Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene


This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500 mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration percentage of 60. The delivery of an antisense ACC oxidase gene into the species C. sinensis mediated by Agrobacterium tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.

Share and Cite:

S. Bunnag and D. Tangpong, "Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene," American Journal of Plant Sciences, Vol. 3 No. 9, 2012, pp. 1336-1340. doi: 10.4236/ajps.2012.39161.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Z. N. Yang, I. L. Ingelbrecht, E. Louzada, M. Skaria and T. E. Mirkov, “Agrobacterium-Mediated Transformation of the Commercially Important Grapefruit Cultivar Rio Red (Citrus paradisi Macf.),” Plant Cell Reports, Vol. 19, No. 12, 2000, pp. 1203-1211. doi:10.1007/s002990000257
[2] H. C. Chaturvedi, S. K. Singh, S. K. Sharma, A. K. Sharma and S. Agnihotri, “Citrus Tissue Culture Employing Vegetative Explants,” Indian Journal of Experimental Biology, Vol. 39, 2001, pp. 1080-1095.
[3] L. González-Candelas, S. Alamar, P. Sánchez-Torres, L. Zacarías and J. F. Marcos, “A Transcriptomic Approach Highlights Induction of Secondary Metabolism in Citrus fruit in Response to Penicillium digitatum Infection,” BMC Plant Biology, Vol. 10, 2010, p. 194. doi:10.1186/1471-2229-10-194
[4] I. L. Eaks, “Physiological Studies of Chilling Injury in Citrus Fruits,” Plant Physiology, Vol. 35, No. 5, 1960, pp. 632-636. doi:10.1104/pp.35.5.632
[5] W. C. Cooper, G. K. Rasmussen and E. S. Waldon, “Ethylene Evolution Stimulated by Chilling in Citrus and Persea sp.,” Plant Physiology, Vol. 44, No. 8, 1969, pp. 1194-1196. doi:10.1104/pp.44.8.1194
[6] S. Ben-Yehoshua and I. L. Eaks, “Ethylene Production and Abscission of Fruit and Leaves of Orange,” Botanical Gazette, Vol. 131, No. 2, 1970, pp. 144-150. doi:10.1086/336525
[7] H. Hyodo, “Ethylene Production by Albedo Tissue of Satsuma Mandarin (Citrus unshiu Marc.) Fruit,” Plant Physiology, Vol. 59, No. 1, 1977, pp. 111-113. doi:10.1104/pp.59.1.111
[8] J. B. Biale, R. E. Young and A. J. Olmstead, “Fruit Respiration and Ethylene Production,” Plant Physiology, Vol. 29, No. 2, 1953, pp. 168-174. doi:10.1104/pp.29.2.168
[9] Y. Aharoni, “Respiration of Oranges and Grapefruit Harvest at Different Stages of Development,” Plant Physiology, Vol. 43, No. 1, 1968, pp. 99-102. doi:10.1104/pp.43.1.99
[10] W. C. Cooper, G. K. Rasmussen and D. J. Hutchison, “Promotion of Abscission of Orange Fruits by Cycloheximide as Related to the Site of Treatment,” Bioscience, Vol. 19, No. 5, 1969, pp. 443-444. doi:10.2307/1294480
[11] A. C. Purvis and C. R. Barmore, “Involvement of Ethylene in Chlorophyll Degradation in Peel of Citrus Fruits,” Plant Physiology, Vol. 68, No. 4, 1981, pp. 854-856. doi:10.1104/pp.68.4.854
[12] E. E. Goldschmidt, M. Huberman and R. Goren, “Probing the Role of Endogenous Ethylene in the Degreening of Citrus Fruit with Ethylene Antagonists,” Plant Growth Regulation, Vol. 12, No. 3, 1993, pp. 325-329. doi:10.1007/BF00027214
[13] E. E. Trebitsh, E. E. Goldschmidt and J. Riov, “Ethylene Induces de Novo Synthesis of Chlorophyllase, a Chlorophyll Degrading Enzyme, in Citrus Fruit Peel,” Proceedings of the National Academy of Science, Vol. 90, No. 20, 1993, pp. 9441-9445. doi:10.1073/pnas.90.20.9441
[14] R. Porat, B. Weiss, L. Cohen, A. Daus, R. Goren and S. Droby, “Effects of Ethylene and 1-Methylcyclopropene on the Postharvest Qualities of ‘Shamouti’ Oranges,” Postharvest Biology and Technology, Vol. 15, No. 2, 1999, pp. 155-163. doi:10.1016/S0925-5214(98)00079-9
[15] A. J. Hamilton, G. W. Lycett and A. J. Grierson, “Antisense Gene That Inhibits the Synthesis of the Hormone Ethylene in Transgenic Plants,” Nature, Vol. 364, 1990, pp. 284-287. doi:10.1038/346284a0
[16] P. W. Oeller, M. W. Li, L. P. Taylor, D. A. Pike and A. Theologis, “Reversible Inhibition of Tomato Fruit Senescence by Antisense RNA,” Science, Vol. 254, No. 5030, 1991, pp. 437-439. doi:10.1126/science.1925603
[17] T. Murashige and D. P. H. Tucker, “Growth Factor Requirement of Citrus Tissue Culture,” Proceedings of 1st Citrus Symposiums, Univ. of Califo, Riverside, Vol. 3, 1969, pp. 1155-1161.
[18] R. A. Jefferson, “Assaying Chimeric Genes in Plants: The GUS Gene Fusion System,” Plant Molecular Biology Reporter, Vol. 5, No. 4, 1987, pp. 387-405. doi:10.1007/BF02667740
[19] J. J. Doyle and J. L. Doyle, “A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue,” Phytochemical Bulletin, Vol. 19, 1987, pp. 11-15.
[20] J. Kochaba, P. Spiegel-Roy and H. Safran, “Adventive Plants from Ovules and Nucelli in Citrus,” Planta, Vol. 106, No. 3, 1972, pp. 237-245. doi:10.1007/BF00388101
[21] F. Carmini, M. C. Tortorici, F. Pasquale, F. G. Crescimanno and F. Pasquale, “Somatic Embryogenesis and Plant Regeneration from Undeveloped Ovules Stigma/ Style Explants of Sweet Orange Navel Group (Citrus sinensis (L.) Osb.),” Plant Cell, Tissue and Organ Culture, Vol. 54, No. 3, 1998, pp. 183-189. doi:10.1023/A:1006113731428
[22] K. Datta, R. Valazhahan and N. Oliva, “Overexpression of the Cloned Rice Thaumatin-Like Protein (PR-5) Gene in Transgenic Rice Plants Enhanced Environmental Friendly Resistance to Rhizoctonia solani Causing Sheath Bright Disease,” Theoretical and Applied Genetics, Vol. 98, No. 6-7, 1999, pp. 1138-1145. doi:10.1007/s001220051178
[23] S. Sriskandarajah, B. P. Goodwin and J. Spiers, “Genetic Transformation of Apple Scion Culture ‘Delicious’ via Agrobacterium tumefaciens,” Plant Cell, Tissue and Organ Culture, Vol. 36, No. 3, 1994, pp. 317-329. doi:10.1007/BF00046089

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.