Abstract
Genetic modification using Agrobacterium rhizogenes has been an important biotechnological tool for the improvement of secondary metabolite production in plant species over the past four decades. The tropical vine Pentalinon andrieuxii, a native plant from the Yucatán Peninsula, is known to produce the triterpene betulinic acid and the novel tri-nor-sesquiterpene urechitol A and is used in the Mayan traditional medicine to treat leishmaniasis-derived lesions. Agrobacterium rhizogenes strain ATCC 15834 was used to infect leaf and hypocotyl explants of P. andrieuxii to generate 14 transformed plant lines with increased production of the terpenoids betulinic acid and urechitol A. Insertion of the transgenes was confirmed through PCR amplification of the rolA, rolB, and rolC genes. Betulinic acid and urechitol A were detected and quantified using standardized high-performance liquid chromatography and gas chromatography analyses, respectively. A significant increase in the terpenoid content was observed in some of the transgenic plants with up to 11.37 and 3.35 times more betulinic acid and urechitol A, respectively, when compared with the wild-type control. These new transgenic lines are promising tools to further the studies and knowledge about the biosynthesis of terpenoids in P. andrieuxii, especially regarding the biosynthetic origin of the rare urechitols.
Similar content being viewed by others
References
Ali-Seyed M, Jantan I, Vijayaraghavan K, Bukhari SNA (2016) Betulinic acid: recent advances in chemical modifications, effective delivery, and molecular mechanisms of a promising anticancer therapy. Chem Biol Drug Des 87:517–536. https://doi.org/10.1111/cbdd.12682
Bettini PP, Marvasi M, Fani F, Lazzara L, Cosi E, Melani L, Mauro ML (2016) Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants. J Plant Physiol 204:27–35. https://doi.org/10.1016/j.jplph.2016.07.010
Bian ZH, Yang QC, Liu WK (2015) Effects of light quality on the accumulation of phytochemicals in vegetables produced in controlled environments: a review. J Sci Food Agric 95:869–877. https://doi.org/10.1002/jsfa.6789
Bonhomme V, Laurain-Mattar D, Lacoux J, Fliniaux MC, Jaquin-Dubreuil A (2000) Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only. J Biotechnol 81:151–158. https://doi.org/10.1016/S0168-1656(00)00287-X
Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotech Adv 26:318–324. https://doi.org/10.1016/j.biotechadv.2008.03.001
Bulgakov VP, Khodakovskaya MV, Labetskaya NV, Chernoded GK, Zhuravlev YN (1998) The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures. Phytochemistry 49:1929–1934. https://doi.org/10.1016/S0031-9422(98)00351-3
Burgos-May Y, Avilés-Berzunza E, Peña-Rodríguez LM, Godoy-Hernández G (2018) Genetic transformation of Pentalinon andrieuxii tissue cultures. In: Loyola-Vargas V, Ochoa-Alejo N (eds) Plant cell culture protocols, Methods in molecular biology, vol 1815. Humana Press, New York, NY, pp 475–492
Carvalho SD, Schwieterman ML, Abrahan CE, Colquhoun TA, Folta KM (2016) Light quality dependent changes in morphology, antioxidant capacity, and volatile production in sweet basil (Ocimum basilicum). Front Plant Sci 7:1–14. https://doi.org/10.3389/fpls.2016.01328
Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempé J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434
de Oliveira BH, Santos CAM, Espíndola APDM (2002) Determination of the triterpenoid, betulinic acid, in Doliocarpus schottianus by HPLC. Phytochem Anal 13:95–98. https://doi.org/10.1002/pca.628
Dilshad E, Cusido RM, Palazon J, Ramirez-Estrada K, Bonfill M, Bushra M (2015) Enhanced artemisinin yield by expression of rol genes in Artemisia annua. Malar J 14:1–10. https://doi.org/10.1186/s12936-015-0951-5
Dilshad E, Zafar S, Ismail H, Waheed MT, Cusido RM, Palazon J, Mirza B (2016) Effect of Rol genes on polyphenols biosynthesis in Artemisia annua and their effect on antioxidant and cytotoxic potential of the plant. Appl Biochem Biotechnol 179:1456–1468. https://doi.org/10.1007/s12010-016-2077-9
Domínguez-Carmona DB, Escalante-Erosa F, García-Sosa K, Ruiz-Pinell G, Gutierrez-Yapu D, Chan-Bacab MJ, Giménez-Turba A, Peña-Rodríguez LM (2010) Antiprotozoal activity of betulinic acid derivatives. Phytomedicine 17:379–382. https://doi.org/10.1016/j.phymed.2009.08.002
Georgiev MI, Agostini E, Ludwig-Müller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotech 30:528–537. https://doi.org/10.1016/j.tibtech.2012.07.001
Hiebert-Giesbrecht MR, Escalante-Erosa F, García-Sosa K, Dzib GR, Calvo-Irabien LM, Peña-Rodríguez LM (2016) Spatio-temporal variation of terpenoids in wild plants of Pentalinon andrieuxii. Chem Biodivers 13:1521–1526. https://doi.org/10.1002/cbdv.201600085
Hiebert-Giesbrecht MR, Novelo-Rodríguez CY, Dzib GR, Calvo-Irabien LM, von Arx G, Peña-Rodríguez LM (2018) Herb-chronology as a tool for determining the age of perennial forbs in tropical climates. Botany 96:73–78. https://doi.org/10.1139/cjb-2017-0167
Li J, Goto M, Yang X, Morris-Natschke SL, Huang L, Chen CH, Lee KH (2016) Fluorinated betulinic acid derivatives and evaluation of their anti-HIV activity. Bioorganic Med Chem Lett 26:68–71. https://doi.org/10.1016/j.bmcl.2015.11.029
Matveeva TV, Sokornova SV (2018) Agrobacterium rhizogenes-mediated transformation of plants for improvement of yields of secondary metabolites. In: Pavlov A, Bley T (eds) Bioprocessing of plant in vitro systems. Reference Series in Phytochemistry. Springer, Cham, pp 161–202
Mauro ML, Costantino P, Bettini PP (2017) The never ending story of rol genes: a century after. Plant Cell Tissue Organ Cult 131:201–212. https://doi.org/10.1007/s11240-017-1277-5
Morales JF (2009) Estudios en las Apocynaceae neotropicales XXXIX : revisión de las Apocynoideae y Rauvolfioideae de Honduras. DARWINIANA 66:217–262. https://doi.org/10.3989/ajbm
Pan L, Lezama-Davila CM, Isaac-Marquez AP, Calomeni EP, Fuchs JR, Satoskar AR, Kinghorn D (2012) Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii. Phytochemistry 82:128–135. https://doi.org/10.1016/j.phytochem.2012.06.012
Pavlova OA, Matveyeva TV, Lutova LA (2013) Rol-genes of Agrobacterium rhizogenes. Ecol Genet 11:59–68. https://doi.org/10.17816/ecogen11159-68
Peña-Rodríguez LM, Yam-Puc A, Knispel N, Schramek N, Huber C, Graßberger C, Ramírez-Torres F, Escalante-Erosa F, García-Sosa K, Hiebert-Giesbrecht MR, Chan-Bacab MJ, Godoy-Hernández G, Bacher A, Eisenreich W (2014) Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3′-R-hydroxy)-stearate in Pentalinon andrieuxii. J Org Chem 79:2864–2873. https://doi.org/10.1021/jo402677w
Phillips GC, Collins GB (1979) In vitro tissue culture of selected legumes and plant regeneration from callus cultures of red clover. Crop Sci 19:59–64. https://doi.org/10.2135/cropsci1979.0011183X001900010014x
Pulido MT, Serralta L (1993) Lista anotada de las plantas medicinales de uso actual en el Estado de Quintana Roo. México, Centro de Investigaciones de Quintana Roo
Qiao X, Sai L, Chen X, Xue L, Lei J (2019) Impact of fruit-tree shade intensity on the growth, yield, and quality of intercropped wheat. PLoS One 14:1–17. https://doi.org/10.1371/journal.pone.0203238
Riker AJ, Banfield WM, Wright WH, Keitt GW, Sagen HE (1930) Studies on infectious hairy root of nursery apple trees. J Agric Res 41:507–540
Sarkar S, Ghosh I, Roychowdhury D, Jha S (2018) The effects of rol genes of Agrobacterium rhizogenes on morphogenesis and secondary metabolite accumulation in medicinal plants. In: Kumar N (ed) Biotechnological approaches for medicinal and aromatic plants: conservation. Genetic Improvement and Utilization. Springer Singapore, Singapore, pp 27–51
Shkryl YN, Veremeichik GN, Bulgakov VP, Tchernoded GK, Mischenko NP, Fedoreyev SA, Zhuravlev YN (2008) Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol Bioeng 100:118–125. https://doi.org/10.1002/bit.21727
Sumiya T, Ishigami K, Watanabe H (2010) Stereoselective total synthesis of (+/−)-urechitol A. Angew Chem Int Ed Engl 49:5527–5528. https://doi.org/10.1002/anie.201002505
Tapia-Tussell R, Quijano-Ramayo A, Rojas-Herrera R, Larque-Saavedra A, Perez-Brito D (2005) A fast, simple, and reliable high-yielding method for DNA extraction from different plant species. Mol Biotechnol 31:137–139. https://doi.org/10.1385/MB:31:2:137
Yam-Puc A, Avilés-berzunza E, Chan-Bacab MJ, Peña-Rodríguez LM, Godoy-Hernández G (2012a) Agrobacterium-mediated transient transformation of Pentalinon andrieuxii Müll. Arg. Adv Biosci Biotechnol 3:256–258. https://doi.org/10.4236/abb.2012.33035
Yam-Puc A, Chee-González L, Escalante-Erosa F, Arunachalampillai A, Wendt O, Sterner O, Godoy-Hernández G, Peña-Rodríguez LM (2012b) Steroids from the root extract of Pentalinon andrieuxii. Phytochem Lett 5:45–48. https://doi.org/10.1016/j.phytol.2011.09.004
Yam-Puc A, Escalante-Erosa F, García-Sosa K, Ramírez-Torres FG, Chan-Bacab MJ, Eisenreich W, Huber C, Knispel N, Godoy-Hernández G, Peña-Rodríguez LM (2013) A case of mistaken identity: lupeol-3-(3-0-R)-hydroxy-stearate can be mistakenly identified as lupeol acetate when only analyzed by GC–MS. Phytochem Lett 6:649–652
Yam-Puc A, Escalante-Erosa F, Pech-Lopez M, Chan-Bacab MJ, Arunachalampillai A, Wendt OF, Sterner O, Peña-Rodríguez LM (2009) Trinorsesquiterpenoids from the root extract of Pentalinon andrieuxii. J Nat Prod 72:745–748
Acknowledgments
The authors wish to thank Claudia Guadalupe Torres-Calzada for PCR analyses and Karlina García-Sosa for technical support.
Funding
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT)-México (grant numbers CB2013/223404 and CB2015/257915). M.R.H.G. wishes to thank CONACYT for scholarship no. 297661.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Editor: Gregory Phillips
Rights and permissions
About this article
Cite this article
Hiebert-Giesbrecht, M.R., Avilés-Berzunza, E., Godoy-Hernández, G. et al. Genetic transformation of the tropical vine Pentalinon andrieuxii (Apocynaceae) via Agrobacterium rhizogenes produces plants with an increased capacity of terpenoid production. In Vitro Cell.Dev.Biol.-Plant 57, 21–29 (2021). https://doi.org/10.1007/s11627-020-10101-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11627-020-10101-z