An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus

Chuanfu An, Vladimir Orbović, Zhonglin Mou

Citrus Research and Education Center, University of Florida, Lake Alfred, USA.
Department of Microbiology and Cell Science, University of Florida, Gainesville, USA.

DOI: 10.4236/ajps.2013.411265                

Abstract

Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transferring genes across species, the presence of selectable marker genes, and bacteria-derived vector backbone sequences have raised considerable health and environmental concerns. Intragenic vector system-based intragenesis/cisgenesis is a new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the associated shortcomings. We report here the development of an intragenic vector by assembling a T-DNA-like fragment and a buffering sequence following the left border from Citrus clementina into the backbone of the binary vector pCB302. Recovery of citrus regenerants is performed under non-selective conditions and positive intra-/cisgenic regenerants were identified through PCR analysis. Transformation efficiencies obtained in Arabidopsis and “Duncan” grapefruit were ~3% and ~0.67%, respectively, demonstrating the potential of the system for development of “foreign DNA-free” intra-/cisgenic citrus cultivars.

Keywords

Intragenesis/Cisgenesis; Citrus; Selectable Marker; Transgenesis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	A. J. Conner, T. R. Glare and J. P. Nap, “The Release of Genetically Modified Crops into the Environment: II. Overview of Ecological Risk Assessment,” Plant Journal, Vol. 33, No. 1, 2003, pp. 19-46. http://dx.doi.org/10.1046/j.0960-7412.2002.001607.x
[2]	R. B. Flavell, E. Dart, R. L. Fuchs and R. T. Fraley, “Selectable Marker Genes: Safe for Plants?” Nature Biotechnology, Vol. 10, No. 2, 1992, pp. 141-144. http://dx.doi.org/10.1038/nbt0292-141
[3]	B. Darbani, A. Eimanifar, C. N. Stewart and W. N. Camargo, “Methods to Produce Marker-Free Transgenic Plants,” Biotechnology Journal, Vol. 2, No. 1, 2007, pp. 83-90. http://dx.doi.org/10.1002/biot.200600182
[4]	M. Malnoy, E. E. Boresjza-Wysocka, J. L. Norelli, M. A. Flaishman, D. Gidoni and H. S. Aldwinckle, “Genetic Transformation of Apple (Malus x domestica) without Use of a Selectable Marker Gene,” Tree Genetics & Genomes, Vol. 6, No. 3, 2010, pp. 423-433. http://dx.doi.org/10.1007/s11295-009-0260-7
[5]	A. J. Conner, P. J. Barrell, S. J. Baldwin, A. S. Lokerse, P. A. Cooper, A. K. Erasmusn, J. P. Nap and J. M. E. Jacobs, “Intragenic Vectors for Gene Transfer without Foreign DNA,” Euphytica, Vol. 154, No. 3, 2007, pp. 341-353. http://dx.doi.org/10.1007/s10681-006-9316-z
[6]	C. M. Rommens, M. A. Haring, K. Swords, H. V. Davies and W. R. Belknap, “The Intragenic Approach as a New Extension to Traditional Plant Breeding,” Trends in Plant Science, Vol. 12, No. 9, 2007, pp. 397-403. http://dx.doi.org/10.1016/j.tplants.2007.08.001
[7]	A. Ballester, M. Cervera and L. Peña, “Selective Marker-Free Transgenic Orange Plants Recovered under Non-Selective Conditions and through PCR Analysis of All Regenerants,” Plant Cell, Tissue and Organ Culture, Vol. 102, No. 3, 2010, pp. 329-336. http://dx.doi.org/10.1007/s11240-010-9737-1
[8]	C. Xiang, P. Han, I. Lutziger, K. Wang and D. J. Oliver, “A Mini Binary Vector Series for Plant Transformation,” Plant Molecular Biology, Vol. 40, No. 4, 1999, pp. 711-717. http://dx.doi.org/10.1023/A:1006201910593
[9]	S. B. Gelvin, “Agrobacterium-Mediated Plant Transformation: The Biology behind the ‘Gene-Jockeying’ Tool,” Microbiology and Molecular Biology Reviews, Vol. 67, No. 1, 2003, pp. 16-37. http://dx.doi.org/10.1128/MMBR.67.1.16-37.2003
[10]	C. M. Rommens, O. Bougri, H. Yan, J. M. Humara, J. Owen, K. Swords and J. Ye, “Plant-Derived Transfer DNAs,” Plant Physiology, Vol. 139, No. 3, 2005, pp. 1338-1349. http://dx.doi.org/10.1104/pp.105.068692
[11]	S. J. Clough and A. F. Bent, “Floral Dip: A Simplified Method for Agrobacterium-Mediated Transformation of Arabidopsis thaliana,” Plant Journal, Vol. 16, No. 6, 1998, pp. 735-743. http://dx.doi.org/10.1046/j.1365-313x.1998.00343.x
[12]	V. Orbovic and J. W. Grosser, “Citrus: Sweet Orange (Citrus sinesis L. Osbeck ‘Valencia’) and Carrizo Citrange (Citrus sinesis (L.) Osbeck x Poncirus trifoliate (L.) Raf.),” In: K. Wang, Eds., Agrobacterium Protocol-Methods in Molecular Biology, Humana Press, New York, 2006, pp. 177-189.
[13]	T. Vanblaere, I. Szankowski, J. Schaart, H. Schouten, H. Flachowsky, G. A. Broggini and C. Gessler, “The Development of a Cisgenic Apple Plant,” Journal of Biotechnology, Vol. 154, No. 4, 2011, pp. 304-311. http://dx.doi.org/10.1016/j.jbiotec.2011.05.013
[14]	A. Ballester, M. Cervera and L. Peña, “Evaluation of Selection Strategies Alternative to nptII in Genetic Transformation of Citrus,” Plant Cell Report, Vol. 27, No. 6, 2008, pp. 1005-1015. http://dx.doi.org/10.1007/s00299-008-0523-z
[15]	M. G. C. Costa, W. C. Otoni and G. A. Moore, “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 Report, Vol. 21, No. 4, 2002, pp. 365-373. http://dx.doi.org/10.1007/s00299-002-0533-1