Potential for introducing cold tolerance into papaya by transformation with C-repeat binding factor (CBF) genes

  • S. A. Dhekney
  • R. E. Litz
  • D. A. Moraga Amador
  • A. K. Yadav
Biotechnology/Genetic Transformation/Functional Genomics


Papaya (Carica papaya L.) production is affected by low temperatures that occur periodically in the subtropics. The C-repeat binding factor (CBF) gene family is known to induce the cold acclimation pathway in Arabidopsis thaliana. Embryogenic papaya cultures were induced from hypocotyls of “Sunrise Solo” zygotic embryos on semisolid induction medium. The CBF 1/CBF 3 genes along with the neomycin phosphotransferase (NPT II) gene were placed under the control of the CaMV 35 S promoter and introduced into a binary vector pGA 643. Embryogenic cultures were transformed with Agrobacterium strain GV 3101 harboring pGA 643. After selection of transformed embryogenic cultures for resistance to 300 mg l−1 kanamycin, somatic embryo development was initiated and transgenic plants were regenerated. The presence of the CBF transgenes in regenerated plants was confirmed by Southern blot hybridization. The papaya and the related cold-tolerant Vasconcella genomes were probed for the presence of cold inducible sequences using polymerase chain reaction (PCR). Possible cold inducible sequences were present in the Vasconcella genome but were absent in the Carica genome.


Agrobacterium tumefaciens Carica Genetic transformation Somatic embryo 



The authors are grateful for support provided by USDA/CSREES research project on papaya biotechnology, to Dr. Michael Thomashow and Dr. Sarah Gilmour of Michigan State University who provided the CBF constructs, and to Dr. Nirmal Joshee (FVSU) for assistance with molecular biology techniques.


  1. Altschule, S. F.; Madden, T. L.; Schäffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402; 1997.CrossRefGoogle Scholar
  2. Aradhya, M. K.; Manshardt, R. M.; Zee, F.; Morden, C. W. A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region. Genet. Resour. Crop Evol. 46:579–589; 1999.CrossRefGoogle Scholar
  3. Burrow, M. D.; Chlan, C. A.; Sen, P.; Murai, N. High frequency generation of transgenic tobacco plants after modified leaf disk co-cultivation with Agrobacterium tumefaciens. Plant Mol. Biol. Rep. 8:124–139; 1990.Google Scholar
  4. Cai, W.; Gonsalves, C.; Tennant, P.; Fermin, G.; Sounz, M. Jr.; Sarinadu, N.; Jan, F. Y.; Zhu, H. Y. A protocol for efficient transformation and regeneration of Carica papaya L. In Vitro Cell Dev. Biol., Plant 35:61–69; 1999.Google Scholar
  5. Cheng, Y. H.; Yang, J. S.; Yeh, S. D. Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with carborundum. Plant Cell Rep. 16:127–132; 1996.Google Scholar
  6. Clark, M. S.; Gu, H. Y.; Qu, L. J. Plant Molecular Biology—A laboratory manual (Chinese translation) in DNA purification from polysaccharide rich cells. Protocols in Protozoology D-31; 1989.Google Scholar
  7. Fitch, M. M.; Manshardt, R. M.; Gonsalves, D.; Slightom, J. L.; Sanford, J. C. Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep. 9:189–194; 1990.Google Scholar
  8. Fitch, M. M.; Manshardt, R. M.; Gonsalves, D.; Slightom, J. L. Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep. 12:245–249; 1993.CrossRefGoogle Scholar
  9. Fowler, S.; Thomashow, M. F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14:1675–1690; 2002.PubMedCrossRefGoogle Scholar
  10. Gilmour, S. J.; Seblot, A. M.; Salazar, M. P.; Everard, J. D.; Thomashow M. F. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124:1854–1865; 2000.PubMedCrossRefGoogle Scholar
  11. Gilmour, S. J.; Zarka, D. G.; Stockinger, E. J.; Salazar, M. P.; Houghton, J. M.; Thomashow, M. F. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J. 16:433–442; 1998.PubMedCrossRefGoogle Scholar
  12. Gonsalves, D. Control of papaya ringspot virus in papaya: A case study. Annu. Rev. Phytopathol. 36:415–437; 1998.PubMedCrossRefGoogle Scholar
  13. Hsieh, T. H.; Lee, J. T.; Yang, P. T.; Chiu, L. H.; Charng, Y. Y.; Wang, Y. C.; Chan, M. T. Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol. 129:1086–1094; 2002.PubMedCrossRefGoogle Scholar
  14. Jaglo-Ottosen, K. R.; Gilmour, S. J.; Zarka, D. G.; Schabenberger, O.; Thomashow, M. F. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280:104–106; 1998.PubMedCrossRefGoogle Scholar
  15. Jaglo-Ottosen, K. R.; Kleff, S.; Amundsen, K. L.; Zhang, X.; Haake, V.; Zang, J. Z.; Deits, T.; Thomashow, M. F. Components of the Arabidopsis C-repeat/Dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol. 127:910–917; 2001.CrossRefGoogle Scholar
  16. Jorgensen, R. A.; Cluster, P. D.; English, J.; Que, Q.; Napoli, C. A. Chalcone synthase cosuppression phenotypes in petunia flowers: Comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31:957–973; 1996.PubMedCrossRefGoogle Scholar
  17. Kasuga, M.; Liu, Q.; Miura, S.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol. 17:287–291; 1999.PubMedCrossRefGoogle Scholar
  18. Levitt, J. In: Responses to Environmental Stresses: Chilling, Freezing, and High Temperature stress. Vol. 1. Academic Press, New York; 1980.Google Scholar
  19. Magdalita, P. M.; Yabut-Perez, E. M.; Mendoza, V. N.; Villegas, V. N.; Botella, J. R. Towards transformation, regeneration and screening of papaya containing Antisense ACC synthase Synthase gene. Abstract S42. 10th International Association of Plant Tissue Culture and Biotechnology Congress, Plant Biotechnology 2002 and Beyond: A showcase, Orlando, FL, June 23–28; 2002.Google Scholar
  20. Matzke, M. A.; Matzke, A. J. M.; Mittelsten, M. S. Inactivation of repeated genes- DNA-DNA interactions? In Paszkowski, J., ed. Homologous Recombination and Gene Silencing in Plants. Dordrecht: Kluwer Academic Publishers, 271–307; 1994.Google Scholar
  21. McCafferty, H. R. K.; Moore, P. H.; Zhu,Y. J. Towards improved insect resistance in papaya. Abst # 688. Annual Meeting of the American Society of Plant Biologists; 2003.Google Scholar
  22. Murashige, T.; Skoog, F. A revised medium for rapid growth of and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  23. Owens, C. L.; Thomashow, M. F.; Hancock, J.; Iezzoni, A. CBF1 orthologs in sour cherry and strawberry and the heterologous expression of CBF1 in strawberry. J. Am. Soc. Hortic. Sci. 127:489–494; 2002.Google Scholar
  24. Sakai, A.; Larcher, W. (eds). Frost Survival of Plants: Responses and Adaptation to Freezing Stress. Berlin Heidelberg New York: Springer; 1987.Google Scholar
  25. Stockinger, E. J.; Gilmour, S. J.; Thomashow, M. F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. U. S. A. 94:1035–40; 1997.PubMedCrossRefGoogle Scholar
  26. Thomashow, M. F. Plant Cold Acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:571–599; 1999.PubMedCrossRefGoogle Scholar
  27. Yeh, S. D.; Bau, H. J.; Cheng, Y. H.; Yu, T. A.; Yang, J. S. Greenhouse and field evaluations of coat-protein transgenic papaya resistant to papaya ringspot virus. Acta Hortic. 461:321–328; 1998.Google Scholar
  28. Ying, Z.; Xia, Y.; Davis, M. J. A new method for obtaining transgenic papaya plants by Agrobacterium-mediated transformation of somatic embryos. Proc. Fla. State Hort. Soc. 112:201–205; 1999.Google Scholar

Copyright information

© The Society for In Vitro Biology 2007

Authors and Affiliations

  • S. A. Dhekney
    • 1
  • R. E. Litz
    • 1
  • D. A. Moraga Amador
    • 2
  • A. K. Yadav
    • 3
  1. 1.Tropical Research and Education CenterUniversity of FloridaHomesteadUSA
  2. 2.Center for BiotechnologyUniversity of FloridaGainesvilleUSA
  3. 3.Agricultural Research StationFort Valley State UniversityFort ValleyUSA

Personalised recommendations