ORIGINAL PAPER

Extensive citrus triploid hybrid production by 2x 3 4x sexualhybridizations and parent-effect on the lengthof the juvenile phase

P. Aleza • J. Juarez • J. Cuenca • P. Ollitrault •

L. Navarro

Received: 24 February 2012 / Revised: 26 April 2012 / Accepted: 4 May 2012

� Springer-Verlag 2012

Abstract The citrus fresh market demands the production

of seedless citrus fruits, as seedy fruits are not accepted by

consumers. The recovery of triploid plants has proven to be

the most promising approach to achieve this goal, since

triploids have very low fertility, are generally seedless and

do not induce seeds in other cultivars by cross pollination.

Triploid plants can be recovered by 2x 9 4x sexual

hybridization. In this work, we present an effective meth-

odology to recover triploid plants from 2x 9 4x hybrid-

izations based on in vitro embryo rescue, ploidy level

analysis by flow cytometry and genetic origin of triploid

plants. The pollen viability of diploid and tetraploid citrus

genotypes was analyzed by comparing the pollen germi-

nation rate in vitro. The pollen viability of tetraploid

(doubled-diploid) genotypes is generally reduced but suf-

ficient for successful pollination. Triploid embryos were

identified in normal and undeveloped seeds that did not

germinate under greenhouse conditions. The influence of

parents and environmental conditions on obtaining triploid

plants was analyzed and a strong interaction was noted

between the parents and environmental conditions. The

parental effect on the length of the juvenile phase was also

demonstrated through observations of a large number of

progeny over the last 15 years. The juvenile phase length

of the triploid hybrids obtained with ‘Fortune’ mandarin as

female parent and tetraploid ‘Orlando’ tangelo as male

parent was shorter than the juvenile phase obtained with

a clementine as female parent and tetraploids of ‘Nova’,

‘W. Leaf’ and ‘Pineapple’ male parents.

Key message Effective methodology to recover citrus

triploid plants from 2x 9 4x sexual hybridizations and the

parental effect on the length of the juvenile phase.

Keywords Diploid � Tetraploid � Mandarin � Culture

tissue � Embryo rescue � Flow cytometry � SSR markers

Introduction

Seedlessness is one of the most important characteristics for

mandarin on the fresh fruit market. Parthenocarpy, an

essential trait for seedless fruit production, is present in citrus

germplasm. The creation of triploid plants is an important

breeding strategy for the development of new seedless citrus

commercial varieties (Navarro et al. 2002a; Ollitrault et al.

2008). Indeed, triploid plants generally give rise to aneuploid

gametes which exhibit very low fertility (Otto and Whitton

2000). Predominantly trivalent and a high number of bivalent

and univalent associations are formed during meiosis in cit-

rus triploid plants (Cameron and Frost 1968). Moreover, the

abortion of megasporogenesis during the period between the

embryo-sac first division and the fecundated egg cell is

common (Fatta del Bosco et al. 1992). For this reason, citrus

triploid plants are generally sterile, although they can occa-

sionally produce fruits with very few seeds and are capable

of inducing seed formation in fruits of other cultivars.

Communicated by W. Harwood.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-012-1286-0) contains supplementarymaterial, which is available to authorized users.

P. Aleza � J. Juarez � J. Cuenca � P. Ollitrault � L. Navarro (&)

Centro de Proteccion Vegetal y Biotecnologıa, Instituto

Valenciano de Investigaciones Agrarias (IVIA), Ctra.

Moncada-Naquera km 4.5, 46113 Moncada, Valencia, Spain

e-mail: lnavarro@ivia.es

P. Ollitrault

CIRAD, UMR AGAP, Avenue Agropolis, TA A-75/02,

34398 Montpellier, France

123

Plant Cell Rep

DOI 10.1007/s00299-012-1286-0

Triploid citrus plants can be recovered directly from

crosses between two diploid genotypes resulting from the

union of a 2n megagametophyte and a haploid pollen (Esen

and Soost 1971a) or by hybridization between diploid and

tetraploid parents (Esen and Soost 1971b). In 2x 9 2x

hybridizations, the frequency of 2n gametes is generally

low (Esen and Soost 1971a) and extensive breeding pro-

grams based on this type of hybridization require very

effective methodologies for embryo rescue and the ploidy

evaluation of large progeny populations (Aleza et al.

2010a). Implementation of extensive citrus breeding pro-

grams based on 2x 9 4x hybridizations requires (1) the

production of tetraploid genotypes to serve as male parents,

(2) an effective methodology for the recovery of triploid

citrus plants from seeds that do not germinate under

greenhouse conditions, and (3) an accurate and rapid ploidy

level analysis system.

In citrus germplasm, apomictic and non-apomictic

genotypes can be found (Frost and Soost 1968). The

majority of citrus genotypes are apomictic, with the

exception of all citron (Citrus medica L.), pummelo

(C. grandis (L.) Osb.), clementine (C. clementina Hort. ex

Tan.) and some mandarins. Tetraploidization by the chro-

mosome doubling of nucellar cells is a frequent event in

apomictic citrus, and tetraploid genotypes arising from

chromosome doubling are widely used as male parents in

breeding programs (Aleza et al. 2011). In non-apomictic

genotypes, tetraploid plants are not found in the citrus

germplasm but can be artificially produced. Aleza et al.

(2009) develop a new methodology based on in vitro shoot-

tip grafting combined with treatment of the micro-grafted

shoot-tips with colchicine and oryzalin to achieve chro-

mosome doubling and a dechimerization procedure assis-

ted by flow cytometry. Stable tetraploid plants of different

mandarins have been developed for use as male and female

parents in interploid hybridizations. Somatic hybridization

is another technique that permits tetraploid plant produc-

tion (Grosser et al. 2000, 2010; Grosser and Gmitter 2011).

Tetraploid plants produced in 2x 9 4x hybridizations

(Tachikawa et al. 1961) are also useful as parents for the

production of triploid plants (Williams and Roose 2004).

Since 1996, the Plant Protection and Biotechnology

Center of Instituto Valenciano de Investigaciones Agrarias

(IVIA, Moncada, Spain) has developed an extensive citrus

triploid breeding program based on interploid sexual

hybridizations. The present study analyzes the main factors

that affect the recovery of citrus triploid plants using

2x 9 4x hybridizations, with an aim toward producing new

high-quality seedless triploid mandarins. The various seed

types obtained from 2x 9 4x hybridizations have been

characterized. This study reports upon (1) the factors that

influence the behavior of embryo cultures in vitro, (2) the

ploidy level of regenerated plants from each type of seed,

(3) the effect of parents and environmental conditions on

triploid plant production, (4) the genetic origin of triploid

plants, and (5) the influence of parents on the length of the

juvenile phase.

Materials and methods

Plant material

The genotypes used (Table 1) are from the IVIA citrus

Germplasm Bank of pathogen-free plants (Navarro et al.

2002b). All the genotypes used as female parents are self-

incompatible and non-apomictic. The hybridizations were

conducted over a 10-year period (from 1997 to 2007) and

are part of the breeding program that has been carried out

since 1996 (Navarro et al. 2005).

Pollen viability

The pollen viability of diploid and tetraploid (doubled-

diploid) plants of ‘Willow leaf’ mandarin and ‘Pineapple’

sweet orange was evaluated by analyzing the pollen ger-

mination rate in vitro. A minimum of 20 flowers per

genotype were randomly collected from field-grown plants.

The anthers were removed from flowers collected in pre-

anthesis and dried in petri dishes over silica gel in a des-

iccator. Pollen from fully dehisced anthers was distributed

with a paintbrush onto 5.5-cm petri dishes containing

Table 1 Genotypes used in 2x 9 4x hybridizations

Scientific name

Diploid female parents

‘Bruno’ clementine C. clementina Hort. ex Tan

‘Clemenules’ clementine

‘Fina’ clementine

‘Hernandina’ clementine

‘Marisol’ clementine

‘Tomatera’ clementine

‘Fortune’ mandarin C. clementina 9 C. tangerina

‘Moncada’ mandarin C. clementina 9

(C. unshiu 9 C. nobilis)

Tetraploid male parents

‘Nova’ mandarin C. clementina 9

(C. paradisi 9 C. tangerina)

‘Moncada’ mandarin C. clementina 9

(C. unshiu 9 C. nobilis)

‘Willow leaf’ mandarin C. deliciosa Ten.

‘Orlando’ tangelo C. paradisi 9 C. tangerina

‘Pineapple’ sweet orange C. sinensis (L.) Osb.

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Murashige and Skoog (1962) culture media with 30 g/L

sucrose and 8 g/L Bacto agar. These petri dishes were

placed inside 9-cm petri dishes with a moist piece of filter

paper at 24 ± 1 �C in the dark for 24 h. Pollen was scored

as germinated when the length of the pollen tube exceeded

the diameter of the pollen grain. For each genotype, ger-

mination was recorded by counting a minimum of 700

pollen grains.

Pollination, seed extraction and characterization

Pollination was conducted in trees grown in a large

greenhouse and in the field. Anthers were removed from

flowers collected in pre-anthesis and prepared as described

above. Dried dehisced anthers were stored in small petri

dishes at -20 �C. Flowers were hand pollinated.

Fruits were collected when ripe. Seeds were extracted

and surface sterilized with a sodium hypochlorite solution

(0.5 % active chlorine for 10 min). Seeds were classified

by size and developmental stage. Size was evaluated by

measuring the area (mm2) and the developmental stage was

evaluated using morphological parameters. Seeds were

considered developed when they had a normal appearance,

were totally filled out, and did not contain any malforma-

tion. Seeds were considered undeveloped if they exhibited

incomplete development, were not totally filled out, were

wrinkled or had a split outer integument (Fig. 1). For

measurements, the seeds were washed, dried and uniformly

distributed in 9-cm petri dishes. The petri dishes were

scanned at 150 pp with an Epson� Perfection 4870 Photo

scanner. Images were analyzed using the Matrox� soft-

ware, which provides automatic exact measurements of the

area.

Embryo rescue

Embryo rescue was done following the methodology

described by Aleza et al. (2010a). Embryos were isolated

from undeveloped and normal seeds with the aid of a ste-

reoscopic microscope. These isolated embryos were cul-

tured under aseptic conditions in 9-cm petri dishes

containing Murashige and Skoog culture media with

50 g/L of sucrose, 500 mg/L malt extract, supplemented

with vitamins (100 mg/L of myo-inositol, 1 mg/L pyri-

doxine hydrochloride, 1 mg/L nicotinic acid, 0.2 mg/L

thiamine hydrochloride and 4 mg/L glycine) and 8 g/L of

Bacto agar. After germination, the plants were transferred

to 25 9 1,500-mm test tubes containing the same culture

media without malt extract. Cultures were maintained at

24 ± 1 �C, 60 % humidity and 16 h daily exposure to

40-lE m-2 s-1 illumination.

Ploidy level analysis

Ploidy level analysis by cytogenetic methods is a slow and

inadequate process when large populations of plants need

to be analyzed. Flow cytometry enables accurate and rapid

ploidy level analysis (Ollitrault and Michaux-Ferriere

1992; Navarro et al. 2002a).

The ploidy level was determined by flow cytometry

according to the methodology described by Aleza et al.

(2009). Each sample consisted of a small piece of leaf

(*0.5 mm2) collected from each test tube plant and a

similar leaf piece collected from a diploid control plant.

The samples were chopped together using a razor blade in

the presence of a nuclei isolation solution (High Resolution

DNA Kit Type P, solution A; Partec�, Munster, Germany).

Fig. 1 Different types of seeds

obtained from hybridizations

between diploid ‘Fina’

clementine and tetraploid

‘Willow leaf’ mandarin.

a Undeveloped seeds,

b developed seeds (normal

seeds)

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Nuclei were filtered through a 30-lm nylon filter and

stained with a DAPI (4-6-diamine-2-phenylindole) solution

(High Resolution DNA Kit Type P, solution B; Partec�).

Following a 5-min incubation period, the stained samples

were run in a Ploidy Analyzer (Partec�, PA) flow

cytometer equipped with a HBO 100-W high-pressure

mercury bulb and both KG1 and BG38 filter sets. Histo-

grams were analyzed using the dpac v2.0 software (Par-

tec�), which determines peak position, coefficient of

variation (CV) and the relative peak index of the samples.

Transfer to soil

Triploid plants were transferred to pots containing steam-

sterilized artificial soil mix suitable for growing citrus

(40 % black peat, 29 % coconut fiber, 24 % washed sand,

and 7 % perlite). Pots were enclosed in polyethylene bags

that were closed with rubber bands and placed in a shaded

area within a temperature-controlled greenhouse set at

18–25 �C. After 8–10 days, the bags were opened. After

another 8–10 days, the bags were removed and the plants

were grown under regular greenhouse conditions (Navarro

and Juarez 2007).

Molecular characterization

Twenty-four triploid plants were selected from the

hybridization between ‘Clemenules’ clementine and tetra-

ploid ‘Orlando’ tangelo, 12 from undeveloped seeds and 12

from normal seeds. These plants were analyzed using two

SSR markers heterozygous and with different alleles in

both parents, mCrCIR01E02 (Froelicher et al. 2008) and

Mest104 (Luro et al. Personal Communication).

The extraction of genomic DNA was conducted accord-

ing to Dellaporta and Hicks (1983) with slight modifications.

PCR amplifications were performed using a Thermocycler

ep gradient S (Eppendorf�) in a 10-mL final volume con-

taining 0.8U of Taq DNA polymerase (Fermentas�), 2

ng/mL citrus DNA, 0.2 mM wellRED (Sigma�) dye-

labeled forward primer, 0.2 mM non dye-labeled reverse

primer, 0.2 mM of each dNTP, 109 PCR buffer and

1.5 mM MgCl2. The following PCR program was applied:

denaturation at 94 �C for 5 min followed by 40 repetitions

of the following cycle: 30 s at 94 �C, 1 min at 55 �C, 45 s at

72 �C; and a final elongation step of 4 min at 72 �C.

Capillary electrophoresis was conducted with a CEQTM

8000 Genetic Analysis System (Beckman Coulter Inc.).

The GenomeLabTM GeXP v.10.0 genetic analysis software

was used for data collection and analysis. PCR products

were initially denaturized at 90 �C for 2 min, injected at

2 kV for 30 s and subsequently separated at 6 kV for

35 min. Alleles were sized according to a DNA size stan-

dard (400 bp).

Field evaluation and juvenility study

Triploid plants were cultured in the greenhouse for

approximately 1 year to produce quality budwood for

grafting in the field. Buds were grafted onto ‘Carrizo’

citrange rootstock (C. sinensis 9 P. trifoliata) for field

evaluation at IVIA plots. Flowering was recorded for each

plant.

For the juvenility study, 2,098 triploid hybrids were

analyzed. Of these, 915 arose from hybridizations between

‘Clemenules’, ‘Fina’ and ‘Hernandina’ clementines and

‘Fortune’ mandarin with tetraploid ‘Orlando’ tangelo; 513

arose from hybridizations between ‘Clemenules’, ‘Fina’

and ‘Hernandina’ clementines with tetraploid ‘Pineapple’

sweet orange; 327 arose from hybridizations between

‘Clemenules’, ‘Fina’ and ‘Hernandina’ clementines with

tetraploid ‘Nova’ mandarin; and 343 arose from hybrid-

izations between ‘Clemenules’ and ‘Fina’ clementines with

tetraploid ‘Willow leaf’ mandarin.

Statistical analysis

Pollen germination percentage data were analyzed by

proportion test and number of triploid hybrids that flowered

after 3 and 6 years from grafting were analyzed by the Chi-

square test with 1 degree of freedom and 5 % of signifi-

cance level (v2 = 3.418).

Results and discussion

Pollen viability

The pollen viability of different diploid and tetraploid

citrus genotypes was evaluated by examining the pollen

germination rate in vitro. The highest percentage, 72 %

(774/1,081), was obtained from diploid ‘Willow leaf’

mandarin. By contrast, pollen from autotetraploid ‘Willow

leaf’ mandarin had a 37 % (473/1,280) germination rate,

displaying statistical significance between diploid and

autotetraploid ‘Willow leaf’ mandarins (p \ 0.0001). No

statistical significance was observed (p = 0.1120) between

diploid ‘Pineapple’ sweet orange and its tetraploid (dou-

bled-diploid). These two genotypes displayed, respectively,

the germination rates of 31 % (359/1,164) and 34 % (484/

1,433). The pollen germination rates of the tetraploid

(doubled-diploid) genotypes were similar or less than the

corresponding diploid genotypes, although the viability is

sufficient for these types to be used as male parents in

sexual hybridizations. The pollen viability of other diploid

and tetraploid (doubled-diploid) citrus genotypes (manda-

rin, tangor, grapefruit and sour orange) that were not

included in this study have been tested previously. During

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meiosis in tetraploid genotypes, the pollen mother cells

degenerate before the reduction division much more fre-

quently than those of corresponding diploids (Frost and

Soost 1968). Trivalent and univalent chromosomes are

common in tetraploids’ genotypes although they usually

produce less pollen grains whose chromosome complement

is completely normal (Frost and Soost 1968; Fatta del

Bosco et al. 1999). However, the remaining pollen fertility

is adequate for successful controlled hand-pollination

(Viloria and Grosser 2005; Ollitrault et al. 2008).

Seed characterization and ploidy level

of recovered plants

The seeds obtained from the hybridization of diploid ‘Fina’

clementine with tetraploid ‘Willow leaf’ mandarin were

used to analyze the relationship between seed morphology

and embryo ploidy. From 24 fruits, 114 seeds were

obtained; 12 seeds were developed (normal seeds) and 102

seeds were undeveloped (Fig. 1a, b).

The average area of normal seeds was 89 ± 8 mm2,

whereas the area of undeveloped seeds was 35 ± 15 mm2.

Thus, the undeveloped seeds were 49–75 % smaller than

normal seeds. Embryos were rescued from the 12 normal seeds

and cultured in vitro. Of the five plants obtained, one was

triploid and four were tetraploid. From the 102 undeveloped

seeds, 76 plants were recovered and all of these were triploid.

In 2x 9 4x hybridizations, it is possible to recover

triploid plants from both types of seeds, normal and

undeveloped. Therefore, in our extensive triploid breeding

program, both types of seeds have been routinely used to

recover triploid plants.

Recovery of citrus triploid plants; methodological

considerations

Results on fruit set, and the number and type of seeds

obtained in 2x 9 4x hybridizations are shown in Table 2.

The fruit set average was 61 %, very similar to the fruit set

average obtained in 2x 9 2x hybridizations (59 %) (Aleza

et al. 2010a) and higher than that obtained from 4x 9 2x

hybridizations (37 %) (Aleza et al. 2012). From all of the

hybridizations, 9,090 seeds were obtained. Of these, 12 %

of seeds were normal and 88 % were undeveloped. The

average number of normal seeds per fruit was 1.0 whereas

the average number of undeveloped seeds per fruit was 7.5.

Similar results were observed by Viloria and Grosser

(2005), in 2x 9 4x hybridisations using different lemons,

limes, citrons and limequats as female parents with

autotetraploids and allotetraploids somatic hybrids as male

parents. They also observed different types of seeds in

2x 9 4x hybridizations and a variation on the seed number

and type of seed depending on the male parents.

Embryo rescue from undeveloped seeds, ploidy level

and transfer to soil

From the 7,994 undeveloped seeds, 36 % of seeds were

aborted and 64 % contained embryos (Fig. 2a, b). Using the

embryo rescue technique, the average germination percentage

Table 2 Fruit set, number and type of seeds obtained from 2x 9 4x hybridizations

Diploid female parent Tetraploid

male parent

Number of

pollinated flowers

Number of

fruits set

Number of

developed seeds

Number of

undeveloped seeds

Clemenules Moncada 50 35 14 230

Novaa 100 55 47 573

W. Leafa 165 81 40 816

Pineapplea 185 105 114 550

Orlando 100 53 76 619

Fina Moncada 100 74 44 533

Novaa 100 68 35 371

W. Leafa 150 88 17 573

Pineapplea 150 85 19 304

Hernandina Pineapple 50 31 33 283

Orlandoa 150 115 184 1,210

Bruno Orlando 50 37 20 274

Marisol Orlando 150 95 40 363

Tomatera Nova 50 38 21 242

Moncada W. Leaf 50 21 5 99

Fortune Orlandoa 150 85 387 954

a Date represent the average of 2 years

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was 65 % (Table 3). In all the hybridizations using clemen-

tines as the female parent, the undeveloped seeds had either

one (monoembryonic) (Fig. 2a) or multiple embryos (poly-

embryonic) (Fig. 2b). By contrast, the hybridization between

‘Fortune’ mandarin and tetraploid ‘Orlando’ tangelo pro-

duced undeveloped seeds containing only a single embryo.

Embryos were at the globular to early cotyledonary stage and

variable in size (macroscopically, some embryos were so

small that they were difficult to identify, whereas others

reached a size of approximately 2–3 mm). In polyembryonic

seeds, the embryos were very compact and sometimes sur-

rounded by traces of endosperm. This made it impractical to

individualize and count the embryos precisely without caus-

ing damage. For this reason, all of the embryos of a single

seed were cultivated together (Fig. 2c, d). The global per-

centages of monoembryonic and polyembryonic seeds were

67 and 33 %, respectively (Table 3). The average number of

embryos that germinated from each polyembryonic seed

varied from 1.2 to 2.3 (Table 3).

From the undeveloped seeds, 2,651 plants were

obtained. Of these, 2,636 plants were triploid, eight plants

were diploid, four plants were tetraploid, two plants were

hexaploid and one plant was mixoploid. The diploid, tet-

raploid, hexaploid and mixoploid plants died during the

Fig. 2 a Monoembryonic

undeveloped seed.

b Polyembryonic undeveloped

seed. c Germination of embryo

from monoembryonic

undeveloped seed.

d Germination of multiple

embryos from polyembryonic

undeveloped seed

Table 3 In vitro culture of embryos obtained from undeveloped seeds produced from 2x 9 4x hybridizations

Diploid

female

parent

Tetraploid

male

parent

Number of

seeds with

embryo

Number of

germinated

seedsa

Number of

monoembryonic

seeds

Number of

polyembryonic

seeds

Number of

germinated

embryos

Number of germinated

embryos per

polyembryonic seed

Clemenules Moncada 175 151 106 45 200 2.1

Novaa 476 345 251 94 440 2.0

W. Leafa 577 450 186 264 696 1.9

Pineapplea 368 277 190 87 371 2.1

Orlando 510 387 291 96 476 1.9

Fina Moncada 391 187 84 103 227 1.4

Novaa 269 207 149 58 262 1.9

W. Leafa 367 211 113 98 260 1.5

Pineapplea 180 146 84 62 226 2.3

Hernandina Pineapple 180 138 108 30 145 1.2

Orlandoa 843 408 318 90 491 1.9

Bruno Orlando 80 30 25 5 35 2.0

Marisol Orlando 265 140 110 30 169 2.0

Tomatera Nova 185 131 109 22 142 1.5

Moncada W. Leaf 63 32 22 10 44 2.2

Fortune Orlandoa 201 109 109 0 109 0.0

a Date represent the average of 2 years

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transplant phase or in the greenhouse. The global average

of triploid plants per fruit obtained from the 2x 9 4x

hybridizations was 2.5, varying between 0.3 and 6.6 trip-

loid plants per fruit. The average percentage of survival

during the transplant phase was 78 % (Table 4). Viloria

and Grosser (2005) obtained a high variation in the number

of triploid hybrids recovered from 2x 9 4x hybridizations,

with five genotypes of lemon, the percentage of triploid

hybrids, ranged from 7.6 to 20.4 %.

Aleza et al. (2010b) used SSR markers to demonstrate

that all of the plants recovered from a single undeveloped

seed obtained from 2x 9 4x sexual hybridizations resulted

from cleavage of the original zygotic embryo. Thus, during

the stage of embryo rescue from undeveloped seeds, only

one plant per seed was regenerated. Considering only one

triploid genotype per undeveloped polyembryonic seed, the

general average of triploid genotypes per fruit decreases to

2.1 triploid plants per fruit. This practice reduces the work

necessary during the in vitro stage and avoids costly and

lengthy field evaluation of several plants belonging to the

same genotype. For example, we have obtained 2,636

triploid plants from undeveloped seeds, but only 2,195

different triploid genotypes. Therefore, the duplicate

evaluation of 441 triploid genotypes was avoided.

Embryo rescue from normal seeds, ploidy level

and transfer to soil

From the 1,096 normal seeds obtained from all the 2x 9 4x

hybridizations, 1,061 plants with a general germination

percentage over 97 % were recovered using the embryo

rescue technique (Table 5). All of the normal seeds con-

tained only one well-formed embryo.

Among the 1,061 plants, 279 plants were triploid

(26 %), 423 plants were tetraploid (40 %) and 359 plants

were diploid (34 %). The average number of triploid plants

per fruit obtained from normal seeds varied from 0.06

for ‘Fina’ clementine 9 tetraploid ‘W. Leaf’ mandarin to

0.81 for ‘Hernandina’ clementine 9 tetraploid ‘Pineapple’

sweet orange. The global average of triploid plants per fruit

obtained was 0.26. The average survival percentage in the

transplant phase was 93 % (Table 5).

Efficiency in production of citrus triploid genotypes

The recovery efficiency of citrus triploid genotypes was

calculated as the number of triploid genotypes (one geno-

type per undeveloped seed) per harvested fruit. Both the

types of seeds, undeveloped and normal, were considered.

The number of triploid genotypes obtained per fruit was 2.3

when all the hybridizations were considered. In the

2x 9 2x hybridizations, the recovery efficiency in citrus

triploid plants was dependent on the genotype of the female

parent (Aleza et al. 2010a). With ‘Fortune’ mandarin, 2.5

triploid hybrids per fruit were obtained. By contrast, the

efficiency when clementines were used as the female par-

ent varied from 0.32 to 0.17 (for ‘Hernandina’ and ‘Fina’

clementines, respectively). Therefore, triploid production

efficiencies of 2x 9 2x and 2x 9 4x were very similar for

‘Fortune’ mandarin, while with clementines, the efficiency

Table 4 Plant regeneration, ploidy level and transfer to soil of plants obtained in undeveloped seeds produced from 2x 9 4x hybridizations

Diploid

female

parent

Tetraploid

male parent

Number of

plants obtained

Number of

triploid plants

Number of

tetraploid

plants

Number of

diploid plants

Number of triploid

plants in greenhouse

Number of

triploid

genotypes

Clemenules Moncada 137 136 0 1 100 113

Novaa 366 365 0 0 296 297

W. Leafa 334 334 0 0 243 267

Pineapplea 284 282 0 1 252 219

Orlando 299 299 0 0 187 268

Fina Moncada 110 110 0 0 89 101

Novaa 134 134 0 0 105 112

W. Leafa 167 163 0 4 118 129

Pineapplea 170 169 0 0 153 112

Hernandina Pineapple 128 128 0 0 119 99

Orlandoa 238 237 1 0 160 209

Bruno Orlando 10 10 0 0 6 10

Marisol Orlando 93 91 2 0 67 86

Tomatera Nova 69 68 0 1 61 65

Moncada W. Leaf 19 19 0 0 9 17

Fortune Orlandoa 93 91 1 1 79 91

a Date represent the average of 2 years

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in 2x 9 4x hybridizations was more than eight times

greater than in the 2x 9 2x hybridizations. However, Aleza

et al. (2012) found that 4x 9 2x hybridizations were more

efficient, with an average double than obtained in the

2x 9 4x hybridizations (4.6 triploid plants/genotypes per

fruit).

A well-established, routine embryo rescue protocol is

indispensable for the development of extensive triploid

breeding programs from 2x 9 4x sexual hybridizations. It

requires the isolation of intact undamaged embryos under

aseptic conditions using a dissecting microscope. We have

cultivated embryos from more than 6,220 undeveloped and

normal seeds with a general germination percentage over

71 %, establishing an optimized process for extensive

triploid citrus breeding program based on 2x 9 4x hy-

bridisations although it was lower than that obtained in

2x 9 2x (93 %) (Aleza et al. 2010a, b) and 4x 9 2x

(95 %) hybridizations (Aleza et al. 2012). The recovery of

triploid plants from 2x 9 4x hybridizations is more labor

intensive during the in vitro culture phase compared to

2x 9 2x and 4x 9 2x hybridizations because embryos

from undeveloped seeds are much more difficult to ger-

minate. Moreover, when these embryos do germinate, it is

necessary to differentiate and isolate plantlets from the

same undeveloped seed to avoid competition and promote

plantlet development.

During the transplant phase, differences were observed

between triploid plants recovered from undeveloped seeds

(78 %) and those recovered from normal seeds (93 %).

These differences resulted from the differential develop-

ment of the regenerated plants. Triploid plants recovered

from embryos of undeveloped seeds were fragile and weak

(Fig. 3a), whereas plants recovered from embryos of

Table 5 Plant regeneration, ploidy level and transfer to soil of plants obtained in normal seeds produced from 2x 9 4x hybridizations

Diploid

female

parent

Tetraploid

male parent

Number of

normal seeds

Number of

plants obtained

Number of

triploid plants

Number of

tetraploid plants

Number of

diploid plants

Number of triploid

plants in greenhouse

Clemenules Moncada 14 11 4 7 0 0

Novaa 47 45 27 16 2 26

W. Leafa 40 40 21 19 0 19

Pineapplea 114 111 29 18 64 27

Orlando 76 69 20 20 29 20

Fina Moncada 44 43 7 33 3 5

Novaa 35 35 12 3 20 11

W. Leafa 17 13 5 8 0 5

Pineapplea 19 18 6 7 5 6

Hernandina Pineapple 33 33 25 7 1 23

Orlandov 184 183 53 78 52 48

Bruno Orlando 20 20 6 5 9 6

Marisol Orlando 40 39 7 31 1 7

Tomatera Nova 21 21 12 9 0 12

Moncada W. Leaf 5 4 4 0 0 3

Fortune Orlandoa 387 376 41 162 173 40

a Date represent the average of 2 years

Fig. 3 a Triploid plant recovered from an embryo contained in an

undeveloped seed. b Triploid plant recovered from embryo contained

in a normal seed

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normal seeds were robust and vigorous (Fig. 3b). After

1 month of growth in the greenhouse, triploid plants from

undeveloped seeds grew very well, and differences were no

longer noted compared to the triploid plants from normal

seeds. In 2x 9 2x and 4x 9 2x hybridizations, the average

survival percentage during the transplant phase was 90 %

(Aleza et al. 2010a) and 84 % (Aleza et al. 2012),

respectively.

Effect of parents and environmental conditions

on the recovery of citrus triploid plants

Considering all the crosses, the highest number of triploid

plants per fruit was obtained with ‘Clemenules’ clementine

at 3.8 triploids per fruit. This was followed by ‘Hernan-

dina’ and ‘Tomatera’ clementines with 2.6 and 2.0 triploid

plants per fruit, respectively, ‘Fortune’ mandarin with 1.6

triploid plants per fruit and ‘Fina’ clementine with 1.5

triploid plants per fruit. The triploid production efficiency

was estimated for ‘Clemenules’ and ‘Fina’ clementines that

were crossed with four tetraploid male parents over a

6-year period (Fig. 4). Variations were observed according

to the female and male parents and the pollination years.

For a same year and male parent, ‘Clemenules’ always

produced higher number of triploid plants than ‘Fina’.

Tetraploid ‘Nova’ mandarin male parent produced the

highest number of triploid plants per fruit, whereas the

lowest number of triploid plants per fruit was obtained with

‘W. Leaf’ mandarin in both clementines.

Inter-annual variances were observed for the same

parental combinations and, in some cases, the variances

were very large (Fig. 4). For example, in the cross between

‘Clemenules’ clementine and ‘Nova’ mandarin, the varia-

tion in the number of triploid plants per fruit ranged from

9.1 in 2003 to 1.1 in 2006. Moreover, the ranking of the

male parents was not conserved between years, which

would indicate an important interaction between the male

parent genotype and environmental conditions.

Luro et al. (2004), Viloria and Grosser (2005) and Aleza

et al. (2010a) reported that the male parent and environ-

mental conditions influence the production of citrus triploid

plants. In handmade pollinations conducted between dif-

ferent mandarin genotypes, great differences in the number

of seeds per fruit were observed depending on the male

parents used for the crosses (unpublished data). This could

be related to pollen viability or the citrus gametophytic

incompatibility system (Soost 1965, 1969).

The influence of environmental conditions on plant

reproductive success is well documented. Environmental

conditions can affect pollen production, pollen size, pollen

germination and pollen tube growth rate (Young and

Stanton 1990). The temperature during the progamic phase

represents one of the most important environmental factors

that affect pollen performance. Studies in other fruit trees

recently demonstrated that, besides physiological effects,

the temperature also affects pollen tube population

dynamics. This was shown by pollinating a female geno-

type with pollen from two different genotypes. Thus, the

response to temperature during the reproductive phase is

genotype-dependent (Hedhly et al. 2005a, b).

Genetic origin of triploid plants recovered

from 2x 9 4x hybridizations

Esen and Soost (1971b) proposed that the production of

triploid citrus hybrids from 2x 9 4x hybridizations was

hampered by precocious endosperm development. They

proposed that the 3/4 ploidy ratio of embryo and endo-

sperm caused the induction of seed abortion and endo-

sperm degeneration. Consequently, a high number of

embryos fail after 2x 9 4x hybridization, thus reducing the

frequency of viable triploid hybrid production. Rather than

Fig. 4 Triploid hybrids recovered per fruit over 6 years with diploid ‘Clemenules’ and ‘Fina’ clementines as female parents and five different

male parents

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embryo/endosperm ploidy ratio, diverse studies in Ara-

bidopsis and potato suggest that a main determinant of seed

abortion could be the maternal/paternal contribution to the

endosperm (Adams et al. 2000; Vinkenoog et al. 2003) or

the endosperm balance number (EBN) (Carputo et al.

2003). The results obtained in the present study demon-

strate that normal seeds produced by 2x 9 4x hybridization

can contain triploid embryos. These triploid plants could be

originated as a consequence of unreduced megagameto-

phyte with haploid pollen grain with a most suitable

endosperm/embryo ploidy ratio (3/5) or maternal/paternal

contribution. With the view to solve this question, 24

triploid plants were selected from the hybridization

between ‘Clemenules’ clementine and tetraploid ‘Orlando’

tangelo. Of these plants, 12 were from undeveloped seeds

and 12 from normal seeds. These plants were analyzed

using SSR markers displaying different alleles for both

parents. All the triploid plants from the undeveloped and

normal seeds were hybrids that displayed one allele of the

female parent and two alleles of the male parent (Fig. 5).

These results conclude that these plants were produced

from a fecundation of a haploid ovule and a diploid pollen

grain. Therefore, it appears that about 3.1 % (279/9,090) of

the seeds with triploid embryos developed normally. Esen

and Soost (1971b) previously reported that 1–8 % of the

seeds from different 2x 9 4x sexual hybridizations were

normal.

In this study, tetraploid plants were identified in normal

seeds from all the conducted hybridizations. As a result,

more than 400 tetraploid plants were recovered. These

results support the observations of Cameron and Soost

(1969) as well as the spontaneous production of diploid

megagametophytes and a suitable endosperm/embryo

ploidy ratio or EBN when diploid pollen fertilizes unre-

duced megagametophytes (Ollitrault et al. 2008). Aleza

et al. (2010a) demonstrated that the frequency of unreduced

gametes is an intrinsic characteristic of each genotype.

‘Fortune’ mandarin exhibited a higher frequency of triploid

hybrids per fruit than clementines, with 2.5 and 0.23,

respectively. Results obtained in this work displayed that

‘Fortune’ mandarin produces 1.9 tetraploid plants per fruit

and clementines 0.27 tetraploid plants per fruit, thus

agreeing with the results of 2n gamete production obtained

during 2x 9 2x hybridizations. Genetic analysis with SSR

Fig. 5 Profiles obtained using the Mest104 SSR marker in diploid ‘Clemenules’ clementine, tetraploid ‘Orlando’ tangelo, and a triploid hybrid

recovered from undeveloped seed and a triploid hybrid recovered from normal seed

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markers of several tetraploid plants obtained in 2x 9 4x

hybridizations that were not included here demonstrate this

process (data not shown).

Juvenile characteristics of triploid hybrids

Juvenile traits in citrus plants originating from seeds are

very prominent and often persist for a long time. These

traits include thorniness, vigorous growth, alternate bearing

during early years, physical differences in fruit characters

and slowness to flower and bear fruit (Cameron and Frost

1968). The long juvenile period of citrus trees obtained

from seeds is a major impediment to citrus breeding pro-

grams based on the sexual hybridization.

The juvenility phase of 2,098 triploid hybrids was

studied in this work. All the triploid hybrids displayed

juvenile characteristics. When transplanted to pots in the

greenhouse, the triploid hybrids took approximately 1 year

to produce quality budwood that could be grafted in the

field. 1 year after field grafting, none of the triploids pro-

duced flowers. After 2 years, 22 % of the triploids flow-

ered. This number increased to 43 % after 3 years and

75 % after 5 years. Differences were observed according to

the female and male parents (Figs. 6, 7 and Supplementary

material).

After 3 years of grafting, 61 % of the triploid hybrids

obtained with ‘Fortune’ mandarin as the female parent had

already flowered, whereas only 39–51 % of the triploid

hybrids from three clementine varieties used as female

parents had flowered. This advantage of ‘Fortune’ man-

darin hybrids over clementines ones was maintained in

subsequent years. After 6 years, the flowering percentages

were 90 and 83–84 % for ‘Fortune’ mandarin and clem-

entines, respectively (Fig. 6). In 2x 9 2x hybridizations,

Aleza et al. (2009) reported that triploid hybrids with

‘Fortune’ mandarin as the female parent flowered earlier

than triploid hybrids obtained with clementines. In this

previous work, 6 years after grafting, the flowering per-

centages were 94 and 65 % for ‘Fortune’ mandarin and

clementines, respectively. This result indicates that the

juvenile phase length of triploid hybrids obtained with

‘Fortune’ mandarin as the female parent in 2x 9 2x and

2x 9 4x hybridizations are very similar. However, all the

Fortune hybrids from interploid hybridization were

obtained with tetraploid Orlando. It should be useful to

check if similar results would also be obtained with other

male parents.

When clementines are used as female parents, the

juvenile phase length of the triploid hybrids obtained in

2x 9 2x hybridizations is longer than the juvenile phase of

triploid hybrids recovered in 2x 9 4x hybridizations. This

result suggests that allele doses affect the expression of the

juvenile phase. Indeed, the genomic maternal contribution

is 2/3 and 1/3 on triploid plants arising from 2x 9 2x or

2x 9 4x sexual hybridizations, respectively.

A very clear effect of the male parent on the juvenile

phase length was also observed (Fig. 7). When tetraploid

‘Orlando’ tangelo was used as the male parent, 43 % of the

triploid hybrids flowered 2 years after grafting. This was

followed by tetraploid ‘Nova’ mandarin (15 %), tetraploid

‘W. Leaf’ mandarin (4 %) and tetraploid ‘Pineapple’ sweet

orange (3 %). These differences were maintained 6 years

after grafting; 97 % of the triploid hybrids with tetraploid

‘Orlando’ tangelo had flowered, 89 % with tetraploid

‘Nova’ mandarin, 77 % with tetraploid ‘W. leaf’ mandarin

and 62 % with tetraploid ‘Pineapple’ sweet orange (Fig. 7).

This study demonstrates the effect of the female and

male parents on the length of the juvenile phase in triploid

hybrids obtained in 2x 9 4x sexual hybridizations. A

shorter juvenile period compared with the 2x 9 2x originFig. 6 Evolution of the triploid hybrid percentage that flowered

6 years after grafting obtained with four different female parents

Fig. 7 Evolution of the triploid hybrid percentage that flowered

6 years after grafting obtained with four different male parents

Plant Cell Rep

123

was also observed when clementines were used as female

parents. This observation has a practical interest because

juvenility mainly determines the number of years required

to produce new selected varieties.

From analyses of the fruit seed content, 71 % of triploid

hybrids never produced seeds, 24 % produced between 0.1

and 0.9 seeds per fruit and only 5 % of triploid hybrids

contained more than one seed per fruit. These results val-

idate triploid hybrid production as a strategy to recover

seedless varieties.

Conclusions

Various factors influencing the recovery of citrus triploid

hybrids from 2x 9 4x hybridizations have been analyzed.

The pollen germination rates of tetraploid genotypes are

enough for efficient pollination. Triploid embryos are

found in normal seeds and in undeveloped seeds that are

49–75 % smaller than normal ones. Embryo rescue and

flow cytometry are two indispensable techniques for

extensive citrus triploid breeding programs based on

2x 9 4x hybridizations. Triploid plants recovered from

normal and undeveloped seeds result both from the

fecundation of a haploid ovule and a diploid pollen grain.

An unclear parent effect has been observed in triploid

hybrid production and environmental conditions appear to

be a major determinant of triploid production. This study

demonstrates the effect of the female and male parents on

the length of the juvenile phase in triploid hybrids. At

IVIA, an extensive triploid citrus breeding program based

on such hybridization has been run since 1996. To date,

more than 4,400 triploid hybrids have been recovered from

77 different 2x 9 4x sexual hybridizations. Of these, 2,063

triploid hybrids have been evaluated for fruit quality.

Twenty-two triploid hybrids were pre-selected and one of

them, IVIA-600 triploid (a hybrid between diploid ‘Her-

nandina’ clementine and tetraploid ‘Orlando’ tangelo), was

selected for its high fruit quality, excellent flavor and

production period (Fig. 8). Pathogen-free plants of IVIA-

600 triploid have been obtained by shoot-tip grafting in

vitro, according to the methodology described by Navarro

et al. (1975) and the protection of Plant Breeder’s Rights

has been requested.

Acknowledgments We thank M. Hernandez, J.M. Arregui, C.

Ortega, A. Navarro, V. Ortega, and C. Martı for technical assistance

in the laboratory, and J.A Pina, V. Lloris, J.M. Conchilla, F. Ahuir, D

Conchilla, A. Conchilla, R. Lopez, and F.J. Martı for growing plants

in the greenhouse and field. We also thank Dr. F. Luro from INRA

(France) for providing unpublished SSR markers. This work was

supported by a grant (Prometeo/2008/121) from the Generalitat

Valenciana, Spain and by two grants (AGL2008-00596 and

AGL2011-26490) from the Ministry of Science and Innovation of

Spain-Fondo Europeo de Desarrollo Regional (FEDER).

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