2. mature ovules were preferred as explants to be cultured (Viloria and
Grosser, 2005; Fatta Del Bosco et al., 2007; Aleza et al., 2012). How-
ever, it was challengeable to isolate intact embryos from ovules without
damage. In other words, excision of small embryos under microscope
was really laborious and time-consuming. In our previous work, half-
ovules as explants have been tried to culture in vitro instead of excised
embryos (Xie et al., 2013, 2014b). However, the germination rate of the
ovules in most of the crosses was very low (Xie et al., 2013, 2014b).
Herein, to improve the efficiency of triploid production and ovule
germination rate based on 2x × 4x crosses, an optimized embryo rescue
approach was developed and applied in four 2x × 4x crosses with the
aim to generate more genetically diversified triploid hybrids. To de-
termine the ploidy of plants accurately and rapidly, FCM and chro-
mosome counting were used. SNP genotyping was employed to de-
termine the genetic origin of progenies.
2. Materials and methods
2.1. Plant materials
The diploid cultivars ‘Nadorcott’ tangor (Citrus reticulata Blanco ×
C. sinensis L. Osbeck, polyembryonic), ‘Bendizao’ tangerine (C. succosa
Hort. ex Tanaka, polyembryonic) and ‘Orah’ mandarin (C. reticulata
Blanco, monoembryonic) were selected as seed parents and two allo-
tetraploid somatic hybrids ‘NS’ (‘Nova’ tangelo + ‘Succari’ sweet or-
ange) (Grosser et al., 1992), ‘SD’ (‘Succari’ sweet orange + ‘Dancy’
tangerine) (Grosser and Gmitter, 2011) derived by protoplast fusion
and one doubled diploid ‘4X Ponkan’ (C. reticulata Blanco, Guo et al.,
2016) were used as pollen parents. ‘4X Ponkan’ is a newly flowering
tetraploid and this is the first time to be used as pollen parent to pro-
duce triploid plants. The pollens of ‘NS’ and ‘SD’ were kindly provided
by Dr. Jude Grosser. ‘4X Ponkan’, ‘Nadorcott’ tangor, ‘Bendizao’ tan-
gerine are cultivated in the Institute of Citrus Science, Huazhong
Agricultural University, Wuhan, China. ‘Orah’ mandarin is cultivated in
Guangxi Citrus Research Institute located in Guilin city, Guangxi pro-
2.2. Pollination, embryo rescue, plant regeneration and transplantation
Pollen collection and artificially pollination were conducted ac-
cording to Xie et al. (2013). Young fruits (Fig. 1c) were harvested at 85
days after pollination (DAP) and surface sterilized for 15 min in 75%
alcohol solution. Under aseptic condition, immature seeds were ex-
tracted (Fig. 1d) and classified into developed seeds and undeveloped
seeds. The seed coats were cut into two halves at the antipodal end of
the seeds, and then tore using forceps to expose the intact embryos
(Fig.1e), which is a newly optimized step never applied previously in an
effort to enhance germination rate. The embryo-exposed seeds instead
of isolated embryos were in vitro cultured on germination medium (MT
medium supplemented with 1 mg/L GA3) (Fig. 1f). After seed germi-
nation (Fig. 1g), the embryoids/shoots were transferred on MT medium
supplemented with 0.5 mg/L BA, 0.5 mg/L KT and 0.1 mg/L NAA for
shoot induction (Fig. 1h) or 1/2 MT medium with 0.5 g/L activated
carbon, 0.1 mg/L IBA and 0.5 mg/L NAA for root induction (Fig. 1i). All
in vitro cultures were maintained in a culture chamber at 25 ± 1 ℃
with a 16 h daily exposure to 40 μmol m−2
After getting vigorous roots, the polyploidy plants were transferred
to the plastic tubs containing commercial soil mixture which was steam
sterilized before use (Fig. 1j). To avoid evaporation, each tub was en-
closed by an inverted one. After acclimation for 5–7 days in greenhouse,
all plants were transferred to large plastic pots in greenhouse (Fig. 1k).
2.3. Ploidy level analysis
Flow cytometry (Partec, Cyflow space, Germany) was used to
measure the ploidy of seedlings according to Xiao et al. (2014). A
known diploid plant was used as control. For each sample at least 3000
nuclei were analyzed and histogram was generated automatically by
Flomax software (Partec, Germany). Root-tips of progenies were used
for chromosome counting as described by Wang et al. (2016a) with
slide staining with DAPI (4′-6-diamidino-2-phenylindole). Images were
captured by a fluorescent microscope (Olympus BX61, Japan) with UV
2.4. Genomic DNA extraction and SNP genotyping
Genomic DNA was extracted from young leaves as described by
Cheng et al. (2003). The quality and concentration of stock DNA were
checked with NanoDrop 1000 spectrophptometer (Thermo Scientific,
USA) and each stock DNA was diluted to 50–100 ng/μL for amplifica-
tion. KASP genotyping technology (http://www.lgcgenomics.com) was
used to genotype the polyploid progenies. Six SNP markers (Table S1)
were exploited by aligning the DNA re-sequencing data of two parents
(‘Nadorcott’ tangor vs ‘4X Ponkan’, ‘Orah’ mandarin vs ‘SD’, un-
published) with the genome of sweet orange as reference (Xu et al.,
2013), according to Dreissig et al. (2017). Primers were directly de-
signed from ˜50 nt flanking sequence of the SNP locus using Primer 5.0.
KASP genotyping was performed on a Roche LightCycler 480 instru-
ment (Roche, Switzerland) according to the manipulating guide. Allele
dosage of the progenies was determined as described by Cuenca et al.
Fig. 1. Pipeline of the modified embryo rescue technique. a: 4X male parent; b: 2X female parent; c: young fruits at 85 DAP; d: immature seeds extracted from young
fruits under aseptic condition; e: a undeveloped seeds tore two halves from the antipodal end to expose the embryos; f: seeds on germination medium; g: seeds
germinated after culturing on germination medium; h: embryoids/shoots on shoot induction medium; i: a whole plant on shoot induction medium; j: polyploid
plantlets in small plastic pots for acclimation; k: polyploid plants in large plastic pots in greenhouse.
K.-D. Xie, et al. Scientia Horticulturae 252 (2019) 104–109
3. 3. Results
3.1. High germination rate and shoot regeneration was achieved in four 2x
× 4x crosses via the optimized embryo rescue approach
Four interploid crosses were conducted with ‘Nadorcott’ tangor,
‘Bendizao’ tangerine and ‘Orah’ mandarin as seed parents, whereas ‘4X
Ponkan’, ‘NS’ and ‘SD’ as pollen parents. As shown in Table 1, a total of
971 flowers were pollinated in all crosses, resulting in 341 young fruits
harvested, with an average setting rate of 35.1%. In contrast to the
developed seeds in open-pollinated fruits (Fig. 2a–b down), most of the
seeds from pollinated fruits were undeveloped (Fig. 2a–b upper). By
analyzing their percentage in each cross, we found at least 54.5% of
seeds were undeveloped (Table 1). By applying the newly optimized
embryo rescue approach as illustrated in Fig. 1, totally 1970 of 4863
immature seeds germinated, with the average germination rate as
40.5%. Among these crosses, the average germination rates in the two
crosses using 'Nadorcott’ tangor and ‘Bendizao’ tangerine as seed
parents (54.9% and 50.0%) was higher than that in the crosses with
'Orah’ mandarin as seed parent (30.2% and 31.8%). After the germi-
nated embryoids being cultured for shoot and root induction, 1354
plantlets were finally regenerated from all crosses.
3.2. 401 triploid and 54 tetraploid progenies were obtained as determined
by FCM analysis
Flow cytometry analysis (Fig. 3a–c) showed that 401 triploid pro-
genies were obtained from all regenerated plants (Table 1) with the
average triploid regeneration of 29.6%, varying from 4.8% for ‘Na-
dorcott’ tangor × 4X Ponkan to 57.8% for ‘Bendizao’ tangerine × 4X
Ponkan. In addition, 54 tetraploid plants were also regenerated from
these four crosses (Table 1). Of them, 38 recovered from the crosses
with ‘Nadorcott’ tangor and ‘Bendizao’ tangerine (polyembryonic) as
seed parents and 16 regenerated from the crosses using ‘Orah’ mandarin
(monoembryonic) as seed parent. Chromosome counting (Fig. 3d–f)
was conducted for 2–3 triploids or tetraploids randomly selected from
each cross, which confirmed the ploidy level measured by flow cyto-
metry. All the triploid and tetraploid progenies were transplanted into
the greenhouse (Fig. 2c) and some progenies were already grafted in the
field to accelerate flowering and fruit evaluation.
3.3. Hybrid nature of the selected triploids and two different origins of the
selected tetraploid progenies were confirmed by SNP genotyping
The genetic origin of 17 randomly selected triploids and 20 tetra-
ploid progenies from ‘Nadorcott’ tangor × 4X Ponkan was determined
by a SNP marker (c2-20450078) homozygous for both parents. It
showed that all triploids selected were the hybrids of both parents be-
cause they formed a cluster located between two homozygous parents
(Fig. 4a). Similarly, nine of the tetraploid progenies also proved to be
the hybrids of both parents, whereas for the remaining ones, genome
doubling of nucellar cells of female parent was deduced for their for-
mation due to their sharing one cluster with the female parent (Fig. 4b).
However, for the five tetraploid progenies from ‘Orah’ × NS, the hybrid
origin was verified for all of them using two SNP markers (c3-10903745
and c6-5839066) homozygous for both parents (Fig. 4c). To determine
which parent contributed another genome to these tetraploid hybrids,
their allele dosage was further determined by three SNP markers (c1-
657672, c1-10927046 and c1-12169985) heterozygous for ‘Orah’
mandarin (Fig. 4d). Two copies of one allele inherited from female
parent was observed for all tetraploid progenies at these SNP loci
(Table 2), indicating that all of them came from the fertilization of 2n
megagametophytes of ‘Orah’ mandarin.
Embryo rescue, involving the culture of ovary, ovule or excised
embryos, is a useful in vitro tissue culture technique that has been
widely applied in many plants to overcome the failure of endosperm
development in interspecific, intergeneric and interploid hybridizations
Fruit set, number of seeds and polyploids recovered from the 2x × 4x crosses.
No. seeds obtained No. seeds germinated Germination
No. triploids No. tetraploids
Developed Undeveloped Developed Undeveloped
Nadorcott tangor × 4X
272 133 48.9 438 1,517 380 693 54.9 793 38 37
Bendizao tangerine ×
211 18 8.5 70 84 28 49 50.0 64 37 1
Orah mandarin × NS 289 132 45.7 535 1,310 162 396 30.2 284 188 5
Orah mandarin × SD 199 58 29.1 411 498 99 190 31.8 213 138 11
Total or average 971 341 35.1 1454 3409 669 1301 40.5 1354 401 54
Fig. 2. Seed characterization and polyploid transplantation of ‘Orah’
mandarin × SD. a: the pollinated fruits (upper) and open-pollinated fruits
(down); b: the immature developed and undeveloped seeds (arrow) from pol-
linated fruits (upper) and the seeds (all developed) from open-pollinated fruits;
c: polyploid plants in greenhouse.
K.-D. Xie, et al. Scientia Horticulturae 252 (2019) 104–109
4. (Zhu et al., 2013; Li et al., 2015; Zhang et al., 2018; Yan et al., 2019).
Success of embryo rescue is determined largely by the developmental
stage of embryo and composition of the culture medium (Viloria et al.,
2005; Shen et al., 2011; Li et al., 2015). In citrus, many studies have
been performed to investigate the optimal developmental stage to
conduct embryo rescue (Jaskani et al., 2005; Viloria and Grosser 2005;
Tan et al., 2007; Pérez-Tornero and Porras, 2008), and to optimize the
medium to improve embryo germination rate (Viloria et al., 2005).
Concerning the explants (ovary, ovule or excised embryo), excised
embryos from immature to mature fruits were extensively used in
previous reports (Jaskani et al., 2005; Viloria and Grosser, 2005; Aleza
et al., 2012; Zhu et al., 2013). However, the culture of ovules in citrus
2x × 4x crosses was rarely reported, whereas isolation of embryos is
laborious due to tedious dissection process. Furthermore, it is prone to
damage the embryos when they abort at a very early stage, resulting in
the decrease of embryo germination rate. For this reason, in our pre-
vious work, a half-ovule culture method, in which the micropylar
halves of the immature seeds were in vitro cultured instead of isolated
embryos, was used to simplify the procedure of citrus embryo rescue
(Xie et al., 2013, 2014b). However, the average germination rates
(18.0% for monoembryonic and 30.5% for polyembryonic cultivars)
were much lower than 55–80% as reported by Jaskani et al. (2005) and
65% by Aleza et al. (2012) via isolated embryo culture. In the present
study, by applying the optimized embryo rescue approach (Fig. 1), the
average seed germination rates increased greatly (31.0% for mono-
embryonic and 52.5% for polyembryonic cultivars) compared with our
previous reports. Although the germination rates are still lower than
that reported by other groups via isolated embryo culture, this opti-
mized embryo rescue method is easy to conduct and does not need the
tedious dissection procedure, making it a promising approach for citrus
triploid breeding programs.
As expected, all of the analyzed triploid progenies from ‘Nadorcott’
tangor × 4X Ponkan were proved to be hybrids of both parents.
However, for the tetraploid progenies from the same cross, two types of
tetraploid were present based on SNP genotyping, which is consistent
with our previous study where 2n megagametophyte formation and
chromosome doubling of nucellar cells were verified responsible for the
formation of these tetraploid progenies (Xie et al., 2014a). For the
tetraploid progenies from ‘Orah’ mandarin × NS, the hybrid origin was
demonstrated in all of them and 2n megagametophytes from ‘Orah’
mandarin were responsible for their formation. These results indicated
that 2n megagametophyte formation may play a dominant role in tet-
raploid progeny formation in 2x (monoembryonic) × 4x crosses and an
equally role with chromosome doubling of nucellar cells in tetraploid
progeny formation in apomictic citrus genotypes under same situation.
In most of the countries producing citrus, in addition to fruit see-
diness, the centralized mature period is a another severe problem faced
by citrus industry. Thus, seedlessness and expending the harvesting
period have become the main objectives of many citrus breeding pro-
grams (Aleza at al., 2010b; Grosser and Gmitter, 2011; Cuenca et al.,
2015). Triploid production was considered to be an excellent approach
to achieve above aims simultaneously because triploid plants often
express other desirable traits (Wang et al., 2016b; Yamada and Sato,
2016; Zhou et al., 2017). For example in citrus, production of seedless
varieties with high nutritional value (Sdiri et al., 2012), strong disease
resistance (Viloria et al., 2004), and delayed maturing period (Aleza
et al., 2010a; Cuenca et al., 2010, 2015) has been achieved by triploid
strategy. ‘Bendizao’ tangerine, native in Zhejiang province of China,
was very popular because of high fruit quality, early-maturing and easy
to peel. However, its cultivation areas have sharply decreased owing to
the fruit seediness. ‘Orah’ mandarin and ‘Nadorcott’ tangor are two
“star” cultivars in recent years due to its supreme fruit quality and late-
maturing trait. But excessive seeds greatly reduced its competitiveness
to other new emerging seedless cultivars in fresh-fruit market of China.
Although a less-seeded variant ‘Orri’ has been obtained by gamma ir-
radiation, the seedless trait in ‘Orri’ is not stable (Barry et al., 2015). To
solve this problem, triploid breeding strategy was adopted herein using
these three diploid cultivars as seed parents and the triploid plants
Fig. 3. Ploidy level determination of regenerated plants by flow cytometry analysis and chromosome counting. a-c: The histograms of flow cytometry of diploid
control (peak = 50), triploid (peak = 75) and tetraploid (peak = 100) plants respectively; d-f: chromosome counting of diploid control (2n = 2x = 18), triploid
(2n = 3x = 27) and tetraploid (2n = 4x = 36) plants respectively. Bar = 5 μm.
K.-D. Xie, et al. Scientia Horticulturae 252 (2019) 104–109
5. obtained are valuable germplasm for the selection of new promising
early- or late-mature varieties with seedless fruits.
In conclusion, by applying an optimized embryo rescue method,
numerous triploids together with some tetraploid progenies were re-
covered from four interploid crosses using three elite diploid seedy
cultivars as seed parents. The hybrid origin of triploids and two kinds of
tetraploid progenies were determined using SNP genotyping. The tri-
ploid hybrids obtained are of great value not only for selecting new
seedless cultivars, but also for fundamental researches such as meiotic
inheritance of tetraploid parents (Xie et al., 2015; Kamiri et al., 2018;
Rouiss et al., 2018) and mapping or cloning some resistant genes in
citrus (Cuenca et al., 2013b; Jiang et al., 2016). The tetraploid pro-
genies also hold great potential as parents in citrus triploid seedless
This research was financially supported by the National Natural
Science Foundation of China (nos.31701873, 31820103011), the
Ministry of Education of China (no.IRT_17R45), the Fundamental
Research Funds for the Central Universities of China (2662017PY019)
and the open project of Guangxi Key Laboratory of Citrus Biology
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.scienta.2019.03.038.
Fig. 4. Genotyping data plotted using LightCycler software. (a) 17 triploid progenies and (b) 20 tetraploid progenies from ‘Nadorcott’ tangor × 4X Ponkan mandarin
identified by c2-20450078; (c-d) five tetraploid progenies from ‘Orah’ mandarin × NS identified by c3-10903745 and c1-12169985 respectively. Each dot represents
one sample. ‘Na’, ‘4X-PK’ and ‘4X Auto’ refer to ‘Nadorcott’ tangor, 4X Ponkan mandarin and autotetraploid (doubled diploid) progenies derived from the genome
doubling of nucellar cells of ‘Nadorcott’ tangor.
Genotypes of the tetraploid progenies from ‘Orah’ mandarin × NS determined
by three SNP markers heterozygous for female parent.
SNP locus Genotypes of parents Genotypes of tetraploid progenies
Orah NS ONS1a
ONS2 ONS3 ONS4 ONS5
c1-657672 TG TTTT TTGG TTGG TTGG TTGG TTGG
c1-10927046 CT CCCC CCTT CCCC CCCC CCCC CCCC
c1-12169985 GA GGGG GGAA GGGG GGGG GGGG GGGG
Note: a: ONS1-ONS5 refer to the five tetraploid progenies from ‘Orah’
mandarin × NS.
K.-D. Xie, et al. Scientia Horticulturae 252 (2019) 104–109
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