Agrobacterium-Mediated Transformation of Pomegranate (Punica granatum L.) ‘Yousef Khani’ Using the Gus Reporter Gene

Document Type : Research paper

Authors

1 Department of Horticultural Science, Faculty of Agriculture, Bu-Ali Sina University, Azadegan Boulevard, Hamedan, Iran

2 Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, Karaj, Iran

Abstract

In this study, an efficient Agrobacterium-mediated transformation method was developed for
pomegranate (Punica granatum L.), a difficult-to-transform plant. In vitro shoot segments were
inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector
pBI121 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and β-
glucuronidase (gus) gene as a reporter. After 28 d in WPM selection medium containing 50 mg
L-1 kanamycin, 59 new shoots proliferated. gus analysis was performed on these putative
transgenic shoots, of which 32 stained positive. Positive staining shoots were cut and cultured
in selection medium for 2 subsequent subcultures until final gus analysis. After three months of
the selection period, 6 putative transgenic shoots were obtained. Presence of the gus and nptII
genes was confirmed by polymerase chain reaction. Southern blot analysis confirmed that TDNA
was stably integrated into the genome of three out of six PCR-positive plants. The
transgenic plants were rooted and successfully acclimatized.

Keywords

References

1. Archilletti, T., P. Lauri, and C. Damiano. 1995. Agrobacterium-mediated Transformation of Almond Leaf Pieces. Plant Cell Rep. 14:267– 272.
2. Carroll, D., B. Puget, B. Higbee, M. Quist, O. Magallene, N. Smith, A. Gjerde, and K. Schneider. 2006. Pomegranate Pest Management in the San Joaquin Valley. www. aaie.net/IPMinfo/PomegranatePestManagement_ 002.pdf.
3. Cervera, M., A. Navarro, L. Navarro, L. Pena. 2008. Production of Transgenic Adult Plants from Clementine Mandarin by Enhancing Cell Competence for Transformation and Regeneration. Tree Physiol. 28:55–66.
4. Chauhan, R.P. and K. Kanwar. 2012. Biotechnological Advances in Pomegranate (Punica granatum L.). In Vitro Cellular and Developmental Biology – Plant.
5. Chen, W.X., G.F. Xiao, and Z. Zhu. 2002. Obtaining High Pestresistant Transgenic Upland Cotton Cultivars Carrying Cryiac3 Gene Driven by Chimeric OM Promoter. Acta Bot. Sin. 44:963–970.
6. Christou P. and D.E. McCabe. 1992. Prediction of Germ-Line Transformation Events in Chimeric Transgenic Soybean Plantlets Using Tissue-Specific Expression Patterns. Plant J. 2:283–290.
7. Corredoira, E., D. Montenegro, M.C. San-Jose, A.M. Vieitez, and A. Ballester. 2004. Agrobacterium-mediated Transformation of European Chestnut Embryogenic Cultures. Plant Cell Rep. 23:311–318.
8. Costa, M.G.C., W.C. Otoni, and G.C. Moore. 2002. An Evaluation of Factors Affecting the Efficiency of Agrobacterium-Mediated Transformation of Citrus paradisi (Macf.) and Production of Transgenic Plants Containing Carotenoid Biosynthetic Genes. Plant Cell Rep. 21:365–373.
9. De Vetten N., A.M. Wolters, K. Raemakers, I. Van der Meer, R. Ter Stege, E. Heeres, P. Heeres, and R. Visser. 2003. A Transformation Method for Obtaining Marker-Free Plants of a Cross-Pollinated and Vegetatively Propagated Crop. Nature Biotech. 21:439–442.
10. Domínguez, A., M. Cervera, R.M. Pérez, J. Romero, C. Fagoaga, J. Cubero, M.M. López, J.A. Juárez, L. Navarro, and L. Peña. 2004. Characterisation of Regenerants Obtained under Selective Conditions after AgrobacteriumMediated Transformation of Citrus Explants Reveals Production of Silenced and Chimeric Plants at Unexpected High Frequencies. Molecular Breeding. 14:171–183.
11. DOYLE, J. J. and J. L. DOYLE. 1987. Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin. 19:11–15.
12. Gago, J., J. Grima-Pettenati, and P.P. Gallego. 2011. Vascular-Specific Expression of GUS and GFP Reporter Genes in Transgenic Grapevine (Vitis vinifera L. cv. Albariño) Conferred by the Egccr Promoter of Eucalyptus gunnii. Plant Physiol. Biochem. 49:413-419.
13. Gomez-Lim, M.A. and R.E. Litz. 2004. Genetic Transformation of Perennial Tropical Fruits. In Vitro Cell Dev Biol Plant. 40:442–449.
14. Harjeet, K., S.K. Raina, Srinivasulu and K. Kumar. 1997. Biolistic Transformation of Elite Indica Rice (Oryza sativa L.) Cultivars Through Semi-Solid and Liquid Medium Selection Systems. J. Plant Biochem. Biotech. 6:75–80.
15.Jalikop, S.H. 2010. Pomegranate Breeding. Fruit, Vegetable and Cereal Sci. Biothec. 4:26-34.
16.James, D.J., A.J. Passey, and D.J. Barbara. 1990a. Agrobacterium-Mediated Transformation of the Cultivated Strawberry (Fragaria ×anannassa Duch.) Using Disarmed Binary Vectors. Plant Sci. 69:79-94.
17.James, D.J., A.J. Passey, and D.J. Barbara. 1990b. Regeneration and Transformation of Apple and Strawberry Using Disarmed Ti-Binary Vectors, pp. 239-249. In: G.W. Lycett and D. Grierson (eds.) Genetic Engineering of Crop Plants. Butterworths. London.
18.Jefferson, R.A., T.A. Kavanagh, and M.W. Bevan. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907.
19. Kanwar, K., R. Deepika, and R. Chauhan. 2008. Agrobacterium tumefaciens Mediated Genetic Transformation and Regeneration of Punica granatum L. National Seminar on Physiological and Biotechnological Approaches to Improve Plant Productivity, March 15–18, 2008. p-52.
20. Kim J.W. and T. Minamikawa. 1996. Transformation and Regeneration of French Bean Plants by the Particle Bombardment Process. Plant Sci. 117:131–138.
21. Liu, X. and P.M. Pijut. 2010. Agrobacteriummediated Transformation of Mature Prunus serotina (Black Cherry) and Regeneration of Transgenic Shoots. Plant Cell Tissue Organ Cult. 101:49–57.
22. Lloyd, G.B. and B.H. McCown. 1980. Commercially Feasible Micropropagation of Mountain Laurel, Kalmia latifolia by Use of Shoot Tip Culture. Comb. Proceed. Int. Plant Propa. Soc. 30:421–427.
23. Maheswaran, G.M., M. Welander, J.F. Hutchinson, M.W. Graham, D. Richards. 1992. Transformation of Apple Rootstock M26 with Agrobacterium tumefaciens. J. Plant Physiol. 139:560–568.
24. Mathews, H., V. Dewey, W. Wagoner, R.K. Bestwick. 1998. Molecular and Cellular Evidence of Chimaeric Tissues in Primary Transgenics and Elimination of Chimaerism Through Improved Selection Protocols. Transgenic Res. 7:123–129.
25. Miguel, C.M. and M.M. Oliveira. 1999. Transgenic Almond (Prunus dulcis Mill.) Plants Obtained by Agrobacterium-Mediated Transformation of Leaf Explants. Plant Cell Rep. 18:387–393.
26. Mirkarimi, A. 2000. Biological control of carob moth with mass release of Trichogramma embryophagum Hartig for Pomegranate worm control, the Ectomyelois (Spectrobates) ceratoniae Zell. Iranian J. Agr. Sci. 31:103–110.
27. Naik, S.K. and P.K. Chand. 2011. Tissue Culture-Mediated Biotechnological Intervention in Pomegranate: a Review. Plant Cell Rep. 30:707–721.
28. Niu, X., X. Li, P. Veronese, R.A. Bressan, S.C. Weller, and P.M. Hasegawa. 2000. Factors Affecting Agrobacterium tumefaciens Mediated Transformation of Peppermint. Plant Cell Rep. 19:304–310.
29. Norelli, J.L. and H.S. Aldwinckle. 1993. The Role of Aminoglycoside Antibiotics in the Regeneration and Selection of Neomycin Phosphotransferase-Transgenic Apple Tissue. J. Amer. Soc. Hort. Sci. 118:311–316.
30. Ozturk, N., M.R. Ulusoy, and E. Bayhan. 2005. Pest and Natural Enemy Species Determined in Pomegranate Orchards in the Eastern Mediterranean Region, Turkey. Turkiye Entomoloji Dergisi 29:225–235.
31. Park, S.H., S.C. Rose, C. Zapata, M. Srivatanakul, and R.H. Smith. 1998. Crossprotection and Selectable Marker Genes in Plant Transformation. In Vitro Cell. Dev. Biol. Plant. 34:117–121.
32. Petri, C., H. Wang, N. Alburquerque, M. Faize, L. Burgos. 2008. Agrobacterium-mediated Transformation of Apricot (Prunus armeniaca L.) Leaf Explants. Plant Cell Rep. 27:1317–1324.
33. Polin, L.D., H. Liang, R.E. Rothrock, M. Nishii, D.L. Diehl, A.E. Newhouse, C.J. Nairn, W.A. Powell, and C.A. Maynard. 2006. Agrobacterium Mediated Transformation of American Chestnut [Castanea dentate (Marsh.) Borkh.] Somatic Embryos. Plant Cell Tiss. Organ Cult. 84:69–78.
34. Scorza, R., J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming. 1996. Producing Transgenic ‘Thompson Seedless’ Grape (Vitis vinifera L) plants. J. Amer. Soc. Hort. Sci. 121:616–619.
35. Terakami, S., N. Matsuta, T. Yamamoto, S. Sugaya, H. Gemma and J. Soejima. 2007. Agrobacterium-mediated Transformation of the Dwarf Pomegranate (Punica granatum L. var. nana). Plant Cell Rep. 26:1243–1251.
36. Tohidfar, M., M. Mohammadi, and B. Ghareyazie. 2005. Agrobacterium-mediated Transformation of Cotton (Gossypium hirsutum) Using a Heterologous Bean Chitinase Gene. Plant Cell Tiss. Org. Cult. 83:83–96.
37. Vidal, N., R. Mallon, S. Valladares, A.M. Meijomin, A.M. Vieitez. 2010. Regeneration of Transgenic Plants by Agrobacterium-Mediated Transformation of Somatic Embryos of Juvenile and Mature Quercus robur. Plant Cell Rep. 29:1411–1422.
38. Yao, J.L., D. Cohen, R. Atkinson, K. Richardson, B. Morris. 1995. Regeneration of Transgenic Plants from the Commercial Apple Cultivar Royal-Gala. Plant Cell Rep. 14:407– 412.
39. Yao, J.L., J.H. Wu, A.P. Gleave, and B.A.M. Morris. 1996. Transformation of Citrus Embryogenic Cells Using Particle Bombardment and Production of Transgenic Embryos. Plant Sci. 113:175–183.
40. Yu, C., S. Huang, C. Chen, Z. Deng, P. Ling, and F.G. Gmitter. 2002. Factors Affecting Agrobacterium-Mediated Transformation of Sweet Orange and Citrange. Plant Cell Tiss. Org. Cult. 71:147–155.

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History

  • Receive Date: 29 May 2013
  • Revise Date: 01 September 2013
  • Accept Date: 13 July 2013

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