Agrobacterium Mediated Transformation of Somatic Embryos of Persian Walnut Using fld Gene for Osmotic Stress Tolerance

Authors
M. A. Sheikh Beig Goharrizi 1
A. Dejahang 1
M. Tohidfar 2
A. Izadi darbandi 3
C. Nestor 4
M. R. Hajirezaei 5
K. Vahdati 1
1 Department of Horticulture, Aburaihan Campus, University of Tehran, Pakdasht, Islamic Republic of Iran.
2 Department of Biotechnology, Faculty of New Technologies and Energy Engineering, Shahid Beheshti University, Tehran, Islamic Republic of Iran.
3 Department of Agronomy and Crops Breeding Sciences, Aburaihan Campus, University of Tehran, Pakdasht, Islamic Republic of Iran.
4 Institute of Molecular and Cell Biology of Rosario (IBR-CONICET) - National University of Rosario- Rosario – Argentina.
5 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 03, 06466 Gatersleben, Germany.
Abstract
Somatic embryos of Persian walnut were transformed with two strains of Agrobacterium tumefaciens i.e. LBA4404 and C58, and two plasmids, namely, pBI121 with nptII and gus genes for improving the transformation protocol, and p6u-ubi-FVTI plasmid containing the hpt and fld genes. The transformation frequency was 10%. PCR and RT-PCR analysis proved the presence and expression of the genes. The transgenic and non-transgenic somatic embryos of Persian walnut were exposed to four salinity levels (0, 50, 100, and 200 mM NaCl) and four osmotic stress (0, 1.5, 5, and 10% PEG) levels. After 20 days, the number of survived, secondary and cotyledonary somatic embryos, as well as fresh and dry weights of embryos were evaluated. In addition, the transgenic and non-transgenic regenerated plantlets with 3 leaves and 2.5 cm length were subjected to 200 mM NaCl. In both experiments, the main effects of fld-transformation and stress treatments on evaluated parameters were significant. Transgenic somatic embryos showed no significant differences at 0 and 200 mM NaCl and 0 and 1.5% PEG. Significant differences of transgenic vs. non-transgenic somatic embryos were observed at 50 and 100 mM NaCl and 5 and 10% PEG. Non-transgenic plantlets on medium containing 200 mM NaCl showed complete necrosis and died after 10 days, while transgenic lines continued growth until 45 days. Our results clearly showed that expression of fld gene increased stress tolerance in fld transformant lines of walnut, and also revealed that expression of this specific cyanobacterial protein may provide a powerful tool to improve tolerance to environmental stresses.

Keywords

1. Attree S. M., Moor, D., Sawhney, V. R. and Fowke, L. C. 1991. Enhanced Maturation and Desiccation Tolerance of White Spruce (Picea glauca) Somatic Embryos: Effects of a Non- Plasmolyzing Water Stress and Abscisic Acid. Annal. Bot., 68: 59-525.
2. Blanco, N. E., Ceccoli, R. D., Segretin, M. E., Poli, H. O., Voss, I., Melzer, M., Bravo-Almonacid, F. F., Scheibe, R., Hajirezaei, M. R. and Carrillo, N. 2011. Cyanobacterial Clavodoxin Complements Ferredoxin Deficiency in Knocked-down Transgenic Tobacco Plants. Plant J., 65: 922-935.
3. Chang, S., Puryear, J., and Cairney, J. 1993. A Simple and Efficient Method for Isolating RNA from Pine Trees. Plant Mol. Biol. Rep., 11(2): 113-116.
4. Driver, J. A. and Kuniyuki, A. H. 1984. In-vitro Propagation of Paradox Walnut Rootstock. HortSci., 19: 507–509.
5. Erdner, D. L., Price, N. M., Doucette, G. J., Peleato, M. L. and Anderson, D. M. 1999. Characterization of Ferredoxin and Flavodoxin as Markers of Iron Limitation in Marine Phytoplankton. Marin. Ecol. Prog. Ser., 184: 43–53.
6. Han, K. H., Gordon, M. P. and Strauss S. H. 1996. Cellular and Molecular Biology of Agrobacterium-mediated Transformation of Plants and Its Application to Genetic Transformation of Populus. In: “Biology of Populus”, (Eds.): Stettler, R. F., Bradshaw, H. D., Heilman, Jr. and Hinckley T. M.. NRC Research Press, Canada, PP. 201-216.
7. Hase, T., Schürmann, P. and Knaff, D. B. 2006. The Interaction of Ferredoxin with Ferredoxin-Dependent Enzymes. In: “Photosystem I”, (Ed.): Golbeck, J. H.. Springer, The Netherlands, PP. 477-498.
8. Holtgrefe, S., Bader, K. P., Horton, P., Scheibe, R., von Schaewen, A., and Backhausen, J. E. 2003. Decreased Content of Leaf Ferredoxin Changes Electron Distribution and Limits Photosynthesis in Transgenic Potato Plants. Plant physiol., 133(4), 1768-1778.
9. Hu, H. and Xiong, L. 2014. Genetic Engineering and Breeding of Drought-Resistant Crops. Ann. Rev. Plant Bio., 65: 715-741.
10. Jefferson R. A. 1987. Assaying Chimeric Genes in Plants: The GUS Gene Fusion System. Plant Mol. Biol. Report., 5: 387-405.
11. Kawasaki, T., G. Shimizu, and M. Moritsugu. 1983. Effects of High Concentrations of Sodium Chloride and Polyethylene Glycol on the Growth and Ion Absorption in Plants. II. Multi-compartment Transport Box Experiment with Excised Roots of Barley. Plant Soil, 75:87–93.
12. Knaff, D. B. 2005. Ferredoxin and Ferredoxin-Dependent Enzymes. In: “Dordrecht Oxygenic Photosynthesis: The Light Reactions”, (Eds.): D. R. Ort, and C. F. Yocum, Heichel, I. F. Kluwer Academic Publishers, The Nederlands, PP. 333-361.
13. Krajnakova, J., Gomory, D. and Haggman, H. 2009. Effect of Sucrose Concentration, Polyethylene Glycol and Activated Charcoal on Maturation and Regeneration of Abies cephalonica Somatic Embryos. Plant Cell Tiss. Org. Cult., 96: 251–262.
14. Leple, J. C., Brasiliero, A. C. M., Michel, M. F., Delmotte, F. and Jouanin, L. 1992. Transgenic Poplars: Expression of Chimeric Genes Using four Different Constructs. Plant Cell Rep., 11:137-141.
15. Lotfi, N., Vahdati, K., Kholdebarin, B. and Najafian Ashrafi, E. 2009. Germination, Mineral Composition and Ion Uptake in Walnut under Salinity Conditions. HortSci., 44: 1352-1357.
16. Mayhew, S. G. and Ludwig, M. L. 1975. Flavodoxins and Electron Transferring Flavoproteins. Enz., 12: 57–118.
17. McGranahan, G. H., Driver, J. A. and Tulecke, W. 1987. Tissue Culture of Juglans. Forest. Sci., 26: 261-271.
18. Munns, R. and Tester, M. 2008. Mechanisms of Salinity Tolerance. Ann. Rev. Plant Biol., 59: 651-681.
19. Murray, M. G. and Thompson, W. F. 1980. Rapid Isolation of Molecular Weight Plant DNA. Nucleic Acid Res., 8: 4321-4325.
20. Pueyo, J. J. and Gomez-Moreno, C. 1991. Characterization of the Cross-linked Complex Formed between Ferredoxin-NADP+ Reductase and Flavodoxin from Anabaena PCC 7119. Biochimica et Biophysica Acta (BBA) Bioenergetics., 1059: 149–156.
21. Pueyo, J. J., Gomez-Moreno, C. and Mayhew, S. G. 1991. Oxidation-reduction Potentials of Ferredoxin NADP+ Reductase and Flavodoxin from Anabaena PCC7119 and of Their Electrostatic and Covalent Complexes. Eur. J. Biochem., 202: 1065–1071.
22. Rodriguez, R. L. and Tait, R. C. 1983. Recombinant DNA Techniques: An Introduction. Addison-Wesley, Reading, Massachusetts, PP. 186–187.
23. Sandmann, G., Peleato, M. L., Fillat, M. F., Lazaro M. C. and Gomez-Moreno, C. 1990. Consequences of the Iron-dependent Formation of Ferredoxin and Flavodoxin on Photosynthesis and Nitrogen-fixation on Anabaena Strains. Photosynth. Res., 26: 119–125.
24. Singh, A. K., Li, H. and Sherman L. A. 2004. Microarray Analysis and Redox Control of Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803. Physiologia Plantarum, 120: 27–35.
25. Tognetti, V. B., Monti Marila, R., Valle Estela, M., Carrillo, N. and Smania, A. M. 2007, Detoxification of 2,4-Dinitrotoluene by Transgenic Plants Expressing a Bacterial Flavodoxin. Environ. Sci. Technol., 41: 4071–4076.
26. Tognetti, V. B., Palatnik, J. F., Fillat, M. F., Melzer, M., Hajirezaei, M. R., Valle, E. M. and Carrillo, N. 2006. Functional Replacement of Ferredoxin by a Cyanobacterial Flavodoxin in Tobacco Confers Broad-range Stress Tolerance. Plant Cell Online, 18: 2035-2050.
27. Vahdati, K., Bayat, S., Ebrahimzadeh, H., Jariteh, M. and Mirmasoumi, M. 2008. Effect of Exogenous ABA on Somatic Embryo Maturation and Germination in Persian Walnut (Juglans regia L.). Plant Cell Tiss. Organ Cult., 93: 163–171.
28. Vahdati, K., Jariteh, M., Niknam, V., Mirmasoumi, M. and Ebrahimzadeh, H. 2006. Somatic Embryogenesis and Embryo Maturation in Persian Walnut. Acta Hort., 705: 199–205.
29. Vahdati, K., McKenna, J. R., Dandekar, A. M., Leslie, C. A., Uratsu, S. L., Hackett, W. P., Negri, P. and McGranahan, G. H. 2002. Rooting and Other Characteristics of a Transgenic Walnut Hybrid (Juglans hindsii×J. regia) Rootstock Expressing rolABC. J. Amer. Soc. Hortic. Sci., 127: 724-728.
30. Vasquez-Tello, A., Zuily-Fodil, Y., Pham Thi, A. T., Vieira Da Silva, J. B. 1990. Electrolyte and Pi Leakages and Soluble Sugar Content as Physiological Tests for Screening Resistance to Water Stress in Phaseolus and Vigna Species. J. Exp. Bot., 41:827-832.
31. Verslues, P. E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J. and Zhu J. K. 2006. Methods and Concepts in Quantifying Resistance to Drought, Salt and Freezing, Abiotic Stresses that Affect Plant Water Status. Plant J., 45: 523–539.
32. Wang, W. X., Vinocur, B., Shoseyov, O. and Altman, A. 2001. Biotechnology of Plant Osmotic Stress Tolerance Physiological and Molecular Considerations. Acta Hortic., 560:285–292.
33. Widdel, F. 2007. Theory and Measurement of Bacterial Growth. Di Dalam Grundpraktikum Mikrobiologie, 4.
34. Yousef, N., Pistorius, E. K., Michel, K. P. 2003. Comparative Analysis of idiA and isiA Transcription under Iron Starvation and Oxidative Stress in Synechococcus elongatus PCC 7942 Wld-type and Selected Mutants. Arch. Microb., 180: 471–483.
35. Zecri, M., Parsons, L. R. 1990. Comparative Effects of NaCl and Polyethylene Glycol on Root Distribution, Growth, and Stomatal Conductance of Sour Orange Seedlings. Plant Soil, 129: 137-143.
36. Zheng, M., Doan, B., Schneider, T. D. and Storz, G. 1999. OxyR and SoxRS Regulation of fur. J. Bacteriol., 181: 4639–4643.
37. Zurbriggen, M. D., Tognetti, V. B. and Carrillo, N. 2007. Stress-induced Flavodoxin from Photosynthetic Microorganisms. The Mystery of Flavodoxin Loss from the Plant Genome. IUBMB Life, 59: 355-360.
38. Zurbriggen, M. D., Tognetti, V. B., Fillat, M. F., Hajirezaei, M. R., Valle, E. M. and Carrillo, N. 2008. Combating Stress with Flavodoxin: A Promising Route for Crop Improvement. Trend. Biotech., 26: 531–537.
Journal of Agricultural Science and Technology
Volume 18, Issue 2
March and April 2016
Pages 423-435