533.full

Polyembryony in non-apomictic citrus genotypes

Pablo Aleza1, Jose Juarez1, Patrick Ollitrault1,2 and Luis Navarro1,*1Centro de Proteccion Vegetal y Biotecnologıa, Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra. Moncada-

Naquera km 4.5, 46113 Moncada, Valencia, Spain and 2Unite de Recherche Multiplication Vegetative, Centre de CooperationInternationale en Recherche Agronomique pour le Developpement (CIRAD), Montpellier 34398, France

* For correspondence. E-mail [email protected]

Received: 12 February 2010 Returned for revision: 10 May 2010 Accepted: 17 June 2010 Published electronically: 30 July 2010

† Background and Aims Adventitious embryony from nucellar cells is the mechanism leading to apomixis inCitrus sp. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a conse-quence of 2x × 4x hybridizations and in vitro culture of isolated nucelli. The origin of the plants arising from theaforementioned processes remains unclear.† Methods The genetic structure (ploidy and allelic constitution with microsatellite markers) of plants obtainedfrom polyembryonic seeds arising from 2x × 4x sexual hybridizations and those regenerated from nucellusculture in vitro was systematically analysed in different non-apomictic citrus genotypes. Histological studieswere also conducted to try to identify the initiation process underlying polyembryony.† Key Results All plants obtained from the same undeveloped seed in 2x × 4x hybridizations resulted from clea-vage of the original zygotic embryo. Also, the plants obtained from in vitro nucellus culture were recovered bysomatic embryogenesis from cells that shared the same genotype as the zygotic embryos of the same seed.† Conclusions It appears that in non-apomictic citrus genotypes, proembryos or embryogenic cells are formed bycleavage of the zygotic embryos and that the development of these adventitious embryos, normally hampered,can take place in vivo or in vitro as a result of two different mechanisms that prevent the dominance of theinitial zygotic embryo.

Key words: Citrus, polyembryony, apomixis, embryo cleavage, nucellus, interploid hybridization.

INTRODUCTION

The polyembryony phenomenon was discovered byLeeuwenhoek in 1719, who observed the formation of twoplantlets from the same citrus seed (Batygina andVinogradova, 2007). Later, Strasburger (1878) reported theformation of adventive embryos from citrus nucellar cellsand called this phenomenon nucellar or adventive polyembry-ony. The main classification criteria for polyembryony includethe origin of the initial embryogenic cells, the mode of embryoformation and genetic traits. Based on the cellular origin ofembryogenesis, polyembryony can be divided into two maintypes: gametophytic and sporophytic. Gametophytic polyem-bryony includes apogamety and apospory, whereas sporophy-tic polyembryony encompasses nucellar, integumental,monozygotic cleavage and endospermal polyembryony(Batygina and Vinogradova, 2007). With the exception ofthe latter two, these different forms of polyembryony areassociated with apomixis, defined as asexual reproductionthrough seeds (Savidan, 2000). In citrus, apomictic and non-apomictic genotypes are found in the diploid germplasm(Frost and Soost, 1968). There are three basic taxa fromwhich all cultivated citrus have originated, namelypummelos [Citrus maxima (L.) Osb.], citrons (C. medica L.)and mandarins (C. reticulata Blanco) (Nicolosi et al., 2000).The most widely grown species, like sweet oranges[C. sinensis (L.) Osb.], lemons [C. limon (L.) Burm f.], grape-fruits (C. paradisi Macf.), limes [C. aurantifolia (Christm.)

Swing.], clementines (C. clementina Hort. ex Tan.) and satsu-mas [C. unshiu (Mak.) Marc.], originated from crosses of thetree basic taxa (Nicolosi et al., 2000). Most citrus genotypesare apomictic, with the exception of all citron, pummelo andclementine cultivars and some mandarin hybrids. Sporophyticadventitious embryony is the mechanism leading to facultativeapomixis in citrus. During apomictic citrus seed formation,embryos are initiated directly from the maternal nucellar cellssurrounding the embryo sac containing a developing zygoticembryo (Kobayashi et al., 1981). In vivo, full development ofthe nucellar embryos is endosperm-dependent, and thus priorfertilization is required (Esen and Soost, 1977). Duringembryo sac expansion, nucellar embryos develop alongsidethe zygotic embryo, which may or may not develop fully. Themajority of seedlings arising from polyembryonic seeds corre-spond to the maternal genotype, and are called nucellar seed-lings. However, the frequency with which nucellar seedlingsarise can vary depending on the genotype and environmentalconditions (Khan and Roose, 1988). Zygotic seedlings can bediscriminated from their nucellar counterparts based on isoen-zymes (Iglesias et al., 1974), random amplification of poly-morphic DNA (Luro et al., 1995) and simple sequence repeat(SSR) markers (Ruiz et al., 2000). Also, a major gene linkedto apomixis has recently been identified by Kepiro and Roose(2010), by using amplified fragment length polymorphismmarkers.

Non-apomictic citrus genotypes usually have only onesexual embryo per seed, although occasionally extra embryos

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can arise either from the fission of one fertilized egg or fromtwo or more functional embryo sacs in a single ovule (Frost,1926; Bacchi, 1943; Cameron and Garber, 1968; Frost andSoost, 1968). Frost (1938) described monozygotic polyembry-ony by cleavage of the sexual embryo in hybrids with identicalphenotype. Subsequently, Bacchi (1943) histologicallydescribed two gametophytes in the same ovule of ‘Foster’grapefruit. More recently, on analysing 633 hybrids of citrusrootstocks, Medina-Filho et al. (1993) identified ten monozy-gotic twin hybrids and two dizygotic pairs by isozyme analy-sis. Traditionally, apomictic genotypes are referred to aspolyembryonic and non-apomictic genotypes as monoembryo-nic, although these terms may cause confusion when attempt-ing to distinguish nucellar embryony from sexual twinning(Kepiro and Roose, 2007).

Consistent formation of multiple embryos in seeds of non-apomictic citrus genotypes has been observed in twosystems. In the first case, Oiyama and Kobayashi (1990)reported multiple embryos in seeds of non-apomictic clemen-tines and ‘Iyokan’ mandarin (C. iyo Hort. ex Tan.) pollinatedwith tetraploid ‘Kawano Natsudaidai’ (C. natsudaidaiHayata). In such crosses, different types of seeds are obtainedand multiple small embryos are observed exclusively in par-tially developed seeds containing triploid embryos.Similarly, we have made the same observations in many2x × 4x crosses in our triploid breeding programme;however, the causes underlying this type of polyembryonyremain unknown. Oiyama and Kobayashi (1990) analysedone isoenzymatic system and concluded that all zygoticembryos from the same seed arose from cleavage of the orig-inal zygotic embryo. However, due to the very limited scale ofanalysis, this conclusion requires confirmation, as the possi-bility that polyembryony originates from two or more func-tional embryo sacs in a single ovule (Bacchi, 1943) cannotbe discarded.

The second case concerns embryo production by nucelli iso-lated from developing seeds and cultured in vitro after thezygotic embryo has been discarded. This technique was devel-oped with a view to recovering pathogen-free nucellar plantsof non-apomictic genotypes (Rangan et al., 1968; Esan,1973). Indeed, in vivo nucellar embryony of apomictic geno-types was widely used in the past to obtain virus-free plants,as most pathogens are not transmitted through embryogenesis(Weathers and Calavan, 1959; Roistacher, 1979). Navarroet al. (1985) also used this procedure to recover pathogen-freeplants of several clementine varieties. However, long-termfield evaluation of regenerated plants showed that their pheno-type differed from the original seed source plants. In the afore-mentioned study (Navarro et al., 1985), phenotypic differenceswith the original plants were found in the greenhouse andwhen several plants were recovered from the same nucellusall were alike. Isoenzymatic analysis of plants produced byeach nucellus indicated that plants regenerated from thesame nucellus were identical, but one nucellus differed fromanother and from the maternal plant (Navarro et al., 1985).Although the embryos produced by nucellus cultured in vitroare thought to originate from the nucellus, their true originremains unclear.

The present study aims to answer the following questions.(1) Does pollination with a tetraploid parent systematically

induce the formation of polyembryonic seeds in the non-apomictic citrus genotypes? (2) What is the origin of in vivopolyembryony in 2x × 4x crosses and in vitro embryogenesisfrom isolated nucellus of non-apomictic citrus genotypes?(3) Might these two phenomena have the same biologicalorigin?

To do so, we systematically analysed the genetic structure(ploidy and microsatellite allelic constitution) of plantsobtained from polyembryonic seeds arising from 2x × 4xsexual hybridizations or regenerated from nucellus culture invitro of different non-apomictic citrus genotypes.Histological studies were also conducted to try to identifythe initiation process of polyembryony.

MATERIALS AND METHODS

Hybridizations 2x × 4x

Plant material. The non-apomictic clementine genotypes‘Bruno’, ‘Fina’, ‘Hernandina’, ‘Clemenules’ and ‘Tomatera’were used as female parents. Diploid ‘Nova’ mandarin[C. clementina × (C. paradisi × C. tangerina)] and tetraploid‘Nova’ mandarin, ‘Orlando’ tangelo (C. paradisi ×C. tangerina) and ‘Pineapple’ sweet orange were used asmale parents. The tetraploid parents were previously selectedfrom seedlings of diploid cultivars, which are doubleddiploid arising from chromosome doubling of the maternalgenotype, frequent in citrus (Ollitrault et al., 2008). All geno-types belong to the Citrus Germplasm Bank of the InstitutoValenciano de Investigaciones Agrarias (IVIA).

Pollination and embryo rescue. Between 50 and 150 flowers ofclementines were hand-pollinated with each male parent.Fruits were collected at maturity and seeds were extractedand surface sterilized with a sodium hipochloride solution(0.5 % active chlorine) for 10 min and washed with sterilewater.

Embryos were isolated from undeveloped seeds under asepticconditions with the aid of a stereoscopic microscope and culti-vated on Petri dishes containing the Murashige and Skoog(1962) culture medium with 50 g L21 sucrose, 500 g L21 maltextract supplemented with vitamins (100 mg L21 myo-inositol,1 mg L21 pyridoxine hydrochloride, 1 mg L21 nicotinic acid,0.2 mg L21 thiamine hydrochloride, 4 mg L21 glycine) and8 g L21 Bacto agar (MS culture medium). After germinationplants were transferred to 25 × 150-mm test tubes with MSculture medium without malt extract. Cultures were main-tained at 24+1 8C, 60 % humidity and 16 h daily exposure to40 mE m22 s21 illumination.


Plant material. ‘Clemenules’ clementine and ‘Fortune’ man-darin (C. clementina × C. tangerina) non-apomictic femaleparents and Poncirus trifoliata (L.) Raf. ‘Benecke’ maleparent from the IVIA Citrus Germplasm Bank were used.Intergeneric hybridizations of citrus with P. trifoliata do notinduce problems related to compatibility, seed-set, fruit-setor plant production. Additionally, trifoliate leaves ofP. trifoliata are a dominant trait and constitute a strong mor-phological marker determining the hybrid origin of progenies.

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Pollination and nucellus culture in vitro. One hundred flowers of‘Clemenules’ clementine and 100 flowers of ‘Fortune’ man-darin were hand pollinated with pollen of P. trifoliata‘Benecke’. Fruits were collected 13–15 weeks after pollina-tion and the nucellus of the immature seeds were excised asep-tically by removing both integuments and the zygotic embryoat heart-shaped to early cotyledonary stage of development(Fig. 1). Only nucelli from seeds with clearly visible zygoticembryos were cultured. Nucelli were cultivated in MSculture medium and one nucellus was planted in each 25 ×150-mm culture tube with the chalazal end embedded in themedium. Cultures were kept at 24+ 1 8C, 60 % humidityand 16 h daily exposure to 40 mE m22 s21 illumination.Zygotic embryos extracted from nucelli were also cultivatedseparately in 25 × 150-mm culture tubes containing the MSculture medium and kept under the same conditions.

Transfer to soil

Recovered plants were transferred to pots containing steam-sterilized artificial soil mix suitable for citrus growing (40 %black peat, 29 % coconut fibre, 24 % washed sand and 7 %perlite). Pots were enclosed in polyethylene bags, closedwith rubber bands and placed in a shaded area of atemperature-controlled greenhouse set at 18–25 8C. After 8–10 d, the bags were opened, then 8–10 d later the bags wereremoved and the plants grown under regular greenhouse con-ditions (Navarro and Juarez, 2007).

Ploidy-level analysis

Ploidy level was determined by flow cytometry according tothe methodology described by Aleza et al. (2009). Eachsample comprised a small leaf-piece of the analysed plant(approx. 0.5 mm2) with a similar leaf-piece from a diploidcontrol plant. Samples were chopped using a razor blade in anuclei isolation solution (High Resolution DNA Kit Type P,solution A; Partec, Munster, Germany). Nuclei were filteredthrough a 30-mm nylon filter and stained with a DAPI(4′,6-diamine-2-phenylindol) (High Resolution DNA KitType P, solution B; Partec) solution. After 5 min incubation,stained samples were run in a Ploidy Analyzer (PA) (Partec)flow cytometer equipped with an 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 vari-ation (CV) and the relative ploidy index of the samples.

Genetic analysis

Genetic analysis was carried out using 13 SSR markers(Kijas et al., 1997; Froelicher et al., 2008; Luro et al.,2008). Genomic DNA was extracted according to Dellaportaand Hicks (1983), with slight modifications. PCR amplifica-tions of the samples were performed using a Termocycler epgradient S (Eppendorf) in a final volume of 17 mL containing0.8 U Taq DNA polymerase (Need), 30 ng of citrus DNA,1.25 mM of each dNTP, 5 mM MgCl2, 750 mM Tris-HCl (pH9), 200 mM (NH4)2SO4, 0.001 % (v/v) bovine serumalbumin, and 5 mM reverse and forward primers. The followingPCR programme was applied: denaturation at 94 8C for 5 min;followed by 35 repeats 30 s at 94 8C, 1 min at 50 or 55 8C, and45 s at 72 8C; and a final elongation step of 4 min at 72 8C.PCR products were separated by means of vertical denatura-lized electrophoresis (acrylamide-bis acrylamide 6 %, urea7 M) buffer TBE 0.5× (Tris, acid boric and EDTA 0.5 M,pH 8) in a BioRad DCode, according to the methodologydescribed by Froelicher et al. (2007). The amplified fragmentswere detected by silver staining (Benbouzas et al., 2006).

Parental allelic structure was analysed to select a set ofprimers for each parental combination to discriminatebetween nucellar and zygotic origin, as well as between inde-pendent zygotic origins. Results were processed using clusteranalyses based on the Dice dissimilarity coefficient by theWeighted Neighbour Joining method. All these calculationswere performed with Darwin V.5.0.155 software (Perrieret al., 2003; Perrier and Jacquemoud-Collet, 2006).

Histological characterization

Ovules and seeds from fruits obtained from ‘Clemenules’clementine pollinated with P. trifoliata ‘Benecke’ were col-lected weekly after pollination and fixed in FAA (formal-dehyde, glacial acetic acid and alcohol 50 %). Samples wereembedded in paraffin, cut into 10-mm sections and stainedwith PAS (Periodic acid-Schiff reaction) according to thegeneral methodology described by Hotchkiss (1948).Observations were recorded with an E800 Eclipse Nikonmicroscope.

RESULTS

Identification of molecular markers for unambiguous geneticcharacterization

SSR markers were selected to discriminate between parentalsand to estimate the probability of identity between two inde-pendent zygotic plants and between zygotic and nucellarplants obtained in 2x × 4x and 2x × 2x hybridizations. Fromthe 13 SSR markers analysed, eight indicated important poly-morphism among the parents of the 2x × 4x progenies. Withall the SSR markers used, identical profiles were obtainedfor the different clementine genotypes and consequently,

A B C D

FI G. 1. (A) Developmental stage of a seed used from nucellus culture in vitroextracted 13–15 weeks after pollination. (B) Seed with both integuments par-tially eliminated with the zygotic embryo inside. (C,D) Nucellus without inte-

guments and zygotic embryo extracted at early cotyledonary stage.

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hereafter they are all referred to under the generic name ofclementines.

The markers used are known to be monolocus; therefore, thegenotypes of the diploid plants could be inferred directly fromtheir profiles. Clementines were heterozygous for all themarkers and shared two, one or no alleles with the maleparent according to the markers and the parental combination.The SSR analysis was carried out using polyacrylamide gelelectrophoresis (PAGE) and silver staining. We preferred notto estimate allelic doses in the triploid genotypes. Thus, to cal-culate the probability of identity between two progenies forone locus, we considered that the genotypes ab, aab and abb(where a and b are two alleles at the considered locus) wereindistinguishable. The theoretical probability of identitybetween two independent zygotic plants and between zygoticand nucellar plants for one locus has been estimated for thedifferent allelic combinations without distinction of allelicdose (Table 1, and Supplementary Data, available online).Given that the analysed markers are genetically independent,the probability of observing a zygotic seedling identical tothe nucellar ones was calculated by multiplying the corre-sponding probability for each marker (Table 2). These theor-etical probabilities fluctuated between 0, in the hybridisationof clementine × tetraploid ‘Orlando’ tangelo, and 4 × 1026

in the clementine × tetraploid ‘Nova’ mandarin (Table 2).

The probabilities of identity for two independent zygoticplants were calculated in the same way and oscillatedbetween 2 × 1023 in clementine × tetraploid ‘Orlando’tangelo and 1 × 1023 in the other two hybridizations.

From the 13 SSR markers analysed, seven were used forgenetic analysis of plants recovered via in vitro nucellusculture from intergeneric hybridizations between ‘Clemenules’clementine and ‘Fortune’ mandarin by P. trifoliata ‘Benecke’.These markers clearly differentiated parents with specificalleles of P. trifoliata ‘Benecke’ male parent. The likelihoodof identity between nucellar and zygotic plants was thereforenil. The probability of identity between two independentzygotes was 5 × 1023 for clementine and ‘Fortune’mandarin × P. trifoliata ‘Benecke’, calculated as describedabove.


Fruit- and seed-set, embryo rescue and ploidy level of recoveredplants. Different 2x × 4x hybridizations were performedto study the formation of polyembryonic seeds in 2x × 4xhybridizations in the non-apomictic citrus genotypes(Table 3). Developed and undeveloped seeds were obtainedin all hybridizations (Fig. 2A, B). The number of developedseeds varied between 0.2 and 2.1 per fruit (Table 3).Meanwhile, the number of undeveloped seeds was muchhigher (90 % of total seeds) than developed seeds in allhybridizations, varying between 3.4 and 10.8 per fruit.

In all hybridizations, undeveloped seeds had either one (mono-embryonic) or multiple embryos (polyembryonic) (Fig. 2C, D).Embryos were at globular to early cotyledonarystage and variablein size (macroscopically some embryos were so small that theywere difficult to identify, whereas others reached a size ofapprox. 3 mm). In polyembryonic seeds, embryos were highlycompacted and sometimes surrounded by endosperm traces,which made it practically impossible to individualize and countthem accurately, without damaging them. Consequently, allembryos of a single seed were cultivated together (Table 4,Fig. 2E, F). The smallest proportion (20 %) of polyembryonicseeds corresponded to the pollination between ‘Clemenules’clementine and tetraploid ‘Nova’ mandarin, whereas the

TABLE 1. Theoretical probability of identity between two zygoticgenotypes and between zygotic and nucellar plants obtained in

2x × 4x hybridizations in terms of parental SSR profiles

Female parent Male parent PIZ* PINZ†

ab aaaa / bbbb ½ ½aabb 51/72 5/6aacc / bbcc ¼ 1/12ccdd ¼ 0cccc / dddd ½ 0

* PIZ, probability of identity between two independent zygotic genotypes(without allelic dosis estimation).

† PINZ, probability of identity between nucellar and zygotic plants(without allelic dosis estimation).

TABLE 2. SSR marker selection and probability of identity between two independent zygotic plants and between zygotic and nucellargenotypes obtained in 2x × 4x hybridizations

Parents SSR loci used for each 2x ×4x hybridization

SSR locus Clementine Nova 4x Orlando 4x Pineapple 4x Clementine × Nova 4x Clementine × Orlando 4x Clementine × Pineapple 4x

Ci01C06 ab aacc bbcc bbdd Not used Used UsedCi01C07 ab aacc aacc aadd Used Used Not usedCi02B07 ab bbcc ccdd aadd Used Used UsedCi05A05 ab aacc aacc aacc Used Used UsedCi07C07 ab aaaa cccc bbbb Not used Used UsedCAC 15 ab aabb aacc aaaa Not used Not used UsedTAA 15 ab bbcc bbcc bbdd Used Not used Not usedTAA 41 ab bbcc aadd aadd Used Not used UsedProbability of identity between zygotic and nucellar genotypes 4 × 1026 0 1 × 1025

Probability of identity between two independent zygotic genotypes 1 × 1023 2 × 1023 1 × 1023




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TABLE 3. Fruit-set, number and type of seeds obtained in 2x × 4x hybridizations

Diploid female parent Tetraploid male parent No. of pollinated flowers No. of fruits set No. of developed seeds Number of undeveloped seeds

Fina Nova 50 39 7 242Clemenules Nova 25 13 5 124Hernandina Nova 80 38 15 218Bruno Pineapple 100 65 47 282Tomatera Pineapple 150 60 14 204Fina Orlando 50 46 24 499Clemenules Orlando 100 75 159 742

TABLE 4. In vitro culture of embryos obtained in undeveloped seeds produced in 2x × 4x hybridizations

Diploid femaleparent

Tetraploidmale parent

No. ofmonoembryonic

seeds

No. ofpolyembryonic

seeds

No. ofgerminated

embryosNo. of germinated embryos

per polyembryonic seedNo. of triploid plants per

polyembryonic seed

Fina Nova 95 33 162 2.0 1.3Clemenules Nova 71 18 105 1.9 1.5Hernandina Nova 47 13 74 2.1 1.5Bruno Pineapple 46 24 92 1.9 1.5Tomatera Pineapple 84 78 276 2.5 1.9Fina Orlando 110 70 278 2.4 0.8Clemenules Orlando 221 199 632 2.1 1.0

A

B

E

C

D

F

FI G. 2. (A) Developed seeds obtained in 2x × 4x hybridizations. (B) Undeveloped seeds obtained in 2x × 4x hybridizations. (C) Multiple embryos contained inundeveloped seeds. (D) Monoembryonic undeveloped seed. (E,F) Germination of multiple embryos from undeveloped seeds.




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hybridization between ‘Tomatera’ clementine and tetraploid‘Pineapple’ sweet orange gave the highest proportion of polyem-bryonic seeds (48 %).

The average number of embryos that germinated from eachpolyembryonic seed ranged from 1.9 to 2.5 (Table 4). Thehighest number of regenerated plants per polyembryonicseed was 1.9 in the hybridization between ‘Tomatera’ clemen-tine and tetraploid ‘Pineapple’ sweet orange (Table 4). All theregenerated plants were triploid.

Additionally, ‘Fina’ and ‘Clemenules’ clementines werepollinated with diploid ‘Nova’ mandarin to confirm thepremise that the male parent was not responsible for producingmultiple embryos in 2x × 4x hybridizations. Citrus genotypesare known to occasionally produce unreduced megagameto-phytes, giving rise to triploid embryos in 2x × 2x hybridiz-ations (Cameron and Frost, 1968). In such hybridizations,the size of seeds with triploid embryos was reduced to suchan extent that they could be visually distinguished fromnormal seeds (Fig. 3). At maturity they were fully developedand one-third to one-sixth of the size of normal seeds (Esenand Soost, 1971a). All the small seeds obtained containedonly one embryo and regenerated only one plant per embryoand the ploidy level of all plants was triploid (Table 5).

Genetic analysis. To determine the genetic origin of triploidplants recovered from germination of multiple embryos con-tained in the same undeveloped seed from 2x × 4x hybridiz-ations, 46 triploid plants corresponding to 13 different seedsand their parents were analysed with eight heterozygoticSSR markers for clementine (Table 2). These included the fol-lowing subsets of triploids: 16 triploid plants originating fromfive different undeveloped seeds obtained from hybridizationsbetween clementines and tetraploid ‘Nova’ mandarin; 13 tri-ploid plants originating from four different undevelopedseeds from hybridizations between clementines and tetraploid‘Orlando’ tangelo; and 17 triploid plants obtained from fourdifferent undeveloped seeds produced by hybridizationsbetween clementines and tetraploid ‘Pineapple’ sweet orange.

The same profile was revealed for all triploid plants obtainedfrom the same seed; moreover, it was possible to differentiate allplants regenerated from different seeds and hybridizations(Fig. 4). As an example, Fig. 5 shows the profiles of triploidplants recovered from hybridization between clementines andtetraploid ‘Orlando’ tangelo with the Ci02B07 SSR marker.Clementines displayed two alleles of 162 and 164 nt whereastetraploid ‘Orlando’ tangelo presented two alleles of 160 and170 nt. Plants 3, 4 and 5 (corresponding to the same seed)and 14 and 15 (plants from the same seed) showed the sameprofiles, which comprised two alleles of 160 and 170 ntcoming from tetraploid ‘Orlando’ tangelo and one allele of162 nt corresponding to clementine. Plants 6–13 (correspond-ing to two different seeds) displayed the same profile, compris-ing one allele of 160 nt coming from tetraploid ‘Orlando’tangelo and one allele of 164 nt from clementine.


Fruit- and seed-set, nucellus culture in vitro, embryo rescue andploidy level of recovered plants. Different 2x × 2x hybridiz-ations were performed to study in vitro embryogenesis fromisolated nucelli of non-apomictic citrus genotypes. In thehybridizations between ‘Clemenules’ clementine and‘Fortune’ mandarin with P. trifoliata ‘Benecke’, fruits werecollected 13–15 weeks after pollination. In the hybridizationbetween ‘Clemenules’ clementine and P. trifoliata‘Benecke’, 49 fruits were used, which contained 550 seeds(11.2 seeds per fruit). In hybridization between ‘Fortune’ man-darin and P. trifoliata ‘Benecke’, 19 fruits were used, whichcontained 175 seeds (9.2 seeds per fruit) (Table 6). Theseeds collected were well developed (Fig. 1A).

Nucellus culture in vitro produced embryos, initially by aprocess of direct embryogenesis and later by a process of adven-tive embryogenesis (Fig. 6A, B). From the hybridizationbetween ‘Clemenules’ clementine and P. trifoliata ‘Benecke’,550 normal seeds were obtained and 529 nucelli were isolatedand cultivated (Table 6). From the 550 normal seeds, eight had

TABLE 5. Fruit-set, number, seeds, plants and ploidy level obtained in 2x × 2x hybridizations

Femaleparent

Maleparent

Number pollinatedflowers

Number offruit set

Number smallseeds

Number culturedembryos

Number regeneratedplants

Number triploidplants

Fina Nova 200 148 10 10 10 10Clemenules Nova 100 70 25 25 22 22

10 mm

A

B

FI G. 3. Types of seeds obtained in 2x × 2x hybridization. (A) Developed small seeds with triploid embryos. (B) Normal seeds.




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0 0·1

Clementine

Nova 4x

FinNov 1.2FinNov 1.1


FinNov 2.2


FinNov 2.1

HerNov 1.2HerNov 1.1

HerNov 1.4HerNov 1.3

NulNov 1.2NulNov 1.1

NulNov 2.2NulNov 2.1

0 0·1

Clementine

Orlando 4x

FinOrl 1.2

FinOrl 1.1

FinOrl 1.3

FinOrl 2.2

FinOrl 2.1

FinOrl 2.3

FinOrl 2.4

NulOrl 1.4

NulOrl 1.2

NulOrl 1.3

NulOrl 1.1

NulOrl 2.2

NulOrl 2.1

0 0·1

Clementine

Pineapple 4x

TomPin 1.2TomPin 1.1

TomPin 1.3TomPin 1.4TomPin 1.5TomPin 1.6



BruPin 1.1BruPin 1.2BruPin 1.3

BruPin 2.2

BruPin 2.4BruPin 2.3

BruPin 2.1

C

B

A

FI G. 4. Cluster analysis of SSR data from 2x × 4x progenies based on the neighbour-joining method and Dice dissimilarity. (A) Triploid plants obtained fromundeveloped seeds originating from the pollinations between diploid clementines ‘Fina’ (Fin), ‘Hernandina’ (Her), ‘Clemenules’ (Nul) and tetraploid ‘Nova’mandarin (Nov) male parent. (B) Triploid plants obtained from undeveloped seeds originating from pollinations between diploid clementines (Fin, Nul) andtetraploid ‘Orlando’ tangelo (Orl) male parent. (C) Triploid plants obtained from undeveloped seeds originating from the pollinations between diploid clemen-

tines ‘Bruno’ (Bru), ‘Tomatera’ (Tom) and tetraploid ‘Pineapple’ sweet orange (Pin) male parent.




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more than one embryo (1.5 %) and 13 did not have any embryosand were eliminated. Ninety-eight nucelli (19.0 %) developed,producing a total of 422 embryos, from which 327 plants wereregenerated. All plants obtained had trifoliate leaves (Fig. 6C,D). In parallel, 529 isolated embryos were rescued from thesame seeds and cultivated in vitro. Seventy-four (14.0 %) germi-nated, producing plants with trifoliate leaves (Table 7). Finally,we obtained plants from the isolated zygotic embryos and corre-sponding nucelli from 57 seeds.

From the hybridization between ‘Fortune’ mandarin withP. trifoliata ‘Benecke’, 175 normal seeds were obtained(Table 6). From these seeds, 151 nucelli were isolated and cul-tivated; the remaining 24 seeds without embryos were dis-carded. Twenty-seven (18.0 %) developed. A total of 131embryos were obtained and 93 plants were regenerated, allof which displayed trifoliate leaves (Table 6). At the sametime, 151 embryos were rescued from the same seeds and cul-tivated in vitro. Twenty-two (14.6 %) germinated and

Ci02B07

170 nt

164 nt162 nt160 nt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 c

FI G. 5. Genetic analysis of clementine by tetraploid ‘Orlando’ tangelo with Ci02B07 SSR marker. Genotypes: 1, clementine; 2, tetraploid ‘Orlando’ tangelo;3–5 and 6–9, triploid hybrids obtained from two different undeveloped seeds originating from the hybridization between ‘Fina’ clementine and tetraploid‘Orlando’ tangelo; 10–13, triploid hybrids obtained from the same undeveloped seed originating from the hybridization between ‘Clemenules’ clementineand tetraploid ‘Orlando’ tangelo; 14 and 15, triploid hybrids obtained from the same undeveloped seed originating from the hybridization between ‘Fina’ clem-

entine and tetraploid ‘Orlando’ tangelo; C, PCR negative control.

TABLE 6. In vitro culture of nucellus isolated from seeds obtained in 2x × 2x hybridizations; ploidy level of plants obtained

Femaleparent

Maleparent

No. ofpollinated

flowers

No. ofusedfruits

No. ofseeds

obtained

No. ofculturednucellus

No. ofdevelopednucellus

No. ofembryosobtained

No. ofplant

analysed

No. ofdiploidplants

No. oftriploidplants

Clemenules Benecke 106 49 550 529 98 422 263 260 3Fortune Benecke 100 19 175 151 27 131 65 54 11

TABLE 7. Embryo rescue from seeds obtained in 2x × 2x hybridizations

Femaleparent

Maleparent

Numberobtained seeds

Number of seedswith embryo

Numberculturedembryos

Numbergerminated

embryosNumber

analysed plantsNumber

diploid plantsNumber

triploid plants

Clemenules Benecke 550 529 529 74 66 65 1Fortune Benecke 175 151 151 22 21 18 3

A B C D

FI G. 6. (A,B) Nucellus cultured in vitro with embryo development in the micropilar region. (C) Germination of embryos obtained from nucellus culturedin vitro. (D) Regenerated plants from embryos produced by nucellus cultured in vitro, showing trifoliate leaves.




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produced plants with trifoliate leaves (Table 7). Ultimately, weobtained plants from the isolated zygotic embryos and corre-sponding nucelli from 11 seeds.

From among the 263 plants obtained from nucellus culturein vitro of ‘Clemenules’ clementine seeds, 260 were diploid(98.9 %) and three (1.1 %) were triploid (Table 6). Of the 66plants obtained from germination of isolated zygoticembryos, 65 were diploid (98.5 %) and one was triploid(1.5 %) (Table 7). The three triploid plants regenerated fromthe nucellus culture in vitro and the triploid plant obtainedfrom germination of the isolated embryo belonged to thesame seed.

From among the 65 plants obtained from nucellus culture invitro of ‘Fortune’ mandarin seeds, 54 were diploid (83.1 %)and 11 (16.9 %) were triploid (Table 6). Among the 21plants obtained from the germination of the isolated zygoticembryos, 18 were diploid (85.7 %) and three were triploid(14.3 %) (Table 7). The 11 triploid plants regenerated fromthe nucellus culture in vitro and the three triploid plantsobtained from germination of the embryos isolated insidenucelli belonged to three different seeds.

Genetic analysis. To determine the genetic origin of plantsregenerated from nucellus culture in vitro, seven SSRmarkers, which are heterozygotic for clementine and‘Fortune’ mandarin, were employed. We analysed (1) 53

diploid plants and four triploid plants obtained in the hybrid-ization between ‘Clemenules’ clementine and P. trifoliata‘Benecke’, corresponding to ten different seeds; and (2) 22diploid plants and 14 triploid plants corresponding to sevendifferent seeds obtained in the hybridization between‘Fortune’ mandarin and P. trifoliata ‘Benecke’. All plants ana-lysed had trifoliate leaves and all plants corresponding to thesame seed displayed identical profiles for all hybridizationsat diploid and triploid level. It was possible to differentiateall the groups studied. The cluster analysis (Fig. 7) clearlydemonstrates that identical profiles were found for all seedscorresponding to the plant obtained from the zygotic embryoand plants recovered from nucellus culture in vitro. Resultswith the Mest 419 SSR marker are shown as an example inFig. 8. ‘Clemenules’ clementine showed two alleles of 108and 120 nt whereas P. trifoliata ‘Benecke’ displayed oneallele of 102 nt. All plants from seeds 1, 4, 5, 8 and 9 pre-sented a 108-nt allele of ‘Clemenules’ clementine and102-nt allele of P. trifoliata ‘Benecke’. The other groupsshowed an allele of 120 nt of ‘Clemenules’ clementine andthe 102-nt allele of P. trifoliata ‘Benecke’.

Histology. Twenty-seven seeds obtained from hybridizationbetween ‘Clemenules’ clementine and P. trifoliata ‘Benecke’were analysed at different developmental stages. The presenceof a single embryo was observed in all seeds (Fig. 9A) except

0 0·1

‘Clemenules’

‘Fortune’

‘Benecke’

ForBec 1 (1 zygotic 2x plant / 9 nucellar 2x plants)




NulBec 1 (1 zygotic 3x plant / 3 nucellar 3x plants)













FI G. 7. Cluster analysis of SSR data from 2x × 2x progenies based on the neighbour-joining method and Dice dissimilarity. NulBec and ForBec correspond toplants recovered from nucellus culture in vitro obtained in the pollinations between ‘Clemenules’ clementine (Nul) and ‘Fortune’ mandarin (For) with P. trifoliata‘Benecke’ (Bec). The first number indicates different seeds and in parentheses are the ploidy level of plants obtained from zygotic embryo and number and ploidy

level of plants recovered from nucellus culture in vitro.




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one (Fig. 9B), in which a dominant embryo and other smallembryos were found in the micropilar region of the seed.

DISCUSSION

SSR markers are highly efficient at determining the origin ofembryos in interspecific crosses

All clementines were identical but important polymorphismwas found between clementines and all the genotypes usedas male parents. In citrus, SSR markers can easily distinguishbetween species or cultivars arising from sexual hybridization(Barkley et al., 2006; Luro et al., 2008). Conversely, differen-tiation of genotypes arising from spontaneous mutations isvery difficult, as in the case of clementines (Breto et al.,2003) or sweet oranges (Fang and Roose, 1997). By usingseveral heterozygous markers differentiating the male andfemale parents, we also found good probability of differen-tiation between independent zygotic plants.

Multiple embryos, produced in undeveloped seeds arising fromhybridizations between diploid non-apomictic citrus genotypesand tetraploid pollen, are the result of zygotic embryo cleavage

Thirty-nine per cent of the undeveloped seeds producedmore than one embryo per seed and genetic analysis demon-strated that all plants obtained from the same undevelopedseed were identical and of hybrid origin. Our results thereforeconfirm the proposal made by Oiyama and Kobayashi (1990)after analysis of 2x × 4x progeny with just a single isozymemarker. Consequently, it is clear that these plants resultedfrom cleavage of the original zygotic embryo (Bacchi,1943), rather than the presence of two or more functionalembryo sacs in a single ovule. Indeed, with the selectedmarkers, the probability of finding two independent zygoticembryos with the same profile was very low.

Mest 419

Seed 6

Z Z Z CI P Z

ZZPCIZZZ

Seed 1 Seed 2 Seed 3 Seed 4 Seed 5

Seed 7 Seed 8 Seed 9

120 nt

108 nt

102 nt

120 nt

108 nt

102 nt

FI G. 8. Genetic analysis with Mest 419 SSR marker of diploid plants obtained from nucellus culture in vitro and germination of zygotic embryo produced in‘Clemenules’ clementine and P. trifoliata ‘Benecke’ hybridization. Z, plant obtained from germination of zygotic embryo; the other profiles of each group corre-

spond to plants obtained from nucellus culture in vitro. Cl, ‘Clemenules’ clementine; P, P. trifoliata ‘Benecke’.

A

B

FI G. 9. Histological section of seeds obtained in ‘Clemenules’ clementine byP. trifoliata ‘Benecke’ hybridization and sections stained by PAS. (A) Seedfixed 100 d after pollination. (B) Seed fixed 80 d after pollination. E,zygotic embryo; Ed, dominant embryo; Es, secondary embryo; En, endosperm;

N, nucellus; Te, outer integument; Ti, inner integument.




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Seed polyembryony of non-apomictic citrus genotypes isspecifically induced by pollination with a tetraploid male parent

We have studied plants regenerated from 2x × 4x hybridiz-ations using three different tetraploid male parents.Polyembryony arising from the original zygotic embryooccurred in every case, eliminating the influence of a specificmale parent genotype and demonstrating the link betweeninterploid hybridization (2x × 4x) and multiple embryo devel-opment. In addition, within our triploid breeding programme,we have performed more than 50 different 2x × 4x hybridiz-ations and observed the presence of multiple embryos insidethe same seed in all of them. Furthermore, in the same hybrid-izations, but between two diploid parents, polyembryony wasnever observed in small seeds resulting from the fertilizationof a diploid unreduced megagametophyte by haploid pollen(Cameron and Frost, 1968). Moreover, no polyembryonicseeds were observed in the different 2x × 4x crosses per-formed in the framework of our triploid breeding programme(our unpubl. res.). These observations support the fact thatmultiple embryo production in undeveloped seeds is not a con-sequence of parental genotype or embryo ploidy but a conse-quence of male parent ploidy and thus male parent genotypeexcess at endosperm or embryo level. Esen and Soost(1971b, 1977) have proposed that in this type of hybridizationthe ratio of ploidy level between embryo and endosperm (3/4)generates an incompatibility between the two, and an abnor-mal development of endosperm followed by underdevelop-ment of the embryos. It is possible that these unfavourableconditions for the development of the initial zygotic embryoaffect its dominance, thereby allowing the production ofadditional embryos from proembryonic cells derived fromthe original zygotic embryo. Another hypothesis could beraised related to the parent-of-origin effect. Parent-of-origineffects generate phenotypes that depend on the direction of across and occur frequently during angiosperm seed develop-ment, where maternal influence is most common (Allemanand Doctor, 2000). Genetic mechanisms can contribute toparent-of-origin effects during seed development, includingthe disproportionate maternal contribution to the endosperm,plastidic and cytoplasmic inheritance, expression of genes inthe gametophytes and gametes, and differential expression ofparental alleles in the developing seed (Dilkes and Comai,2004). In interploidy and interspecific crosses tested inmodel organisms such as maize and Arabidopsis, geneticand phenotypic changes observed in the progenies can beexplained as a consequence of the parent-of-origin effect.

Plants recovered by in vitro nucellus culture from non-apomicticcitrus genotypes are zygotic and not nucellar plants

Genetic analysis of plants obtained from nucellus culture invitro and plants recovered from the corresponding zygoticembryo of the same seed demonstrated that all plants obtainedfrom nucellus culture of clementine and ‘Fortune’ mandarinoriginated from the zygotic embryo and not from the nucellus.The present data suggest that proembryonic cells are formed inthe micropilar region as a result of original zygotic embryocleavage. Elimination of the original zygotic embryo and theculture in vitro of the nucellus containing the zygotic

proembryogenic cells allow these to complete developmentproducing embryos. The histological study of developingseeds did not reveal the presence of proembryonic cells, andonly the zygotic embryo was observed. Only in one case didwe find multiple embryos, but these probably correspondedto the rare cases where twin embryos are formed in vivo(Ozsan, 1964; Cameron and Garber, 1968). Esan (1973)made a comprehensive histological study of in vivo develop-ment of citron seeds and obtained similar results. Esan onlyfound evidence of zygotic embryo development and only inone case did he observe several proembryos in the sameseed; however, all surviving in vitro nucellus cultures pro-duced new embryos after the zygotic embryo was discarded.The failure to observe the proposed proembryonic cells inthe histological study may have been due to their low levelof differentiation, which does not enable proper identificationwith the procedures used.

The total lack of regenerated plants with maternal genotypeindicates that nucellar cells of clementine and ‘Fortune’ man-darin do not have embryogenic capacity either in vivo or invitro, in contrast to nucellar cells of apomictic genotypes(Button and Bornman, 1971; Kobayashi et al., 1981).

Our results for clementine and ‘Fortune’ mandarin are inagreement with observations made of plants from non-apomictic genotypes previously produced by nucellus culturein vitro. Rangan et al. (1968) regenerated plants ofpummelo, ‘Ponderosa’ lemon and ‘Temple’ mandarin(C. temple Hort. ex Y. Tan.) and Esan (1973) regeneratedplants of citron. Phenotypic observations indicated that noneof the plants produced by nucellus culture in vitro maintainedthe maternal phenotype (C. N. Roistacher, University ofCalifornia, Riverside, USA, pers. comm.). Navarro et al.(1985) also regenerated plants of several clementine varietiesfrom nucellus culture in vitro with a view to producinghealthy vigorous nucellar plants. The detailed phenotypiccharacterization of these plants in the greenhouse revealedmorphological similarity among plants obtained from thesame nucellus, but phenotypic diversity between plantsarising from different nucelli, while the majority of plants dif-fered from the maternal genotypes. The isoenzymatic analysisconfirmed the phenotypical data. Long-term field evaluation ofthe plants showed that none of them maintained the maternalgenotype (data not published).

All the available data support our conclusion that plants ofnon-apomictic citrus genotypes produced by nucellus culturein vitro arise from somatic embryogenesis of zygotic cellsand not from nucellar cells, as previously believed.

Adventitious polyembryony in non-apomictic citrus genotypes

Many angiosperms and gymnosperms have the potential formonozygotic cleavage embryony (Durzan, 2008). In normalseed development of conifers usually only one embryo domi-nates while the others remain undeveloped at the base of theseed (Durzan, 2008). In Sequoia sempervirens andActinostrobus acuminatus, cell strands in the zygote stratify toform several embryos. The distribution of free nuclei in thezygote determines their subsequent segregation into rudimentsof new sporophytes (Batygina and Vinogradova, 2007). InTorreya, Cephalotaxus, Ephedra and Gnetum (Johansen,




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1950) embryogenesis from the suspensor cells is also possibleand the formation of a special membrane around the zygotegives rise to embryos in Gnetum and Ephedra (Teryokhin,1991, cited by Batygina and Vinogradova (2007)). The potentialfor cleavage polyembryony in conifers is used to develop seedsartificially. Monozygotic cleavage embryony was described inthe genus Paeonia, in which somatic embryos arise from a mul-tinucleated cell structure.

In species producing monoembryonic seeds usually onlyone embryo remains in a mature seed as the result of competi-tive development, while the other dies during the early stages(Batygina, 1989, 1998, 2006). Competition between embryoshas been described in apomictic citrus genotypes wherezygotic embryos develop more slowly than nucellar embryos(Koltunow, 1993). In polyembryonic citrus seeds the favoureddevelopment of nucellar embryos could be due to an earlierinitiation of embryogenesis and to the competition for avail-able nutrients (Wakana and Uemoto, 1987, 1988). In the non-apomictic citrus genotypes such competition should alsoexplain the differences in development between the originalzygotic embryo and the adventitious one. A late initiation ofsecondary embryo formation could explain the completedevelopment of only one embryo. However, another hypoth-esis could relate to hormonal dominance of the initialzygotic embryo or an inhibition of adventitious embryo devel-opment. This kind of hormonal inhibition of embryogenesiswas proposed by Tisserat and Murashige (1977a), who demon-strated that the presence of ovules of the non-apomictic citroninhibited the in vitro embryogenetic process from embryogeniccallus of the apomictic ‘Ponkan’ mandarin (C. reticulata).Moreover, the same authors have shown that endogenouslevels of indolacetic acid, abcisic acid and gibberellin weremuch higher in citron than in the mandarin ovules (Tisseratand Murashige, 1977b).

Practical implications for citrus breeding and sanitation

Our conclusions have important implications for triploidbreeding programmes based on 2x × 4x hybridizations(Starrantino and Recupero, 1981; Navarro et al., 2002;Yoshida et al., 2003) as during the stages of embryo rescuefrom multiple embryos contained in undeveloped seeds it isnecessary to regenerate only one plant per seed. This is of enor-mous practical value because it significantly reduces the work-load during the in vitro stage and avoids costly and lengthy fieldevaluation of several plants belonging to the same genotype.

Moreover, nucellus culture was previously considered suit-able for producing pathogen-free plants and the techniquewas specifically recommended for this purpose (Soost andCameron, 1975). Our results clearly demonstrate that notrue-to-type plant with maternal genotype can be obtained bynon-apomictic nucellus culture. Coincidentally, interest inthis technique has declined since the same research team intro-duced the shoot-tip grafting in vitro technique to producepathogen-free plants without juvenile traits (Navarro et al.,1975), which has been adopted worldwide (Navarro andJuarez, 2007). However, during the last decade, somatichybridization sparked interest in the induction of embryogeniccallus lines of non-apomictic genotypes via nucellus or ovuleculture in vitro (Wu et al., 2005). It is clear that accurate

molecular studies of the induced calli will be necessary toconfirm their maternal origin.

Conclusions

We have demonstrated that in non-apomictic citrus geno-types, embryogenic cells or proembryos are formed by clea-vage of the zygotic embryo. Moreover, the development ofthese adventitious cells or embryos can take place in vivoand in vitro as the result of two different mechanisms thatprevent the dominance of the initial zygotic embryo. Thefirst mechanism involves the mechanical elimination of thedominant zygotic embryo and subsequent culture in vitro ofthe nucellus, thus facilitating the development of embryosfrom embryogenic cells deriving from the zygotic embryo.The second is associated with interploidy 2x × 4xhybridizations.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxford-journals.org and give the theoretical probability of identitybetween two zygotic genotypes and between zygotic andnucellar plants obtained in 2x × 4x hybridizations in termsof parental SSR profiles without allelic dosis estimation.

ACKNOWLEDGEMENTS

This work was financed by the AGL2008-00596-MCI andPrometeo 2008/121 Generalidad Valenciana projects. Wethank Carmen Ortega and Marıa Hernandez for their technicalassistance in the laboratory and J. A. Pina for growing plants inthe greenhouse.

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