全 文 ：Received 20 Nov. 2003 Accepted 1 Mar. 2004
Supported by the National Natural Science Foundation of China (30200189) and International Foundation for Science (D/3001-2).
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1206-1211
Characterization of Nuclear and Cytoplasmic Compositions of Somatic
Hybrid Plants Between Sweet Orange and Sour Orange
LIU Ji-Hong, XU Xiao-Yong, DENG Xiu-Xin
(State Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding,
Huazhong Agricultural University, Wuhan 430070, China)
Abstract: In the present research, flow cytometry (FCM), simple sequence repeat (SSR) and cleaved
amplified polymorphic sequence (CAPS) were employed to analyze somatic hybrid plants derived from
electrofusion between embryogenic protoplasts of sweet orange (Citrus sinensis Osbeck cv. Shamouti)
and leaf-derived protoplasts of sour orange (C. aurantium L.). FCM showed that all of the somatic hybrid
plants had two-fold fluorescence intensity values of the diploid control, indicating that they were tetraploids.
SSR and CAPS were used to characterize the compositions of nuclear and cytoplasmic genomes of the
somatic hybrid plants. As for SSR four primer pairs were tried and two showed polymorphisms between the
fusion parents. With both primer pairs the somatic hybrid plants encompassed DNA bands from both parents,
indicating that they were heterokaryonic hybrids. Amplification with some universal primers, followed by
digestion with restriction endonucleases, could distinguish the fusion parents from each other. As far as
mitochondrial and chloroplast DNA compositions were concerned the somatic hybrid plants shared the
same banding patterns as the embryogenic parents for all of the polymorphic primer/enzyme combinations.
The results herein demonstrated that the somatic hybrid plants inherited their nuclear genome from both
fusion parents, whereas the cytoplasmic genomes were possibly only contributed by the embryogenic
parent. Merits of the analytical methods and nuclear and cytoplasmic inheritance of citrus tetraploid
somatic hybrids, together with their application, are discussed herein.
Key words: citrus; somatic hybrids; flow cytometry (FCM); cleaved amplified polymorphic sequence
(CAPS); simple sequence repeat (SSR)
Citrus is one of the most economically important fruit
crops in many countries. Sustainable development of cit-
rus industry depends on constant breeding of new cultivars.
Compared with other fruit and field crops, however, con-
ventional breeding of citrus faces many reproductive
impediments, such as sexual incompatibility, nucellar
polyembryony, high degree of heterozygosity, female and/
or male sterility and long juvenility period. As a conse-
quence it is difficult, if not impossible, to obtain a variety of
sexual hybrids. In fact, most of current cultivars widely
grown result predominantly from somatic mutation, which
leads to poor genetic diversity and subsequent disadvan-
tages for cultivar improvement. Somatic hybridization via
protoplast fusion can circumvent the above-mentioned bar-
riers and pave the way for creation of novel germplasms.
Since the first intergeneric somatic hybrid was obtained
(Ohgawara et al., 1985), more than 200 somatic hybrids have
been produced worldwide via symmetric and asymmetric
fusion (Guo et al., 1998; 2000; Liu and Deng, 1999; Grosser
et al., 2000; Liu et al., 2002). Protoplast fusion has now
become an integral part of citrus genetics and variety
improvement program (Grosser et al., 2000). Some of the
somatic hybrids hold great potential for commercial culti-
var or rootstock improvement (Grosser et al., 1998a; 1998b;
Grosser and Chandler, 2003). In addition, the somatic hy-
brids are valuable materials for investigations on interac-
tion between nuclear and cytoplasmic genomes.
Sour orange (Citrus aurantium) is used as rootstock in
many citrus-growing regions, due to its resistance to
Phytophthora-induced diseases, tolerance of citrus blight,
and wide adaptation. However, the use of sour orange as
rootstock has been minimized by its susceptibility to citrus
tristeza virus (CTV), which causes rapid tree decline or stem
pitting. Sweet orange, a widely grown cultivar, is reported
tolerant to CTV. But it is unlikely to produce sexual hybrids
between sour orange and sweet orange since both of them
are polyembryonic. With the objective of transferring CTV
tolerance from sweet orange to sour orange, protoplast fu-
sion between these two species was carried out and some
plants have been regenerated (Liu and Deng, 2001). In the
present research, flow cytometry (FCM) and two molecu-
lar markers, simple sequence repeat (SSR) and cleaved
LIU Ji-Hong et al.: Characterization of Nuclear and Cytoplasmic Compositions of Somatic Hybrid Plants Between Sweet
Orange and Sour Orange 1207
amplified polymorphic sequence (CAPS), were employed
to identify the nuclear and cytoplasmic composition of the
regenerated plants. The study is aimed to find out genetic
background of the somatic hybrids and provide detailed
information with regard to nuclear and cytoplasmic
interaction, which can further deepen genetic study for this
interspecific combination.
1 Materials and Methods
1.1 Plant materials
The somatic hybrid plants (Fig.1) were produced by
electrofusion between leaf-derived protoplasts of sour or-
ange (Citrus aurantium L.) and embryogenic protoplasts
of sweet orange (C. sinensis Osbeck cv. Shamouti) (Liu
and Deng, 2001). Embryogenic callus of sweet orange main-
tained on solid Murashige and Tucker basal medium con-
taining 40 g/L sucrose, and leaves of field-grown sour or-
ange plants and the somatic hybrid plants were used in the
present research.
1.2 Genomic DNA extraction
Total DNA was extracted from the callus and the leaves
as described by Liu et al. (2002). The resultant DNA pellet
precipitated with isopropanol was dissolved in 500 µL TE
buffer (10 mmol/L Tris-HCl, 0.1 mmol/L EDTA). The DNA
quality and concentration were analyzed by electrophore-
sis and UV1601 spectrophotometer (Shimadzu, Japan),
respectively. The samples were then diluted to 25 ng/µL
with TE and stored at –20 °C.
1.3 Ploidy analysis
Ploidy of the regenerants was determined by flow
cytometry analysis (FCM) according to the protocol de-
scribed by Liu et al. (2003) with minor modification. The
leaf and callus were chopped with a sharp razor blade and
incubated in 0.5 mL nuclei extraction buffer (Partec HR-A)
for 3 min, followed by filtration with 30 µm Partec CelltricsTM
and staining with 1 mL Partec HR-B solution for 2 min. The
samples were measured on a Partec Flow Cytometer (PA-I,
Münster, Germany) equipped with a high-pressure mer-
cury lamp. The relative fluorescence intensity of diploid
sour orange was used as control.
1.4 SSR analysis
SSR analysis with four primer pairs (Kijas et al., 1997)
was performed as described by Liu et al. (2002) with minor
modification. PCR reaction solution preparation and ampli-
fications were carried out according to previous program
(Liu et al., 2002). An equal volume of loading buffer (98%
formamide, 0.025% bromophenol blue, and 0.05% xylene
cyanol) was added to the amplified products, followed by
denaturation at 94 °C for 4 min. The resulting products
were analyzed on 6% (W/V) denaturing polyacrylamide gels,
which were silver-stained according to the protocol pro-
vided by the manufacturer (Promega, USA).
1.5 CAPS analysis
PCR amplification was performed with five chloroplast
and three mitochondrial universal primer pairs described
by Demesure et al. (1995) in the PTC-200 thermocycler with
reaction solution and amplification program similar to other
reports (Bastia et al., 2001; Liu et al., 2002). After amplifica-
tion an aliquot (6 µL) of the PCR products was digested
with 5 U of restriction enzyme for 3-4 h, followed by elec-
trophoresis in 2% agarose gels containing ethidium bro-
mide (5 µg/mL) at 2.5 V/cm for 2-3 h, which were then
visualized under UV light.
2 Results
2.1 Ploidy determination of fusion parents and
regenerants by FCM
The fluorescence intensity of sour orange was set to 50
(the value in the horizontal axis corresponding to the peak
position, Fig.2a) as control. When the fusion parents and
five of the regenerated plants were tested the fluorescence
intensities were about 50 (data not shown) and 100
(Fig.2b), respectively. Based on the analysis principle of
relative nuclear size for FCM, the regenerated plants could
be confirmed as tetraploids.
2.2 Characterization of nuclear composition by SSR
Out of the four primer pairs used only two (TAA15,
TAA41) revealed polymorphisms between the parents.
When they were employed for the nuclear composition
analysis the regenerated somatic hybrid plants encom-
passed all of the specific bands from both fusion parents
(Fig.3a, b). The banding patterns of the regenerated
Fig.1. Somatic hybrid plants in the soil pots.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041208
somatic hybrid plants were additive integration of the fu-
sion parents, which indicates that the nuclear composition
of both fusion parents have been incorporated into the
somatic hybrid plants. The banding profiles of the somatic
hybrid plants were identical in each primer pair and no varia-
tions could be detected. In combination with the FCM
results, it could be concluded that all of the regenerated
plants were tetraploid heterokaryonic somatic hybrids.
2.3 Characterization of cytoplasmic composition by CAPS
No polymorphism between the fusion parents was ob-
served when the amplified products were electrophoresesed
on the agarose gel prior to digestion with restriction
endonucleases. Only when the PCR products were di-
gested with the restriction endonucleases could some
polymorphic loci be observed. As far as mtDNA was
concerned, one out of 15 primer/enzyme combinations could
distinguish the fusion parents from each other. The band-
ing pattern of the PCR products amplified with 18S-5S RNA
followed by digest with TasⅠ showed that sour orange
and sweet orange had four and three bands, respectively.
They shared two common bands and each had two and
one distinct bands, respectively. Banding patterns of the
somatic hybrid plants were identical to the embryogenic
parent, sweet orange, and the bands characteristic with the
leaf parent, sour orange, were not detected in the somatic
hybrid plants (Fig.4a). As for cpDNA, polymorphisms were
detected with two out of 17 primer/enzyme combinations,
trnH-trnK/HinfⅠ, trnH-trnK/TasⅠ. Similarly, the somatic
hybrid plants shared the same banding profiles as the em-
bryogenic parent, and the specific bands of the leaf parent
were not present in the somatic hybrid plants (Fig.4b). No
variation in banding profiles was detected among the so-
matic hybrid plants with mtDNA and cpDNA. On the basis
of the results available herein it seems that only the em-
bryogenic parent contributed to the cytoplasmic composi-
tion of the somatic hybrid plants.
3 Discussion
Previously, ploidy determination of citrus somatic hy-
brids was mainly performed by hematoxylin staining (Liu
and Deng, 2001; 2002), which is time-consuming and
laborious. In contrast, FCM analysis is labor-effective and
quick. Ploidy determination of one sample can be finished
Fig.2. Ploidy analysis of the somatic hybrid by flow cytometry
(FCM). a and b are the histograms of the control and No. 2 plant
derived from fusion showing relative fluorescence intensity of 50
and about 100, respectively.
Fig.3. Simple sequence repeat (SSR) analysis of the somatic hybrid plants with TAA15 (a) and TAA41 (b). Lanes in both figures from
the left to the right are sweet orange, sour orange and the somatic hybrid plants No. 1-5, respectively.
Fig.4. Cleaved amplified polymorphic sequence (CAPS) analysis of the somatic hybrid plants. a. Banding pattern of mtDNA with 18S-
5S RNA/TasⅠ. Lanes from the left to the right are sour orange, sweet orange, the somatic hybrid plants No.1-5 and 200-bp DNA ladder.
b. Banding pattern of cpDNA with trnH-trnK/HinfⅠ. Lanes from the right to the left are 200-bp DNA ladder, sour orange, sweet orange,
and the somatic hybrid plants No.1-5, respectively.
LIU Ji-Hong et al.: Characterization of Nuclear and Cytoplasmic Compositions of Somatic Hybrid Plants Between Sweet
Orange and Sour Orange 1209
in 5-10 min. Moreover, much more cells can be counted at
one time compared to limited number of cells with desirable
mitotic metaphase in hematoxylin staining method, which
is more reliable for statistics (Ulrich and Ulrich, 1991). Hy-
bridity identification of citrus somatic hybrids was formerly
carried out by isoenzyme, RAPD and RFLP (Kobayashi
et al., 1991; Grosser et al., 1996; Liu and Deng, 2000; Liu et
al., 1999; 2002). Recently SSR and CAPS have been used
for characterization of somatic hybrids of citrus and other
plants (Luro and Ollitrault, 1996, Matthews et al., 1999,
Bastia et al., 2001; Guo et al., 2002; Liu et al., 2002; Lofty
et al., 2003). Based on the results available one conclusion
can be drawn that SSR and CAPS are powerful markers for
identification of nuclear and cytoplasmic origin. Meanwhile,
compared with Southern hybridization, which was widely
employed for nuclear and cytoplasmic analysis previously,
SSR and CAPS have some advantages such as time-spar-
ing for a single gel, good reproducibility, relative safety
and wide range of DNA quantities. However, due to high
conservative nature of the cytoplasmic organelles (Palmer
and Stein, 1986; Schuster et al., 1990), the efficiency of
cytoplasmic composition determination via CAPS is not
always satisfactory. As shown herein, only 2/17 and 1/15
of the cpDNA and mtDNA primer/enzyme combinations
could reveal polymorphisms between the fusion parents,
respectively, which has also been found in other reports
(Liu et al., 2002).
Since the first somatic hybrids were produced, much
work has been done on identifying the nuclear and cyto-
plasmic origin of the hybrids. To date most of the fusions
were performed between leaf-derived protoplasts and em-
bryogenic protoplasts, which led to regeneration of many
tetraploids (Grosser et al., 1998a; 1998b; Guo et al., 1998;
2000; Grosser et al., 2000). For the tetraploid somatic hy-
brids previous reports showed that their nuclear genomes
resulted from complete integration of nuclear materials from
both fusion parents (Motomura et al., 1995; Moriguchi
et al., 1996; Motomura et al., 1996; Guo et al., 2002; Cheng
et al., 2003), which is in accordance with the result herein.
With respect to cpDNA of citrus tetraploid somatic hybrids
it has been demonstrated in many reports that random seg-
regation was observed, namely random uniparental trans-
mission from either fusion parent (Kobayashi et al., 1991;
Grosser et al., 2000; Lofty et al., 2003). Uniparental trans-
mission of cpDNA was possibly caused by organelle loss
during embryogenesis and plant development (Lofty et al.,
2003). Only Moreira et al. (2000a; 2000b) reported that
cpDNA co-existence was detected in somatic hybrids be-
tween Citropsis gilletiana and Succari sweet orange,
Microcitrus australis and Hazzara (Citrus reticulata).
However, as for mtDNA it has been well documented that
nearly all of the somatic hybrids obtained their mtDNA from
the embryogenic parent (Moreira et al., 2000a; 2000b;
Grosser et al., 2000; Cabasson et al., 2001), which is identi-
cal to the results for the combination herein. Constant trans-
mission of mtDNA from the embryogenic parent to the so-
matic hybrids was considered as a necessity for successful
embryogenesis and subsequent plant development of so-
matic hybrid cells (Grosser et al., 1996). Different from so-
matic hybrids of annual crops with frequent mtDNA recom-
bination or rearrangements (Pupilli et al., 2001), few reports
concerning mtDNA recombination or rearrangements are
available in citrus tetraploid somatic hybrids. Motomura et
al. (1995) first reported mtDNA recombination in the so-
matic hybrids between Citrus and Atlantia or Severinia,
followed by Moriguchi et al. (1997) and Moreira et al.
(2000a; 2000b). Interestingly, in the fusion combination be-
tween Mexican lime and Clausena excavata, the mtDNA of
two somatic hybrids originated from either fusion parent
(Lofty et al., 2003). In the present study both cpDNA and
mtDNA were derived from the embryogenic parent, similar
to uniparental transmission of cytoplasm in sexual cross,
which has been reported in other combinations (Yamamoto
and Kobayashi, 1995; Moriguchi et al., 1996; 1997; Moreira
et al., 2000b; Cheng et al., 2003; Lofty et al., 2003). SSR and
CAPS results indicated that all of the genetic components
of sweet orange had been incorporated into the somatic
hybrids. So transfer of CTV tolerance is, in principle, ex-
pected to be fulfilled, but further work is needed to study
the agronomic traits of the somatic hybrids.
Acknowledgements: Thanks should be extended to Dr.
Anna Koltunow at Commonwealth Scientific and Indus-
trial Research Organization (CSIRO), Australia, and Dr.
Takaya Moriguchi at National Institute of Fruit Tree Science,
Japan, for their critical reading of the manuscript.
References:
Bastia T, Scotti N, Cardi T. 2001. Organelle DNA analysis of
Solanum and Brassica somatic hybrids by PCR with “univer-
sal primers”. Theor Appl Genet, 102: 1265-1272.
Cabasson C M, Luro F, Ollitrault P, Grosser J W. 2001. Non-
random inheritance of mitochondrial genomes in Citrus hy-
brids produced by protoplast fusion. Plant Cell Rep, 20: 604-
609.
Cheng Y J, Guo W W, Deng X X. 2003. Molecular characteriza-
tion of the cytoplasmic and nuclear genomes in phenotypi-
cally abnormal Valencia orange (Citrus sinensis) + Meiwa
kumquat (Fortunella crassifolia) intergeneric somatic hybrids.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041210
Plant Cell Rep, 21: 445-451.
Demesure B, Sodiz N, Petit R J. 1995. A set of universal primers
for amplification of polymorphic noncoding regions of mito-
chondrial and chloroplast DNA in plants. Mol Ecol, 4: 129-
131.
Grosser J W, Chandler J L. 2003. New Rootstocks via protoplast
fusion. Acta Hort, 622: 491-497.
Grosser J W, Gmitter F G Jr, Tusa N, Recupero G R, Cucinotta
P. 1996. Further evidence of a cybridization requirement for
plant regeneration from citrus leaf protoplasts following so-
matic fusion. Plant Cell Rep, 15: 672-676.
Grosser J W, Jiang J, Mourao-Fo F A A, Louzada E S, Baergen K,
Chandler J L, Gmitter F G Jr. 1998a. Somatic hybridization,
an integral component of citrus cultivar improvement. Ⅰ.
Scion improvement. Hort Sci, 33: 1057-1059.
Grosser J W, Jiang J, Louzada E S, Chandler J L, Gmitter F G Jr.
1998b. Somatic hybridization, an integral component of cit-
rus cultivar improvement. Ⅱ. Rootstocks improvement. Hort
Sci, 33: 1060-1061.
Grosser J W, Ollitrault P, Olivares-Fuster O. 2000. Somatic hy-
bridization in Citrus: an effective tool to facilitate variety
improvement. In Vitro Cell Dev Biol Plant, 36: 434- 449.
Guo W W, Cheng Y J, Deng X X. 2002. Regeneration and molecu-
lar characterization of intergeneric somatic hybrids between
Citrus reticulata and Poncirus trifoliata. Plant Cell Rep, 20:
829-834.
Guo W-W , Deng X-X , Shi Y-Z . 1998. Optimization of protoplast
electrofusion parameters and interspecific somatic hybrid re-
generation in Citrus. Acta Bot Sin , 40: 417-424. (in Chinese
with English abstract)
Guo W-W, Zhou C-H, Yi H-L , Deng X-X. 2000. Intergeneric
somatic hybrid plants between Citrus and Poncirus trifoliata
and evaluation of their root rot resistance. Acta Bot Sin , 42:
668-672.
Kijas J M H, Thomas M R, Fowler J C S, Roose M L. 1997.
Integration of trinucleotide microsatellites into a linkage map
of citrus. Theor Appl Genet, 94: 701-706.
Kobayashi S, Ohgawara T, Fujiwara K, Oiyama I. 1991. Analysis
of cytoplasmic genomes in somatic hybrids between navel
orange (Citrus sinensis Osb.) and “Murcott” tangor. Theor
Appl Genet, 82: 6-10.
Liu J H, Deng X X. 1999. Regeneration of hybrid calluses via
donor-recipient fusion between Microcitrus papuana and Cit-
rus sinensis. Plant Cell Tiss Organ Cult, 59: 81- 87.
Liu J-H, Deng X-X. 2000. Preliminary analysis of cytoplasmic ge-
nome of diploid somatic hybrid derived from fusion between
rough lemon and Hamlin sweet orange. Acta Bot Sin , 42:
102-104. (in Chinese with English abstract)
Liu J-H , Deng X-X. 2001. Production of somatic hybrids between
sour orange and sweet orange for creation of CTV-resistant
rootstock. Acta Phytophysiol Sin , 27: 473-477.
Liu J-H, Hu C-G, Deng X-X. 1999. Regeneration of diploid inter-
generic somatic hybrid plants between Microcitrus and Citrus
via electrofusion. Acta Bot Sin , 41: 1177-1182.
Liu J H, Pang X M, Cheng Y J, Meng H J, Deng X X. 2002.
Molecular characterization of the nuclear and cytoplasmic
genomes of intergeneric diploid plants from cell fusion be-
tween Microcitrus papuana and rough lemon. Plant Cell Rep,
21: 327-332.
Liu J-H, Zeng S-H, Xu C-Y, Zhang J-E , Deng X-X. 2003. Flow
cytometric analysis of ploidy and pollen viability investigation of
somatic hybrids between Microcitrus papuana and rough
lemon. J Fruit Sci , 20: 251-255. (in Chinese with English
abstract)
Lotfy S, Luro F, Carreel F, Froelicher Y, Rist D, Ollitrault P. 2003.
Application of cleaved amplified polymorphic sequence method
for analysis of cytoplasmic genome among Aurantioideae in-
tergeneric somatic hybrids. J Am Soc Hort Sci, 128: 225-230.
Luro F, Ollitrault P. 1996. A new set of cytoplasmic markers based
on amplification reaction: application for taxonomy and control
of somatic hybridization. Proc Int Soc Citricult, 899-902.
Matthews D, McNicoll J, Harding K, Millam S. 1999. 5 anchored
simple sequence repeat primers are useful for analyzing potato
somatic hybrids. Plant Cell Rep, 19: 210-212.
Moreira C D, Chase C D, Gmitter F G Jr, Grosser J W. 2000a.
Inheritance of organelle genomes in citrus somatic cybrids.
Mol Breeding, 6: 401-405.
Moreira C D, Chase C D, Gmitter F G Jr, Grosser J W. 2000b.
Transmission of organelle genomes in citrus somatic hybrids.
Plant Cell Tiss Organ Cult, 61: 165-168.
Moriguchi T, Hidaka T, Omura M. 1996. Genotype and parental
combination influence efficiency of cybrid induction in Citrus
by electrofusion. Hort Sci, 31: 275-278
Moriguchi T, Motomura T, Hidaka T, Akihama T, Omura M.
1997. Analysis of mitochondrial genomes among citrus plants
produced by the interspecific somatic fusion of “Seminole”
tangelo with rough lemon. Plant Cell Rep, 16: 397-400.
Motomura T, Hidaka T, Moriguchi T, Akihama T, Omura M.
1995. Intergeneric somatic hybrids between Citrus and Atlantia
or Severinia by electrofusion, and recombination of mitochon-
drial genomes. Breeding Sci, 45: 309-314.
Motomura T, Moriguchi T, Akihama T, Hidaka T, Omura M. 1996.
Analysis of cytoplasmic genome in somatic hybrids between
“Hazzara (Abohar)” (Citrus reticulata Blanco) and Microcitrus
australis (Planch.) Swingle. J JPN Soc Hort Sci, 65: 497-503.
Ohgawara T, Kobayashi S, Ohgawara E, Uchimiya H, Ishii S. 1985.
Somatic hybrid plant obtained by protoplast fusion between
Citrus sinensis and Poncirus trifoliata. Theor Appl Genet,
LIU Ji-Hong et al.: Characterization of Nuclear and Cytoplasmic Compositions of Somatic Hybrid Plants Between Sweet
Orange and Sour Orange 1211
(Managing editor: ZHAO Li-Hui)
71: 1-4.
Palmer J D, Stein D B. 1986. Conservation of chloroplast genome
structure among vascular plants. Curr Genet, 10: 823-833.
Pupilli F, Labombarda P, Arcioni S. 2001. New mitochondrial
genome organization in three interspecific somatic hybrids of
Medicago sativa including the parent-specific amplification of
substoichiometric mitochondrial DNA units. Theor Appl Genet,
103: 972-978.
Schuster W, Unseld M, Wissinger B, Brennicke A. 1990. Riboso-
mal protein S 14 transcripts are edited in Oenothera
mitochondria. Nucleic Acids Res, 16: 6897-6973.
Ulrich I, Ulrich W. 1991. High resolution flow cytometry of nuclear
DNA in higher plants. Protoplasma, 165: 212-215.
Yamamoto M, Kobayashi S. 1995. A cybrid plant produced by
electrofusion between Citrus unshiu (satsuma mandarin) and
C. sinensis (sweet orange). Plant Tiss Cult Lett, 12: 131-137.