ORIGINAL PAPER

Recovery of citrus triploid hybrids by embryo rescue and flowcytometry from 2x 3 2x sexual hybridisation and its applicationto extensive breeding programs

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

Luis Navarro

Received: 22 March 2010 / Revised: 27 May 2010 / Accepted: 14 June 2010 / Published online: 6 July 2010

� Springer-Verlag 2010

Abstract Seedlessness is one of the most important

characteristics for mandarins for the fresh-fruit market and

mandarin triploid hybrids have this trait. Citrus triploid

plants can be recovered by 2x 9 2x sexual hybridisations as

a consequence of the formation of unreduced gametes at

low frequency. Triploid embryos are found in small seeds

that do not germinate under greenhouse conditions.

Extensive breeding programs based on this type of

hybridisation require very effective methodologies for

embryo rescue and ploidy evaluation. In this work, we

describe an effective methodology to recover triploid

hybrids from 2x 9 2x hybridisations based on in vitro

embryo rescue and ploidy level determination by means of

flow cytometry. The influence of parents and environmental

conditions on obtaining triploid hybrids has been analysed.

The strongest effect was associated with the genotype of the

female parent while a strong interaction was found between

the male parent genotype and environmental conditions.

The effect of the female parent genotype on the length of the

juvenile phase was also demonstrated by observing a large

number of progenies over the last 10 years. The method-

ology described here has enabled us to obtain over 4,000

triploid hybrids so far, of which 13 have been protected in

the European Union and two are being extensively planted

by citrus growers to establish new commercial plots. These

triploid hybrids have been analysed with simple sequence

repeats markers to differentiate all the new triploid varieties

and their parents, and thus molecular identification will help

defend plant breeders’ rights.

Keywords Triploid � Embryo rescue � Flow cytometry �2x 9 2x sexual hybridisation � Juvenility � Mandarin

Introduction

Citrus are the most extensively produced fruit-tree crop in

the world (FAO 2009). In the Mediterranean area, citrus

fruits are primarily produced for the fresh-fruit market.

Spain is the main producer in the area with a surface of

330,000 ha and a production about 6.3 million tons.

Seedlessness is one of the most important characteristics

for mandarins sold on the fresh-fruit market as consumers

do not accept seedy fruits. The creation of triploid hybrids

is an important breeding strategy to develop new seedless

citrus commercial varieties (Ollitrault et al. 2008). Cyto-

genetic studies have shown that during meiosis of citrus

triploid hybrids, trivalent, bivalent and univalent associa-

tions are formed (Cameron and Frost 1968) producing

sterile gametes. Moreover, Fatta Del Bosco et al. (1992)

have shown that the abortion of megasporogenesis from the

first divisions of the embryo sac in the fertilised egg cell is

frequent. For this reason, citrus triploid hybrids are gen-

erally sterile, although they can occasionally produce fruits

with very few seeds. Triploid plants are generally consid-

ered as an evolutionary dead-end, since they generally give

rise to aneuploid gametes with very low fertility (Otto and

Communicated by W. Harwood.

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

UPR amelioration genetique des especes a multiplication

vegetative, Centre de Cooperation Internationale en Recherche

Agronomique pour le Developpement (CIRAD), Avenue

Agropolis TA A-75/02, 34398 Montpellier Cedex 5, France

123

Plant Cell Rep (2010) 29:1023–1034

DOI 10.1007/s00299-010-0888-7

Whitton 2000). However, horticultural methods of grafting

allow the clonal vegetative propagation of triploid citrus.

During the past few years, new triploid genotypes have

been obtained from 2x 9 4x and 4x 9 2x hybridisations in

USA, Japan and Italy (Williams and Roose 2004; Toku-

naga et al. 2005; Reforgiato-Recupero et al. 2008), and

some of these cultivars are now starting to be commercially

propagated.

Citrus triploid hybrids can be recovered by 2x 9 2x

(Cameron and Frost 1968; Esen and Soost 1971a; Geraci

et al. 1975), 2x 9 4x (Esen and Soost 1971b; Oiyama et al.

1981; Starrantino and Recupero 1981) and 4x 9 2x

(Cameron and Burnett 1978; Esen et al. 1978; Aleza et al.

2009) sexual hybridisations. In the case of 2x 9 2x

hybridisation, triploid hybrids arise from the union of a 2n

megagametophyte with haploid pollen (Esen and Soost

1971a, 1973; Geraci et al. 1975). The frequency of such

events is generally low (Cameron and Frost 1968; Esen and

Soost 1971a; Geraci et al. 1975) and extensive breeding

programs based on this type of hybridisation require very

effective methodologies for embryo rescue and ploidy

evaluation of large progenies.

Esen and Soost (1971a) were the first to indicate that

triploid embryos are preferentially found in seeds that are

between 1/3 and 1/6 smaller than normal seeds; moreover,

these small seeds generally do not germinate in conven-

tional greenhouse conditions. The in vitro culture of whole

seeds with their integuments can improve germination

rates, although still at relatively low germination percent-

ages (Ollitrault et al. 1996). In rare cases, triploid hybrids

can be found in conventional greenhouse seedlings, as was

the case for ‘Winola’ mandarin (Vardi et al. 1991) and

‘A-12’ mandarin (Bono, personal communication).

In the past, the implementation of extensive triploid

projects based on 2x 9 2x hybridisation was also ham-

pered by the triploid selection step among the progenies.

Indeed, ploidy level analysis by cytogenetic methods is a

slow and inadequate process when large populations of

plants have to be analysed. By contrast, techniques for

ploidy level analysis by flow cytometry (Ollitrault and

Michaux-Ferriere 1992; Ollitrault et al. 1996; Navarro

et al. 2002a) enabled ploidy level to be determined both

accurately and very rapidly.

In this work, we present the methodological improve-

ments that have enabled us to establish an optimised pro-

cess for extensive triploid citrus breeding from 2x 9 2x

hybridisations, with the aim to produce new high-quality,

late-maturing and seedless triploid mandarins. The differ-

ent types of seeds obtained from 2x 9 2x hybridisations

have been characterised and small seed in vitro culture was

compared with embryo rescue method. We have analysed

the different factors that influence the behaviour of

embryos cultured in vitro, the ploidy level of plants

regenerated from each type of seed and how parents

influence triploid hybrid recovery using this technology.

Our breeding program gave rise to over 4,000 triploid

hybrids, from which 13 varieties have been protected and

fingerprinted by simple sequence repeats (SSR) markers,

thus contributing to the protection of breeders’ rights.

Materials and methods

Plant material

All the genotypes used (Table 1) are included in the Citrus

Germplasm Bank of pathogen-free plants of the Instituto

Valenciano de Investigaciones Agrarias (IVIA, Moncada,

Spain) (Navarro et al. 2002b). All the genotypes used as

female parents are self-incompatible and non-apomictic.

The hybridisations were done over a 10-year period (from

1996 to 2006) and are part of the breeding program carried

Table 1 Genotypes used in 2x 9 2x hybridisations

Scientific name

Female parents

Clementines

‘Clemenules’

‘Fina’

‘Hernandina’

‘Marisol’ Citrus clementina Hort. ex Tan.

‘Fortune’ mandarin C. clementina 9 C. tangerina

‘Moncada’ mandarin C clementina 9 (C. unshiu 9 C. nobilis)

Male parents

Clementines

‘Clemenules’

‘Fina’

‘Hernandina’ C. clementina

‘Chandler’ pummelo C. grandis 9 C. grandis

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

‘Fortune’ mandarin C. clementina 9 C. tangerina

‘Kara’ mandarin C. unshiu 9 C. nobilis

‘Moncada’ mandarin C clementina 9 (C. unshiu 9 C. nobilis)

‘Nova’ mandarin C. clementina 9 (C. paradisi 9

C. tangerina)

‘Willow leaf’ mandarin C. deliciosa Ten.

‘Chandler’ pummelo C. grandis 9 C. grandis

‘Pink’ pummelo Citrus grandis (L.) Osb.

Tangors

‘Ellendale’

‘Murcott’

‘Nadorcott’

‘Ortanique’ C. reticulata 9 C. sinensis

1024 Plant Cell Rep (2010) 29:1023–1034

123

out since 1995 (Navarro et al. 2005). In addition, we made

a hybridisation between ‘Clemenules’ clementine (Citrus

clementina Hort. ex Tan.) female parent with ‘Pink’

pummelo (C. grandis (L.) Osb.) male parent to describe

and characterise seeds obtained in 2x 9 2x hybridisations.

Pollination, seed extraction and characterisation

Pollinations were carried out in trees grown in a large

screenhouse and in the field. Anthers of the male parents

were removed from flowers collected in pre-anthesis and

dried in Petri dishes over silica gel in a desiccator. Dried

dehisced anthers were stored in small Petri dishes at

-20�C. Flowers were hand-pollinated.

Fruits were collected when ripe and seeds were extracted

and surface sterilised with a sodium hypochlorite solution

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

size and developmental stage. Size was evaluated by mea-

suring the area (mm2) and developmental stage was eval-

uated by morphological parameters. Seeds were considered

developed when they had a normal appearance, totally filled

out, and without any malformation. Seeds were considered

undeveloped when had incomplete development, not totally

filled out, wrinkled and with split outer integument (Fig. 1).

Seeds were washed, dried and uniformly distributed in

a 9-cm Petri dishes, making sure that seeds did not

touch each other. Petri dishes were scanned at 150 pp in an

Epson Perfection 4870 Photo scanner. Images were ana-

lysed with the Matrox software, which gives automatic

exact measurements.

Embryo rescue and plant recovery

In experiments to determine the influence of integuments

on germination, intact small seeds and embryos isolated

with the aid of a stereoscopic microscope from small seeds

were cultured under aseptic conditions in Petri dishes

containing the Murashige and Skoog (1962) culture media

with 50 g/L sucrose, 500 mg/L malt extract and supple-

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

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

thiamine hydrochloride, 4 mg/L glycine) and 8 g/L Bacto

agar (MS culture media). After germination, plants were

transferred to 25 9 150 mm test tubes with the same cul-

ture 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 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 with a similar leaf piece

taken from a diploid control plant. 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). Nuclei were fil-

tered through a 30-lm nylon filter and stained with a DAPI

(4,6-diamine-2-phenylindol) (High Resolution DNA Kit

Type P, solution B; Partec�) solution. Following a 5-min

incubation period, 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. Histograms were analysed using the

dpac v2.0 software (Partec�), which determines peak

position, coefficient of variation (CV) and the relative

ploidy index of the samples.

Transfer to soil

Triploid plants were transferred to pots containing steam-

sterilised artificial soil mix suitable to grow citrus (40%

black peat, 29% coconut fibre, 24% washed sand and 7%

perlite). Composition was developed in our group to grow

citrus in the greenhouse. Pots were enclosed in polyethyl-

ene bags that were closed with rubber bands and placed in a

shaded area in a temperature-controlled greenhouse set at

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

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

grown under regular greenhouse conditions (Navarro and

Juarez 2007).

Field evaluation

Triploid plants were cultured in the greenhouse for

approximately 1 year to produce quality budwood to graft

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

Fig. 1 Different types of seeds obtained in ‘Clemenules’ clementine

by ‘Pink’ pummelo hybridisation. a Developed small seeds,

b undeveloped seeds, and c developed seeds (normal seeds)

Plant Cell Rep (2010) 29:1023–1034 1025

123

rootstock (C. sinensis 9 P. trifoliata) for field evaluation at

IVIA plots. Flowering was recorded for each hybrid and

when they started fruiting, a sample of ten mature fruits

was collected and data were taken on diameter, weight,

fruit shape, rind adherence to flesh, pulp texture, flavour of

the juice and a general final evaluation. Triploid hybrids

selected during the first evaluation were later described

following the guidelines to conduct tests for distinctness,

uniformity and stability for Citrus L. Group 1 mandarins,

from the International Union for the Protection of New

Varieties of Plants (UPOV 2009).

Molecular characterisation

Thirteen triploid plants obtained by 2x 9 2x hybridisations

were selected for their high quality (Table 2). These trip-

loid plants and their parents were analysed with 13 SSR

markers (Kijas et al. 1997; Froelicher et al. 2008; Luro

et al. 2008) heterozygotic for clementine and ‘Fortune’

mandarin. The extraction of genomic DNA was done

according to Dellaporta and Hicks (1983) with slight

modifications. After a M13 tailed PCR reaction (Schuelke

2000), genetic analysis was performed in a capillary-array

sequencer CEQTM 800 System (Beckamn Coulter� Inc.,

Fullerton, CA) and results were analysed with Genome-

LabTM GeXP Genetic Analysis System software. A cluster

analysis was done with Darwin V.5.0.155 program (Perrier

et al. 2003; Perrier and Jacquemoud-Collet 2006) accord-

ing to the Weighted Neighbour-Joining method, using Dice

dissimilarity coefficient.

Results

Seed description and ploidy level of recovered plantlets

Two hundred and ninety-nine seeds from 40 fruits were

obtained from the pollination between ‘Clemenules’

clementine and ‘Pink’ pummelo. From 299 seeds, 252 were

developed (Fig. 1a, c) and 47 were undeveloped (Fig. 1b).

In developed seeds, we clearly identified two different

groups, 12 small seeds (Fig. 1a) and 240 normal seeds

(Fig. 1c).

Average area of normal seeds was 78 ± 11 mm2,

whereas area of small seeds was 34 ± 8 mm2, between 52

and 62% smaller than normal seeds. Average area of

undeveloped seeds was 56 ± 17 mm2, 18–42% smaller

than normal seeds (Table 3).

Normal seeds were germinated in a greenhouse and all

regenerated plantlets were diploid. Embryos were rescued

from the 12 small seeds, and cultured in vitro. Twelve

plantlets were obtained, of which three were diploid and

nine triploid. Only 13 of the 47 undeveloped seeds con-

tained embryos (27.7%) and only five plantlets, all diploid,

were recovered after embryo culture (Table 3). Embryos

contained in small seeds were well developed, with two

cotyledons and a well-formed embryonary axis (Fig. 2a).

This characteristic was observed systematically in all 44

hybridisations performed in the breeding program, and

triploid plants were recovered exclusively from embryos

rescued from the small developed seeds.

Recovery of citrus triploid plants

Comparison between in vitro germination of intact seeds

and embryo rescue

Small seeds obtained in fruits of open pollinated ‘Fortune’

mandarin were cultured in vitro with and without tegu-

ments. The germination percentage of intact small seeds

with teguments was 42.4% (25/59) slightly higher than the

33% obtained by Ollitrault et al. (1996) for small clem-

entine seeds, whereas germination of isolated embryos was

84% (42/50). Henceforth, in vitro embryo rescue was used

systematically in the breeding program.

Fruit set, plant regeneration, ploidy level

and transfer to soil

Results of embryo rescue, plant regeneration, ploidy level

and transfer to soil of plants recovered in 2x 9 2x

hybridisations are shown in Table 4. The data correspond

to more than 8,800 pollinations done between 1996 and

2006.

Table 2 Selected triploid hybrids obtained in 2x 9 2x hybridisations

and its parents

Female parent Male parent Triploid hybrids

‘Fortune’

mandarin

‘Murcott’ tangor ForMur 96-019

ForMur 96-023

ForMur 96-081

(Garbı’ mandarin)

ForMur 96-086

‘Ellendale’ tangor ForEll 96-023

ForEll 96-058

ForEll 96-060

Open pollination For? 95-087

For? 95-097

‘Kara’ mandarin ForKar 96-037

(‘Safor’ mandarin)

ForKar 96-073

‘Willow leaf’ mandarin ForMco 96-082

‘Willits Newcom’

clementine

Open pollination Cwn? 95-001

1026 Plant Cell Rep (2010) 29:1023–1034

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Fruit set average was 59%. Average number of small

seeds per fruit varied between 0.23 with ‘Fina’ clementine

and 3.0 with ‘Fortune’ mandarin. In total, 4,400 small

seeds were obtained and 4,110 (93%) of them contained

only one embryo per seed (Fig. 2a), whereas the other

seeds were aborted. From the 4,110 embryos cultured in

vitro, 3,816 plants were recovered. Average germination

percentage was 93% using the embryo rescue technique,

fluctuating between 83% for ‘Marisol’ clementine and 98%

for ‘Fina’ clementine (Fig. 2b, c).

The average number of plants obtained from small seeds

per fruit was 0.22 for ‘Fina’ clementine and ‘Moncada’

mandarin, whereas for ‘Fortune’ mandarin it was 2.6 plants

per fruit.

From the 3,816 plants obtained, 315 were diploid (8.3%)

and 3,497 were triploid (91.6%). Also four pentaploid

plants were obtained, two from ‘Clemenules’ clementine

and two from ‘Fortune’ mandarin female parents.

The average survival percentage in the transplant phase

was as high as 90% and the plants obtained were generally

robust and vigorous (Fig. 2e).

Factors affecting recovery efficiency of citrus

triploid hybrids

Female parent

Recovery efficiency in citrus triploid plants was calculated

as the number of triploid plants per harvested fruit. Triploid

hybrids were obtained most efficiently with ‘Fortune’

mandarin (Table 4), with an average of 2.54 triploid

hybrids per fruit. The efficiency in clementines varied

between 0.32 with ‘Hernandina’ and 0.17 with ‘Fina’.

‘Moncada’ mandarin gave a low efficiency, with 0.12

triploid hybrids per fruit. The efficiency of ‘Fortune’

mandarin was eight times higher and more than 20 times

Table 3 Type and size of seeds

obtained in ‘Clemenules’

clementine and ‘Pink’ pummelo

hybridisation and number and

ploidy level of plants recovered

Type of seeds No. of seeds Average area (mm2) Obtained plants Ploidy level

Diploid Triploid

Developed seeds 96 78 ± 11 96 96 0

Developed small seeds 12 34 ± 8 12 3 9

Undeveloped seeds 47 56 ± 17 5 5 0

Fig. 2 a Small seed of

‘Fortune’ mandarin with one

embryo. b, c Germinated

embryos of ‘Fortune’ mandarin

rescued from small seeds.

d In vitro triploid plant obtained

from a hybridisation between

‘Fortune’ mandarin and

‘Hernandina’ clementine.

e Triploid plants obtained from

2x 9 2x hybridisations

transplanted and cultivated in

the greenhouse

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higher than ‘Hernandina’ clementine and ‘Moncada’

mandarin, respectively.

Male parent and environmental conditions

The triploid production efficiency was estimated for ‘For-

tune’ mandarin and three clementine parents crossed with

four male parents over 8 years (Table 5). Among the

clementine varieties and ‘Fortune’ mandarin, differences

were observed concerning the male parents. ‘Kara’ man-

darin male parent gave the lowest number of triploid

hybrids per fruit with all female parents, whereas the

highest number of triploid hybrids per fruit was obtained

with ‘Willow leaf’ mandarin and ‘Comuna’ sweet orange

male parents for all female parents. Inter-annual variances

have been estimated for the parental combinations for

which data of at least 3 years were available. This variance

is important and masks a potential male effect for the

crosses with clementine varieties. For example, in the

hybridisation between ‘Clemenules’ clementine and ‘Kara’

mandarin, inter-annual variance was ±0.02, whereas in the

hybridisation between ‘Hernandina’ clementine and ‘Kara’

mandarin was ±0.18 triploid hybrids per fruit.

To obtain a better estimation of the environmental effect

and its interaction with the male parent, we evaluated the

number of triploid plants recovered per fruit of all the

crosses done with ‘Fortune’ mandarin over 5 years

(Fig. 3). A strong variability was observed between years

for the same parental combinations. For example, in the

cross between ‘Fortune’ mandarin and ‘Willow leaf’

mandarin, the variation in the number of triploid hybrids

per fruit varied from 1.5 in 1996 to 6.6 in 1998. Variability

Table 4 Embryo rescue, plant regeneration and ploidy level of plants obtained in 2x 9 2x hybridisations

Female

parent

No.of

male

parents

No. of

pollinated

flowers

No. of

fruits

set

No. of

small

seeds set

No. of

embryos

cultured

No. of

obtained

plants

No. of

diploid

plants

No. of

triploid

plants

No. of

triploid

plants per fruit

No. of triploid

plants in

greenhouse

Clemenules 10 1,880 1,194 384 376 353 81 270 0.20 ± 0.14 229

Fina 10 2,300 1,690 386 371 364 76 288 0.17 ± 0.08 268

Hernandina 7 1,830 940 502 444 382 78 304 0.32 ± 0.25 275

Marisol 4 350 276 112 112 93 9 84 0.30 ± 0.15 60

Fortune 10 2,247 1,000 2,986 2,777 2,598 59 2,537 2.54 ± 1.50 2,374

Moncada 3 200 116 30 30 26 12 14 0.12 ± 0.12 14

Table 5 Effect of male parent in recovery citrus triploid plants by 2x 9 2x hybridisations

Female

parent

Male

parent

No. of

pollinate

flowers

No. of

fruits

No. of small

seeds set

No. of cultured

embryos

No. of

obtained

plants

No. of

triploid

plants

No. of triploid

plants per fruit

Inter-annual

variance

Fina Comuna 150 122 28 28 28 22 0.18 –

Clemenules Comuna 200 158 56 54 48 40 0.25 –

Hernandina Comuna 400 260 144 121 107 91 0.35 ±0.20

Fortune Comuna 150 96 249 243 227 219 2.28 –

Fina Kara 200 121 10 10 10 8 0.07 –

Clemenules Kara 550 254 19 19 19 11 0.04 ±0.02

Hernandina Kara 300 174 81 64 50 35 0.20 ±0.18

Fortune Kara 200 72 169 157 149 145 2.01 ±1.02

Fina W. leaf 350 247 50 51 50 39 0.16 ±0.04

Clemenules W. leaf 100 77 23 21 19 17 0.22 –

Hernandina W. leaf 450 244 133 124 99 78 0.32 ±0.20

Fortune W. leaf 350 177 716 693 657 642 3.63 ±2.34

Fina Murcott 200 134 30 28 26 21 0.16 ±0.01

Clemenules Murcott 280 199 58 58 54 42 0.21 ±0.07

Hernandina Murcott 280 152 57 48 42 25 0.16 ±0.07

Fortune Murcott 297 126 355 326 317 312 2.48 ±0.47

Hybridisations were done in eight different years (1996, 1997, 1998, 1999, 2001, 2002, 2003 and 2006)

1028 Plant Cell Rep (2010) 29:1023–1034

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was also observed between hybridisations performed in a

same year with different male parents. For example, for the

1996 hybridisations, the number of triploid hybrids per

fruit oscillated between 1.4 and 3.3 triploids per fruit for

hybridisations with ‘Comuna’ sweet orange and ‘Ellendale’

tangor, respectively. Moreover, the rank of the different

male parents was not conserved between years which

would indicate an important interaction between the geno-

type of the male parent and the environmental conditions.

Selection of citrus triploid hybrids

From over 4,000 triploid hybrids obtained in the IVIA

triploid program based on 2x 9 2x hybridisations (Navarro

et al. 2005), 3,600 triploid hybrids have already flowered

and 1,998 have been evaluated. In this work, we have

analysed more than 730 triploid hybrids arising from

hybridisations between ‘Fortune’ mandarin as female parent

and ‘Willow leaf’ mandarin, ‘Murcott’ and ‘Ellendale’

tangors and ‘Comuna’ sweet orange as male parents as well

as hybridisations between ‘Clemenules’ and ‘Hernandina’

clementines with ‘Comuna’ sweet orange.

All the triploid hybrids displayed juvenile characteris-

tics. Triploid hybrids transplanted to pots in the greenhouse

took approximately 1 year to produce quality budwood that

could be grafted in the field. One year after field grafting,

none produced flowers, after 2 years 17% flowered, after

3 years 51% flowered and after 5 years 81% of the triploid

hybrids flowered (Fig. 4). Furthermore, clear differences were

observed between the female parents (Fig. 5). After 3 years of

grafting, more than 50% of the triploid hybrids obtained with

‘Fortune’ mandarin had already flowered, whereas only

25–30% of the triploid hybrids from clementines had done so.

This advantage of ‘Fortune’ mandarin was observed in the

following years, and after 7 years flowering percentages were,

respectively, 90 and 65% for ‘Fortune’ mandarin and clem-

entines. Moreover, the observations for the progenies obtained

from hybridisation between ‘Fortune’ mandarin and four male

parents suggest that a slight effect was exerted by the male

parent on the length of the juvenile phase. ‘Willow leaf’

mandarin seems to promote earlier flowering, while ‘Murcott’

tangor progenies appear to flower the latest.

From the 1,998 triploid hybrids evaluated for fruit

quality, 99 were pre-selected and 13 (Table 2; Fig. 6) were

finally selected for their high quality and excellent flavour

and breeder’s rights have been protected.

Genetic analysis

Genetic analysis with the SSR markers differentiated all

protected triploid hybrids and their parents, and confirmed

the origin of all hybrids analysed (Fig. 7). Triploid hybrids

Fig. 3 Triploid hybrids

recovered by fruit in 5 years

with ‘Fortune’ mandarin as

female parent and ten different

male parents

Fig. 4 a Triploid hybrid of

‘Fortune’ mandarin 9 ‘Willow

leaf’ mandarin that flowered

after 4 years grafted in the field.

b Triploid hybrid of ‘Fortune’

mandarin 9 ‘Willow leaf’

mandarin that not produced

flowers and c Control ‘Fortune’

mandarin at the same flowering

time

Plant Cell Rep (2010) 29:1023–1034 1029

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obtained with ‘Fortune’ mandarin as female parent and

‘Murcott’ and ‘Ellendale’ tangors and ‘Kara’ and ‘Willow

leaf’ mandarins as male parents clustered with the male

parent, which indicates the importance of inter-parental

diversity in order to differentiate triploid hybrids corre-

sponding to different families.

The genetic distances between triploid hybrids of ‘For-

tune’ mandarin with ‘Murcott’ and ‘Ellendale’ tangors and

‘Kara’ mandarin were minor respect ‘Fortune’ mandarin

than male parents. This characteristic indicates that genetic,

and probably phenotypic, distances between triploid

hybrids are shorter with the female parent, producing

diploid gametes, than with the male parent, producing

haploid gametes.

Discussion

Origin of triploid progenies

In this work, in which we performed in vitro culture of

more than 4,100 embryos rescued from small but well

developed seeds obtained in 44 different hybridisations, we

have demonstrated that triploid embryos are mostly found

in seeds between 52 and 62% smaller than normal seeds.

This finding is in agreement with the observation of Esen

and Soost (1971a), who found that triploid embryos were

only contained in seeds between 1/3 and 1/6 smaller than

normal seeds, in hybridisations between three female par-

ents pollinated with one male parent. Histological studies

done by Esen and Soost (1971a, 1973) demonstrated that

small seeds contained triploid embryos with pentaploid

endosperms, indicating that triploid embryos originate

from unreduced megagametophytes. In addition, these

authors proposed that the three to five ratio between the

ploidy level of embryos and endosperm was responsible for

seed size reduction, since pentaploid endosperms grow

more slowly and stop developing prematurely. Luro et al.

(2000) proposed from the analysis of maternal heterozy-

gosity restitution that Second Division Restitution (SDR)

should be the mechanism responsible for unreduced

gametes in clementine, whereas First Division Restitution

was proposed for sweet orange by Chen et al. (2008).

Formation of unreduced gametes is a common phenome-

non in species of Lilium (Zhou et al. 2008), Solanum

(Peloquin et al. 2008) and Vaccinium (Lyerene et al. 2003).

Recovery of citrus triploid plants: methodological

considerations

Comparison between in vitro germination of intact seeds

and embryo rescue clearly demonstrates that embryo res-

cue is an essential technique for triploid hybrid recovery.

Embryo rescue is a difficult technique since it requires

isolation of intact undamaged embryos under aseptic con-

ditions and requires the aid of dissecting microscope and

tools, in order to obtain high rates of germination. A well-

established routine embryo rescue protocol is indispensable

to develop extensive breeding programs in citrus triploid

hybrids from 2x 9 2x hybridisations. We have cultivated

over 4,100 embryos, obtained from 24 different non-apo-

mictic citrus genotypes, with a germination percentage

over 90%, demonstrating the high efficiency of our routine

protocol.

Fig. 5 Bloom evolution of triploid hybrids obtained by 2x 9 2x

hybridisations

Fig. 6 Two triploid hybrids

pre-selected with their parents

obtained in 2x 9 2x

hybridisations. a ‘Garbı’

mandarin. Triploid hybrid

obtained from a cross between

‘Fortune’ mandarin and

‘Murcott’ tangor. b ForMco

96-082. Triploid hybrid

obtained from a cross between

‘Fortune’ mandarin and

‘Willow leaf’ mandarin

1030 Plant Cell Rep (2010) 29:1023–1034

123

Obtained plants had diploid (8.3%), triploid (91.6%) and

very occasionally pentaploid ploidy levels (0.1%). Oiyama

and Kobayashi (1991) also obtained some pentaploid plants

from small seeds in 2x 9 2x hybridisations and indicated that

they might have arisen from the union of double unreduced

female gametes with normal reduced male gametes.

Survival percentage of transplanted triploid plants pro-

duced in 2x 9 2x hybridisations was higher than 90%

following our methodology. It is essential to master this

acclimatisation phase for the implementation of large

triploid breeding programs.

Recovery of citrus triploid hybrids; biological

and environmental considerations

The results obtained in this work clearly demonstrate that

the main factor determining the frequency of recovery

citrus triploid hybrids is the genotype of the female parent.

In this context, ‘Fortune’ mandarin displayed higher fre-

quency of triploid hybrids per fruit than clementines and

‘Moncada’ mandarin. It confirms the results of Geraci et al.

(1975) who observed average values of triploid hybrids per

fruit between six and zero from nine non-apomictic citrus

genotypes pollinated with the same male parent. Frequency

of unreduced gametes is an intrinsic characteristic of each

genotype. In peach cultivars, frequency can range from 0 to

50% (Dermen 1938) and in potato, it is under genetic

control (Mok and Peloquin 1975). The frequency of unre-

duced gametes can be quantified by screening genotypes

before starting large-scale breeding programs with 2x 9 2x

hybridisations. At a practical level, this characteristic

implies that a different number of pollinations must be

performed for each genotype to recover a progeny of a

given number. For example, to obtain 100 triploid hybrids

in the greenhouse, more than 650 pollinations would have

to be performed with ‘Hernandina’ clementine, whereas

Fig. 7 Dendogram obtained by

Dice distance and tree

constructed by Weighted

Neighbour-Joining method

using 13 SSR markers

corresponding to registered

triploid plants obtained by

2x 9 2x hybridisations and

their parents

Plant Cell Rep (2010) 29:1023–1034 1031

123

with ‘Fortune’ mandarin the pollination of just 96 flowers

would be enough.

The use of in vitro embryo rescue and ploidy level

assessment by flow cytometry gave highly efficient

recovery of citrus triploid hybrids from small seeds and

enabled low unreduced gamete frequencies, such as ‘Fina’

clementine and ‘Moncada’ mandarin (0.17 and 0.12 trip-

loid hybrids per fruit, respectively), to be exploited for the

development of triploid breeding programs.

Geraci et al. (1975) observed that the production of

diploid female gametes was significantly affected by the

male parent. However, our observations over 10 years of

hybridisation suggest an important interaction between

male parent and environmental conditions on triploid pro-

duction. Luro et al. (2004) also showed that environmental

conditions dramatically affect the frequency of recovered

triploids. It could be hypothesised that for the same number

of 2x gametophytes potentially available, the production of

triploids will be influenced by the ability of the pollen to

perform successful fecundation. This ability is dependent

on (1) the quality of pollen determined by the male geno-

type, but also environmental conditions; (2) the compati-

bility level between female and male parents; and (3)

environmental effects on pollen tube germination. Studies

in a few species have shown that environmental conditions

affect pollen development (Young and Stanton 1990;

Johannsson and Stephenson 1998), as well as pollen tube

growth (Stephenson et al. 1992). One of the most important

environmental factors that could affect pollen performance

is the temperature during the pregamic phase. Indeed,

temperature has been shown to affect pollen germination

(Elgersma et al. 1989; Shivanna et al. 1991), and pollen

tube kinetics in the style (Lewis 1942; Jefferies et al. 1982;

Elgersma et al. 1989). Bono et al. (2006) demonstrated

that, within the mandarin group, there are great differences

in the number of seeds per fruit depending on the parents

used for the crosses. For example, the average number of

seeds per fruit in ‘Clemenules’ clementine was around two

when pollinated with ‘Ortanique’ tangor, and 30 with

‘Moncada’ mandarin. These observations could be related

with the gametophytic incompatibility system identified in

citrus (Soost 1965, 1969).

Juvenile characteristics of triploid progenies

Juvenile traits in citrus plants originating from seeds are

very prominent and often persist for a long time. They

include thorniness, vigorous growth, alternate bearing in

early years, physical differences in fruit characters and

slowness to flower and to bear fruit (Cameron and Frost

1968). The long juvenile period of citrus trees obtained

from seeds is the major impediment for citrus breeding

programs based on sexual hybridisations.

In this work, we demonstrate for the first time the effect

of the female and male parents on the length of the juvenile

period in progenies. This characteristic is of great interest,

since the triploid hybrids can be evaluated more quickly,

allowing extensive triploid breeding programs based on the

2x 9 2x strategy, since it determines the number of years

required to produce new varieties. In this context, it is very

important to evaluate the juvenile period, as well as the

frequency of unreduced gametes of the genotypes included

in Germplasm Banks. This information will allow for a

much better selection of parents for triploid breeding

programs.

Selection of citrus triploid hybrids

We have protected breeder’s rights in Spain for 13 new

triploid varieties for their high fruit quality. Two of them,

‘Garbı’ mandarin (Aleza et al. 2010) and ‘Safor’ mandarin

(Cuenca et al. 2010), have also been protected in the

European Union (EU), Morocco, Egypt, South Africa and

Turkey; moreover, a US Plant Patent has been requested.

Pathogen-free plants of ‘Garbı’ and ‘Safor’ mandarins have

been obtained by shoot-tip grafting in vitro, according to

the methodology described by Navarro et al. (1975) and

healthy budwood has been released to 22 Spanish nurser-

ies, which have signed propagation agreements and are

already producing certified plants. These are the first

varieties recovered in genetic breeding programs from the

2x 9 2x controlled hybridisation strategy.

Complete differentiation between the 13 new triploid

varieties was obtained using 13 SSR markers. The effi-

ciency of such markers for hybrid identification in sexual

diploid progeny had been demonstrated previously.

Molecular identification should contribute to defending

plant breeders’ rights, preventing possible fraud, and pro-

vides traceability from the field to sale points. In addition,

it is a very useful tool in certification programs as illegal

propagations can be detected and possible mistakes or

confusions avoided in nursery identification.

Nowadays, SSR markers are being used as a legally

recognised technique in the defence of breeders’ rights.

Furthermore, our group has performed more in-depth and

detailed genetic studies that can analyse the genetic and

phenotypic structures of the triploid hybrids obtained from

2x 9 2x, 2x 9 4x and 4x 9 2x hybridisations.

Conclusions

This work explores the different factors influencing the

recovery of citrus triploid hybrids from 2x 9 2x hybridi-

sations. Triploid hybrids are found in developed seeds that

are between 52 and 62% smaller than normal seeds.

1032 Plant Cell Rep (2010) 29:1023–1034

123

Embryo rescue and flow cytometry are two indispensable

techniques for extensive citrus triploid breeding programs

based on 2x 9 2x hybridisation. The frequency of unre-

duced megagametophyte production depends on the

maternal genotype, and should be known before starting

extensive breeding programs. ‘Fortune’ mandarin displays

a higher frequency of this phenomenon than clementines

and ‘Moncada’ mandarin. An unclear male parent effect

has been observed in triploid production hybrids, as well

as effects of environmental conditions. In this work, we

have demonstrated for the first time the effect of the

female and male parents on the length of the juvenile

period of the progenies. At IVIA, an extensive breeding

program of citrus triploid hybrids based on such hybrid-

isation has been running since 1995. To date, we have

obtained over 4,000 hybrids and 13 new triploid varieties

have been protected; furthermore, triploid ‘Garbı’ and

‘Safor’ mandarins, obtained in this program, are being

planted extensively by citrus growers to establish new

commercial plots.

Acknowledgments This work is jointly financed by the AGL2008-

00596-MCI and Prometeo 2008/121 Generalidad Valenciana projects.

We thank C. Ortega, A. Navarro, V. Ortega, C. Martı, J.M. Arregui

and M. Hernandez for their technical assistance in the laboratory and

J.A Pina, V. Lloris, J.M. Conchilla, F. Ahuir, D Conchilla, A. Con-

chilla, R. Lopez and F.J. Martı for growing plants in the greenhouse

and in the field.

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