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Scientia Horticulturae 138 (2012) 101–107

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Scientia Horticulturae

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olyembryony and seedling emergence traits in apomictic citrus

undan Kishore ∗, Monika N., Rinchen D., Boniface Lepcha, Brijesh PandeyCAR Research Complex for NEH Region, Sikkim Centre, Gangtok 737102, Sikkim, India

r t i c l e i n f o

rticle history:eceived 5 November 2011eceived in revised form 26 January 2012ccepted 31 January 2012

eywords:

a b s t r a c t

Highest polyembryony, range of embryo, clutch size and frequency of quintuplets, sextuplets and hep-tuplets were observed in Citrus jambhiri. However, quadruplet and triplet embryos were more frequentregardless the genotypes. Embryos were mainly arranged at micropylar region which were characterizedby asynchronous developments. Moreover, their biomass partitioning significantly varied with morpho-types. C. jambhiri also had highest CVE and uncertainty of emergence. However, it had minimal emergence

synchronousmbryosorphotypes

olyembryonyynchronization index

time (34.4 ± 1.8 days) and synchronization index. Additionally, it had highest frequency of triplet andquadruplet seedlings and maximal embryo to seedling conversion rate. Synchronization index showedsignificantly negative correlation with the number of seedlings and frequency of multiple seedlings.Emergence rate and time were not influenced with the intensity of embryony in citrus genotypes. More-over staggered seedling emergence gives an indication of bet-hedging strategy which helps in betterseedling survival in a temporally variable environment.

. Introduction

Polyembryony in citrus is a type of facultative apomixis whereinimultaneous development of embryos of nucellar origin co-existsn the same seed containing sexual embryo. Nucellar tissues afterifferentiation enter into the pathway of embryogenesis whoseevelopment is endosperm-dependant and the resulting seedlingsre of the same genotype as the female parent (Koltunow et al.,995; Kepiro and Roose, 2007). The presence of nucellar and zygoticmbryoids in the same seed indicates heterogeneity of embry-nal population and unlike the normal seeds, different processes ofmbryoidogenesis take place (Koltunow, 2000). Nucellar embryosre generally oriented in the micropylar region of ovule but theyay also be localized in the lateral and chalazal parts (Batygina andinogradova, 2007).

On the basis of cellular origin of embryogenesis, polyembry-ny may be divided into two types: gametophytic and sporophytic.ametophytic polyembryony (n) includes apogamety (formationf embryos from gametophytic cells; synergids and antipodals,xcepting egg cell) and apospory (formation of unreduced embryoacs from somatic cells), whereas sporophytic polyembryony (2n)ncompasses nucellar and integumental embryony (Batygina and

inogradova, 2007). In citrus, sporophytic nucellar embryony isommon that implies the development of extranumerary embryos

∗ Corresponding author at: DRWA, Baramunda, Bhubaneswar 751003, Odisha,ndia. Tel.: +91 674 2386241; fax: +91 674 2386242.

E-mail address: kkhort2003@yahoo.com (K. Kishore).

304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2012.01.035

© 2012 Elsevier B.V. All rights reserved.

from nucellar cell of ovule without forming extra embryo sac(Savidan, 2000).

Most citrus genotypes exhibit polyembryony with the exceptionof Citrus medica, Citrus maxima, Temple tangelo and Clementine cul-tivars (Kobayashi et al., 1981). The number of embryos that developfrom nucellar cell decides the clutch size of embryo and in turn themorphotypes. The expression of the various morphotypes occurs atdifferent frequencies under the influence of gene and environment.In polyembryonic condition multiple sibling embryos are exposedto competition right from their development (Uma Shaanker andGaneshaiah, 1997) which continues even after seed germinationwhen the gamellar seedlings (originated from polyembryonic seed)struggle to establish themselves in very close proximity (Hotchkisset al., 2008). Germination measurements are indicatives of seedvigour and in this dynamic process of energy utilization the ger-minability, germination rate and synchrony of emergence aregreatly influenced (Mendes-Rodrigues et al., 2010). Seed vigour,a physiological characteristics determined by genotype and modi-fied by the environment, changes the seedling emergence rate andgerminability in citrus species.

Nucellar embryony is considered to be an essential trait forthe propagation of genetically uniform healthy plants. Moreoverit enhances its rapid fixation in the progeny and is effectivelyused as a method of clonal selection in raising quality citrus plantsincluding rootstock (Xiang and Roose, 1988). Although, quantum ofstudies has been made on polyembryony of citrus, there is a dearth

of information about the morphotypes and emergence traits ofcitrus genotypes. Therefore the present study aims to answerthe following questions. (1) How polyembryony and its morpho-types vary with genotype? (2) What is the pattern of biomass

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artitioning between embryos under different morphotypes? (3)ow does emergence pattern vary with genotypes? (4) What is

he level of correlation among emergence traits?To do so, we studied polyembryony, morphotypes, variation in

iomass of embryos, emergence traits in different genotypes andorrelation coefficient between different emergence traits.

. Materials and methods

Studies were conducted during 2008–2011 at the research farmf Indian Council of Agricultural Research (ICAR) Sikkim Cen-re, Gangtok, Sikkim, India located at an altitude of 1300 m amsllatitude – 27◦20′N; longitude – 88◦04′E). Twelve citrus geno-ypes: Sikkim mandarin (Citrus reticulate Blanco), Thorny mandarinCitrus deliciosa Ten.), Kinnow mandarin (Citrus nobilis Lour × C.eliciosa Ten.), Cleopatra mandarin (Citrus reshni hort. ex Tanaka),alamondin (Citrus medurensis Lour.); Nova (Clementine (C. retic-late) × Orlando (C. X paradisi × C. reticulata)), Minneola (C. Xaradisi × C. reticulata), Thornton (C. X paradisi × C. reticulata), Par-amatta (Citrus sinensis L. Osbek), Parson Brown (C. sinensis L.sbek), Smooth Flat Seville (Citrus aurantium L.) and rough lemon

Citrus jambhiri Lush.), a major rootstock, were evaluated for theirolyembryony, morphotypes and emergence traits.

.1. Polyembryony

Matured fruits of all the genotypes were collected in 2008, 2009nd 2010 during October–November from citrus germplasm blocknd seeds were extracted by hand and washed in running wateror 5 min. To study frequency of polyembryony, embryony andmbryo range, more than 100 seeds of each genotype were dis-ected with needle and forceps every year and embryo numbersere counted under stereo-zoom microscope (Leica 30×). On the

asis of the number of seeds containing more than one embryo,ercentage of polyembryony was obtained. Similarly the range ofmbryos (1, 2, . . ., n) in polyembryonic seeds was worked by count-ng the minimum and maximum number of embryos. Clutch sizeaverage number of embryos) in polyembryonic seeds of genotypesas calculated dividing total number of embryos by number ofolyembryonic seeds. Similarly clutch size in different genotypeas obtained by dividing total number of embryos by total number

f seeds (polyembryonic and monoembryonic).

.2. Morphotypes of polyembryony

On the basis of number of embryos (1, 2, 3, . . .), embryony wasivided into different morphotypes: singlet (one embryo/seed),uplet (two embryos/seed), triplet (three embryos/seed), quadru-let (four embryos/seed), quintuplet (five embryos/seed), sextupletsix embryo/seed), heptuplet (seven embryos/seed), octupletseight embryos/seed) and so on and percent occurrence wasorked out on the basis of frequency of different morphotypes in

eeds of each genotype (n = 100) in 2008, 2009 and 2010.

.3. Spatial distribution and biomass partitioning of embryos

Spatial distribution of embryos was studied in polyembryoniceeds of each genotypes (n = 25) by observing the arrangementsf embryos under stereo zoom microscope (Leica, 40×) andxpressed in percentage (mean of all genotypes). The region aroundilum (point of attachments between seed and ovary) was con-idered as the micropylar region, whereas other regions were

on-micropylar. Embryos were categorized into four developmen-al stages; matured, torpedo, heart and globular as defined by

arilyn et al. (1993). For biomass partitioning, weight of embryosnder different morphotypes for each genotype was taken and

lturae 138 (2012) 101–107

expressed as the mean of all the genotypes. Under each morpho-types of each genotype 12 seeds were taken for measurement.

2.4. Morphotypes of multiple seedlings and emergence traits

Freshly extracted seeds were properly washed in running waterand treated with carbandazime 50WP (0.2%) for 15 min followedby shade drying for 24 h. We used three replicates of 100 seeds foreach genotype in 2008, 2009 and 2010. Seeds were sown in pro-trays (25 cells) containing mixture of soil, sand and vermicompostin equal proportion and kept inside a polyhouse (100 m2, quonset-shape, temperature and humidity control system, UV stabilizedpolythene 200 �m thick, in built cooling and misting systems) tostudy the emergence measurements and morphotypes of multi-ple seedlings. The temperature and relative humidity under thepolyhouse were 25 ◦C and 75%, respectively during the study. Mor-photypes of multiple seedlings were worked out on the basis ofnumbers of seedling(s)/seed: singlet (one seedling/seed), duplet(two seedlings/seed), triplet (three seedlings/seed), quadruplet(four seedlings/seed), quintuplet (five seedlings/seed), sextuplet(six seedlings/seed), heptuplet (seven seedlings/seed) and so onand expressed as percentage.

Emergence traits were evaluated in terms of germinability, ini-tial and final emergence time, mean emergence rate, coefficient ofvelocity of emergence, uncertainty and synchronization index in2009, 2010 and 2011 following the methods of Ranal and Santana(2006). Germinability (G) was calculated on the basis of protrusionof the embryo. Initial emergence time (ti), i.e. time of emergence offirst seedling and final emergence time (tf), i.e. time for last seedlingemergence were considered to evaluate the duration of the emer-gence process (Labouriau, 1970). The mean emergence rate (t) wascalculated as the weighted mean of the emergence time wherethe number of seeds emerged in the intervals of time was usedas weight. Coefficient of velocity of emergence (CVE) expressesthe rate of seedling emergence per day. Uncertainty of emer-gence (U) measures the degree of uncertainties associated with thedistribution of the relative frequency of germination whereas syn-chronization index (Z) measures the synchrony of germination andevaluates the degree of overlapping in emergence among seedlingsof one population. Further details on mathematical expression,intermediate expression calculus, sense and application of thesegermination measurements can be found in the studies of Ranaland Santana (2006) and Ranal et al. (2009).

2.5. Embryo to seedling conversion rate

The embryo to seedling conversion rate was worked out everyyear for each genotype dividing average number of embryos byaverage number of seedlings/seed and expressed as percentages.

2.6. Statistical analysis

Data (means of three years) from the experiments were sub-jected to one way analysis of variance (ANOVA), Duncan’s multiplerange test (DMRT) at P = 0.05 to compare the means of variablesusing SPSS statistical package (11.5 version). The correlation testamong variables was done by Pearson’s correlation (r).

3. Results

3.1. Polyembryony

Initial visual analysis indicated the highest polyembryony in C.jambhiri (>90%) followed by Sikkim mandarin (Table 1), whereas C.medurensis had the lowest percentage of polyembryony. However,all the genotypes were highly polyembryonic in nature. Differences

K. Kishore et al. / Scientia Horticulturae 138 (2012) 101–107 103

Table 1Polyembryony, range of embryo and clutch size in apomictic citrus genotypes.

Citrus genotypes No. of seedsdissected

Polyembryony (%) Range of embryos inpolyembryonic seeds

Clutch size (no.)

Min. Max. Polyembryonic seed Total seed

Sikkim mandarin 112 86.6 ± 5.9c 2 9 3.24 ± 0.42b 3.06 ± 0.34b

Thorny 108 79.6 ± 4.8d 2 7 2.81 ± 0.22c 2.64 ± 0.21d

Kinnow 113 81.4 ± 5.7cd 2 9 3.21 ± 0.31b 3.08 ± 0.30b

Nova 102 83.2 ± 5.8b 2 6 3.18 ± 0.27b 3.02 ± 0.28b

Minneola 110 75.4 ± 4.6d 2 7 2.75 ± 0.24d 2.42 ± 0.26e

Thornton 107 72.9 ± 4.4e 2 6 2.21 ± 0.20 1.87 ± 0.24g

Parramatta 106 81.1 ± 5.3cd 2 8 3.05 ± 0.27bc 2.94 ± 0.29c

Parson Brown 105 78.0 ± 5.1d 2 8 2.90 ± 0.31c 2.71 ± 0.33d

Smooth Flat Seville 112 80.3 ± 5.4cd 2 8 3.15 ± 0.27bc 2.98 ± 0.27c

C. jambhiri 115 91.4 ± 6.6a 2 14 3.48 ± 0.47a 3.26 ± 0.42a

C. reshni 104 75.0 ± 5.1d 2 8 2.64 ± 0.23d 2.52 ± 0.21e

C. medurensis 106 69.8 ± 4.2e 2 6 2.28 ± 0.18e 2.04 ± 0.19f

DM sed on the DMR tests (P = 0.05).

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15.4% (Fig. 5). A similar trend of biomass partitioning was observed

ata are means ± standard error.eans followed by the same letter in each column are not significantly different ba

oncerning maximal range of embryos showed highest in C. jamb-iri whereas C. medurensis had the minimum range of embryos. Asxpected, genotypes showed significant difference in clutch size inolyembryonic seeds as well as in total seed which in turn influ-nced the seedling production across the genotypes. Maximumlutch size was recorded in C. jambhiri whereas Thornton and C.edurensis had relatively small clutch size.

.2. Morphotypes of polyembryony

Polyembryony differs significantly in terms of morphotypesFig. 1). Quadruplets and triplets were more frequent (65–70%)han other forms of morphotypes. However, frequency varied withenotypes. The frequency of duplets ranged 10–30% in differentenotypes with the maximal in C. medurensis and Thornton. Theighest frequency of occurrence of quintuplets, sextuplets and hep-uplets was observed in C. jambhiri.

.3. Spatial distribution and biomass partitioning of embryos

In more than 90% of the polyembryonic seeds, embryos werepatially arranged at the micropylar region (Fig. 2). However,n some of the cases embryos were arranged at non-micropylaregions. A progressive embryonic development was observed inost of the polyembryonic seeds and embryos were at differ-

nt developmental stages; matured, torpedo, heart and globularFig. 3). However, the frequency of occurrence of heart and globulartages increased with embryony.

ig. 1. Frequency of occurrence of morphotypes of polyembryony in citrus geno-ypes.

Fig. 2. Embryos at micropylar region.

Partitioning of biomass among embryos under different embry-onic class showed significant variation (Figs. 4 and 5). In most of theduplets more than 90% of biomass was acquired by first embryowhile second embryo had significantly low biomass. In triplets,the biomass partitioning among three embryos was 64.7, 22.9 and

in quadruplets, quintuplets, sextuplets and heptuplets. A propor-tional change in the biomass was observed with embryo size andits stage of development.

Fig. 3. Embryos at different stages: M, matured; H, heart stage; G, globular.

104 K. Kishore et al. / Scientia Horticulturae 138 (2012) 101–107

Fig. 4. Variation in biomass partitioning among embryos. (a) Triplet; (b) quadruplet; and (c) sextuplet.

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.4. Emergence measurements and morphotypes of multipleeedlings

Genotypes exhibited variation in emergence measurementsTable 2). The germinability varied 61.8–84.6% among citrusenotypes. C. jambhiri exhibited highest germinability whereas,hornton had the lowest. Emergence time varied significantly withome of the genotypes. The earliest emergence was observed in C.ambhiri whereas Smooth Flat Saville took relatively longer period.t is evident from data that emergence in citrus begins after 25–30

ays and continues up to 60 days under controlled environment.he minimal mean emergence time was recorded in C. jambhirind maximal in Smooth Flat Saville. The highest CVE, uncertaintyf emergence (U) and the lowest synchronization index (Z) due

able 2mergence traits of apomictic citrus genotypes.

Citrus genotype GGerminability (%)

ti (days) tf (days) t (days)

Sikkim mandarin 78.3 ± 4.2b 29.2 ± 2.4bc 52.2 ± 3.6c 38.2 ± 2.14b

Thorny 73.8 ± 3.8c 31.4 ± 3.3b 57.2 ± 2.1ab 40.4 ± 2.3ab

Kinnow 79.4 ± 3.6b 30.3 ± 2.3b 53.6 ± 2.9c 39.1 ± 1.9ab

Nova 80.6 ± 3.9b 29.4 ± 2.8bc 58.4 ± 2.2ab 41.2 ± 1.8a

Minneola 72.7 ± 3.1c 31.8 ± 3.1b 60.3 ± 2.0a 41.6 ± 1.6a

Thornton 61.8 ± 2.6e 30.5 ± 3.4b 59.4 ± 2.2ab 40.5 ± 2.4ab

Parramatta 66.4 ± 2.4de 32.5 ± 2.7b 54.7 ± 3.6c 39.8 ± 2.4ab

Parson Brown 71.4 ± 3.5c 32.3 ± 2.6b 57.5 ± 3.2ab 42.6 ± 2.1a

Smooth Flat Seville 69.8 ± 7.2d 36.7 ± 3.3a 62.8 ± 3.5a 44.5 ± 2.4a

C. jambhiri 84.6 ± 4.3a 26.6 ± 2.1c 49.2 ± 2.1d 34.4 ± 1.8c

C. reshni 78.8 ± 4.1b 32.3 ± 3.6b 52.4 ± 3.6c 42.8 ± 2.6a

C. medurensis 68.6 ± 3.8d 35.7 ± 3.7a 53.2 ± 3.7c 43.6 ± 2.3a

ata are means ± standard error, ti , initial emergence time; tf , final emergence time; t, memergence; Z, synchronization index.eans followed by the same letter in each column are not significantly different based on

bryos under different morphotypes.

to asynchronous emergence (Fig. 6) were recorded in C. jambhiri.Whereas C. medurensis showed maximal synchrony in emergence(Fig. 7).

Significant variation in mean number of seedlings and frequencyof multiple seedlings was observed across the genotypes whichranged 1.28–2.24/seed and 18.2–69.7%, respectively (Table 2). Thehighest number of seedling and frequency of multiple seedlingswas obtained in C. jambhiri, whereas C. medurensis exhibited lowest.

3.5. Morphotypes of multiple seedlings

Like embryo, seedlings also emerged in different combinations;duplets, triplets, quadruplet, quintuplet and sextuplets whichshowed variation with genotype (Fig. 8). Except C. jambhiri, Sikkim

CVE (day−1) U (bit) Z Mean number ofseedlings/seed

Multipleseedlings (%)

0.19 ± 0.01b 4.2 ± 0.54b 0.19 ± 0.08e 1.72 ± 0.14b 55.3 ± 4.0b

0.12 ± 0.01e 3.8 ± 0.42c 0.27 ± 0.02cd 1.33 ± 0.16d 50.4 ± 3.8bc

0.16 ± 0.02c 4.1 ± 0.62b 0.23 ± 0.21d 1.58 ± 0.14c 49.2 ± 4.1bc

0.18 ± 0.02b 4.3 ± 0.51b 0.22 ± 0.09d 1.45 ± 0.17cd 41.4 ± 3.8d

0.12 ± 0.01e 3.8 ± 0.47c 0.31 ± 0.07c 1.39 ± 0.11d 51.7 ± 4.3b

0.12 ± 0.01e 3.0 ± 0.39de 0.34 ± 0.08ab 1.32 ± 0.15d 41.1 ± 4.1c

0.14 ± 0.01d 3.7 ± 0.34c 0.22 ± 0.07d 1.55 ± 0.14c 49.3 ± 3.7c

0.14 ± 0.02d 3.6 ± 0.03c 0.35 ± 0.04ab 1.38 ± 0.16d 46.1 ± 4.4c

0.10 ± 0.01f 3.7 ± 0.73c 0.36 ± 0.09a 1.53 ± 0.18c 51.3 ± 3.9b

0.23 ± 0.01a 4.8 ± 0.94a 0.12 ± 0.06f 2.24 ± 0.14a 69.1 ± 5.3a

0.14 ± 0.02d 3.4 ± 0.63d 0.26 ± 0.04cd 1.41 ± 0.12cd 52.2 ± 4.7b

0.10 ± 0.01f 3.1 ± 0.68de 0.38 ± 0.08a 1.28 ± 0.14d 40.8 ± 4.3d

an emergence time; CVE, coefficient of velocity of emergence; U, uncertainty of the

the DMR tests (P = 0.05).

K. Kishore et al. / Scientia Horticulturae 138 (2012) 101–107 105

Fig. 6. Asynchronous emergence of duplet, triplet and quadruplet seedlings in C. jambhiri.

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andarin and Kinnow, multiple seedlings were predominantlyuplets. The frequency of triplets and quadruplets was significantlyigh in C. jambhiri and Sikkim mandarin. However, quintuplets andextuplets were also recorded in C. jambhiri.

.6. Embryo to seedling conversion rate

The conversion rate of embryo to seedling indicated signifi-ant difference across the genotype with the variation of 41–69%.. jambhiri had the maximal conversion rate, whereas Nova had

Fig. 8. Frequency of occurrence of differen

Fig. 9. Conversion rate of polyembryony into multiple seedlings in citrus genotypes.

the minimal. However, in most of the genotypes about 50% of theembryos were converted into seedlings (Fig. 9).

3.7. Correlation studies

Germinability showed a positive and significant correlation withthe CVE (r = 0.786**, P = 0.01). However, it was negatively correlatedwith the initial emergence time (r = −0.598*, P = 0.05). Mean emer-

gence time exhibited positive and significant correlation with theinitial emergence time whereas it was negatively and significantlycorrelated with CVE (r = −0.750**, P = 0.01) and frequency of occur-rence of multiple seedlings (r = −0.681*, P = 0.05). A positive and

t morphotypes of multiple seedlings.

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ignificant correlation was observed between uncertainty and theumber of seedlings/seed (r = 0.806**, P = 0.01) and frequency ofccurrence of multiple seedlings (r = 0.695*, P = 0.01). In contrary,ynchronization index was negatively and significantly correlatedith the number of seedlings/seed (r = −0.789**, P = 0.01) and fre-

uency of occurrence of multiple seedlings (r = −0.681*, P = 0.05).

. Discussion

.1. Polyembryony and morphotypes

Variation in polyembryony and clutch size across the citrusermplasm may be due to the complex interrelation betweenenotype and environment. Batygina (1999) stated that the pres-nce of morphogenetic and morphophysiological correlation isne of the causes of embryoidogenesis which leads to sporo-hytic polyembryony. Moreover the presence of dominant factoror polyembryony, ploidy level and polar transport of auxins playsmportant roles in the development of extranumerary embryo andetermines its symmetry (Savidan, 2000; Grossniklaus et al., 2001;ischer et al., 1997; Jaskani et al., 2005). Furthermore improperndosperm development leads to early breakdown of nucellarmbryos since endosperm serves as a conduit for nutrients androwth factors from the maternal tissue to the embryo (Gupta et al.,996; Yeung and Meinke, 1993).

Findings on clutch size and embryo range are not in consistentith Andrade-Rodriguez et al. (2005) who observed an average

lutch size of 4.7 in C. reshni which clearly indicates the impactf location on frequency of occurrence of multiple embryos. Theignificant variation in the frequency of clutch size might be due totress changes in the environmental conditions which acts as a fac-or for change in the genetic developmental program of cells or theirormonal status, which affect morphogenetic and morphophysio-

ogical correlations in development of the embryo and surroundingeed structures (Batygina and Vinogradova, 2007). On the otherand, Ganeshaiah et al. (1991) observed a maternal and offspringontrol over the frequency of polyembryony and stated that forarginal benefit of resources offsprings favour brood reduction,hereas mother prefers polyembryony which resulted in an active

onflict over number of offspring.Studies of the mechanisms underlying induction and regula-

ion of polyembryony are essential not only for the fundamentalroblems of biology, such as population aspects of reproduction,ut also for practical purposes of genetic selection. The expres-ion of morphotypes suggests complex influence of a number ofiotic and abiotic factors. The high frequency of occurrence ofuadruplets, quintuplets, sextuplets and heptuplets suggests highate of polyembryony. Batygina and Vinogradova (2007) suggestedhe embryologeny, embroidogeny and types of apomixis influence

orphotypes. Whereas Onovo et al. (2009) observed variation inorphotypes of fluted pumpkin due to abiotic factors.

.2. Spatial distribution and biomass partitioning of embryos

Embryo does not follow any universal pattern of distributionnside seed. Martinez-Gomez and Gradziel (2003) observed a typi-al basal to distal orientation of almond embryos and partitioningf more biomass at distal end than that of basal end. However, inome of the seeds equal distribution of mass was also recorded. Inontrary, the orientation of embryos in citrus was quite differentnd they were mostly located at micropylar region regardless of

he embryo number. Moreover, biomass partitioning also showedifferent trend. In most of the duplets, embryos were distinct inheir developmental stages and biomass was centralized to onembryo only. In other morphotypes decentralization of biomass

lturae 138 (2012) 101–107

was observed and the first embryo possessed about half of thebiomass and rest of the biomass was distributed among otherembryos. Reduction in the biomass with the increase in the numberof embryo was an indicative of sequential development of nucel-lar embryos and ruled out simultaneous adventive embryogeny inseed. Moreover, sequential development of embryos seems an indi-cation of bet-hedging strategy to avoid completion for resourcesand space inside the seed (Ladd and Cappuccino, 2005).

Findings suggested that citrus embryos were mainly arrangednear micropyle. However, positioning of nucellar embryos is notfixed and they may be arranged near micropyle or away frommicropyle. The reason for arrangement of nucellar embryos ofcitrus at micropylar region could be due to their endosperm-dependant growth habit. The presence of endosperm promotes thedevelopment of adventives embryos at the micropylar end, butsuppresses their development towards chalazal end. The degreeof suppression is directly related to the distance of embryo fromthe micropylar end (Wakana and Uemoto, 1987).

4.3. Emergence measurements

Seed emergence studies are important not only for the usefulinformation they contribute to seed technology, but also for theirhelp in understanding the ecophysiology of plant species (Kondoet al., 2002). Findings clearly indicate that polyembryony does notseem to be a constraint for germinability and emergence whichcoincides with Scalon et al. (2003) who observed in Bombacopsisglabra. In contrary, Mendes-Rodrigues et al. (2011) reported thatpolyembryony was a limiting factor for germinability and initialseedling growth in E. pubescence. Difference in germination traitsindicates difference in germination strategy among citrus geno-types. High CVE and low mean emergence time in C. jambhiri mayreflect high rate of imbibitions and fast enzymatic hydrolysis oftesta which makes it soft enough to allow embryo protrusion. HighCVE and low mean emergence time in C. jambhiri in spite of highrate of embryony clearly suggest that the intensity of embryonydoes not always determine the emergence rate and time. However,in Inga sp., CVE and emergence time decreased with the increase inembryony (Mendes-Rodrigues et al., 2008). Lower synchrony andhigher uncertainty of emergence with the increase in the clutchsize may be viewed as relative dormancy and bet-hedging strategywhich are considered as a way to spread germination over timeand thus reduce resource competition between siblings and in turnprovides better chance of survival in a temporally variable environ-ment (Carvalho et al., 2005; Ladd and Cappuccino, 2005; Blanchardet al., 2010). However, staggered emergence time is a plastic traitthat shows variation with environment.

5. Conclusion

A simple perspective that emerges from this study is that majorrootstock (C. jambhiri) and leading mandarin cultivar (Sikkim man-darin) are highly polyembryonic with more clutch size. Moreoverthey produce significantly high number of multiple seedlings whichin turn provide better chance of rapid fixation in the progenyand propagation of genetically uniform healthy plants. Embryoswere present in different morphotypes which are responsible forasynchronous embryo development and disproportional biomasspartitioning. Emergence traits varied significantly with genotypesand morphotypes. On the other hand polyembryony did not seemto affect emergence process in apomictic citrus. Asynchronous

embryo development and seedling emergence clearly give an indi-cation of bet-hedging strategy and relative dormancy which indeedincreases the probability of seedling survival due to reductionsin competition for resources and space, as well as increases the

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robability of adding individuals to the next generation. Persis-ence of somatic population represents an important reproductivelternative with potential evolutionary advantages and needs moreetailed study along with the genetic heterogeneity of citrus seedsince it provides better scope for crop improvement and conserva-ion of biological diversity.

cknowledgements

Authors are thankful to Dr. H. Rahman, Joint Director, Dr. B.C.eka, Head and Dr. Ashok Kumar, Scientist for extending help inonducting studies. We thank Director, and Dr. N.K. Meena, NRCor Orchid, Pakyong, Sikkim for providing facilities of stereo-zoom

icroscope. The correction made in the manuscript by Dr. H.K. Das,RWA, Bhubaneswar is duly acknowledged.

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