Efficient Transformation of Solanum tuberosum cv. Spunta Mediated by Agrobacterium tumefaciens Using Hygromycin as a Selective Agent

Cecilia Decima Oneto; Joel Coronel; Leonardo Storani; Matt Nailor; Sergio Feingold; Gonzalez Matias; Gabriela Massa

doi:10.67690/fdh5p957

Efficient Transformation of Solanum tuberosum cv. Spunta Mediated by Agrobacterium tumefaciens Using Hygromycin as a Selective Agent

Authors

Décima Oneto, C
Coronel, J.

Author
Storani, L.

Author
M. Nailor

Translator
Feingold, S.

Author
Gonzalez M. N.

Author
Massa, G. A.

Author

DOI:

https://doi.org/10.67690/fdh5p957

Abstract

Plant genetic transformation is a biotechnological tool with a high impact on plant breeding, since it offers the possibility of introducing new traits in crops to fulfill the demands of production and consumption. The most widely used transformation strategy in potatoes is based on the integration of genes via A. tumefaciens. The objective of the present work was to develop a transformation protocol for Solanum tuberosum cv. Spunta mediated by A. tumefaciens using hygromycin as a selective agent. For this, explants of potato cv. Spunta were transformed using A. tumefaciens with a binary vector containing the hygromycin resistance transgene (hpt) under the control of the NOS promoter. Molecular and phenotypic determinations were performed to validate both the efficiency of the transformation protocol and the performance of hygromycin as a selection agent. Sixty-two regenerated shoots were obtained from explants inoculated with A. tumefaciens (62% regeneration), of which 30 plants were positive for the hpt gene. A thirty percent transformation efficiency, calculated as the number of transgenic plants / the number of explants inoculated, was obtained using this protocol. The present work presents a simple and efficient protocol for the genetic transformation of potato cv. Spunta by A. tumefaciens using hygromycin as a selective agent.

Author Biographies

Décima Oneto, C
- PhD from the University of Buenos Aires, Biotechnology
- Researcher at the National Institute of Agricultural Technology
Coronel, J.

Joel Coronel is a researcher affiliated with Valencian International University (VIU), Spain, with research experience in plant biotechnology, genetic transformation, and agricultural biotechnology. His published work includes experimental research on Agrobacterium-mediated genetic transformation of potato, conducted in association with the Laboratory of Agrobiotechnology at Argentina’s National Institute of Agricultural Technology (INTA), EEA Balcarce.
Storani, L.

Leonardo Storani is a biotechnology researcher affiliated with the Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Balcarce, and CONICET in Argentina. He holds a degree in Biotechnology from the National University of General San Martín (UNSAM) and has research experience in plant biotechnology, molecular biology, genetic engineering, plant tissue culture, abiotic-stress tolerance, and CRISPR/Cas9 genome editing. His published work includes genetic transformation and genome editing of potato, including research aimed at reducing enzymatic browning and improving crop traits.
M. Nailor

Sindonologist and gerontologist specializing in the biology of aging and disease, Matt Nailor holds a Doctor of Divinity (D.D). His research focuses on the evolutionary theory of aging, which accounts for senescence through mutation accumulation, antagonistic pleiotropy, and disposable soma theory. This work engages origins science, with particular attention to molecular aging, mutation rates and accumulation, biological evolution, genetic variation, and historical linguistics.
Feingold, S.

Sergio Feingold, PhD, is Coordinator of the National Biotechnology Program at Argentina’s Instituto Nacional de Tecnología Agropecuaria (INTA). He is an Agricultural Engineer and holds a Master of Science in Plant Production and a Doctorate in Biological Sciences from the University of Buenos Aires (UBA). His research focuses on agricultural biotechnology, plant genomics, molecular genetics, crop improvement, and related applications of biotechnology.
Gonzalez M. N.

Researcher while affiliated with Instituto Nacional de Tecnología Agropecuaria and other places.
Massa, G. A.

Gabriela Massa is an Argentine biotechnologist and researcher at the National Institute of Agricultural Technology (INTA) and CONICET, with expertise in plant biotechnology, genetics, genomics, and crop improvement. Her research has focused particularly on the genetic improvement of potato and other crops, including the application of modern molecular techniques and CRISPR/Cas gene editing to develop varieties with improved agricultural and nutritional characteristics. She is also involved in university teaching and has contributed to numerous scientific publications in plant genetics and agricultural biotechnology.

References

AHLOOWALIA, B.S. 1982. Plant regeneration from callus culture in potato. Euphytica. 31, 755-759.

AHMED, H.A.; BARPETE, S.; AKDOGAN, G.; AYDIN, G.; SANKAT, C.; OZCAN, S. 2018. Efficient regeneration and mediated genetic transformation of potato (L.). Feb-Fresenius Environmental Bulletin, 3020.

AN, G.; COSTA, M.; HA, S. 1990. Nopaline Synthase Promoter Is Wound Inducible and Auxin Inducible. American Society of Plant Physiologists. The Plant Cell vol. 2, 225-233.

BAKHSH, A. 2020. Development of efficient, reproducible and stable Agrobacterium-mediated genetic transformation of five potato cultivars. Food Technology and Biotechnology, 58(1), 57-63.

BARPETE, S.; OGUZ, M.C.; ÖZCAN, S.F.; ANAYOL, E.; AHMED, H.A. 2015. Effect of temperature on germination, seed vigor index and seedling growth of five Turkish cotton (Gossypium hirsutum L.) cultivars. Fresen. Environ. Bull. 24, 2561-2566.

BEAUJEAN, A.; SANGWAN, R.S.; LECARDONNEL, A.; SANGWAN-NORREEL, B.S. 1998. Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. Journal of Experimental Botany, 49(326), 1589-1595.

BETTANY, A.; DALTON, S.; TIMMS, E.; DHANOA, M.; MORRIS, P. 2002. Effect of selectable gene to reporter gene ratio on the frequency of co-transformation and co-expression of uidA and hpt transgenes in protoplast-derived plants of tall fescue. Plant Cell, Tissue and Organ Culture, 68(2), 177-186.

BIRCH, R. 1997. Plant transformation: problems and strategies for practical application. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:297-326.

BIRCHLER, J. 2011. Plant Chromosome Engineering: Methods and Protocols. Methods in Molecular Biology, vol. 701, DOI 10.1007/978-1-61737-957-4_1.

BORNA, R.S.; HOQUE, M.I.; SARKER, R.H. 2010. Agrobacterium mediated genetic transformation for local cultivars of potato (Solanum tuberosum L.) using marker genes. Plant Tiss. Cult. Biotechno. 20, 145-155.

BRUCE, M.A.; SHOUP RUPP, J.L. 2019. Agrobacterium-Mediated Transformation of Solanum tuberosum L., Potato. In: KUMAR, S.; BARONE, P.; SMITH, M. (eds.). Transgenic Plants. Methods in Molecular Biology, vol. 1864. Humana Press, New York.

CAÑEDO, V.; CISNEROS, F. 2004. Clones de papa transformadas con la toxina de Bacillus thurigensis (Berliner) contra la polilla de la papa, Phthorimea operculella (Zeller). I. Transformación de clones de papa y verificación de la presencia del gen cry1A (b). Revista Peruana de Entomología 44:89-93.

CAVATORTA, J.; PEREZ, K.; GRAY, S.; VAN ECK, J.; YEAM, I.; JAHN, M. 2011. Engineering virus resistance using a modified potato gene. Plant Biotechnology Journal, 9(9), 1014-1021.

CHAKRABORTY, N.; CHAKRABORTY, P.; SEN, M.; BANDOPADHYAY, R. 2020. Choice of explant for plant genetic transformation. Biolistic DNA Delivery in Plants. Humana, New York. 107-123 pp.

CINGEL, A.; VINTERHALTER, B.; VINTERHALTER, D.; DRAGOSAVAC, D.A.; SMIGOCKI, A.; NINKOVI, S. 2010. Agrobacterium-mediated transformation of two Serbian potato cultivars (Solanum tuberosum L. cv. Draga_evka and cv. Jelica). Afr. J. Biotechno. 9, 4644-4650.

CRAZE, M.; BATES, R.; BOWDEN, S.; WALLINGTON, E.J. 2018. Highly Efficient Agrobacterium Mediated Transformation of Potato (Solanum tuberosum) and Production of Transgenic Microtubers. Current Protocols in Plant Biology, 3(1), 33-41.

DI RIENZO, J.; CASANOVES, F.; BALZARINI, M.; GONZÁLEZ, L.; TABLADA, M.; ROBLEDO, C. 2012. Programa Estadístico InfoStat, version 2012, para Windows. Grupo InfoStat, Universidad Nacional de Córdoba, Argentina.

DOUCHES, D.S.; WESTEDT, A.L.; ZARKA, K.; SCHROETER, B.; GRAFIUS, E.J. 1998. Breeding, cultivars, rootstocks, & germplasm resources: potato transformation to combine natural and engineered resistance for controlling tuber moth. HortScience, 33(6), 1053-1056.

FAO. Estadísticas. (Available: http://www.fao.org/statistics/es/ accessed: 05 September 2017).

HALTERMAN, D.; GUENTHNER, J.; COLLINGE, S.; BUTLER, N.; DOUCHES, D. 2016. Biotech potatoes in the 21st century: 20 years since the first biotech potato. American Journal of Potato Research, 93(1), 1-20.

HAYMES, K. 1996. Mini-prep method suitable for plant breeding programs. Plant Mol. Biol. Rep. 14: 280-284.

HEERES, P.; SCHIPPERS, R.M.; JACOBSEN, E.; VISSER, R.G.F. 2002. Transformation of a large number of potato varieties: genotype-dependent variation in efficiency and somaclonal variability. Euphytica. 124, 13-22.

HUARTE, M.; CAPEZIO, S. 2013. Cultivo de papa. (Available: http://inta.gob.ar/sites/default/files/script-tmp-inta-_huarte_capezio_papa2013.pdf accessed: August 2017).

IMBO, M.C.; BUDAMBULA, N.; MWEU, C.M.; MULI, J.K.; ANAMI, S.E. 2016. Genetic transformation of sweet potato for improved tolerance to stress: a review. Advances in Life Science and Technology, Vol. 49.

KASHANI, K.; JAVARAN, M.J.; MOHEBODINI, M.; MOIENI, A.; SHEIKI, M.; ABAD, D. 2012. Regeneration and Agrobacterium-mediated transformation of three potato cultivars (Solanum tuberosum cv. Désirée, Agria and Marfona) by human proinsulin gene. AJCS 6(7):1212-1220.

KHATUN, A.; HASAN, M.M.; BACHCHU, M.A.A.; MONIRUZZAMAN, M.; NASIRUDDIN, K.M. 2012. Agrobacterium-mediated genetic transformation of potato (Solanum tuberosum L.) var. Cardinal and Heera. The Agricult. Scientific J. Krishi Found. 10, 81-86.

LAGUNES, F.E. 2009. Transformación genética de ajo (Allium sativum L.) mediante Agrobacterium tumefaciens. Tesis, Magíster en Recursos Genéticos y Productividad, mención en Ciencias. Colegio de Postgraduados Campus Montecillo, Texcoco, Edo. de México. 95 p.

LÓPEZ, A.; CHAPARRO, A. 2007. A system for transformation of potato plants (Solanum tuberosum sp. andigena var. Pastusa Suprema) mediated through Agrobacterium tumefaciens. Agronomía Colombiana 25(1):16-25.

MASSON, J.; LANCELIN, D.; BELLINI, C.; LECERF, M.; GUERCHE, P.; PELLETIER, G. 1989. Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer. Theoretical and Applied Genetics, 78(2), 153-159.

M'HAMDI, M.; ROUVIERE, C.; ROJAS-BELTRAN, J.; DU JARDIN, P. 2003. Optimisation de la transformation génétique de la pomme de terre par Agrobacterium tumefaciens. Utilisation de la résistance à l'hygromycine comme marqueur sélectif. Biotechnol. Agron. Soc. Environ. 7 (3-4):183-188.

MILLAM, S. 2007. Potato (Solanum tuberosum L.). Agrobacterium Protocols, Volume 2, 25-35.

MONSERRAT, E.; MARFA, V.; MELÉ, E.; MESSENGUER, J. 2001. Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in co-cultivation. Plant Cell Tissue and Organ Culture 65: 211-220. DOI: http://dx.doi.org/10.1023/A:1010630726444.

MURASHIGE, T.; SKOOG, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497.

NADAKUDUTI, S.S.; STARKER, C.G.; VOYTAS, D.F.; BUELL, C.R.; DOUCHES, D.S. 2019. Genome editing in potato with CRISPR/Cas9. In: Plant Genome Editing with CRISPR Systems. Humana Press, New York. 183-201 pp.

OKADA, Y.; SAITO, A.; NISHIGUCHI, M.; KIMURA, T.; MORI, M.; HANADA, K.; MURATA, T. 2001. Virus resistance in transgenic sweetpotato [Ipomoea batatas L. (Lam)] expressing the coat protein gene of sweet potato feathery mottle virus. Theoretical and Applied Genetics, 103(5), 743-751.

ORBEGOZO, J.; ROMAN, M.; RIVERA, C.; TOVAR, J.C.; PÉREZ, W.; GAMBOA, S.; GHISLAIN, M. 2013. Agrotransformación y evaluación de la resistencia a Phytophthora infestans en Solanum tuberosum L. variedad Désirée. Revista Peruana de Biología, 20(3), 205-210.

ORBEGOZO, J.; ROMAN, M.; RIVERA, C.; GAMBOA, S.; TOVAR, J.; FORBES, G.; GHISLAIN, M. 2016. Rpi-blb2 gene from Solanum bulbocastanum confers extreme resistance to late blight disease in potato. Plant Cell, Tissue and Organ Culture (PCTOC), 125(2), 269-281.

PANDEY, S.; MISHRA, A.; PATEL, M.K.; JHA, B. 2013. An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.). Applied Biochemistry and Biotechnology, 171(1), 1-9.

PLACKETT, A.; HUANG, L.; SANDERS, H.; LANGDALE, J. 2014. High-efficiency stable transformation of the model fern species Ceratopteris richardii via microparticle bombardment. Plant Physiology, 165(1), 3-14.

ROMÁN, M.L.; RIVERA, C.; ORBEGOZO, J.; SERNA, F.; GAMBOA, S.; PEREZ, W.; GHISLAN, M. 2015. Resistencia a Phytophthora infestans linaje clonal EC-1 en Solanum tuberosum mediante la introducción del gen RB. Revista Peruana de Biología, 22(1), 63-70.

ROMANO, A.; RAEMAKERS, K.; BERNARDI, J.; VISSER, R.; MOOIBROEK, H. 2003. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes. Transgen. Res. 12, 461-473.

SUROV, T.; AVIV, D.; ALY, R.; JOEL, D.M.; GOLDMAN-GUEZ, T.; GRESSEL, J. 1998. Generation of transgenic asulam-resistant potatoes to facilitate eradication of parasitic broomrapes (Orobanche spp.), with the sul gene as the selectable marker. Theoretical and Applied Genetics, 96(1), 132-137.

VAN DEN ELZEN, P.; TOWNSEND, J.; LEE, K.; BEDBROOK, J. 1985. A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Molecular Biology, 5(5), 299-302.

WATERER, D.; BENNING, N.T.; WU, G.; LUO, X.; LIU, X.; GUSTA, M.; GUSTA, L.V. 2010. Evaluation of abiotic stress tolerance of genetically modified potatoes (Solanum tuberosum cv. Desiree). Molecular Breeding, 25(3), 527-540.

WENDT, T.; HOLM, P.; STARKER, C.; CHRISTIAN, M.; VOYTAS, D.; BRINCH-PEDERSEN, H.; HOLME, I. 2013. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants. Plant Molecular Biology, 83(3), 279-285.

Publication Note

This article is an English translation of a study that was peer-reviewed and previously published, in Spanish, as an open-access article. The data, results, figures and conclusions are those of the original authors; the text has been translated and the figures redrawn in English. The original was published as:

Décima Oneto, C.; Coronel, J.; Storani, L.; González, M.N.; Feingold, S.; Massa, G.A. (2020). Transformación eficiente de Solanum tuberosum cv. Spunta mediada por Agrobacterium tumefaciens utilizando higromicina como agente selectivo. RIA: Revista de Investigaciones Agropecuarias, 46(2), 248-257.

Original Spanish article (open access): https://www.researchgate.net/publication/344292216

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