3 Biotech

, 8:410 | Cite as

Containment evaluation, cold tolerance and toxicity analysis in Osmotin transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby)

  • Vikas Yadav PatadeEmail author
  • Harsahay Meena
  • Atul Grover
  • Sanjay Mohan Gupta
  • M. Nasim
Original Article


The present study reports engineered cold tolerance and toxicity analysis in genetically modified tomato (Solanum lycopersicum L. cv. Pusa Ruby) developed through constitutive over expression of Nicotiana tabacum Osmotin gene. Rate of seed germination, seedling establishment and growth remained unaffected in the transgenic tomato in response to a low temperature (15 °C) treatment, but were significantly (P ≤ 0.05) reduced in the wild type. At reproductive stage, the wild type plants failed to recover at the low temperature (4.0 °C) treatment for 10 days but the transgenic plants survived successfully without any leaf senescence or other visible chilling injury symptoms. The quantitative transcript expression analysis confirmed up regulation of the transgene by 55% in the transgenic plants on cold treatment for 2 h whereas, the transcripts were not detected in the wild type. Containment evaluation under normal environmental conditions revealed similar morphology in both the transgenic and wild type tomato plants however an average fruit yield was higher in the transgenic plants (725.91 ± 39.27 g) than the wild type (679.84 ± 28.80 g). The composition of mature fruits in terms of element content was at par in both the transgenic and wild type except significantly higher Ca and Mg contents in the transgenic fruits than that of the wild type. Further, acute and sub-acute toxicity tests conducted in the adult female Wister rats revealed no mortality or significant changes in general and psychological behaviour, at par food intake and body weight and, normal biochemical, and hematological parameters for animals fed with the wild type or transgenic tomato fruits as compared to the control group, confirming its safety for animal consumption.


Genetic engineering Cold stress Germination TaqMan® probe Toxicity 



Authors are grateful to Prof. KC Bansal, National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India for providing binary construct with the Osmotin gene.

Author contributions

VYP conceived and designed the study. VYP, AG and SMG carried out cold tolerance analysis, containment evaluation, transcript expression and statistical analysis. HSM conducted toxicity tests. First draft of the manuscript was prepared by VYP, with inputs from HSM, AG and SMG. MN arranged funds and approved final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

13205_2018_1432_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 34 KB)
13205_2018_1432_MOESM2_ESM.ppt (2 mb)
Supplementary material 2 (PPT 2033 KB)


  1. Das M, Chauhan H, Chhibbar A, Rizwanul Haq QM, Khurana P (2011) High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by constitutive and inducible expression of tobacco Osmotin. Transgenic Res 20:231–246CrossRefGoogle Scholar
  2. Domı´nguez E, Cuartero J, Fernandez-Munoz R (2005) Breeding tomato for pollen tolerance to low temperatures by gametophytic selection. Euphytica 142:253–263CrossRefGoogle Scholar
  3. EPA (2000) Mammalian toxicity assessment guidelines for protein plant pesticides. FIFRA Scientific Advisory Panel. Environmental Protection Agency, ArlingtonGoogle Scholar
  4. Goel D, Singh AK, Yadav V, Babbar SB, Bansal KC (2010) Overexpression of Osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L). Protoplasma 245:133–141CrossRefGoogle Scholar
  5. Goyary D (2009) Transgenic crops, and their scope for abiotic stress environment of high altitude: biochemical and physiological perspectives. DRDO Sci Spectrum 195–201Google Scholar
  6. Jain NC (1986) Schalm’s Veterinary Hematology. 4th ed. Lea and Febiger, 600. Washington square, Philadelphia, USAGoogle Scholar
  7. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using a real-time quantitative PCR and the 2– ∆∆CT method. Methods 25:402–408CrossRefGoogle Scholar
  8. Mantri NL, Ford R, Coram TE, Pang ECK (2010) Evidence of unique and shared responses to major biotic and abiotic stresses in chickpea. Environ Exp Bot 69(3):286–292CrossRefGoogle Scholar
  9. Natt MP, Herrick CA (1952) A new blood diluent for counting erythrocytes and leucocytes of the chicken. Poult Sci 31:735–738CrossRefGoogle Scholar
  10. Ntatsi G, Savvas D (2014) Growth, yield, and metabolic responses of temperature-stressed tomato to grafting onto rootstocks differing in cold tolerance. J Amer Soc Hort Sci 139(2):230–243Google Scholar
  11. Parkhi V, Kumar V, Sunilkumar G, Campbell LM, Singh NK et al (2009) Expression of apoplastically secreted tobacco Osmotin in cotton confers drought tolerance. Mol Breed 23:625–639CrossRefGoogle Scholar
  12. Patade VY (2012) Development of a rapid, reliable and sensitive real time PCR method based on probe hybridisation for validation of transgenic plants. DRDO Technol Spectrum (Supplement). 31–36Google Scholar
  13. Patade VY, Bhargava S, Suprasanna P (2012) Transcript expression profiling of stress responsive genes in response to short-term salt or PEG stress in sugarcane leaves. Mol Biol Rep 39:3311–3318CrossRefGoogle Scholar
  14. Patade VY, Khatri D, Kumari M, Grover A, Gupta SM, Ahmed Z (2013) Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes. SpringerPlus 2:117CrossRefGoogle Scholar
  15. Patade VY, Khatri D, Kumari M, Grover A, Gupta SM, Damke S, Ahmed Z (2014) Simple and efficient method for transgenic confirmation. Natl Acad Sci Lett 37(1):87–90CrossRefGoogle Scholar
  16. Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologists programmers. In: Krawetzs S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
  17. Sarad N, Rathore M, Singh NK, Kumar N (2004) Genetically engineered tomatoes: new vista for sustainable agriculture in high altitude regions. In: Proceedings of the Fourth international crop science congress Brisbane, AustraliaGoogle Scholar
  18. Subramanyam K, Sailaja KV, Subramanyam K, Rao DM, Lakshmidevi K (2011) Ectopic expression of an Osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L). Plant Cell Tiss Org Cult 105:181–192CrossRefGoogle Scholar
  19. Subramanyam K, Arun M, Mariashibu TS, Theboral J, Rajesh M, Singh NK, Manickavasagam M, Ganapathi A (2012) Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections. Planta 236:1909–1925CrossRefGoogle Scholar
  20. Van der Ploeg A, Heuvelink E (2005) Influence of sub-optimal temperature on tomato growth and yield: a review. J Hort Sci Biotechnol 80:652–659CrossRefGoogle Scholar
  21. Weber RLM, Wiebke-Strohm B, Bredemeier C et al (2014) Expression of an osmotin-like protein from Solanum nigrum confers drought tolerance in transgenic soybean. BMC Plant Biol 14:343CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Vikas Yadav Patade
    • 1
    Email author
  • Harsahay Meena
    • 1
  • Atul Grover
    • 1
  • Sanjay Mohan Gupta
    • 1
  • M. Nasim
    • 1
  1. 1.Defence Institute of Bio-Energy ResearchNainitalIndia

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