Pollen grain micromorphological characterisation of four plum (Prunus domestica L.) genotypes from two different breeding programmes (Fruit Research Institute, Serbia, and Hohenheim, Germany) was performed using scanning electron microscopy (SEM). The studied characteristics were pollen size and shape, colpus length, mesocolpium width as well as exine characteristics. All genotypes had tricolpate pollen grains and striate exine ornamentation, with more parallel longitudinal ridges. The most significant differences among the analysed genotypes concerned the length of the pollen grain and exine sculpturing. The genotypes 'Pozna Plava', 'Presenta' and 'Čačanska najbolja' had the largest pollen grains (> 57 µm), whereas 'Hanita' showed a smaller pollen size, below 54 µm. 'Pozna Plava' had perprolate pollen in the equatorial view (L/W ratio 2.06), while the other three genotypes had prolate shaped pollen. By analysing the pollen morphological characteristics of the plum varieties in this paper, primarily the shape, size, length of colpi and, most importantly, the sculpture of the exine, each variety showed a specificity which can be used together with other morphological characteristics for description and characterisation

Cerović, Radosav

Glišić, Ivana

Milošević, Nebojša

Nikolić, Dragan

Radičević, Sanja

Đorđević, Milena

Proceeding of the Bulgarian Academy of Sciences

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Using Scanning Electron Microscopy to Characterise Plum (Prunus domestica L.) Genotypes

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Доклади на Българската академия на наукитеComptes rendus de l’Académie bulgare des SciencesTome 73, No 10, 2020BIOLOGYBotanyUSING SCANNING ELECTRON MICROSCOPYTO CHARACTERISE PLUM (Prunus domestica L.)GENOTYPESMilena Dordević#, Radosav Cerović∗, Dragan Nikolić∗∗,Sanja Radičević, Ivana Glǐsić, Neboǰsa Milošević(Submitted by Academician V. Golemansky on April 9, 2020)AbstractPollen grain micromorphological characterisation of four plum (Prunus do-mestica L.) genotypes from two different breeding programmes (Fruit ResearchInstitute, Serbia, and Hohenheim, Germany) was performed using scanningelectron microscopy (SEM). The studied characteristics were pollen size andshape, colpus length, mesocolpium width as well as exine characteristics. Allgenotypes had tricolpate pollen grains and striate exine ornamentation, withmore parallel longitudinal ridges. The most significant differences among theanalysed genotypes concerned the length of the pollen grain and exine sculp-turing. The genotypes ‘Pozna Plava’, ‘Presenta’ and ‘Čačanska najbolja’ hadthe largest pollen grains (> 57 µm), whereas ‘Hanita’ showed a smaller pollensize, below 54 µm. ‘Pozna Plava’ had perprolate pollen in the equatorial view(L/W ratio 2.06), while the other three genotypes had prolate shaped pollen.By analysing the pollen morphological characteristics of the plum varieties inthis paper, primarily the shape, size, length of colpi and, most importantly,the sculpture of the exine, each variety showed a specificity which can be usedtogether with other morphological characteristics for description and charac-terisation.Key words: European plum, pollen grains, morphology, exine character-istics, sculpture, SEM#Corresponding author.This work was conducted with the financial support of Ministry of Education, Science andTechnological Development of the Republic of Serbia, Agreement No 451-03-68/2020-14/200215.DOI:10.7546/CRABS.2020.10.081390Introduction. Systematically, Prunus domestica L. (European plum) be-longs to the family Rosaceae L., subfamily Prunoideae, and is the most significantfruit species in Serbia. It is mostly used for various forms of industrial and do-mestic processing as well as for fresh consumption [1].To ensure high fruit set, it is important to know the quality of pollen geno-types that are planted in the orchard. Also, studying the characteristics of pollenfrom specific genotypes and cultivars is important for breeding programmes [2].Differences in pollen grain size and surface ultrastructure are a useful tool fordistinguishing species and cultivars of fruit trees [3]. Pollen grain at the time ofmaturity has three principal strata, an outer (exine) wall, an inner wall (intine)and a pollen coat consisting of components that fill the sculptured cavity of thepollen exine [4]. However, subsequent research excludes the possibility of deter-mining varieties based on the sculpturing of the exine, and this determination canbe applied only at the species level [5].So far in several Prunus genotypes: plum [5], apricot [6], peach and nec-tarine [3], sour cherry [7], sweet cherry [8,9] micromorphological characterizationhas been done using SEM.Exines contain pores and ridges and, based on their layout, the ornamentationof the exine can be meshed, granular, grooved, striated, spined or smooth [10]. Theexine also varies in the number, distribution and architecture of the apertures thatinterrupt it, since apertures are diverse across taxa, families, species and evenwithin a single plant [11].The intention of the present work is to describe the pollen morphology of fourplum genotypes, from two different breeding programmes (Fruit Research Insti-tute, Serbia and Hohenheim, Germany), growing in the western region of Serbia.The major plant breeding programme in the Fruit Research Institute, Čačak, wasestablished to breed new cultivars of European plum, using autochthonous andintroduced genotypes as the parental combinations within the planned hybridis-ation. This programme produced ‘Čačanska Najbolja’ and ‘Čačanska Lepotica’,which were recognised in 1975. Both genotypes derived from the crossing combi-nations ‘Wangenheims Frühzwetsche’ × ‘Požegača’. At the University of Hohen-heim, ‘Hanita’ was the first plum cultivar, introduced in 1991, from the crossing of‘President’ × ‘Auerbacher’. This cultivar showed sharka symptoms on few leavesonly, while fruit were not affected. ‘Presenta’ was introduced as a late-ripeningvariety, derived from the crossing combinations ‘Ortenauer’ × ‘President’ [12].We applied SEM to develop an identification tool for germplasm characteri-sation and preservation as well as for breeding.Material and methods. To study the pollen grains of four plum geno-types (‘Čačanska Najbolja’, ‘Pozna Plava’, ‘Hanita’ and ‘Presenta’) growing inthe experimental plum orchard near Čačak, Serbia, fresh pollen material fromliving plants was used. The flower buds of all analysed genotypes were taken atthe later balloon stage. Sepals and petals were removed, and anthers were col-C. R. Acad. Bulg. Sci., 73, No 10, 2020 1391lected into petri dishes and kept at room temperature until anther dehiscence.Dry pollen grains were placed into glass bottles with silica gel and stored in arefrigerator at +4 ◦C until required for SEM evaluation.For the SEM study, pollen grains were mounted using the brush directly onmetallic stubs, using double-sided adhesive tape, and coated with gold in a sput-tering chamber (BAL-TEC SCD 005 Sputter Coater). Observation of the preparedsamples was carried out with a scanning electron microscope (SEM) JEOL JSM-7100F (Tokyo, Japan) at a magnification of 2000× for the whole pollen grainand at 15 000× for the section of the exine pattern. For each genotype, 30 pollengrains were observed to determine the pollen size, and the following morphologicalcharacteristics were analysed: polarity, symmetry, aperture (number, length andbreadth width, mesocopium width), pollen grain shape (polar and equatorial),pollen size (polar diameter – length, equatorial diameter – width, length/widthratio) and characteristics of exine (ornamentation, number of muri at 10×10 µm,width of the muri and grooves). We followed the terminology of Erdtman [13].Data were statistically analysed using analysis of variance (ANOVA). Themeans were separated using Fisher’s LSD test for P ≤ 0.05.Results. Pollen grains of all analysed genotypes were symmetric, isopolarand trizonocolpate (with three germinal furrows or colpi). Colpi were positioned atthe meridian, along the pollen grain, and symmetrically arranged at a distance of120◦ in the equatorial view (Fig. 1). The mean of the equatorial diameter rangedfrom 28.22 µm in ‘Hanita’ up to 29.84 µm in ‘Čačanska Najbolja’ (Table 1). Theshape of pollen grains was mainly prolate (P/E = 1.92–1.98), except for the ‘PoznaPlava’ genotype, which had perprolate pollen grains (P/E = 2.06). In the polarT a b l e 1Chief pollen grain characteristics of the analyzed plum genotypesCultivars P E P/E‘Pozna Plava’ 60.14± 0.50a∗ 29.11± 0.47ab 2.06± 0.038a‘Čačanska Najbolja’ 57.66± 0.38b 29.84± 0.38a 1.92± 0.020b‘Hanita’ 53.99± 0.27c 28.22± 0.30b 1.92± 0.021b‘Presenta’ 58.42± 0.49b 29.59± 0.29a 1.98± 0.019abCultivars LC WC WM‘Pozna Plava’ 50.47± 0.49a 2.53± 0.16ns 18.98± 0.30a‘Čačanska Najbolja’ 48.54± 0.50b 2.42± 0.12ns 16.82± 0.75bc‘Hanita’ 44.93± 0.38d 2.18± 0.14ns 15.84± 0.65c‘Presenta’ 49.77± 0.53c 2.25± 0.19ns 17.79± 0.52ab∗The values are statistically significant at P ≤ 0.05 by LSD test; ns – non significant. Meanvalues followed by the different lower-case letters in column represent significant differences; P –length of polar axis; E – length of equatorial diameter; P/E – ratio of polar axis and equatorialdiameter; LC – length of colpi; WC – width of colpi; WM – width of mesocolpi. All measurementsin µm1392 M. Dordević, R. Cerović, D. Nikolić et al.Fig. 1. Pollen shape and look of colpi analyzed genotypes: A–C. ‘Pozna Plava’; D–F. ‘ČačanskaNajbolja’; G–I. ‘Hanita’; J–L. ‘Presenta’. Scale bars – 10 µm (A, B, D, E, G, H, J, K), 5 µm(C, F, I, L)view, pollen shape was triangular-obtuse-convex, while in the equatorial view, itwas elliptic-accuminate-acute. All pollen grains were tricolpate.The highest length (µm) and width (µm) of colpi, as well as the highestmesocolpium width (µm), were determined in ‘Pozna Plava’ genotypes, while thelowest values were obtained for the ‘Hanita’ genotype (50.47, 2.53, 18.98 and 44.93,2.42, 16.82, respectively).Regarding exine sculpture, the tectum surface ornamentations in all analysedgenotypes were with muri and separated with grooves (striate) (Fig. 2).C. R. Acad. Bulg. Sci., 73, No 10, 2020 1393Fig. 2. Egine patterns of analysed genotypes: A. ‘Pozna Plava’; B. ‘Čačanska Najbolja’; C.‘Hanita’; D. ‘Presenta’. Scale bars – 1 µmMuri width ranged between 0.39 (‘Hanita’) and 0.63 µm (‘Pozna Plava’) (Ta-ble 2). The muri in all analysed genotypes covered the surface of the pollengrain and were parallel to the colpi, separated by shorter (0.34 µm in ‘Hanita’and‘Presenta’) or larger grooves (‘Pozna Plava’ and ‘Čačanska Najbolja’, 0.55 and0.44 µm, respectively). Near the colpi and at the end, muri were sometimescurved and branched; this exine formation was most pronounced in ‘Pozna Plava’T a b l e 2Tectum surface ornamentations values in analyzed genotypesCultivars WM WG Number of muriin 100 µm2‘Pozna Plava’ 0.63± 0.014a 0.55± 0.015a 10.33± 0.38c‘Čačanska Najbolja’ 0.49± 0.011b 0.44± 0.008b 14.33± 0.44b‘Hanita’ 0.39± 0.007d 0.34± 0.009c 16.66± 0.09a‘Presenta’ 0.43± 0.01c 0.34± 0.01c 16.33± 0.17a∗The values are statistically significant at P ≤ 0.05 by LSD test; ns – non significant. Meanvalues followed by the different lower-case letters in column represent significant differences;WM – width of muri; WG – width of grooves. All measurements in µm1394 M. Dordević, R. Cerović, D. Nikolić et al.and ‘Presenta’ genotypes. The number of muri at an area of 100 µm2 ranged from10.33 (‘Pozna Plava’) to 16.66 (‘Hanita’). In all genotypes, small-sized perfora-tions were noticed.Apart from the length of the polar axis, all other analysed pollen morpho-logical characteristics showed the highest values in the ‘Pozna Plava’ genotype,while in ‘Hanita’, except from the number of muri in 100 µm2, the other analysedcharacteristics showed the lowest values.Discussion. Pollen grain size was less than 0.1 mm and varied significantlyamong plant species. The pollen of entomophilic plants, in addition to beinglarger than the pollen of anemophilic plants, is characterised by the exine beingdifferently sculptured and with varying ornamentation [14]. So far, results haveshown that the quality, morphology and architecture of pollen grains, along withthe chemical composition, are strongly influenced by high temperatures [15].In this paper, pollen length ranged from 53.99 to 60.14 µm, while pollen widthranged from 28.22 to 29.84 µm. Our data agreed with those reported by [16] and [5].Based on the obtained values, all pollen grains can be classified as large grains.Based on the obtained values of the L/W ratio, pollen grains of the genotypes‘Čačanska Najbolja’, ‘Hanita’ and ‘Presenta’ are prolate (L/W ≤ 2), while thepollen grain of the genotype ‘Pozna Plava’ is perpolate (2.06). Pollen grain lengthin species of the family Rosaceae varies from 32.10 to 59.45 µm, with a widthfrom 22.2 to 35.20 µm, depending on the species. According to measurementson Prunus domestica, pollen grains have a flattened spherical shape [17], althoughmost authors describe it as a prolate shape [3,16]. In other Prunus species, accord-ing to L/W ratio, prolate shape of pollen was determined in apricot [18], peach [10],while in sweet cherry it was described as prolate [9] and as perprolate [8].Analysis of colpi showed that they extended the length of the grain, rangingfrom 44.93 (‘Hanita’) to 50.47 (‘Pozna Plava’). This result is in accordance withthe previous finding that colpus length spans 80–90% of the distance betweenpoles [19].Analysing pollen morphology in a number of individuals of the genus Prunus,different authors outline the significance of examining exine sculptures, the mostvariable morphological characteristic of the pollen surface [16,18]. The highest vari-ability of the tested parameters was observed in pollen of plum genotypes in thestudy of the micro-morphological characteristics of pollen in 13 autochthonousvarieties and three substrates within four species of Prunus in Sicily (apricot,cherry, plum, peach) [3]. Pollen grains of ‘Pozna Plava’ had the largest width ofmuri and ridges and therefore the smallest number of muri per 100 µm2 of equa-torial area, while the ‘Hanita’ variety had the smallest width of muri and groovesand the highest number of muri per 100 µm2 of equatorial area. In the genotype‘Čačanska Najbolja’, width of the muri and grooves was approximately the same,while in the genotype ‘Presenta’, the width of the muri was slightly larger thanthe width of the furrow. The longest and most parallel ridges, extending almost5 C. R. Acad. Bulg. Sci., 73, No 10, 2020 1395to the poles, were noticed in the pollen grains of the genotype ‘Hanita’. In theother three genotypes, the muri were short to medium long, with branching atthe ends. Examining 13 autochthonous plum cultivars in the territory of Bosnia,no correlation was found between ornamentation and basic phenotypic character-istics of the examined cultivars. A positive correlation between the variability ofthe exine ornamentation and the fruit shape of analysed the plum varieties wasobserved [3]. The same authors state that the existence of high variability inthe examined parameters of the analysed plum genotypes can be related to theirhybrid origin.Conclusions. Examination of the pollen grains in four plum genotypesshowed differences in length, width, length of the colpi and tectum surface or-namentation. Pollen shape was mostly prolate to perprolate. In all analysedgenotypes, pollen grain was tricolpate. Sculpturing provides a diagnostic tool bywhich taxa can be identified, usually at the generic level, but also frequently atthe specific level. By analysing the pollen morphological characteristics of theplum varieties in this paper, primarily the shape, size, length of colpi and, mostimportantly, the sculpture of the exine, each variety showed a specificity whichcan be used together with other morphological characteristics for description andcharacterisation.REFERENCES[1] Glišić I., R. Cerović, N. Milošević, M. Dordević, S. Radičević (2012)Initial and final fruit set in some plum (Prunus domestica L.) hybrids under differentpollination types, Genetika, 44, 583–593.[2] Sharafi Y. (2011) In vitro pollen germination in stone fruit tree of Rosaceaefamily, Afr. J. Agric. Res., 6, 6021–6026.[3] Geraci A., P.V. Vicenza, M.P. Pasquale, S. R. Rosario (2012) Investiga-tion on the pollen morphology of traditional cultivars of Prunus species in Sicily,Acta Soc. Bot. Pol., 81, 175–184.[4] Edlund A.F., R. Swanson, D. Preuss (2004) Pollen and stigma structure andfunction: the role of diversity in pollination, Plant Cell, 16, S84–S97.[5] Mićić N. (1988) Pollen morphology of plum, J. Yugoslav Pomology, 84/85, 173–181.[6] Asma B.M. (2008) Determination of pollen viability, germination ratios and mor-phology of eight apricot genotypes, Afr. J. Biotechnol., 7, 4269–4273.[7] Nyéki J., P. Sótonyi, Z. Szabó, E. Felhosné-Váczi, F. Csillag (1996) Ascanning electron microscopy survey of pollen grains of sour cherry cultivars, ActaHortic., 410, 133–135.[8] Radičević S., D. Nikolić, R. Cerović, M. Dordević (2013) In vitro pollengermination and pollen grain morphology in some sweet cherry (Prunus avium L.)cultivars, Rom. Biotech. Lett., 18, 8341–8349.1396 M. Dordević, R. Cerović, D. Nikolić et al.[9] Nikolić D., D. Milatović (2016) Pollen morphology of some sweet cherry culti-vars observed by scanning electron microscopy, Acta Hortic., 1139, 369–374.[10] Evrenosoрlu Y., A. Misirli (2009) Investigations on the pollen morphology ofsome fruit species, Turk. J. Agric. For., 33, 181–190.[11] Mignot A., H. Coss, I. Dajoz, C. Leuret, J. P. Henry et al. (1994) Pollenaperture polymorphism in the Angiosperms: importance, possible causes and con-sequences, Acta Bot. Gallica, 141, 109–122.[12] Hartmann W. (1993) Plum breeding at Hohenheim, Acta Hortic., 359, 55–62.[13] Erdtman G. (1952) Pollen Morphology and Plant Taxonomy—Angiosperms,Stockholm, Almqvist and Wiksell, 539 pp.[14] Iwanami Y., T. Sasakuma, Y. Yamada (1988) Pollen: Illustrations and scanningelectronmicrograps, Berlin, Springer-Verlag.[15] Hedhly A., J. I. Hormaza, M. Herrero (2008) Global warming and sexualplant reproduction, Trends Plant Sci., 14, 30–36.[16] Čalić D., N. Devrnja, M. Kostić, I. Kostić (2013) Pollen morphology, viabil-ity, and germination of Prunus domestica cv. Požegača, Sci. Hortic., 155, 118–122.[17] Gilani S.A., R.A. Qureshi, A.M. Khan, D. Potter (2010) Morphologicalcharacterization of the pollens of the selected species of genus Prunus Linn. fromNorthern Pakistan, Afr. J. Biotechnol., 9, 2872–2879.[18] Arzani K., M.A. Nejatian, G. Karimzadeh (2005) Apricot (Prunus arme-niaca) pollen morphological characterization through SEM, using the multivariateanalysis, New Zeal. J. Crop Hort., 33, 381–388.[19] Hebda R. J., C.C. Chinnappa (1994) Studies on pollen morphology of Rosaceae,Acta Bot. Gallica, 141, 183–193.Department for Fruit Breeding and PomologyFruit Research InstituteKralja Petra I/932000 Čačak, Serbiae-mail: mdjordjevic@institut-cacak.orgsradicevic@institut-cacak.orgiglisic@institut-cacak.orgnmilosevic@institut-cacak.org∗University of BelgradeInnovation Centre at Faculty ofTechnology and Metallurgy11120 Belgrade, Serbiae-mail: radosav.cerovic@gmail.com∗∗Faculty of AgricultureUniversity of Belgrade11080 Belgrade-Zemune-mail: nikolicd@agrif.bg.ac.rsC. R. Acad. Bulg. Sci., 73, No 10, 2020 1397

Using scanning electron microscopy to characterise plum (Prunus domestica L.) genotypes

Pollen grain micromorphological characterisation of four plum (Prunus domestica L.) genotypes from two different breeding programmes (Fruit Research Institute, Serbia, and Hohenheim, Germany) was performed using scanning electron microscopy (SEM). The studied characteristics were pollen size and shape, colpus length, mesocolpium width as well as exine characteristics. All genotypes had tricolpate pollen grains and striate exine ornamentation, with more parallel longitudinal ridges. The most significant differences among the analysed genotypes concerned the length of the pollen grain and exine sculpturing. The genotypes Pozna Plava', Presenta' and 'Caanska najbolja' had the largest pollen grains ( gt  57 mu m), whereas `Hanita' showed a smaller pollen size, below 54 mu m. Pozna Plava' had perprolate pollen in the equatorial view (LAW ratio 2.06), while the other three genotypes had prolate shaped pollen. By analysing the pollen morphological characteristics of the plum varieties in this paper, primarily the shape, size, length of colpi and, most importantly, the sculpture of the exine, each variety showed a specificity which can be used together with other morphological characteristics for description and characterisation

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Using scanning electron microscopy to characterise plum (prunus domestica l.) Genotypes

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Using scanning electron microscopy to characterise plum (Prunus domestica L.) genotypes

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