Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 127, Issue 2, pp 417–423 | Cite as

Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

  • Sarika Gupta
  • Joyce Van Eck
Original Article


Tomato (Solanum lycopersicum) has rapidly become a valuable model species for a variety of studies including functional genomics. A high-throughput method to obtain transgenic lines sooner than standard methods would greatly advance gene function studies. The goal of this study was to optimize our current transformation method by investigating medium components that would result in a decreased time for recovery of transgenics. For this study, 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings were infected with the Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium with indole-3-acetic acid (IAA) at concentrations of either 0.05 or 0.1 mg/l decreased the recovery time for transgenic lines by 6 weeks as compared to our standard medium that contains zeatin as the only plant growth regulator. We observed 50 and 54 % transformation efficiency on plant regeneration medium containing 0.05 and 0.1 mg/l IAA, respectively. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in earlier root development than medium that did not contain IAA. Addition of IAA to the plant regeneration and rooting media did not have any negative effects on plant development. Recovery of transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time and resources needed to complete investigations of gene function.


Agrobacterium tumefaciens Indole-3-acetic acid Solanaceae Solanum pimpinellifolium Tomato 



We thank Cynthia Du for assistance with part of the experimental process. We thank Cynthia Du, Patricia Keen, and Michelle Tjahjadi for their critical review of the manuscript. Support for this work was through a grant from the National Science Foundation Plant Genome Research Program (IOS-1237880).

Author contributions

SG and JVE designed the experiments, SG performed the experiments, SG and JVE wrote the manuscript. Both authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, Conrad LJ, Gelvin SB, Jackson DP, Kausch AP, Lemaux PG, Medford JI, Orozco-Cárdenas ML, Tricoli DM, Van Eck J, Voytas DF, Walbot V, Wang K, Zhang ZJ, Stewart CN Jr (2016) Advancing crop transformation in the era of genome editing. Plant Cell 28:1510–1520PubMedPubMedCentralGoogle Scholar
  2. Bombarely A, Menda N, Tecle IY, Buels RM, Strickler S, Fischer-York T, Pujar A, Leto J, Gosselin J, Mueller LA (2011) The Sol Genomics Network ( growing tomatoes using Perl. Nucleic Acids Res 39:1149–1155CrossRefGoogle Scholar
  3. Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen P-Y, Wang C-K, Soong S-C, To K-Y (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breeding 11:287–293CrossRefGoogle Scholar
  5. Chyi Y-S, Phillips GC (1987) High efficiency Agrobacterium-mediated transformation of Lycopersicon based on conditions favorable for regeneration. Plant Cell Rep 6:105–108PubMedGoogle Scholar
  6. Consortium TTG (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635–641CrossRefGoogle Scholar
  7. Dan Y, Zhang S, Matherly A (2016) Regulatoin of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation. Plant Cell Tissue Organ Cult. doi: 10.1007/s11240-016-1045-y Google Scholar
  8. Emmanuel E, Levy AA (2002) Tomato mutants as tools for functional genomics. Curr Opin Plant Biol 5:112–117CrossRefPubMedGoogle Scholar
  9. Fillatti JJ, Kiser J, Rose R, Comai L (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio Technol 5:726–730CrossRefGoogle Scholar
  10. Frary A, Earle ED (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep 16:235–240PubMedGoogle Scholar
  11. Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jurgens G (2003) Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426:147–153CrossRefPubMedGoogle Scholar
  12. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gonzali S, Mazzucato A, Perata P (2009) Purple as a tomato: towards high anthocyanin tomatoes. Trends Plant Sci 14:237–241CrossRefPubMedGoogle Scholar
  14. Gubis J, Lajchova Z, Farago J, Jurekova Z (2004) Effect of growth regulators on shoot induction and plant regeneration in tomato (Lycopersicon esculentum Mill.) Biologia 59:405–408Google Scholar
  15. Gupta S, Rashotte AM (2012) Down-stream components of cytokinin signaling and the role of cytokinin throughout the plant. Plant Cell Rep 31:801–812CrossRefPubMedGoogle Scholar
  16. Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S (2015) CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochem Biophys Res Commun 467:76–82CrossRefPubMedGoogle Scholar
  17. Jones JBJ (1998) Tomato plant culture: in the field, greenhouse, and home garden. CRC Press LLC, Boca RatonCrossRefGoogle Scholar
  18. Martel C, Vrebalov J, Tafelmeyer P, Giovannoni JJ (2011) The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner. Plant Physiol 157:1568–1579CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mccormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Leaf disk transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81–84CrossRefPubMedGoogle Scholar
  20. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  21. Nguyen HP, Chakravarthy S, Velasquez AC, McLane HL, Zeng L, Nakayashiki H, Park DH, Collmer A, Martin GB (2010) Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana. Mol Plant Microbe Interact 23:991–999CrossRefPubMedGoogle Scholar
  22. Overvoorde P, Fukaki H, Beeckman T (2010) Auxin control of root development. Cold Spring Harb Perspect Biol 21:1–16Google Scholar
  23. Park SH, Morris JL, Park JE, Hirschi KD, Smith RH (2003) Efficient and genotype-independent Agrobacterium—mediated tomato transformation. J Plant Physiol 160:1253–1257CrossRefPubMedGoogle Scholar
  24. Petersson SV, Johansson AI, Kowalczyk M, Makoveychuk A, Wang JY, Moritz T, Grebe M, Benfey PN, Sandberg G, Ljung K (2009) An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis. Plant Cell 21:1659–1668CrossRefPubMedPubMedCentralGoogle Scholar
  25. Pitzschke A, Hirt H (2010) New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation. EMBO J 29:1021–1032CrossRefPubMedPubMedCentralGoogle Scholar
  26. Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B (1999) An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99:463–472CrossRefPubMedGoogle Scholar
  27. Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin-cytokinin interactions in the control of shoot branching. Plant Mol Biol 69:429–435CrossRefPubMedGoogle Scholar
  28. Somerville C, Koornneef M (2002) A fortunate choice: the history of Arabidopsis as a model plant. Nat Rev Genet 3:883–889CrossRefPubMedGoogle Scholar
  29. Stepanova AN, Robertson-Hoyt J, Yun J, Benavente LM, Xie DY, DoleZal K, Schlereth A, Jurgens G, Alonso JM (2008) TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 133:177–191CrossRefPubMedGoogle Scholar
  30. Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431CrossRefPubMedGoogle Scholar
  31. Sun W, Xu X, Zhu H, Liu A, Liu L, Li J, Hua X (2010) Comparative transcriptomic profiling of a salt-tolerant wild tomato species and a salt-sensitive tomato cultivar. Plant Cell Physiol 51:997–1006CrossRefPubMedGoogle Scholar
  32. Van Eck J, Kirk DD, Walmsley AM (2006) Tomato (Lycopersicum esculentum). In: Wang K (ed) Methods in molecular biology, Agrobacterium protocols, vol 343. Humana Press Inc., Totowa, pp 459–473Google Scholar
  33. Xu C, Liberatore KL, MacAlister CA, Huang Z, Chu YH, Jiang K, Brooks C, Ogawa-Ohnishi M, Xiong G, Pauly M, Van Eck J, Matsubayashi Y, van der Knaap E, Lippman ZB (2015) A cascade of arabinosyltransferases controls shoot meristem size in tomato. Nat Genet 47:784–792CrossRefPubMedGoogle Scholar
  34. Yasmeen A (2009) An improved protocol for the regeneration and transformation of tomato (cv Rio Grande). Acta Physiol Plant 31:1271–1277CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.The Boyce Thompson InstituteIthacaUSA

Personalised recommendations