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Home , Diospyros virginiana, Persimmon

Modern Phytomorphology 3: 45–50, 2013

Pollen characteristics in some

Olga Grygorieva 1*, Ján Brindza 2, Radovan Ostrovský 2, Svetlana Klymenko 1, Olga Grabovetska 3

Abstract. Aim of the work was to study the general characteristics and essential morphological traits of pollen grains viz the size, shape of pollen grains and number, form and position of apertures in L.f., D. virginiana L., D. lotus L. species and interspecies hybrid of D. virginiana × D. kaki. Significant differences were detected among the tested species and the interspecific hybrid as well as between individual genotypes ofD. lotus, especially in the equatorial axis.

Key words: Diospyros kaki, , Diospyros lotus, pollen grains; morphological traits, hybrids

1 M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, 1 Timiryazevska str., Kyiv, 01014, Ukraine; * [email protected] 2 Slovak University of Agriculture, Nitra, 2 Tr. A. Hlinku, Slovak Republic 3 Experimental field «Novokakhovske» Nikita Botanical Garden – National Science centre NAS of Ukraine, Nova Kakhovka, Ukraine

Introduction variability and reproduction capacity in local climatic conditions. It is known that the Genus Diospyros belongs to an extensive flowers are prevailingly unisexual, family consisting of 7 genera. only exceptionally being bisexual. It means Yonemori et al. (2000) listed over 400 species that the bears both, the male and female forming the genus Diospyros and several of these flowers. Sometimes the dioecism occurs. species are of economic importance. The mild Many individuals have only female flowers climatic region is suitable for growing and use and the can arise without fertilization in horticulture of three persimmon species – – by and they are seedless. Diospyros kaki L.f., D. virginiana L. and D. lotus L. Flowering biology complexity is for these (Bellini & Giordani 2005). species symptomatic, and it brings questions In Slovakia, persimmon species are cultivated concerning several topics like the pollen grains in botanical gardens as well as by many small characteristics designated in palinology as growers. The present knowledge on growing sporoforms. Information on the genus Diospyros of these originally exotic clearly show pollen grain traits occurs scarcely in a few papers the possibility of successful cultivation in local (Erdtman 1966; Kodela 2006; Geeraerts climatic conditions, preferably in the southern et al. 2009). part of Slovakia. Research work and most studies The morphological characteristics of pollen of these species were concentrated for several grains are applied for taxonomic classification years in the Slovak University of Agriculture in purposes, further for phylogenetic evaluation Nitra in close cooperation with the National of plant species and detection of hybrid and Botanic Garden of the Ukraine National Academy polyploid forms (Erdtman 1966; 1986; of Sciences in Kiev (Grygorieva et al. 2009). Erdtman & Roger 2007). For practical reasons, concerning the Then aim of this work was determination of use and distribution of mentioned plant pollen grains general characteristics and basic species in Slovakia, it is quite important to morphological traits in selected species from learn the biological peculiarities connected the genus Diospyros including the variability of with flowering as well as with interspecific examined species. © The Author(s), 2013 46 Modern Phytomorphology 3 (2013) Material and methods that pollen grains are radially symmetrical, isopolar and according to the localization of The pollen grains morphology was studied apertures they are zono-tricolporate. on Diospyros kaki, D. virginiana, D. lotus and Three compound apertures are according interspecies hybrid D. virginiana × D. kaki. to distribution equidistant. The size, the shape We have evaluated the basic morphological of pollen grains and the number of apertures characteristics of tested persimmon species are documented on Fig. 1. Pollen grains pollen grains – the size and shape, position, possess three prolonged apertures – colpi number and form of apertures (aperture – which are narrowed at poles. Additional pole the thinned region of the sporoderma – wall is in the colpus middle at the equatorial centre pollen grain, usually function as sites of the (circular equatorial aperture). This classification germination). The pollen grain size has been correlates with literature data (Sowunmi 1995; determined by measuring the length of polar Kodela 2006; Geeraerts et al. 2009). In some axis (P – the straight line between the distal pollen grains were observed annuluses – an area and proximal poles of a pollen grain) and length of the exine surrounding a pore with thickened equatorial axis (E – pollen width, the distance periphery or operculum – part of exine over the between the poles in equatorial part of pollen). pore (Fig. 1). The exine membrane in an area of The shape index (SI – the ratio of the length the colpus is granulated. From cytological point of the polar axis and equatorial axis) allowed of view the pollen grains of most Diospyrus are determination of the pollen grains shape. bicellular with exception of D. kaki and D. lotus The pollen grains are classified according to with unicellular pollen grains (Поддубная- shape index into 9 shape classes (Dostál et al. Арнольди 1982). It means that generative cell 1966). The pollen size (length of polar axis and division on sperm cells is accomplished in the equtorial axis – width of pollen) and shape (SI – pollen pocket. P/E) were measured in micrometers. An important morphological characteristic Above mentioned morphological traits were is the size of pollen grains. Our data showed that measured and evaluated on 40–60 pollen grains the pollen grains of D. kaki, D. virginiana, D. lotus (in polar position) of each species. Electron and interspecies hybrid D. virginiana × D. kaki microscopes Carl Zeiss EVO LS 15 and are from medium size to large accordingly to РЕММА-102 (SELMI) enabled to distinguish Dostál et al. (1966). The length of polar axis and identify the pollen surface – structure, (P) measured in the mentioned three species special ornamentation, upmost thickness of (Tab. 1) varied from 42.25 to 55.41 μm and exine – sexine and aperture membrane structure. the width of equatorial axis (E) was in range Morphological traits were evaluated of 21.40‑32.47 μm. The highest values of mean on pollen samples collected in 2010 from length of polar axis (71.19 μm) and equatorial female flowers during full flowering from axis (33.14 μm) was recorded in pollen of the the persimmon of species D. virginiana, interspecies hybrid D. virginiana × D. kaki. cultivated in Kyiv (Ukraine), D. lotus – in Differences in values of the both indicators Arborétum Mlyňany (Slovakia) and D. kaki of pollen grain size (P and E) between this – in Nitra (Slovakia). Pollen samples released hybrid and other tested species were statistically from dry male flowers were further dried under significant. laboratory conditions. For microscopic studies The significance of differences was observed on morphological characteristics the dry pollen also in the mean length of polar and equatorial was used. axes in all analyzed species with exception of mean length of polar axis in D. kaki and D. lotus. Results and discussion The values of the coefficient of variation were in the range of 3.63-6.50% for polar and in the Pollen grains of the genus Diospyros are range of 6.15-7.56% – for equatorial axes. It solitery, defined as monades. This study showed suggests a low variability in pollen grains size of Grygorieva O., Brindza J., Ostrovský R., Klymenko S., Grabovetska O 47

A B

C D

Fig. 1. Pollen grains of Diospyros species in different position:A – D. lotus; B – D. virginiana; C – D. kaki; D – D. virginiana × D. kaki. studied species, despite statistically significant that pollen grains size varies considerably in one intraspecific variability, illustrated in Tab. 1 individual plant even (Ostrolucká & Križo (P = 48.00-51.39 μm and E = 23.56-27.78 μm). 1989). Shape index (SI) of pollen grain depends Results of morphological analysis of on parameters of polar (P) and equatorial (E) pollen in selected genotypes of D. lotus from axis. Shape index (the P/E ratio) of tested Arborétum Mlyňany (Tab. 2) confirmed species varied from 1.75 in D. kaki to 2.18 – in a higher intraspecific variability of pollen D. virginiana (Tab. 2). grains size as evidenced by the values of the According to the values of shape index coefficients of variation for polar (9.76-21.64%) the pollen grains can be classified as prolate and equatorial (7.09-9.29%) axes. It was found, or prolate-spheroidal. The shape indexes 48 Modern Phytomorphology 3 (2013)

Table 1. The measured pollen morphological traits ofDiospyros species: min – minimum value; max – maximum value; mean – arithmetic mean; SI – shape index (P/E); SEmean – standard error; CV – coefficient of variation (%). Homogeneous Species min max mean SE CV% SI mean groups P – polar axis (μm) D. kaki 43.97 54.06 48.73 0.91 6.50 – c D. lotus 42.50 50.50 48.00 0.50 3.63 – c D. virginiana 49.28 55.41 51.39 0.59 4.00 – bc D. virginiana × D. kaki 63.17 80.54 71.19 0.48 6.30 – a E – equatorial axis (μm) D. kaki 24.58 32.47 27.78 0.57 7.20 1.75 b D. lotus 23.97 31.17 26.06 0.56 7.56 1.84 bc D. virginiana 21.40 25.94 23.56 0.41 6.15 2.18 d D. virginiana × D. kaki 30.20 36.51 33.14 0.50 5.00 2.14 a ranging from 1.77 to 1.83 in the genotypes of method (Ostrolucká & Križo 1989). These D. lotus confirmed certain degree of identity factors can significantly influences on the substantiating thus the classification to shape pollen size. For example Sowunmi (1995) class of prolonged pollen grains, i.e. with the reported for D. mespiliformis Hochst. ex A.D.C. vertical axis longer than equatorial one. In the mean value for polar axis length (P) of 42.4 comparison with the data of Geeraerts et al. μm (in range 39.0-46.0 μm) and the equatorial (2009) our results concerning the shape indexes axis (E) of 31.2 μm (27.0-33.0 μm). Whereas of tested species are generally higher. On the Geeraerts et al. (2009) for the same species other hand, the data shown in Table 1 and the found the mean values for the length P – 32.3 values of shape indexes (1.28-1.33) presented in μm and E – 24.8 μm. There were found also Tab. 3 verify that the pollen grains of Diospyros significant different values of the mentioned representatives are prolonged sporoformes traits in D. lotus and D. virginiana presented with exception of D. lotus (Erdtman 1966; in our study as well as in the study published Geeraerts et al. 2009). Interesting values of by Geeraerts et al. (2009). The lengths of shape index (1.79-1.82) for D. kaki are reported polar and equatorial axes were larger in pollen by Evrenosoglu & Misirli (2009). However, samples collected on localities of Slovakia. it is consistent with our result for this species Nevertheless, in the paper of Geeraerts et al. (1.75) given in Table 1. (2009) an extensive knowledge on pollen Morphological traits variability could be morphological diversity was accumulated for influenced by the environmental factors, such different persimmon species. as temperature, humidity and nutrition. Kodela (2006) reports the evaluations In case of pollen grain size, except the above for D. australis (R. Br.) Hiern., where the mean mentioned factors, the inner factors are also length of polar axis reached 36.9 μm and the of importance – especially the chromosomes equatorial one – 29.7 μm. Our results suggest a number. Polyploid species are characterized possible polymorphism of pollen obtained from by larger pollen grains with increased aperture individual genotypes of a given species grown in numbers. Pollen size could be influenced different ecological and geographical conditions, even by different methodical procedures and respectively. Colour of pollen grains is taken as approaches to pollen samples processing e.g. the a relevant trait from taxonomic point of view. pollen elaboration by acetolysis, using various The tested samples of persimmon pollen were agents like acetocarmine, KOH or processed of different yellow shades. The exine sculpture without any chemicals, applying the dry pollen is also an important morphological trait, but its Grygorieva O., Brindza J., Ostrovský R., Klymenko S., Grabovetska O 49

Table 2. The measured pollen morphological traits of selected genotypes ofDiospyros lotus from Arborétum Mlyňany. Abbreviations see in Tab 1.

Genotype min max mean CV% SI Homogeneous groups P – polar axis (μm) DL – AM 1 10.07 63.44 44.37 21.64 – a DL – AM 2 26.92 54.09 46.93 9.76 – ab DL – AM 5 22.93 54.22 44.03 11.20 – a E – equatorial axis (μm) DL – AM 1 6.06 34.63 25.01 19.29 1.77 a DL – AM 2 16.46 34.00 25.57 9.34 1.83 ab DL – AM 5 12.17 30.70 24.44 7.09 1.80 b

Table 3. Literature data on pollen morphological traits in some Diospyros species: P – polar axis; E – equatorial axis. Other abbreviations see in Tab 1.

Plant species axis min max mean SI References D. kaki L.f. – G1 P 51.6 53.7 52.6 1.79 Evrenosoglu & Misirli (2009) E 29.0 29.5 29.4 D. kaki L.f. – G2 P 50.8 54.2 52.6 1.82 Evrenosoglu & Misirli (2009) E 28.4 29.6 28.9 D. abyssinica (Hiern) F. White P – – 22.9 1.18 Geeraerts et al. (2009) E – – 11.4 D. kirkii Hiern P – – 31.6 1.05 Geeraerts et al. (2009) E – – 30.2 D. lotus L. P – – 50.6 0.98 Geeraerts et al. (2009) E – – 51.5 D. squarrosa Klotzsch P – – 37.4 1.48 Geeraerts et al. (2009) E – – 25.3 D. virginiana L. P – – 41.4 1.33 Geeraerts et al. (2009) E – – 31.2 P – – 59.0 1.28 Erdtman 1966 E – – 46.0 structural elements could be observed in detail Diospyros are described by Geeraerts et al. only by SEM. Our SEM studies have shown (2009) and Kodela (2006). that the sexine of tested species (Fig. 1) is not smooth but sculptured. There is domination Conclusions of finely striation (striate), scarring (scabrate) or wrinkles, more markedly in D. virginiana, Experimental studies were oriented on also with occurrence of perforations. Also morphological characterization of pollen grains significantly was distinguishedD. kaki, its sexina (sporoforms) of D. kaki, D. virginiana, D. lotus is irregularly baculiform. Similar characteristics and interspecies hybrid D. virginiana × D. kaki of sculpture elements for some species of with special attention paid to the size and 50 Modern Phytomorphology 3 (2013) shape of pollen grains. Statistically significant References differences in the length of polar and equatorial axes identifying their size and shape were Поддубная-Арнольди В.А. 1982. Характеристика detected between investigated taxa. According семейств покрытосеменных растений по to values of shape index, the pollen grains can be цитоэмбриологическим признакам [Characteristics included into the shape class verified as prolate of the Angiosperm families by cytoembriological features]. Наука, Москва. (in Russian). or prolate-spheroidal. Pollen grains of tested Bellini E., Giordani S. 2005. Germplasm and breeding species are radially symmetric and isopolar. of persimmon in Europe. Acta Horticult. 685: 65–69. Taking into account the appertures’ shape and Dostál J., Futák J., Novák F.A. 1966. Flora of Slovakia. their number they were classified as zono- Vol. I. Press SAS, Bratislava. (in Slovak). tricolporate. Erdtman G. 1966. Pollen morphology and plant Our observations showed that not only . Vol. I. Angiosperms. 3rd ed. Hafner pollen size, but also the type and the number Publishing Company, New York. of apertures together with microstructural Erdtman G. 1986. Pollen morphology and plant characteristic of exine sculpture are important taxonomy: An introduction to palynology. E. Brill, Leiden. morphological traits, which are characteristic Erdtman G., Roger P. 2007. An introduction to pollen for each species. Certain polymorphism analysis. Read Books, Wodehause. of pollen morphological traits in Diospyros Evrenosoglu Y., Misirli A. 2009. Investigation on the species was detected. Analyses of tested pollen morphology of some species. Turk. J. Agric. species oriented to pollen grains morphology For. 33: 181–190. are of importance from various viewpoints – Geeraerts A., Raeymaekers J.A.M., Vinckier S., evolution and taxonomy of the genus provides Pletser A., Smets E., Huysmans S. 2009. new information and improves the knowledge Systematic palynology in Ebenaceae with focus on about interesting plants, which despite of their Ebenoideae: Morphological diversity and character evolution. Rev. Palaebot. Palynol. 153 (3-4): 336–353. exotic origin become a fully deserved concern of Grygorieva O., Klymenko S., Brindza J., Tóth D., the Slovakia growers’ community. This is closely Derevjanko V., Grabovecka O. 2009. connected to the fact, that persimmon has an Morphometrical analysis of Diospyros lotus population extending economical importance leading to an in the Mlyňany Arboretum, Slovakia. Acta Horticult. increased exploitation in practice especially as 833: 145–150. fruit species suitable for the branch of ecological Kodela P.G. 2006. Pollen morphology of some rainforest agriculture. taxa occuring in the Illawarra region of New South Wales, Australia. Telopae 11 (3): 346–389. Acknowledgements Ostrolucká M.G., Križo M. 1989. Biológia samčích reprodukčných orgánov druhov rodu Quercus L. [Biology of male reproduction organs in species of the This work has been supported by the genus Quercus L.]. Veda, Bratislava. (in Slovak). „Excellence Center for Agrobiodiversity Sowunmi M.A. 1995. Pollen of Nigerian plants. Grana Conservation and Benefit (ECACB)“ project 34 (2):120–141. implemented under the Operational Programme Yonemori K., Sugiura A., Amada M. 2000. Research and Development financed by Persimmon genetics and breeding. Plant Breed. Rev. European Fund for Regional Development, 19: 191–225. ITMS 26220120015.