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Romanian Biotechnological Letters Vol. 18, No.3, 2013 Copyright © 2013 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 18, No. 3, 2013 8341

In vitro pollen germination and pollen grain morphology in some sweet cherry (Prunus avium L.) cultivars

Received for publication, September 5, 2012

Accepted, February 9, 2013

SANJA RADIČEVIĆ1*, DRAGAN NIKOLIĆ2, RADOSAV CEROVIĆ1, MILENA ĐORĐEVIĆ1 1Department of Pomology and Breeding, Fruit Research Institute, Kralja Petra I 9, 32000 Čačak, Serbia 2Department of Fruit Science and Viticulture, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade – Zemun, Serbia E-mail: [email protected] *Corressponding author: Sanja Radičević, Department of Pomology and Breeding, Fruit Research Institute, Kralja Petra I 9, 32000 Čačak, Serbia; phone: +381 32 221 413; fax: +381 32 221 391; e-mail: [email protected]

Abstract

In vitro pollen germination on agarose-sucrose medium, and pollen grain morphology by scanning electron microscopy, were investigated in four sweet cherry (Prunus avium L.) cultivars (‘Karina’, ‘Kordia’, ‘Regina’ and ‘Summit’). The highest percentage of in vitro pollen germination over a three-year period was observed in ‘Regina’ (46.60±3.58%), and the lowest in ‘Kordia’ (26.68±1.59%). The results suggested a major influence of cultivars specificities which induced the different response to experimental conditions among years.

The differences in morphological characters of pollen grains – size and shape (length, width and length/width - L/W ratio), germinal furrows (number and shape, length, width, distance between furrows) and exine ornamentation (the number of ridges in 100 μm2 of exine, ridges and stria width) were significant enough to separate and identify the investigated cultivars. When all parameters were considered together, the pollen of each cultivar exhibited a unique pattern. The results also suggested a relationship between pollen grains properties and origin of the studied sweet cherry cultivars. The most significant differences were observed among ‘Summit’ (North American cultivar) and the other three European cultivars investigated. The highest similarity in pollen grain characteristics was observed between ‘Karina’ and ‘Regina’, originating from the same parental combination.

Key words: Prunus avium L., pollen viability, SEM, pollen grain, morphological characterization Introduction Pollen grains represent a highly reduced haploid male gametophyte generation in flowering plants consisting of only two or three cells when released from the anthers [1]. In breeding work of various fruit species, and specifically when determining cultivar composition, it is of utmost importance to use a suitable method for determining pollen viability. The three major methods for analyzing pollen include descriptive studies, viability tests and physiological tests [2]. Standard viability tests involve both in vitro and in vivo pollen germination as well as the direct determination of viability of non-germinated pollen grains using diverse chemical tests [3]. The examination of validity of seven pollen viability tests in Prunus species showed that two of the tests – in vitro germination in hanging drop as well as on agarose-sucrose medium – suggested the highest level of validity and high correlation in respect of assessment of pollen viability [4].

SANJA RADIČEVIĆ, DRAGAN NIKOLIĆ, RADOSAV CEROVIĆ, MILENA ĐORĐEVIĆ

8342 Romanian Biotechnological Letters, Vol. 18, No. 3, 2013

Pollen quality in different fruit species is one the key aspects of reproduction process which to a great extent influences its efficacy and consequently realization of the cropping potential. Pollen quality varies among cultivars within a particular species [5]. As Pirlak [6] reports, good in vitro pollen germination in sweet cherry and apricot cultivars grown on some localities and at a particular incubation temperatures can be the first indicator of good adaptability to agro-environmental conditions, from the standpoint of regularity of phases within the fertilization process. On the other hand, the importance of pollen morphology in clarifying the classification of many plants has been recognized from taxonomists, paleobotanists and breeders. Examination of pollen by scanning electron microscope (SEM) of different tree fruits revealed differences in pollen size, coarseness, and depth of ridges and striation of exine [7, 8]. Pollen ultrastructure has been used to distinguish different cultivars in apple [9], pear [10], strawberry [11], walnut [12], pecan [13], chestnut [14], grape [15], and olive [16]. Authors reported differences in exine patterns between some cultivars, but not all cultivars exhibited large differentiated patterns. The evaluation of pollen morphological characteristics can be an adequate method for cultivar identification in Prunus species and their cultivars [7, 17, 18, 19]. Pollen morphological characterization and determination of several Prunus genotypes using SEM in apricot [20], sour cherry [21] and almond [22, 23, 24] was done. The present study was therefore undertaken to examine in vitro pollen germination in four sweet cherry cultivars holding prominent position in commercial plantations across Europe and globally, and which are gaining importance in the Balkan region. The results obtained can be beneficial in the breeding work when using the cultivars as pollenizers, as well as in trials dealing with reproductive biology in sweet cherries. The study also aims to characterize pollen morphology in the studied sweet cherry cultivars as it has not as yet been characterized using SEM. This is for the objective of developing a plant material identification tool, which is of great importance for both germplasm certification and breeding purposes. Materials and Methods Plant material and experimental design Four sweet cherry (Prunus avium L.) cultivars were used in this study viz., ‘Karina’, ‘Regina’ (originating from Germany), ‘Kordia’ (Czech Republic) and ‘Summit’ (Canada). The trial was carried out near Čačak (43°53' N; 20°21' E; 350 m above the sea), Western Serbia, which is a typical area for fruit cultivation in Serbia. Climate is temperate continental, with an average annual temperature of 10.85°C and an average temperature over growing period (April–October) of 16.8°C. Total annual rainfall rate is 748.4 mm. Cultivars were grafted on Gisela 5 rootstock. The trial of plant material was established in 2005, in an experimental orchard in a randomized block design with three trees in three replications, at distance 4.0 × 1.5 m. In vitro pollen germination test

For the examination of pollen viability in the cultivars studied in vitro pollen germination test on agarose-sucrose medium was used. The study was conducted over 2008–2010. Branches with flowers in late balloon phenophase were used for the testing. Some 300 flowers per each cultivar were taken in three replications, each replication involving 100 flowers from all sides of three cherry trees. The anthers were collected in laboratory and were kept in paper boxes for 24–48h at a temperature of 20ºC, i.e. by the time the anthers burst



open and released pollen grains. Pollen of each cultivar was plated in three Petri dishes on nutrition medium (1% agar and 12% sucrose). After the 24 hour incubation at 20ºC the germinated pollen grains were counted in three fields of view under the microscope OLYMPUS BX61 (light regime). A single field of view included about 100 pollen grains. Pollen grains that germinated exceeding their radius were considered as germinating grains [2]. Germination percentage by years of study was determined as the average for three replications (the total of nine microscopic fields of view). Pollen grain morphological characterization This experiment was conducted in season 2009. Some 300 flowers of each cultivar at late balloon stage were collected in three replications and placed into paper bags (one replication included 100 flowers from all sides of three cherry trees). Anthers were placed in Petri dishes and dried at room temperature until burst and pollen dusting. Thirty pollen grains (three samples) from each cultivar were observed; the sample included ten randomly collected pollen grains obtained from the mixture of pollen of three mature cherry trees. Pollen was collected in small vials and stored at 3–5ºC in desiccators containing silica-gel until the required SEM analysis. Flowers, anthers and pollen were carefully manipulated so as to avoid any contamination of samples by pollen. Small quantities of pollen grains were placed by fine brush on the double-sided transparent tape mounted on the microscope stage. Pollen samples were coated with 0.02 μm gold alloys in a sputter-coater BAL-TEC SCD 005 and monitored at 15 kV with a JEOL JSM-6390LV SEM. Damaged or poorly positioned pollen grains were excluded. Pollen grains were observed and photographed at 2,000X (whole grain) and 15,000X (for exine pattern characteristics). The following parameters were monitored: pollen shape and size (length, width and length/width - L/W ratio), germinal furrows characteristics (number and shape, length, width, distance between furrows), and exine ornamentation characteristics (the number of ridges in 100 μm2 of exine, ridge and stria width). Data analysis

Standard error was calculated for the measured parameters. The data were statistically analyzed using two-factor and one-factor analysis of variance (ANOVA) for in vitro pollen germination testing and investigation of pollen morphology using SEM, respectively. The significance of differences among mean values was determined by Duncan’s multiple range test at P<0.05. Data analysis was done by using SPSS statistical software package, Version 8.0 for Windows (SPSS. Inc., Chicago, IL). Results and Discussions In vitro pollen germination

The average values of in vitro pollen germination recorded over the three-year period were highest in ‘Regina’ (46.60%) (Table 1). The other cultivars had relatively low average pollen germination (not exceeding 30%), the lowest being in ‘Kordia’ (26.68%). Analysis of variance showed that variability factors and their interactions had a highly significant influence on pollen germination in the cultivars studied (Table 1). Pollen germination varied among years of study, maximum and minimum values not being uniform among years. Thus the highest in vitro pollen germination in ‘Regina’ was recorded in season 2010 (68.15%), while ‘Karina’ performed poorest in the same year (20.31%). Therefore, the occurrence of opposite tendencies and the lack of parallelism, were observed among major factors and their modalities. The effect of the factors studied on in vitro pollen germination cannot be



considered as independent, i.e. the differences among cultivars observed by years of study depended on interaction effects among variability factors.

There are reports in literature on the occurrence of variation in in vitro pollen germination in sweet cherry cultivars by years [25]. Regularity in microsporogenesis is directly associated with vitality and in vitro pollen germination [26], therefore the different factors that affect pollen formation and their varying impact on certain cultivars can induce non-uniform germination. In addition, the tendencies above may also be due to the experimental conditions affecting in vitro pollen germination, such as incubation temperature and composition of the medium, along with the different, genotype-induced requirements of each cultivar studied in relation to the specific conditions in the trial. It is well known that the temperatures within the range of 15–20°C are generally optimal for in vitro pollen germination in apricot [27] and sour cherry [28] cultivars. Optimal temperature for in vitro pollen germination may depend on flowering time of the cultivars (early or late) [6]. Beyhan & Karakaş [29] report on the best in vitro pollen germination in seven sweet cherry cultivars on medium containing 20% sucrose concentration, whereas the results obtained on media supplemented with 15% and 10% sucrose content were poorer, and in agreement with our results. The results obtained for in vitro pollen germination of ‘Kordia’ and ‘Summit’ are in agreement with some earlier results relative to in vitro pollen germination done in the same cultivars under climate conditions of Western Serbia [30].

Table 1. Percentage of in vitro pollen germination in sweet cherry (Prunus avium L.) cultivars

In vitro pollen germination (%)

‘Karina’ 28.27±1.73 b* ‘Kordia’ 26.68±1.59 b ‘Regina’ 46.60±3.58 a

Cultivar(A)

‘Summit’ 28.87±2.02 b 2008 31.07±1.56 b 2009 29.65±1.34 b Year

(B) 2010 37.10±3.48 a

2008 36.83±0.88 b 2009 27.67±2.17 cde‘Karina’ 2010 20.31±1.55 ef 2008 34.49±1.85 bc 2009 20.79±1.66 ef ‘Kordia’ 2010 24.76±1.76 def2008 34.48±0.50 bc 2009 37.18±2.59 b ‘Regina’ 2010 68.15±0.83 a 2008 18.49±1.10 f 2009 32.95±2.46 bcd

A × B

‘Summit’ 2010 35.17±2.63 bc

*Mean values followed by the different lower-case letters in column represent significant differences at P < 0.05 according to Duncan’s multiple range test Pollen grain morphological characterization All the sweet cherry cultivars studied were described as having monad pollen, elliptical, with iso-polar symmetric and three ectoapertures (tricolporate) (Figure 1).



Generally, the pollen morphology was similar among the cultivars (Table 2). Polar diameter (length) varied from 51.08 μm (‘Regina’) to 53.14 μm (‘Summit’), as well as equatorial diameter (width) from 24.09 μm (‘Regina’) to 26.06 μm (‘Summit’). The comparison of pollen grain diameter among ‘Summit’ and the other cultivars revealed significant differences. Differences in L/W ratio were greater, ranging from 2.039 (‘Summit’) to 2.126 (‘Karina’). L/W ratio did not distinguish all the cultivars, as ‘Karina’ was not significantly different from ‘Regina’. Pollen shape in all the studied sweet cherry cultivars was identified as perprolate (L/W ratio >2) [31].

Figure 1. Pollen grain (equatorial and polar view) in sweet cherry (Prunus avium L.) cultivars: a -‘Karina’; b - ‘Kordia’; c - ‘Regina’ and d - ‘Summit’ Table 2. Morphological characteristics of pollen grains in sweet cherry (Prunus avium L.) cultivars

Pollen grain diameters Cultivar

Length (μm) Width (μm) L/W ratio ‘Karina’ 52.01±0.27b* 24.48 ±0.35 b 2.126±0.020 a ‘Kordia’ 51.26±0.44 b 24.68±0.58 b 2.079±0.031 ab ‘Regina’ 51.08± 0.31b 24.09±0.26b 2.121±0.010 a ‘Summit’ 53.14±0.31a 26.06±0.13 a 2.039±0.010 b

*Mean values followed by different lower-case letters in columns represent significant differences at P<0.05 according to Duncan’s multiple range test



Germinal furrows extended the length of the grain. Germinal furrow length ranged from 43.08 μm (‘Regina’) to 45.38 μm (‘Summit’), while germinal furrow width varied only slightly, ranging from 1.33 μm (‘Regina’) to 1.45 μm (‘Summit’) (Table 3). The distance between furrows was the smallest in ‘Summit’ (13.94 μm) and greatest in ‘Regina’ (14.83 μm). Non-significant results were obtained by analysis of germinal furrow width. As for germinal furrow length and distance between furrows, ‘Karina’ was not significantly different from ‘Regina’ and ‘Kordia’ respectively. Table 3. The characteristics of germinal furrows of the pollen grains in sweet cherry (Prunus avium L.) cultivars

Germinal furrows characteristics

Cultivar Length (μm) Width (μm) Distance between

furrows (μm) ‘Karina’ 43.78±0.71 b 1.40±1.15 a 14.22±0.23 ab ‘Kordia’ 44.21±0.54 ab 1.35±0.24 a 14.07±0.18 ab ‘Regina’ 43.08±0.21 b 1.33±0.10 a 14.83±0.28 a ‘Summit’ 45.38±0.19 a 1.45±0.26 a 13.94±0.29 b

*Mean values followed by different lower-case letters in columns represent significant differences at P<0.05 according to Duncan’s multiple range test The exine ridging was generally longitudinal (Figure 2), with more parallel (‘Karina’) or less parallel ridges (‘Summit’). The ornamentation of pollen surface can be classified as striate. Number of ridges crossing 100 μm2 of exine equatorial region ranged from 18.67 (‘Regina’) to 20.33 (‘Summit’) (Table 4). Mean ridge widths varied slightly, ranging from 0.36 μm (‘Summit’) to 0.40 μm (‘Karina’ and ‘Kordia’). Mean striae widths were found to be close to the mean of ridge, width being the smallest (0.45 μm) in ‘Summit’ and the greatest (0.56 μm) in ‘Karina’. Not significant results were obtained by ridge width analysis. ‘Kordia’ did not significantly differ from ‘Karina’ in number of ridges in 100 μm2 of exine, as well as from ‘Summit’ with regard to striae width. The pits (perforations less than 1 μm in diameter) are more pronounced at exine surface in ‘Kordia’ than in other three cultivars, particularly ‘Regina’, which is almost without pits (Figure 2).

Figure 2. Pollen exine pattern in sweet cherry (Prunus avium L.) cultivars: a - ‘Karina’; b - ‘Kordia’; c - ‘Regina’ and d - ‘Summit’



The size of pollen grain is generally less than 0.1 mm in diameter, but there is wide variation among plant species [32]. Although pollen shape of different species varies, a great majority of pollen is basically spherical or oval. The pollen shape of Prunus species is described as truncate to elliptical in almond [24]; cylindrical in peach and plum [8]; elliptical or obtuse-triangular in apricot [20]. According to L/W ratio, the pollen shape of studied sweet cherry cultivars is more elongated than in other Prunus species – apricot [8, 20], peach and plum [8], almond [24] and sour cherry [21].

Table 4. Exine pattern characteristics in sweet cherry (Prunus avium L.) cultivars

Cultivar No. of ridges in 10 μm

Mean ridge width (μm)

Mean stria width (μm)

‘Karina’ 20.00±0.58 ab* 0.40±0.019 a 0.56±0.013 a ‘Kordia’ 20.00±0.58 ab 0.40±0.003 a 0.46±0.007 b ‘Regina’ 18.67±0.33 b 0.39±0.007 a 0.49±0.044ab ‘Summit’ 20.33±0.33 a 0.36±0.024 a 0.45±0.032 b

* Mean values followed by different lower-case letters within a column represent significant differences at P<0.05 according to Duncan’s multiple range test Analysis of measured parameters indicated that morphological variability was lower in sweet cherry than in other Rosaceae species such as apple [9] and strawberry [11], which may be due to its lower ploidy level (only 16 chromosomes). Sorkheh & al. [24] obtained similar results in diploid almond cultivars. Previous results obtained in apple, plum, peach, strawberry and raspberry cultivars suggested direct relationship between pollen size and ploidy level [17, 11]. Mulas & al. [23] showed stability of pollen traits; ultrastructural morphology of almond pollen does not vary according to rootstock used, irrigation or geographical location. The differences in morphological characters of pollen grains and exine pattern characteristics were significant enough to separate and identify studied sweet cherry cultivars. ‘Summit’ had the largest pollen grains, the lowest L/W ratio, so its shape was less elongated; the germinal furrow length and width were the highest, while the distance between germinal furrows was the lowest. Pollen grains in ‘Summit’ showed very narrow ridges and the highest number of ridges in 100 μm2 of exine. ‘Kordia’ was pitted over the entire surface of pollen grains; its pollen grains were more similar to those in ‘Karina’ and ‘Regina’ than to pollen grains in ‘Summit’. The highest L/W ratio and stria width was observed in ‘Karina’. ‘Regina’ had the smallest pollen grains, the lowest germinal furrows length and width, the greatest distance between germinal furrows and the lowest number of ridges. The results also suggested the relationship between pollen grains characteristics and origin of the studied sweet cherry cultivars. The most significant differences are among the ‘Summit’ (North American cultivar) and other three European cultivars (‘Kordia’, ‘Karina’ and ‘Regina’). The highest similarity of pollen grain characteristics was observed in ‘Karina’ and ‘Regina’, which are not just of the same geographic origin, but also from the same parental combination (both were derived from the cross of ‘Schneiders Späte’ × ‘Rube’). Differences between these cultivars are significant in distance between germinal furrows, number of ridges and particularly in the stria width. Lisek & Rospara [33], using ISSR-PCR technique to determine the genetic similarity among 24 sweet cherry cultivars, reported the highest level of genetic similarity between ‘Karina’ and ‘Regina’. Conclusions



The examination of in vitro pollen germination in the studied sweet cherry (Prunus avium L.) cultivars suggested a major impact of cultivars specificities which induced the different response to non-uniform environmental factors by years. The investigations inferred that the best adaptability to experimental conditions showed ‘Regina’. Pollen grains examination by SEM indicated that several morphological parameters (size, shape, exine characteristics) can be used to distinguish pollen from the studied sweet cherry genotypes, as well as to trace the origins of pollen by honeybees in commercial orchards and study gene flow. Although the investigations indicated minor differences among cultivars, given all the parameters, the pollen of each cultivar exhibited a unique pattern. Pollen morphological characterization, along with other morphological characteristics of sweet cherry trees, could be of great importance for identification studies (plant breeders, gene banks), and helpful in studying gene flow. Acknowledgements

This work was conducted under Research Project TR31064-Development and preservation of genetic potential of temperate zone fruits, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. References

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