Abstract
Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency.
Key Message
A significant improvement in regeneration of transgenic tomato through identification of key regeneration factors and robust screening is described, enabling high throughput genetic engineering in tomato.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Auer CA, Motyka V, Březinová A, Kamínek M (1999) Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol Plant 105:141–147. https://doi.org/10.1034/j.1399-3054.1999.105121.x doi
Barker SJ, Stummer B, Gao L, Dispain I, O’Connor PJ, Smith SE (1998) A mutant in Lycopersicon esculentum Mill. with highly reduced VA mycorrhizal colonization: isolation and preliminary characterisation. Plant J 15:791–797
Broertjes C, Keen A (1980) Adventitious shoots: do they develop from one cell? Euphytica 29:73–87. https://doi.org/10.1007/bf00037251
Broertjes C, Haccius B, Weidlich S (1968) Adventitious bud formation on isolated leaves and its significance for mutation breeding. Euphytica 17:321–344. https://doi.org/10.1007/bf00056233
Chaudhry Z, Rashid H (2010) An improved Agrobacterium mediated transformation in tomato using hygromycin as a selective agent. Afr J Biotechnol 9:1882–1891
Cruz-Mendívil A, Rivera-López J, Germán-Báez LJ, López-Meyer M, Hernández-Verdugo S, López-Valenzuela JA, Reyes-Moreno C, Valdez-Ortiz A (2011) A simple and efficient protocol for plant regeneration and genetic transformation of tomato cv. Micro-Tom from leaf explants. HortScience 46:1655–1660
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469. https://doi.org/10.1104/pp.103.027979
Dan Y, Yan H, Munyikwa T, Dong J, Zhang Y, Armstrong CL (2006) MicroTom—a high-throughput model transformation system for functional genomics. Plant Cell Rep 25:432–441. https://doi.org/10.1007/s00299-005-0084-3
Dan Y, Zhang S, Matherly A (2016) Regulation of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation. Plant Cell Tissue Organ Cult 127:229–236. https://doi.org/10.1007/s11240-016-1045-y
Fillatti JJ, Kiser J, Rose R, Comai L (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Nat Biotechnol 5:726–730
Frary A, Earle ED (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep 16:235–240
Fuentes AD, Ramos PL, Sánchez Y, Callard D, Ferreira A, Tiel K, Cobas K, Rodríguez R, Borroto C, Doreste V, Pujol M (2008) A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors. Biotechnol J 3:1088–1093. https://doi.org/10.1002/biot.200700187
Ganoza MC, Kiel MC (2001) A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes. Antimicrob Agents Chemother 45:2813–2819. https://doi.org/10.1128/aac.45.10.2813-2819.2001
Gerszberg A, Hnatuszko-Konka K, Kowalczyk T, Kononowicz AK (2015) Tomato (Solanum lycopersicum L.) in the service of biotechnology. Plant Cell Tissue Organ Cult 120:881–902. https://doi.org/10.1007/s11240-014-0664-4
Gupta S, Van Eck J (2016) Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines. Plant Cell Tissue Organ Cult 127:417–423. https://doi.org/10.1007/s11240-016-1063-9
Karami O, Esna-Ashari M, Kurdistani GK, Aghavaisi B (2009) Agrobacterium-mediated genetic transformation of plants: the role of host. Biol Plant 53:201–212
Khanna H, Daggard G (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436. https://doi.org/10.1007/s00299-002-0529-x
Khoudi H, Nouri-Khemakhem A, Gouiaa S, Masmoudi K (2009) Optimization of regeneration and transformation parameters in tomato and improvement of its salinity and drought tolerance. Afr J Biotechnol 8:6068–6076
Khuong TTH, Crété P, Robaglia C, Caffarri S (2013) Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number. Plant Cell Rep 32:1441–1454. https://doi.org/10.1007/s00299-013-1456-8
Larkan NJ, Lydiate DJ, Parkin IAP, Nelson MN, Epp DJ, Cowling WA, Rimmer SR, Borhan MH (2013a) The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1. New Phytol 197:595–605. https://doi.org/10.1111/nph.12043
Larkan NJ, Ruzicka DR, Edmonds-Tibbett T, Durkin JMH, Jackson LE, Smith FA, Schachtman DP, Smith SE, Barker SJ (2013b) The reduced mycorrhizal colonisation (rmc) mutation of tomato disrupts five gene sequences including the CYCLOPS/IPD3 homologue. Mycorrhiza 23:573–584. https://doi.org/10.1007/s00572-013-0498-7
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technology 9:963–967. https://doi.org/10.1038/nbt1091-963
Lu J, Chen R, Zhang M, da Silva JA, Ma G (2013) Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi. J Plant Physiol 170:1202–1211. https://doi.org/10.1016/j.jplph.2013.03.019
Matsukura C, Aoki K, Fukuda N, Mizoguchi T, Asamizu E, Saito T, Shibata D, Ezura H (2008) Comprehensive resources for tomato functional genomics based on the miniature model tomato micro-tom. Curr Genomics. https://doi.org/10.2174/138920208786241225
McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81–84
Nguyen AH, Hodgson LM, Erskine W, Barker SJ (2016a) An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes. Plant Cell Tissue Organ Cult 127:623–635. https://doi.org/10.1007/s11240-016-1087-1
Nguyen AH, Wijayanto T, Erskine W, Barker SJ (2016b) Using green fluorescent protein sheds light on Lupinus angustifolius L. transgenic shoot development. Plant Cell Tissue Organ Cult 127:665–674. https://doi.org/10.1007/s11240-016-1079-1
Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735. https://doi.org/10.1007/s00425-002-0922-2
Pinto MdS, Abeyratne CR, Benedito VA, Peres LEP (2017) Genetic and physiological characterization of three natural allelic variations affecting the organogenic capacity in tomato (Solanum lycopersicum cv. Micro-Tom). Plant Cell Tissue Organ Cult 129:89–103. https://doi.org/10.1007/s11240-016-1159-2
Prihatna C (2018) Functional analysis of Rmc locus of tomato involved in mycorrhizal symbiosis and Fusarium wilt tolerance. Ph.D. Thesis, The University of Western Australia
Prihatna C, Barbetti MJ, Barker SJ (2018a) A novel tomato Fusarium wilt tolerance gene. Front Microbiol 9:1226. https://doi.org/10.3389/fmicb.2018.01226
Prihatna C, Larkan NJ, Barbetti MJ, Barker SJ (2018b) Tomato CYCLOPS/IPD3 is required for mycorrhizal symbiosis but not tolerance to Fusarium wilt in mycorrhiza-deficient tomato mutant rmc. Mycorrhiza 28:495–507. https://doi.org/10.1007/s00572-018-0842-z
Sivankalyani V, Takumi S, Thangasamy S, Ashakiran K, Girija S (2014) Punctured-hypocotyl method for high-efficient transformation and adventitious shoot regeneration of tomato. Sci Hort 165:357–364. https://doi.org/10.1016/j.scienta.2013.11.034
Tilney-Bassett RA (1986) Plant Chimeras. Edward Arnold (Publishers) Ltd., London
Acknowledgements
The first author acknowledges the financial assistance of an International SIRF Scholarship funded jointly by the Australian Government and The University of Western Australia. The authors sincerely thank Dr. Kirk W. Feindel, Centre for Microscopy, Characterization and Analysis (CMCA), The University of Western Australia, for scientific and technical assistance and the use of the facilities of the CMCA, The University of Western Australia, a facility funded by the University, State, and Commonwealth Governments. The authors sincerely thank Dr. Mark Waters (School of Chemistry and Biochemistry, The University of Western Australia) for the Gateway vectors pHGWFS7 and pDONR221, Dr. Nicholas Larkan (Armatus Genetics Inc., 450 Melville St, Saskatoon, SK S7J 4M2, Canada) for the vector pMDC32-RmcCDS, and Dr. Daniel Ruzicka (Donald Danforth Plant Science Center, St. Louis, United States) for the vector pEarleyGate-Solyc08g075770.
Author information
Authors and Affiliations
Contributions
CP designed and performed the research, collected the data, analysed and interpreted the data, and drafted the manuscript. SJB and MJB supervised and offered guidance on all aspects of the work and contributed to the writing and review of the manuscript. RC performed and assisted the plant tissue culture works.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Jose M. Segui-Simarro.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Prihatna, C., Chen, R., Barbetti, M.J. et al. Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato. Plant Cell Tiss Organ Cult 137, 473–483 (2019). https://doi.org/10.1007/s11240-019-01583-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-019-01583-w