American Journal of Plant Sciences, 2013, 4, 2131-2137
Published Online November 2013 (http://www.scirp.org/journal/ajps)
http://dx.doi.org/10.4236/ajps.2013.411265
Open Access AJPS
2131
An Efficient Intragenic Vector for Generating Intragenic
and Cisgenic Plants in Citrus
Chuanfu An1, Vladimir Orbović2, Zhonglin Mou1*
1Department of Microbiology and Cell Science, University of Florida, Gainesville, USA; 2Citrus Research and Education Center,
University of Florida, Lake Alfred, USA.
Received September 2nd, 2013; revised October 2nd, 2013; accepted October 23rd, 2013
Copyright © 2013 Chuanfu An et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transfer-
ring 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/cisgene sis is a
new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the as-
sociated 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 und er non-selective conditions and positiv e intra-/cisgenic regen-
erants were identified through PCR analysis. Transfor mation efficiencies obtained in Arabidopsis and “Duncan” grape-
fruit 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
1. Introduction
Genetic improvement of citrus (Citrus spp.) by conven-
tional breeding is hindered by incompatibility, apomixes,
heterozygosity, and length y juvenile p eriod. Transgenesis
has become a promising tool to directly introduce desir-
able traits into elite genotypes without altering existing
genetic background. However, the release of genetically
modified (GM) crops has raised considerable health and
environmental concerns mainly due to the transferring of
genes across wide taxonomic boundaries and the pres-
ence of selectable marker genes [1,2]. With the accumu-
lating knowledge of the structure and function of genes
from crop species, delivering useful alleles of native
genes into crossable plant species or genotypes has be-
come possible. In addition, a number of strategies have
been developed to remove marker genes from transgenic
plants [3,4]. However, currently preferred Agrobacte-
rium-mediated plant genetic transformation and elimina-
tion of selectable marker genes largely rely on prokaryote-
derived vector systems [5]. With this method, foreign
genes or sequences still remain in the GM plants.
Intragenic vector system-based intra-/cisgenesis com-
bines the benefits of traditional breeding and genetic en-
gineering, but circumvents many of their problematic
issues [5,6]. It involves iden tifying functional eq u ivalents
of vector components from target or crossable plant spe-
cies and using these DNA sequences to assemble vector
for plant transformation. Native genes from a sexually
compatible species can be delivered into elite cultivars
by the intragenic vector system in a single step without
linkage drag and, most importantly, without the incorpo-
ration of “foreign DNA” [6].
In citrus, although there is a report on recovering se-
lectable marker-free transgenic orange plant under
non-selective conditions, no intra-/cisgenesis experiment
has been conducted due to the lack of an intragenic vec-
tor system [7]. We present here the development of a
“foreign DNA-free” intragenic vector system, pUFCI
(University of Florida Citrus Intragenic), by assembling
T-DNA-like fragments with functional equivalents of
T-DNA border sequen ces from C. clementina and recov -
ery of positive intra-/cisgenic regenerants through PCR
*Corresponding a uthor.

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus 2132
analysis. The transformation efficiencies obtained in both
Arabidopsis and “Duncan” grapefruit indicate its great
potential for citrus genetic improvement.
2. Materials and Methods
2.1. Construction of Citrus Intragenic Vector
pUFCI
Citrus-derived T-DNA-like regions were identified by
BLAST searching the C. clementina genome sequence in
Citrus Genome Database (http://www.citrusgenomedb.
org/) using a T-DNA left border (LB) sequence
(GTTTACACCACAATATATCCTGCCA) as a query
[5]. Scaffold_89 (an assembled genomic DNA sequence),
which harbors the first seven nucleotides of the LB se-
quence, prospective cloning sites, and the last seven nu-
cleotides of a right border (RB) sequence, was selected.
The rest of the LB sequence was obtained from Scaf-
fold_2 and fused with the above T-DNA-like fragment
through PCR amplification using primers CcF1 and
CcR1 (Table 1). PCR products were directly ligated into
the pGEM-T Easy vector and verified by sequencing.
The plasmid DNA was digested with PstI and EcoRI to
Table 1. Sequences of the primers used in this study.
Primer Sequence (5’ - 3’)
CcF1 GCTGCAGTTTACCCGCCAATATATCCTGTC
ATATTTTGAAACCAATATCAGAG
CcR1 GGAATTCGGGCTAAGGCGGCAGTTCGGCG
ATGGAGGTGGCAGGATATATTGTGGTGTA
AACGAGATGTTTGTTACTTATAGGAAACG
VecF2 GCTGCAGCCGGAATTCATACAGGCAGCCC
ATCAGTCC
VecR1 GCTGCAGCTAAGAGAAAAGAGCGTTTATT
AGAATAATCG
C-intra-F CAAGAGGACAAGAGTCTATCC
C-intra-R TGAGGATGAAGACCTGAACG
Ct-check-F TTCAGGTCTTCATCCTCACG
Ct-check-F2 ATCGAGCACACACCATCATG
Ct-check-R2 AGCCTTAGGTTGTGACAGTG
Ct-check-R3 CGTTGGAGTGGAGTAATCAG
Ct-check-R4 TTTGTAAAGCGAGGAGCAGG
Ct-check-R5 TCTCTTGCCTCAGTTCAAGG
IntraVecRBF1 GTTTACCCGCCAATATATCCTG
IntraVecRBF2 TCATATTTTGAAACCAATATCAGAG
IntraVecRBR AGGTATCAGCATCTAACATCC
CtF2 ACGGAGTTCGGTTTGTTGTC
CtR2 AGCCTCAAGAGAGTTGCTAG
EF1F AAGCCCATGGTTGTTGAGAC
EF1R CAACAGCAAACTGGTGGAAG
BamHI-BarF GTGGATCCCCCGGGCTGCAGG
SpeI-BarR GACTAGTGGTCGACGGTATCGATAAGC
produce a sticky end fragment. The binary vector
pCB302 was used as the template to generate the origin
of replication and the nptIII expression cassette by PCR
using primers VecF2 and VecR1 (Table 1) [8]. The PCR
products were digested with PstI and EcoRI and ligated
to the above sticky end citrus-derived T-DNA-like frag-
ment to produce an intermediate vector pUFCI-1. To
delete the duplicated SacI restriction site within the
T-DNA-like region, pUFCI-1 was digested with SacI and
self-ligated to further produce pUFCI-2. To avoid inte-
gration of vector backbone sequences beyond the LB into
recipient plant genome during T-DNA transfer, a ~3 kb
fragment from C. clementina genome (scaffold_34) was
inserted into pUFCI-2 between the LB and the vector
backbone through the EcoRI site [9,10]. The direction of
the added fragment was identified by PCR using primers
Ct-check-F and Ct-check-F2 or Ct-check-R2 (Figure 1
and Table 1). The sequence of the C. clementina-derived
T-DNA-like region and the buffering fragment outside of
the LB in the final citrus intragenic vector, pUFCI, was
confirmed by sequencing and shown in Figure 2. Three
restriction sites, SpeI, SacI, and BamHI, in the T-DNA-
like region can be utilized for gene cloning.
2.2. pUFCI-Bar Plasmid Construction,
Arabidopsis Transformation, and Selection
A pair of primers BamHI-BarF and SpeI-BarR (Table 1)
were used to amplify the entire exp ression cassette of th e
Bar gene from the binary vector pCB302 [8]. The PCR
products were digested with BamHI and SpeI, and then
ligated into the BamHI and SpeI sites of pUFCI. The re-
sulting plasmid was introduced into the Agrobacterium
tumefaciens strain GV3101(pMP90) by electroporation
and transformed into the Arabidospsis thaliana (L.)
Heynh. ecotype Columbia (Col-0) following the floral
dip method [11]. Transformants were identified by spray-
ing T1 seedlings with Basta.
2.3. Citrus Transformation and Positive
Regenerant Identification
The empty vector was introduced into the A. tumefaciens
strain EHA105 by electroporation and transformed into
‘Duncan’ grapefruit following the protocol described
previously [12]. No selection pressure was exerted on
explants. Regenerated shoots from individual transfor-
mation events were screened by PCR using primers
C-intra-F and C-intra-R (Table 1). A characteristic ~500
bp PCR product was u sed to iden tify p os itive regenerants.
The LB and RB integration sites were mapped by PCR
amplification using a set of primer pairs (Figure 1).
3. Results and Discussion
To facilitate development of intra-/cisgenic citrus culti-
ars, we have constructed an intragenic vector, pUFCI, v
Open Access AJPS

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus
Open Access AJPS
2133
Figure 1. Schematic diagrams of the C. clementina -derived T-DNA-like regions and the positions of the PCR primers used in
this study. LB: left border; RB: right border. The primer pairs CcF1/CcR1 and CtF2/CtR2 were used to amplify the
T-DNA-like fragment and the GC rich buffering fragment from C. clementina, respectively. The vector backbone was gener-
ated by PCR amplification from the binary vector pCB302 using primers VecF2 and VecR1. The redundant SacI site in the
T-DNA-like region was deleted. The primers C-intra-F and C-intra-R were used for identification of positive regenerants.
Ct-check-F, Ct-check-F2, and Ct-check-R2 were used to identify the orientation of the GC rich fragment. Ct-check-F,
Ct-check-R2, Ct-check-R3, Ct-check-R4, and Ct-check-R5 were used to map the integration site outside of the LB. In-
traVecRBR, IntraVecF1, and IntraVecF2 were used to check the integration site of the RB. Three restriction sites, SpeI, SacI,
and BamHI, between the RB and LB are available for gene cloning.
Figure 2. Sequence of the T-DNA-like region and the buffering fragment in pUFCI. All nucleotides in the T-DNA-like region
and the ~3 kb buffering fragment outside of the left border were assembled from C. clementina. The nucleotides from the C.
clementina genome are italicized, with the T-DNA borders in blue and the three unique restriction sites (SpeI, SacI, and
BamHI) in green. The asterisk (*) in the right border indicates the site of T-strand initiation for T-DNA transfer to plants.
The nucleotide fragment shown in the figure was linked to the backbone of the binary vector pCB302 using the PstI and
EcoRI sites (in bold at each end).
which bears a T-DNA-like region with functional equiva-
lents of T-DNA border sequences from C. celementina
(Figure 3). Within this region three restriction sites (SpeI,
SacI, and BamHI) can be used for cloning native genes
from sexually compatible citrus species.
In the new citrus intragenic vector, we designed a sim-
ple PCR amplification strategy for identification of posi-
tive regenerants using the size difference caused by dele-
tion of the duplicated SacI restriction site within the
T-DNA-like region. For the empty vector, positive re-
generants gave a specific ~500 bp fragment together with
a ~1 kb fragment amplified from the recipient “Duncan”

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus 2134
Figure 3. Construction of the citrus intragenic vector pUFCI. LB: left border, RB: right border. Explanation is detailed in
2.1.
grapefruit genome (Figure 4(B)). When a gene cassette
of interest is ligated into pUFCI and introduced into re- cepient citrus genome, positive regenerants can be easily identified through PCR amplification using a gene specific
Open Access AJPS

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus 2135
Figure 4. Characterization of a representative intra-/cisgenic citrus plant generated with the empty pUFCI vector. (A) PCR
confirmation of the presence of the respective DNA in the three DNA samples used in (B)-(D); The pUFCI plasmid DNA (V)
was amplified with the primers Ct-check-F2 and Ct-check-R4, yielding a 185 bp fragment. The wild-type “Duncan” grape-
fruit DNA (D) and the cisgenic “Duncan” grapefruit DNA (C) were amplified with primers for the citrus EF1  gene, pro-
ducing a ~300 bp fragment. M: 1 kb DNA marker; (B) A characteristic ~500 bp fragment was amplified from V and C, but
not from D, whereas a ~1 kb fragment was amplified from D and C, but not from V, confirming the integration of the pUFCI
T-DNA-like fragment into the recipient “Duncan” grapefruit genome; (C) No vector backbone sequences adjacent to the RB
were integrated in the intra-/cisgenic plant. The forward primers IntraVecRBF1 and IntraVecRBF2 anneal to the first 22
nucleotides of the RB and the citrus-derived sequence including the last three nucleotides of the RB, respectively, and the
reverse primer IntraVecRBR is located inside the T-DNA-like region. PCR products were amplified with IntraVecRBF2 and
IntraVecRBR but not with IntraVecRBF1 and IntraVecRBR from C, indicating that the T-DNA transfer in the in-
tra-/cisgenic plant initiated in the RB; (D) No vector backbone sequences adjacent to the buffering sequence on the LB side
were integrated into the intra-/cisgenic plant. Ct-check-F is the forward primer located in the T-DNA-like region, and
Ct-check-R2, Ct-check-R3, Ct-check-R4, and Ct-check-R5 are reverse primers located at different positions in the buffering
sequence. PCR products were amplified from V but not from C with the primers Ct-check-F1 and Ct-check-R5, indicating
that the T-DNA transfer in the intra-/cisgenic plant ended between primers Ct-check-R4 and Ct-check-R5.
primer in combination with a primer annealing to the
T-DNA-like region. Transferring and integration of
T-DNA into the plant genome by Agrobacteria initiate
from the RB and end at the LB. However, integration of
binary vector backbone sequences especially the se-
quence outside of the LB in transgenic plants is a com-
mon phenomenon [5]. To decrease the possibility of the
presence of non-citrus sequences in the regenerants, we
added a ~3 kb C. clementina-originated DNA fragment
outside of the LB to serve as a buffering sequence. It
permits a tolerance towards truncations beyond the LB
without interfering with the concept of gene transfer
without “foreign DNA”. As expected, integration of the
pUFCI T-DNA-like region into the ‘Duncan’ grapefruit
genome started at the RB without integration of any
backbone sequences (Figure 4(C)). Our characterization
of a representative intra-/cisge nic citrus plant showed the
presence of DNA sequences beyond the LB. However, it
stopped before the end of the buffering sequence (Figure
4(D)). Therefore, no bacteria-derived backbone DNA
was introduced into the regenerated citrus plant. These
results demonstrate the possibility of using pUFCI to
Open Access AJPS

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus 2136
Figure 5. The pUFCI vector mediates efficient genetic transformation in Arabidopsis. A. thaliana ecotype Col-0 plants were
transformed with Agrobacteria carrying the plasmid pUFCI-Bar or the T-DNA binary vector pCB302 and the T1 seedlings
were sprayed with Basta. Untransformed Col-0 plants were used as negative controls. The average transformation efficien-
cies were calculated from eight replicated experiments. Green seedlings are transgenic plants
generate “foreign DNA-free” intra-/cisgenic citrus plants.
To test the transformation efficiency of the intragenic
vector, we cloned the expression cassette of the Bar gene
into pUFCI and transformed Arabidopsis with the result-
ing plasmid pUFCI-Bar. Basta screening gave an average
transformation efficiency of ~3% in the A. thaliana eco-
type Col-0, which is comparable to the efficiency of
pCB302 and other widely used binary vectors for Arabi-
dopsis transformation (Figure 5) [8]. These results indi-
cate that the C. clementina-originated sequences in
pUFCI do not contain silencing components. Further-
more, we tested transformation efficiency of pUFCI in
citrus. An average transformation efficiency of ~0.67%
was achieved in our three independent transformation
experiments. Similar levels of transformation efficiency
were observed in the production of intra-/cisgenic apple
plants [13]. Based on our current transformation protocol,
the efficiency is acceptable for generating “foreign
DNA-free” intra-/cisgenic plants in citrus [7,14,15].
4. Conclusion
pUFCI is a versatile intragenic vector that can generate
intra-/cisgenic citrus and transgenic Arabidopsis with
high frequencies. Identificatio n of positiv e intra-/cisg enic
citrus regenerants can be accomplished by simple PCR
analysis. Considering the current citrus transformation
protocol and the availability of the citrus genome se-
quences for candidate gene identification, developing
intra-/cisgenic citrus plants with genes of interest using
pUFCI is applicable for future citrus genetic improve-
ment.
5. Acknowledgements
This research was supported by grants from the Citrus
Research and Development Foundation and National
Science Foundation (IOS-0842716) awarded to ZM.
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, “Se-
lectable Marker Genes: Safe for Plants?” Nature Bio-
technology, 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 & Ge-
nomes, 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
Open Access AJPS

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus 2137
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 Transfor-
mation: 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 De-
velopment of a Cisgenic Apple Plant,” Journal of Bio-
technology, 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 Trans-
formation 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 Agro-
bacterium-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
Open Access AJPS