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Scientia Horticulturae 165 (2014) 36–43

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epa ge: www.elsev ier .com/ locate /sc ihor t i

ollen tube performance in assessment of compatibilityn olive (Olea europaea L.) cultivars

abriela Vuletin Selaka,∗, Julián Cuevasb, Smiljana Goreta Bana, Slavko Pericaa

Department of Plant Sciences, Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, CroatiaDepartamento de Agronomía, Campus de Excelencia Internacional Agroalimentario (ceiA3), Universidad de Almería, La Canada de San Urbano, s/n, 04120lmería, Spain

r t i c l e i n f o

rticle history:eceived 10 September 2013eceived in revised form 25 October 2013ccepted 29 October 2013

eywords:ross-compatibilityertilizationliveollen germinationollen tube growth

a b s t r a c t

Most olive cultivars are partially self-incompatible. However, the mechanisms of self-incompatibility arestill unclear and the choice of pollinizers for the main olive cultivars becomes difficult especially afterthe constant introduction of foreign cultivars in new environmental conditions. In order to determinecompatibility relationships and to select suitable pollinizers for the main Croatian cultivars, pollen ger-mination, pollen tube growth and fertilization percentage have been assessed in ‘Lastovka’ and ‘Oblica’,as well in the Italian cultivar ‘Leccino’ following self- and cross-pollination treatments in 2009 and 2010seasons. Massive pollen tube growth, higher pollen tube growth rates, earlier and greater amount offertilization were found for cross-pollination compared to self-pollination. The flowering periods of thestudied cultivars overlapped in a high degree in both years. In ‘Lastovka’, cross-pollination was moreefficient than self-pollination regarding pollen germination, tube growth and fertilization. Index of self-

elf-incompatibility incompatibility (ISI), calculated as the ratio of fertilization following self-pollination to that followingcross-pollination, was 0.4 and 0.5 in 2009 and 2010, respectively, which categorize this cultivar as par-tially self-incompatible. Higher ISI was found for ‘Leccino’ (0.7 in 2009; 1.0 in 2010) and ‘Oblica’ (0.5in 2009; 0.7 in 2010). However, most cross-pollination treatments provided higher fertilization lev-els than self-pollination, proving that cross-pollination was also beneficial for ‘Leccino’ and ‘Oblica’.Inter-incompatibility reactions between cultivars were not observed.

. Introduction

In the Mediterranean Basin, Croatia included, olive has tradi-ionally been cultivated in mono-cultivar orchards. However, alear increase in fruit set under cross-fertilization has recently beenocumented for many cultivars in different regions (Lavee and Datt,978; Cuevas et al., 2001; Lavee et al., 2002; Moutier, 2002; Vuletinelak et al., 2011). As a result, the inclusion of compatible polliniz-rs of simultaneous flowering is presently recommended to ensure

good fruit set (Cuevas et al., 2009).Different researchers have attempted to identify compatible

ross combinations in olive by measuring fruit set in response to

ross-pollination (Morettini, 1972; Cuevas et al., 2001; Lavee et al.,002; Moutier, 2002; Vuletin Selak et al., 2011). In a few cases,hey have complemented these studies with pollen tube growth

Abbreviations: DAP, days after pollination; ISI, index of self-incompatibility; PDC,ollen donor cultivar.∗ Corresponding author. Tel.: +385 21 434 436; fax: +385 21 316 584.

E-mail addresses: Gabriela.Vuletin.Selak@krs.hr,abriela@krs.hr (G. Vuletin Selak).

304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.10.041

© 2013 Elsevier B.V. All rights reserved.

tests (Bradley and Griggs, 1963; Cuevas and Polito, 1997; Wu et al.,2002; Seifi et al., 2011). Recently, molecular markers offer the pos-sibility to study compatibility by determining the paternity of theseedlings produced in pollination assays (Mookerjee et al., 2005;Díaz et al., 2006; Pinillos and Cuevas, 2009). These seed paternityexperiments have some flaws. For instance, pollination success isstrongly affected by the proximity of the pollen source, and a lowpercentage of seeds fathered by a given cultivar does not precludeits compatibility with the main cultivar, only its secondary role incomparison to another, perhaps overrepresented, pollen donor.

The progamic phase of reproduction in flowering plants encom-passes all processes from pollination to fertilization. This phaseis regulated by pollen-pistil interactions (Linskens, 1986) whichinvolve pollen tubes growth through the pistil on their way towardsthe ovule. Pollen tube growth studies have been employed tostudy compatibility relationships in many temperate zone andMediterranean fruit crops such as pear (Shaheen et al., 2011), apri-cot (Burgos et al., 1993; Egea and Burgos, 1996), plum (Nikolic

and Milatovic, 2010), mandarin (Gómez Alvarado et al., 2004),almond (Ben-Njima and Socias i Company, 1995; Ortega et al., 2002;Dicenta et al., 2002), cherry (Cerovic and Ruzic, 1992; Hedhly et al.,2005), Citrus species (Distefano et al., 2012) and olive (Bradley

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G. Vuletin Selak et al. / Scient

nd Griggs, 1963; Cuevas and Polito, 1997; Vuletin Selak et al.,013). Unfortunately, the information about the most successful,ross-compatible combinations of olive cultivars grown in Croatiand other growing regions is contradictory and scanty, especiallyegarding newly-introduced foreign cultivars (Vuletin Selak et al.,011). ‘Lastovka’ and ‘Oblica’ are the most popular olive oil culti-ars in Croatia. ‘Leccino’ is a productive Italian cultivar producingil of extraordinary flavour and taste. Cultivar Leccino is extensivelylanted in newly orchards to enhance Croatian olive oil production.

In view of the fact that pollen tube growth has been just occa-ionally used in studies on reproductive compatibility in olive,nd in order to establish reliable parameters to test self- andross-(in)compatibility, pollen-pistil interaction (pollen germina-ion, pollen tube growth and fertilization percentage) has beenssessed in the olive cultivars grown in Croatia. The cultivars

Lastovka’ and ‘Oblica’, as well the Italian cultivar ‘Leccino’ weressessed following self- and cross-pollination treatments. ‘Leccino’erved both as pollen acceptor and pollen donor in the experimentsith the aim to study its compatibility with long-established Cro-

tian cultivars. Pollen-pistil interaction appeared as a reliable modeo estimate compatibility in olive cultivars and therefore has impor-ance for worldwide olive growing which is experiencing changesn cultivars and growing regions.

. Materials and methods

.1. Experimental orchard and plant material

The experiments were conducted during 2009 and 2010 using5-year-old ‘Lastovka’, ‘Leccino’, and ‘Oblica’ olive trees growing in

multi-varietal orchard located in the surrounding area of Split,roatia (43◦31′42′′N, 16◦31′3′′E). In both experimental years, fourrees of each cultivar were selected for their high level of flower-ng and denoted as pollen acceptors. Cultivars ‘Lastovka’, ‘Leccino’,Levantinka’, ‘Oblica’, and ‘Pendolino’ were used as pollen donorsnd their pollen grains were applied in the experiments. ‘Oblica’ ishe most widespread olive cultivar in Croatia, suitable for both, oilnd table olive production, whereas cultivars ‘Lastovka’, ‘Leccino’,

Levantinka’, and ‘Pendolino’ are appropriate for oil production.he flowering periods of all cultivars were assessed according toarranco et al. (1994) in both seasons. All trees were managed

ollowing standard commercial practices.In 2009, the means of the daily air temperatures during full

loom (May 17–21) ranged from 24.3 to 26.5 ◦C. In the next year,he full bloom dates were from May 22 to May 27 and the means ofhe daily temperatures then were somewhat lower, between 19.9nd 21.8 ◦C.

.2. Pollination procedure

On each of four replicate trees of the cultivars denoted asollen acceptors, a high number of flowers were emasculated athe white balloon stage just before petals opening and coveredith paper bags to prevent uncontrolled pollination. Consistentower age was assured by removing the open and the immatureowers at the date of emasculation. The day after emasculationas considered the date at which experimental flowers were open

nd receptive. On this day, all emasculated flowers were hand-ollinated with pollen grains from different pollen donor cultivarsnd bagged again to prevent undesirable open pollination. ‘Las-ovka’, ‘Leccino’, ‘Levantinka’, ‘Oblica’, and ‘Pendolino’ were used

s pollen sources in the different cross-pollination treatments. Theollen was collected into Petri dishes at the day pollinations wereerformed, and applied on recipient flowers using a paint brush.he self-pollination treatment was performed pollinating the

ticulturae 165 (2014) 36–43 37

emasculated flowers at anthesis with their own pollen. Germina-tion of pollen applied in pollination assays was tested in vitro on agermination medium proposed by Perica et al. (2001). Pollen via-bility was assessed by fluorochromatic reaction test (Pinillos andCuevas, 2008) as an additional evidence of the quality of pollen.Pollen germination ranged from 4.0% to 24% in 2009 and from 2.3%to 10.0% in 2010 whereas pollen viability ranged from 94.9% to 98.0in 2009 and from 91.9% to 93.8% in 2010.

2.3. Assessment of pollen performance in vivo

Flowers of each pollination treatment (20 per sampling date)were collected every day from 1 to 7 d after pollination and fixed inFAA (formalin, acetic acid, and 70% ethanol at a ratio 1:2:17) for atleast 24 h, then washed with running water and softened with 0.8 MNaOH (Cuevas et al., 1994). After softening, the stigma and the styleof each pistil were cut apart from the ovary with transverse sectionat the end of the style, and then the ovules dissected from the ovary.The separated pistil organs were next stained with 0.1% aniline bluein phosphate buffer and examined under microscope (Axioskop 2plus, Carl Zeiss, Jena, Germany) equipped with UV excitation filters(FT 425 nm, LP 450 nm) to determine pollen germination, pollentube growth, and fertilization levels.

Pollen germination was analyzed on the intact stigma surface ofeach experimental flower previously stained with aniline blue. Thenumbers of adhered and germinated pollen grains were counted,and the percentage of pollen germination calculated. Pollen tubegrowth was analyzed on the squashed flowers and expressed asthe percentage of the flowers with the most advanced pollen tubereaching the base of the style. The fertilization level was expressedas the percentage of fertilized flowers. An experimental flower wasconsidered fertilized when the ovule penetration by a pollen tubewas observed in at least one of the four ovules developed withinthe ovary according to the procedures proposed by Cuevas et al.(1994). A self-incompatibility index (ISI) was calculated as the ratioof fertilization following self-pollination to that following cross-pollination as previously proposed for fruit set data (Zapata andArroyo, 1978).

2.4. Data analysis

The data for the percentage of pollen germination, pollen tubegrowth, and fertilization were arc sin transformed and then sub-jected to analyses of variance using generalized linear modelsin SAS software (SAS Institute Inc., Cary, NC, USA). Differencesbetween groups were determined by the LSD test considering val-ues of P ≤ 0.05 to be statistically significant.

3. Results

3.1. Pollen germination on the stigma following cross-pollinationversus self-pollination

Pollen of different donors was applied to the stigmas of therecipient cultivars and the percentage of in vivo pollen germinationwas considered as a measure of compatibility between differentolive cultivars. Higher pollen germination was found followingcross-pollination than after self-pollination in ‘Lastovka’ (Table 1),‘Leccino’ (Table 2) and ‘Oblica’ (Table 3) in most of the crosses per-formed in 2009 and 2010 (Fig. 1).

The percentage of pollen germination declined with timeelapsed from pollination (Tables 1–3). Thus, the highest percent-

ages of pollen germination were always found 1 d after pollinationin flowers of all recipient cultivars in all pollination treatments.Since pollination was only performed once at anthesis, and theflowers were rebagged to avoid undesirable free pollination, this

38 G. Vuletin Selak et al. / Scientia Horticulturae 165 (2014) 36–43

Table 1Pollen germination (%) of pollen donor cultivars in flowers of ‘Lastovka’ trees fol-lowing self- (בLastovka’) and cross-pollination (בLevantinka’) in 2009 and 2010.

DAPa (d) Pollen germination (%)

2009 2010

Pollen donor cultivar Pollen donor cultivar

Lastovka (self) Levantinka Lastovka (self) Levantinka

1 19.7 ab Bc 32.4 a A 28.4 a B 43.0 a A2 16.6 a B 29.9 a A 24.9 a A 26.0 b A3 8.7 b B 24.2 a A 14.5 b A 20.1 b AxPDCy 15.0 B 27.8 A 22.6 B 29.7 A

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.

c

diappba‘pst3cct(

Table 2Pollen germination (%) of pollen donor cultivars in flowers of ‘Leccino’ trees follow-ing self- (בLeccino’) and cross-pollination (בOblica’; בLastovka’; בLevantinka’;בPendolino’) in 2009 and 2010.

DAPa (d) Pollen germination (%)

Pollen donor cultivar

Leccino (self) Oblica Lastovka Levantinka Pendolino

20091 18.8 ab Ac 19.4 a A 18.8 a A 23.4 a A 30.1 a A2 13.3 a A 14.9 a A 13.6 ab A 15.7 ab A 20.2 ab A3 13.1 a A 14.9 a A 8.5 b A 10.3 b A 14.1 b AxPDCy 15.0 B 16.4 B 13.6 B 16.5 B 21.5 A

20101 16.2 a A 24.3 a A – – –2 8.6 ab B 19.1 ab A – – –3 2.9 b B 13.5 b A – – –xPDC 8.8 B 19.2 A – – –

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.c Mean values (n = 20) followed by different upper-case letters in each row indi-

Fs

c Mean values (n = 20) followed by different upper-case letters in each row indi-ate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

ecline only can be interpreted as the evidence of an increas-ngly difficult evaluation of pollen germination. Stigma cloggingfter heavy hand-pollination and fluorescence fading are the mostrobably causes of this artefact. In consequence, the values ofollen germination observed 1, 2 and 3 d after pollination wille considered as the most reliable measurements and comparedmong treatments. Cross-pollination of ‘Lastovka’ flowers with

Levantinka’ pollen resulted with higher germination of cross-ollen in 2009 (32.4%) and 2010 (43.0%) when compared toelf-pollen (19.7% in 2009 and 28.4% in 2010) 1 d after pollina-ion (DAP) (Table 1). These relations were maintained also 2 and

DAP in both years, although the significant differences were

onfirmed just for 2009. In 2009, pollen of various pollen donorultivars was applied to the stigmas of flowers from ‘Leccino’rees. The highest pollen germination was recorded for ‘Pendolino’30.1%) 1 DAP (Table 2). However, germination percentage for

ig. 1. Pollen germination and tube growth on the stigma 1 d after self-pollination (a) anelf-pollination (c); pollen tube growth and competition in the lower part of the style foll

cate significant differences at P ≤ 0.05 by the LSD test.y PDC = pollen donor cultivar.

‘Pendolino’ was not significantly different from the percentagefor ‘Levantinka’ (23.4%), ‘Oblica’ (19.4%), ‘Lastovka’ (18.8%) andself-pollen (18.8%) as recorded for two later dates after pollina-tion. In 2010, the germination percentage of self-pollen ‘Leccino’1 DAP (16.2%) was lower but not significantly than the percent-age for cross-pollination with ‘Oblica’ (24.3%). However, pollengermination of self-pollen ‘Leccino’ was significantly lower (8.6%and 2.9%) than the percentage recorded for cross-pollination with

‘Oblica’ (19.1% and 13.5%) 2 and 3 DAP, respectively. In ‘Oblica’flowers, the lowest pollen germination was observed for self-pollination (17.1%) 1 DAP in 2009 (Table 3). The percentage for

d cross-pollination (b); pollen tube growth in the lower part of the style followingowing cross-pollination (d).

G. Vuletin Selak et al. / Scientia Horticulturae 165 (2014) 36–43 39

Table 3Pollen germination (%) of pollen donor cultivars in flowers of ‘Oblica’ trees follow-ing self- (בOblica’) and cross-pollination (בLastovka’; בLeccino’; בLevantinka’;בPendolino’) in 2009 and 2010.

DAPa (d) Pollen germination (%)

Pollen donor cultivar

Oblica (self) Lastovka Leccino Levantinka Pendolino

20091 17.1 ab Ac 17.2 a A 22.7 a A 29.7 a A 26.4 a A2 8.6 a C 5.1 b C 14.1 a BC 23.2 a AB 28.9 a A3 10.5 a B 5.2 ab B 15.4 a AB 20.0 a AB 21.9 a AxPDCy 12.3 C 10.8 C 17.8 B 24.2 A 25.7 A

20101 52.5 a B 38.2 a B 40.6 a B 72.2 a A –2 25.9 b B 20.6 b BC 12.8 b C 61.8 a A –3 22.5 b A 21.6 b A 14.6 b A 24.8 b A –xPDC 33.4 B 26.8 C 22.9 C 52.9 A –

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.c

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Table 4Percentage of flowers with pollen tubes at the end of the style in ‘Lastovka’ flowersfollowing self- (בLastovka’) and cross-pollination (בLevantinka’) in 2009 and 2010.

DAPa (d) Pollen tube growth (%)

2009 2010

Pollen donor cultivar Pollen donor cultivar

Lastovka (self) Levantinka Lastovka (self) Levantinka

1 0 bb Bc 75.0 a A 10.3 b A 54.0 bc A2 19.8 ab B 100.0 a A 19.0 b A 42.9 c A3 41.3 a B 100.0 a A 29.4 ab B 89.7 ab A4 43.7 a B 95.2 a A 35.3 ab B 95.2 a A5 34.1 a B 88.9 a A 65.9 a B 100.0 a A6 59.5 a A 93.3 a A 69.0 a B 100.0 a A7 40.5 a A 90.0 a A 65.9 a B 100.0 a AxPDCy 34.1 B 91.8 A 42.1 B 83.1 A

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.

pollen or with pollen of ‘Lastovka’ in 2009. Cross-pollinations with‘Leccino’, ‘Levantinka’ or ‘Pendolino’ resulted with significantlyhigher pollen tube growth rates (76.6%; 91.0%; 96.8%, respectively).In 2010, pollination of ‘Oblica’ flowers with pollen of ‘Levantinka’

Table 5Percentage of flowers with pollen tubes at the end of the style in ‘Leccino’flowers following self- (בLeccino’) and cross-pollination (בOblica’; בLastovka’;בLevantinka’; בPendolino’) in 2009 and 2010.

DAPa (d) Pollen tube growth (%)

Pollen donor cultivar

Leccino (self) Oblica Lastovka Levantinka Pendolino

20091 81.0 ab Ac 84.1 a A 69.8 b A 95.2 a A 90.5 a A2 66.7 a B 84.9 a AB 55.6 b B 100.0 a A 95.2 a A3 91.1 a A 94.4 a A 80.2 ab A 100.0 a A 100.0 a A4 95.2 a A 100.0 a A 74.6 ab A 100.0 a A 95.2 a A5 86.9 a A 100.0 a A 94.4 ab A 100.0 a A 100.0 a A6 95.2 a A 100.0 a A 100.0 a A 100.0 a A 100.0 a A7 93.3 a A 100.0 a A 100.0 a A 100.0 a A 100.0 a AxPDCy 87.1 B 94.8 A 82.1 B 99.3 A 97.3 A

20101 75.6 b A 74.6 b A – – –2 90.5 ab A 69.0 b A – – –3 90.5 ab A 89.7 ab A – – –4 88.9 ab A 94.4 ab A – – –5 100.0 a A 94.4 ab A – – –6 100.0 a A 100.0 a A – – –7 100.0 a A 100.0 a A – – –xPDC 92.2 A 88.9 A – – –

a DAP = days after pollination.b

Mean values (n = 20) followed by different upper-case letters in each row indi-ate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

elf-pollination was not significantly different than the percent-ge recorded for cross-pollination with ‘Lastovka’ (17.2%), ‘Leccino’22.7%), ‘Levantinka’ (29.7%) and ‘Pendolino’ (26.4%). Two DAP,he differences between pollen donors were more pronouncednd a lower rate of germination was recorded for self-pollination8.6%). The highest percentage of pollen germination was recordedor ‘Pendolino’ (28.9%) and ‘Levantinka’ (23.2%) as pollen donors.hree DAP, the highest pollen germination was recorded again forross-pollination with ‘Pendolino’ (21.9%) and ‘Levantinka’ (20.0%),owever these germination percentages were not significantly dif-

erent from the percentage recorded for ‘Leccino’ (15.4%). In 2010,ross-pollination with ‘Levantinka’ resulted in the highest germi-ation (72.2% 1 DAP and 61.8% 2 DAP) on the stigmas of ‘Oblica’owers (Table 3).

.2. Pollen tube growth in the style following cross-pollinationnd self-pollination

After pollen germination on the stigma, pollen tubes continuedo grow through the style of the recipient cultivar. The dynamic ofollen tube growth was very different between self-pollination andross-pollination regardless the cross-pollen donors. Pollen tuberowth under cross-pollination treatments was massive, but verycarce under self-pollination (Fig. 1).

In both seasons, higher percentages of flowers with the pollenube at the end of the style were observed in most cross-pollinationreatments than with self-pollination (Tables 4–6). In spite of theigh number of pollen tubes growing in the stigma and upper partf the style, the number of tubes reaching the ovary was commonlyne, rarely two or a few more. The time required for the pollen tubef self-pollinated ‘Lastovka’ to reach the end of the style in at least0% of the flowers was higher (5–6 DAP) than for cross-pollinatedowers (1 DAP). Also the percentages of flowers with pollen tube athe end of the style were on average considerable higher observedfter cross-pollination (91.8% in 2009; 83.1% in 2010) than afterelf-pollination (34.1% in 2009; 42.1% in 2010) (Table 4).

The growth of pollen tubes in the style of ‘Leccino’ flowers wasery fast. At least 50% of the flowers had a pollen tube at the endf the style 1 d after pollination in the two years, both in self- and

ross-pollinations (Table 5). In 2009, the growth of pollen tubesf self-pollinated ‘Leccino’ was a slightly lower (87.1%) comparedo cross-pollinations with ‘Levantinka’, ‘Pendolino’ and ‘Oblica’99.3%, 97.3% and 94.8%, respectively). In the following season, the

c Mean values (n = 20) followed by different upper-case letters in each row indi-cate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

rate of growth of pollen tubes following self- and cross-pollinationwas equalized.

In both years, at least 50% of ‘Oblica’ flowers had at leastone pollen tube at the end of the style 3 d after self-pollinationwhereas equal or higher percentages were reached earlier aftercross-pollinations indicating also a more rapid growth rate of thepollen tubes in these crosses (Table 6). The highest percentagesof ‘Oblica’ flowers with pollen tube at the end of the style werefound after cross-pollination with ‘Pendolino’ (96.8%) in 2009 andwith ‘Levantinka’ (89.0%) in 2010 (Table 6). The pollen tube growthrate in ‘Oblica’ styles was the lowest when pollinated with its own

Mean values (n = 20) followed by different lower-case letters in each columnindicate significant differences at P ≤ 0.05 by the LSD test.

c Mean values (n = 20) followed by different upper-case letters in each row indi-cate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

40 G. Vuletin Selak et al. / Scientia Horticulturae 165 (2014) 36–43

Table 6Percentage of flowers with pollen tubes at the end of the style in ‘Oblica’flowers following self- (בOblica’) and cross-pollination (בLastovka’; בLeccino’;בLevantinka’; בPendolino’) in 2009 and 2010.

DAPa (d) Pollen tube growth (%)

Pollen donor cultivar

Oblica (self) Lastovka Leccino Levantinka Pendolino

20091 20.0 cb Cc 30.2 c BC 70.8 b AB 80.2 b A 88.9 a A2 41.9 bc B 44.4 bc B 58.3 b B 95.2 ab A 94.4 a A3 52.4 bc C 80.0 b BC 55.6 b C 95.2 ab AB 100.0 a A4 54.8 bc B 62.5 bc B 76.7 b B 83.3 ab AB 100.0 a A5 58.3 bc C 100.0 a A 75.0 b BC 83.3 ab ABC 94.4 a AB6 80.0 b B – 100.0 a A 100.0 a A 100.0 a A7 100.0 a A – 100.0 a A 100.0 a A 100.0 a AxPDCy 58.2 D 63.4 D 76.6 C 91.0 B 96.8 A

20101 29.4 b A 9.5 b A 5.6 c A 38.9 b A –2 34.9 b B 80.2 a AB 68.3 ab AB 100.0 a A –3 66.7 ab A 81.0 a A 55.6 b A 94.4 a A –4 74.6 ab A 73.0 a A 88.9 ab A 89.7 ab A –5 89.7 ab A 85.7 a A 72.2 ab A 100.0 a A –6 100.0 a A 100.0 a A 100.0 a A 100.0 a A –7 90.0 ab A 100.0 a A 100.0 a A 100.0 a A –xPDC 69.3 B 75.6 B 70.1 B 89.0 A –

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.

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Table 8Fertilization percentage (%) in flowers from ‘Leccino’ trees following self-(בLeccino’) and cross-pollination (בOblica’; בLastovka’; בLevantinka’;בPendolino’) in 2009 and 2010.

DAPa (d) Fertilization percentage (%)

Pollen donor cultivar

Leccino (self) Oblica Lastovka Levantinka Pendolino

20091 28.6 cb Ac 50.0 c A 55.6 bc A 55.6 b A 51.6 b A2 42.9 bc A 65.1 bc A 35.7 c A 50.8 b A 74.6 ab A3 55.4 abc A 68.3 abc A 80.2 abc A 79.4 ab A 95.2 a A4 67.3 abc A 94.4 ab A 65.1 bc A 65.9 ab A 90.5 a A5 82.7 ab A 100.0 a A 94.4 ab A 95.2 a A 100.0 a A6 85.7 ab A 100.0 a A 95.2 ab A 100.0 a A 100.0 a A7 93.3 a A 100.0 a A 100.0 a A 100.0 a A 100.0 a AxPDCy 65.1 D 82.5 AB 75.2 C 78.1 BC 87.4 A

20101 0 c A 15.1 c A – – –2 66.7 b A 42.1 bc A – – –3 73.8 b A 78.5 b A – – –4 61.1 b A 68.3 b A – – –5 88.9 ab A 77.8 ab A – – –6 100.0 a A 88.9 ab A – – –7 100.0 a A 100.0 a A – – –xPDC 70.1 A 67.2 A – – –

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.c

(Table 7). Similarly, self-pollination resulted in lower percentagesof fertilization in 2009 than due to most of the cross-pollinationtreatments in ‘Leccino’ (Table 8) and in ‘Oblica’ in 2009 and 2010

Table 9Fertilization percentage (%) in flowers from ‘Oblica’ trees following self- (בOblica’)and cross-pollination (בLastovka’; בLeccino’; בLevantinka’; בPendolino’) in 2009

c Mean values (n = 20) followed by different upper-case letters in each row indi-ate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

esulted with the highest and most rapid pollen tube growth rate;owever, there was no significant difference in the growth rateetween self- versus cross-pollination when ‘Lastovka’ and ‘Lec-ino’ were used as pollen donors.

.3. Fertilization success following cross-pollination andelf-pollination

After the pollen tubes have reached the base of the style, theyntered the ovary and continued with the growth towards thevules. Ovule penetration by a pollen tube always occurred via the

icropyle and generally in only one of the four ovules each olive

ower has. Fertilization, expressed in percentage of fertilized flow-rs, was significantly different among the various pollen donors inoth 2009 and 2010 (Tables 7–9).

able 7ertilization percentage (%) in flowers from ‘Lastovka’ trees following self-בLastovka’) and cross-pollination (בLevantinka’) in 2009 and 2010.

DAPa (d) Fertilization percentage (%)

2009 2010

Pollen donor cultivar Pollen donor cultivar

Lastovka (self) Levantinka Lastovka (self) Levantinka

1 0 bb Ac 12.5 b A 0 b A 0 c A2 15.1 ab B 55.6 a A 9.5 ab A 27.0 bc A3 30.2 a A 55.6 a A 15.1 ab A 50.0 ab A4 19.8 ab B 54.8 a A 29.4 ab A 69.8 ab A5 15.1 ab B 61.1 a A 45.2 a A 72.2 ab A6 26.2 a B 73.3 a A 34.1 a B 89.7 a A7 26.2 a B 70.0 a A 51.6 a A 72.2 ab AxPDCy 18.9 B 54.7 A 26.4 B 54.4 A

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

ndicate significant differences at P ≤ 0.05 by the LSD test.c Mean values (n = 20) followed by different upper-case letters in each row indi-

ate significant differences at P ≤ 0.05 by the LSD test.y PDC = pollen donor cultivar.

Mean values (n = 20) followed by different upper-case letters in each row indi-cate significant differences at P ≤ 0.05 by the LSD test.

y PDC = pollen donor cultivar.

In flowers of ‘Lastovka’ trees, the fertilization percent-age was significantly lower after self-pollination (18.9% in2009; 26.4% in 2010) than after cross-pollination with ‘Lev-antinka’ pollen (54.7% in 2009; 54.4% in 2010) in both seasons

and 2010.

DAPa (d) Fertilization percentage (%)

Pollen donor cultivar

Oblica (self) Lastovka Leccino Levantinka Pendolino

20091 10.0 bb Ac 0 b A 16.7 b A 15.1 c A 9.5 c A2 17.8 ab A 27.8 ab A 20.8 b A 63.7 abc A 49.2 bc A3 9.5 b B 60.0 a AB 22.2 b AB 42.1 bc AB 61.9 ab A4 9.5 b B 37.5 ab AB 23.3 b AB 33.3 bc AB 65.1 ab A5 41.7 ab A 80.0 a A 50.0 b A 66.7 abc A 60.0 abc A6 60.0 ab A – 100.0 a A 80.0 ab A 100.0 a A7 70.0 a A – 100.0 a A 100.0 a A 100.0 a AxPDCy 31.2 C 41.1 C 47.6 B 57.3 AB 63.7 A

20101 0 b A 0 c A 0 c A 0 c A –2 15.1 ab A 31.0 bc A 29.4 bc A 35.7 b A –3 41.3 ab A 41.3 ab A 27.8 bc A 59.5 b A –4 50.0 a A 63.5 ab A 66.7 ab A 73.8 ab A –5 55.2 a A 66.7 ab A 66.7 ab A 79.4 ab A –6 70.0 a A 71.4 ab A 100.0 a A 83.3 ab A –7 70.0 a A 84.6 a A 100.0 a A 100.0 a A –xPDC 43.1 B 51.2 B 55.8 A 61.7 A –

a DAP = days after pollination.b Mean values (n = 20) followed by different lower-case letters in each column

indicate significant differences at P ≤ 0.05 by the LSD test.c Mean values (n = 20) followed by different upper-case letters in each row indi-

cate significant differences at P ≤ 0.05 by the LSD test.y PDC = pollen donor cultivar.

G. Vuletin Selak et al. / Scientia Horticulturae 165 (2014) 36–43 41

Fig. 2. Flowering periods in ‘Lastovka’, ‘Leccino’, ‘Levantinka’, ‘Oblica’ and ‘Pendolino’ during 2009 (horizontal lines) and 2010 (diagonal lines); black filled area denotes fullb

(eiwccfwdirtws(

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loom period.

Table 9). The highest fertilization percentage for ‘Leccino’ flow-rs was recorded after cross-pollination with ‘Pendolino’ pollenn 2009 (87.4%); however, the highest percentage for fertilization

as not significantly different from the percentage observed afterross-pollination with ‘Oblica’ pollen (82.5%). In 2010, no signifi-ant differences were found between the fertilization percentagesollowing self-pollination of ‘Leccino’ flowers and cross-pollinationith ‘Oblica’ pollen. Cross-pollination of ‘Oblica’ flowers with ‘Pen-olino’ pollen resulted with the highest percentage of fertilization

n 2009 (63.7%) and cross-pollination with ‘Levantinka’ polleneached a similar percentage (57.3%) (Table 9). In season 2010,he fertilization percentage in ‘Oblica’ flowers was the highesthen ‘Levantinka’ (61.7%) and ‘Leccino’ (55.8%) were used as pollen

ources. Self-fertilization was significantly reduced in both seasonsTable 9).

Self-incompatibility index was used for assessing the degreef self-incompatibility in cultivars meaning the higher value ofSI higher degree of self-compatibility was shown by the geno-ype. The ISI calculated for ‘Lastovka’ in 2009 and 2010 was 0.4nd 0.5, respectively. Higher values of ISI (less self-incompatibilityxpressed) were found for ‘Leccino’ (0.7 in 2009 and 1.0 in 2010)nd ‘Oblica’ (0.5 in 2009 and 0.7 in 2010) than for ‘Lastovka’. Inter-ncompatibility reactions between the studied cultivars were notbserved in any process from pollen germination to fertilization.

The differences between the percentages of flowers with pollenube at the end of the style and those reaching the ovule indicateshat further impediment for pollen tube growth occurred at thentrance of the ovary or within the ovary (Tables 4–9).

.4. Flowering periods of studied cultivars

The flowering periods of the studied cultivars overlapped toome degree in both experimental years (Fig. 2). In 2009, ‘Leccino’,Levantinka’, ‘Oblica’, and ‘Pendolino’ had simultaneous floweringnd full bloom was coincident; however, the flowering period fin-shed one day earlier in ‘Levantinka’ and ‘Pendolino’. In both years,

Lastovka’ started flowering before the other cultivars. In 2010, theowering period was sparser; however, ‘Leccino’ and ‘Pendolino’,wo Italian cultivars recently introduced in Croatia, overlapped inowering in a high degree.

4. Discussion

Many flowering plants have evolved genetically self-incompatibility (SI) mechanisms to prevent self-fertilizationand promote out-crossing (De Nettancourt, 1977). According toLinskens (1975), the incompatibility may become apparent on thestigma so that pollen do not germinate, as in sporophytic SI (SSI), orin the style, as in gametophytic SI (GSI) when pollen tube growthis arrested in the style. The genetic of SI in olive remain largelyunknown in part due to olive allopolyploid origin. In our study,the self-incompatibility response of the olive cultivars ‘Lastovka’,‘Leccino’, and ‘Oblica’ was apparent as scarce pollen tube growth.Incompatible tubes burst or simply ceased growth shortly aftertubes had penetrated the stigma surface as it was previouslyreported for ‘Manzanillo’ (Cuevas and Polito, 1997). Inhibition ofpollen tube growth shortly after germination after self-pollinationsuggests SI in olive is of the gametophytic type, even though thesite of self-pollen rejection takes place mostly in the stigma, asin most species having SSI. However recent studies question thisclassification and suggest that SSI system occurs in olive (Bretonand Bervillé, 2012; Collani et al., 2012). In the first study, thegenetic mode of inheritance of SI is deciphered and the authorssuggest that SI in olive may corresponds to the sporophytic model.A molecular approach failed to find the genes responsible for GSIin the second study. Furthermore, the identification of the maingenes known to play a crucial role in SSI (S-locus receptor kinaseand S-locus glycoprotein, the female determinants; and S-locuscysteine rich protein, the male determinant) indicates that SSI maybe present in olive.

Although the expression of SI in the olive and hence the conse-quences on yield greatly depends on environmental and growingconditions (Bradley and Griggs, 1963), the inter-planting of com-patible pollinizers of simultaneous blooming is advisable. Unfortu-nately, the information on the successful cultivars combinations issometimes not available and a reliable method to test incompati-bility relationships between cultivars is still incomplete, since olive

S-alleles have not been identified. Based on the evidence that pollentube performance was affected by the pollen genotype, we sortedout the most successful combinations of cultivars from all crossingsperformed in our study. Fertilization was significantly enhanced

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2 G. Vuletin Selak et al. / Scient

n ‘Lastovka’ when pollinated by ‘Levantinka’. The most efficientollen donors for ‘Leccino’ were ‘Pendolino’ and ‘Oblica’. ‘Leccino’as also an efficient pollen donor for ‘Oblica’ confirming the reci-rocity in compatibility relationships, although the most efficientollen donors for ‘Oblica’ were ‘Levantinka’ and ‘Pendolino’. Theesults on successful crossings mentioned above are in agreementith results on fruit set observed earlier in a study carried out on

live cultivars grown in Croatia (Vuletin Selak et al., 2011). Simulta-eous flowering, important criteria for the selection of pollinizers,as confirmed for all compatible crosses in both seasons (Fig. 2).

Pollen-pistil interaction, which includes pollen germination,ollen tube growth rate and pollen competition, is an importantomponent of fertilization success in flowering plants (Hedhlyt al., 2005). Pollen tube behaviour was efficiently used to studyin)compatibility relationships in many fruit species (Burgos et al.,993; Ben-Njima and Socias i Company, 1995; Dicenta et al., 2002;rtega et al., 2002; Sanzol and Herrero, 2002; Hedhly et al., 2005;istefano et al., 2009). In our study, pollen-pistil interaction was

tudied and pollen tube growth rate was considered as a fastrocedure to assess compatibility between different olive culti-ars grown in Croatia. There are three main structures along theollen tube pathway: stigma, style and ovary that can act as rejec-ion sites for self-pollen. The stigma provides the environmentn which pollen grains germinate; however, it can also constrainollen germination and pollen tube growth. The last statement wasonfirmed in our study since percentages of germination in vivoiffered among pollen donors and lower rates were ascribed to self-ollination. It seems as self-incompatibility mechanisms in olivere already active inhibiting pollen germination in some degree.onetheless, pollen germination was not limiting for reproductive

uccess under self-pollination since it was not completely avoided.Incompatibility reactions can become evident at different levels

f the style in flowers of different fruit species. In Citrus species,emperature influence can change the place of incompatibilityxpression (Distefano et al., 2012). In sweet cherry, stigma appearso buffer genetic differences among pollen donor genotypes, how-ver further pollen tube growth in the style clearly distinguishedale genotypes according to efficiency of their pollen tubes in

eaching the base of the style of recipient cultivar (Hedhly et al.,005).

Important differences in pollen tube growth rate through thetyle were observed between different pollen donors in olive. Aeduction in pollen tube number started after germination in thetigma, but reached its highest expression in the growth of theollen tubes in the stigma and in the style. Stigma and style thusppears as the main sites for pollen genotype selection, althoughhe length of the style in olive flower is very short. Anothereduction was evident after pollen tubes entered the ovary. Conse-uently, the percentages of fertilized flowers were lower than theercentages of flowers in which at least one pollen tube crossedhe style. The amount of reduction between percentages for bothypes of pollination (self- and cross-) in ‘Lastovka’ ranged between5.2% and 37.1%, between 9.9% and 22.1% for ‘Leccino’ and 26.2%nd 33.1% for ‘Oblica’. However, high correlation coefficient (r = 0.9)as found between the percentages of flowers with pollen tubes

eaching the ovary and of fertilization indicating that predictionsf the most successful combinations of cultivars are possible at ear-ier events than fertilization. Besides the pollen-pistil interactions,he specific pistil structures can regulate the growth rate of pollenubes on their way to ovules. Pollen tubes in olive flowers reachedhe ovule 1–2 d after pollination. However, the pollen tubes in theeach flowers reached the base of the style 7 d after pollination and

ertilization took place 12 d later (Herrero and Arbeloa, 1989).

Incompatibility reaction of each olive cultivar was calculatedy determining their ISI values. Olive cultivars showing ISI val-es consistently lower than 0.1 are classified as completely

ticulturae 165 (2014) 36–43

self-incompatible (Androulakis and Loupassaki, 1990). In our study,the quoted ISI values classified both Croatian cultivars (‘Lastovka’and ‘Oblica’) as partially self-incompatible since a positive responseto cross-pollination was evident in both years. Cross-pollinationimproved fertilization levels in ‘Leccino’ flowers in 2009, but not in2010. Pollen germination on ‘Leccino’ flowers was similar in all pol-lination treatments in both years; pollen tube growth was also fastin self-pollinated flowers of ‘Leccino’. Based on fruit set measure-ments, ‘Leccino’ was reported as self-incompatible by Tombesi et al.(1982). In earlier study, lower ISI ranging from 0.1 to 0.3 was foundfor ‘Leccino’ under Croatian conditions when fruit set data wereused in calculation (Vuletin Selak et al., 2011). Inter-incompatibilityreactions between the studied cultivars were not observed, how-ever such reactions were confirmed for olive in particular crossesby Seifi et al. (2011).

In conclusion, the results on pollen-pistil interaction after con-trolled pollinations provided an excellent, reliable, and quick meansto test compatibility for the most important olive cultivars. Pollentube behaviour distinguished pollen donors according to readinessof their pollen to germinate or to develop tubes and accomplish fer-tilization when applied to stigma of the recipient cultivar, and couldbe used to assess pollinizer’s effectiveness. Nonetheless, pollenproduction and viability are also important and should be eval-uated when selecting the best pollen donors. Moreover, in olive,a non-alternate bearing habit is also a desirable character in thepollinizers in order to provide a reliable source of pollen to the mainvariety.

Acknowledgements

The authors would like to acknowledge the support of theCroatian Ministry of Science, Education and Sports (grant no.091-0910468-0166), the Croatian Ministry of Agriculture, and theSplit-Dalmatia County. We gratefully acknowledge the contrib-utions of Ana Vidak and thank her for technical assistance.

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