306. https://doi.org/10.1139/cjb-2014-0251
Trevisan MTS et al (2016) Composition of essential oils and ethanol extracts of the leaves of Lippia species: identification, quantitation and antioxidant capacity. Rec Nat Prod 10:485–496
Urrea A, Castrillón P, Monsalve Z (2010) Propagación in vitro y desdiferenciación tisular en Lippia dulcis. Rev Actual Biol 31:21–29
Ziv M (1991) Vitrification: morphological and physiological disorders of in vitro plants. In: Debergh PC, Zimmerman RH (eds) Micropropagation technology and application. Springer, Dordrecht, pp 45–69
Acknowledgements
The authors gratefully acknowledge the Universidade Federal de Pernambuco for support and Conselho Nacional de Pesquisa (CNPq) for a scholarship granted to the first author.
Author information
Authors and Affiliations
Contributions
The authors claim to be responsible for preparing the manuscript “Controlling hyperhydricity in micropropagated plants of Lippia grata (Verbenaceae), a native species of a dry seasonal tropical forest with pharmacological potential” being the first author (LPN) participated in the data collection, data analysis and redaction of the article; these author (LMS) participated in the introduction and in vitro cultivation of plant material, histological analysis and data analysis; the third author (MBM) participated in the realization of histological and biochemical analyses and elaboration of the article; the fourth author (CCA) provided the plant material, contributed information for the species studied and on the technique of micropropagation; the fifth author (TRC) participated in the interpretation of biochemical data and review the article; the sixth author (CU) guided every step of the work and participated in the draft in grand review of the project and of the article.
Corresponding author
Rights and permissions
About this article
Cite this article
Palhares Neto, L., de Souza, L.M., de Morais, M.B. et al. Controlling hyperhydricity in micropropagated plants of Lippia grata Schauer (Verbenaceae), a native species of a dry seasonal tropical forest with pharmacological potential. Braz. J. Bot 41, 529–538 (2018). https://doi.org/10.1007/s40415-018-0476-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40415-018-0476-6