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Pollen morphology, viability, and germination of Prunus domestica cv. Požegača
Scientia Horticulturae 155 (2013) 118–122
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Pollen morphology, viability, and germination of Prunus domestica cv. Poˇzegaˇca ´ c´ a,∗ , N. Devrnja a , I. Kostic´ b , M. Kostic´ c D. Cali a Department of Plant Physiology, Institute for biological Research “Siniˇsa Stankovi´c”, University of Belgrade, Despot Stefan Blvd. 142, 11 060 Belgrade, Serbia b Institute for Multidisciplinary Research, University of Belgrade, University of Belgrade, Kneza Viˇseslava 1,11 000, Belgrade, Serbia c Institute for Medicinal Plant Research “Dr Josif Panˇci´c”, Tadeuˇsa Koˇs´cuˇska 1, 11 000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 25 December 2012 Received in revised form 12 March 2013 Accepted 13 March 2013 Keywords: Pollen germination Pollen morphology Pollen nuclear status Pollen tube growth Poˇzegaˇca plum Viability
a b s t r a c t The pollen morphology and exine ornamentation of an important autochthon plum cultivar (Prunus domestica cv. Poˇzegaˇca L.), which has horticultural, medicinal and alimentary uses, was examined in detail using both light and scanning electron microscope (SEM). Acetocarmine and fluorescein diacetate were used to assess the pollen viability of four Poˇzegaˇca plum genotypes. Pollen viability varied from 67% in genotype Pdp1 to 99% in genotype Pdp4. Also, pollen nucleus status was determined, and binucleate mature pollens were detected. The effect of polyethylene glycol-PEG (10, 15 and 20%, w/v) on pollen germination and tube growth was evaluated. Overall, the inclusion of PEG in the medium improved both pollen germination and tube growth. Regardless of the PEG concentration genotype has an effect on germination and length of pollen tubes. Genotype has a pronounced effect on germination and length of pollen tubes, regardless of the PEG concentration used. Genotype Pdp4 had the highest germination percentage and the highest pollen tube length on all media when compared to the other three genotypes. The highest pollen germination (96%) and a tube length (822 m) of genotype PdP4 was attained in media with 20% PEG. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Poˇzegaˇca plum (Prunus domestica L., Rosaceae), is an important autochthon plum cultivar of Serbia, that is considered to be of high quality throughout the world. This cultivar has an ideal ratio of acids and sugars in it’s fruit, and is irreplaceable in brandy and jam production. However, this cultivar is extremely susceptible to Plum pox virus (PPV) which has caused its distribution to greatly diminish. Plum pox, also known as sharka, is the most devastating viral disease of stone fruit from the genus Prunus. Wild and ornamental species of Prunus may also become infected by some strains of the virus (Dunez and Sutic, 1988). The virus is transmitted by aphids and by the transfer of infected plant material to new locations. Plum pox poses no danger to consumers, but it can ruin the marketability of stone fruit by causing acidity and deformities. The only way to manage the disease is to destroy all infected trees, which has caused significant economic losses. Many studies have investigated the responses of pollen germination and pollen tube growth to a variety of factors such as
∗ Corresponding author. Tel.: +381 11 2078 366; fax: +381 11 2761 433. ´ c). ´ E-mail address: [email protected] (D. Cali 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.03.017
temperature (Dag et al., 2000; Wolukau et al., 2004; Acar and Kakani, 2010; Alcaraz et al., 2011; Matsumoto et al., 2012), sucrose (Vaknin et al., 2003; Alcaraz et al., 2011; Devrnja et al., 2012), polyethilenglycol (Sakhanokho and Rajasekaran, 2010). Many cultivars and genotypes with unfavorable pollen have been reported by breeders and researchers previously. For instance, some of the cultivars/genotypes have sterile pollen or pollen with a low germination percentage (Du et al., 2006; Koyuncu and ´ 2010). Therefore, studying Tosun, 2005; Nikolic´ and Milatovic, the characteristics of pollen from specific genotypes and cultivars is important for breeding programs (Sharafi, 2011). Many studies have investigated pollen viability and germination in peach, plum, sour cherry and other species and cultivars. Many studies have investigated pollen viability and germination in peach, plum, sour cherry and other species and cultivars, using different testing methods to determine the pollen viability (Botu et al., 2002; Herrero and Arbeloa, 1989). Results indicated that viability, germination and tube growth of pollens varied significantly according to species, cultivar, and even test that had been used Du et al. (2006), Hedhly et al. (2004), Koyuncu and Tosun (2005), and Sharafi (2011). The objective of this work was to determine viability, nuclear status, germination and tube growth capacity of pollens in four genotypes of Poˇzegaˇca plum.
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2. Materials and methods
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DAPI- treated microspores were examined under the Zeiss Axiovert fluorescent microscope equipped with a camera.
2.1. Plant materials 2.5. In vitro pollen germination and tube growth The study was conducted on four genotypes (Pdp1, Pdp2, Pdp3, Pdp4) of Poˇzegaˇca plum growing near Belgrade, Serbia. 2.2. Scanning electron microscopy The pollen samples were collected from fresh flowers, and processed for scanning microscopy without the usual fixation and dehydration procedures. In the SEM studies, the pollen grains taken from the anthers were placed directly on the stubs, and were covered with a thin layer of gold (ion sputtering coating) in a BALETECSCD 005 Sputtering Device, imaging at 15 kV, using a JSM-6390 LV (JEOL, Tokyo, Japan) scanning electron microscope. The detailed surface ornamentation and the aperture characteristics were examined under the SEM, and the microphotographs were taken. The measurements were made in micrometer (m). Scanning electron micrograph (SEM) images were taken on Poˇzegaˇca plum pollen grains at 190 × , 450 × , 1400 × , 1800 × and 10000 × (Fig. 1A-F). 2.3. Pollen viability test Anthers were longitudinal cutting and stained with 1% acetocarmine solution. Carmine solution was prepared in 45% acetic acid. Acetocarmine treated anthers were observed under DMRB microscope from Leica (Wetzlar, Germany). A rapid method with fluorescein diacetate -FDA (HeslopHarison and Heslop-Harison, 1970) was used to determine pollen viability after 1 and 24 h. FDA (2 mg L−1 ) dissolved in acetone was diluted by 0.5 M sucrose solution (1:1). Anthers were longitudinally rejected and free pollen grains were stained with 1–2 drops of FDA.
For the pollen germination test, the hanging drop technique was employed following published procedures (Deng and Harbaugh, 2004) with some modifications as described below. A “basic” liquid medium containing 1.2 M sucrose, 0.3 g L−1 calcium nitrate [Ca(NO3 )2 ], 0.10 g L−1 boric acid (H3 BO3 ), 0.1 g L−1 potassium nitrate (KNO3 ), and 0.2 g L−1 magnesium sulfate (MgSO4 ·7H2 O) was used. The effect of polyethylene glycol (PEG) concentrations (10, 15, and 20%, w/v) on pollen germination and tube growth was evaluated in four Poˇzegaˇca plum genotypes. PEG-free medium was used as a control. For the germination test with the PEGbased media, both pollen germination and pollen tube growth were recorded after a 24 h incubation period. Three replicates (slides) were used for each genotype. A pollen grain was considered to have germinated when pollen tube length equaled or exceeded the grain diameter. Germination percentage was determined by dividing the number of germinated pollen grains by the total number of pollen grains per field of view and multiplying by 100. To measure pollen tube growth, samples were prepared as described above. To measure pollen tube growth, samples were prepared as described above, and the slides were observed under the Zeiss Axiovert light microscope equipped with a camera. 2.6. Statistical analysis The measurements of the diameter, viability and nuclei status were taken on 600 pollen grains. The means were separated using Fisher’s LSD post hoc test for P ≤ 0.05. 3. Results 3.1. Scanning electron microscopy
2.4. Pollen nucleus status The number of pollen nuclei was determined by 4’, 6-diamidino2-phenylindole - DAPI (Coleman and Goff, 1985). The content of anthers was removed by squeezing and was stained with 1–2 drops of DAPI (1 g ml−1 ) solution, prepared in distilled water. FDA and
For the pollen morphological characterization different aspects of the pollens were studied including the shape, length, width, length and width ratio, number of colpi, presence or absence of spines and exine sculpturing in the polar and equatorial view. Shape of pollen is triangular-obtuse-convex in the polar view while
Fig. 1. Scanning electron micrographs of stamen, anters and pollen grains. (A) Stamen with mature anther and filament. Pollen emerging from mature anthers. (B) Cross section of anther. (C) Densely packed pollen grains. (D) Individual pollen grains in different views. (E). Equitorial view of pollen grain with a specific ornamental exine. (F) Striata detail of exine with pores.
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Fig. 2. Flower morphology, anthers and pollen photographed under a light and SEM microscope. Morphology of flower (A) and anthers (B). (C) Lengthwise dissection of acetocarmine stained anthers. Darker (viable) colored and lighter (nonviable, arrow) pollen grains. (D) Natural view of undyed pollen. (E) Viable and nonviable (arrow) pollen grains treated with FDA. (F) Binucleate pollen with a vegetative (V) and generative (G) nucleus staining by DAPI.
the shape of pollen is elliptic-acuminate-acute in the equatorial view. Pollen class is trizonocolpateobtus-triangular. The sculpturing of exine surface is striate. The number of colpi is three while the spines are absent. The horizontal distance (width) in the polar view is 54.37 ± 0.05 m, while the vertical distance (length) is 47.98 ± 0.11 m. The length - width ratio in the polar view is 1.13 m. The horizontal distance (width) in the equatorial view is 25.00 ± 0.37 m while the vertical distance (length) is 20.00 ± 0.43 m. The length - width ratio in the equatorial view is 0.80 m. Exine architecture of mature pollen grains taken at 10,000 × shown in Fig. 1F. Pori are elongated and also with a distinct margin. The porus latitude is smaller than the colpus latitude. One to three perforations were noticed on one square m of exine (Fig. 1F). 3.2. Pollen viability test Acetocarmine treated anthers originating from opened flowers (Fig. 2A, B) showed many viable pollen grains with different size, morphology and viability (Fig. 2C). Even pollen grains which are not acetocarmine treated showed different shape and size (Fig. 2D). The pollen grains are densely packed, therefore many more can fit inside of the anther, which was identified by cross sections of the anthers in both light and SEM microscopy. The pollen viability test was determined for 600 pollen grains of Poˇzegaˇca plum by FDA. Pollen viability was different between different genotype after a 1 and 24 h staining procedure. Viability is increased from genotype Pdp1, Pdp2, Pdp3 to genotype Pdp4 from 67, 73, 80 to 99% (Fig. 2E). Pollen grains have the same viability after both 1 and 24 hours. 3.3. Pollen nucleus status The number of pollen nuclei was determined with DAPI. All investigated pollen grains were binucleate (Fig. 2F).
3.4. In vitro pollen germination and tube growth The length of pollen tubes on PEG media was measured after 24 hours under a light microscope. The effect of PEG on pollen germination and tube growth varied according to concentration and time. Pollen grains from genotype Pdp 4 had the highest germination percentage on all media (Fig. 3A-B and 4). The highest germination percentage (96%) was obtained in a media with 20% PEG for genotype Pdp 4 (Fig. 3B). The germination percentage steadily improved with increasing PEG concentration for all genotype. The greatest influence of increased PEG concentrations was observed for genotype PdP1, where in comparison there was a very low germination rate for the control (1%). Overall in comparison to the control there was a 15 times greater germination potential for germination on 20% PEG (15%). Genotype Pdp4 had a greater germination percentage on PEG free medium than other three genotypes on the best medim with 20% PEG (Fig. 4). Genotype Pdp4 had 6.4 times greater germination on medium with 20% PEG compared to genotype Pdp1. Also, genotype Pdp4 had 2.13 times greater germination compared to genotype Pdp2 and 1.48 times in relation to genotype Pdp3 on same medium (Fig. 4). The influence of genotype and PEG was observed for longer pollen tubes, therefore pollen tube growth linearly increased from gen Pdp1 to gen Pdp4. A similar trend exists at higher PEG concentration. The highest pollen tube length (822 m) of genotype Pdp4 was obtained in medium containing 20% PEG (Fig. 4B; Fig. 5).
4. Discussion Since there are no reports on the pollen morphology, viability, and germination of Poˇzegaˇca plum, the present report gives an account of the palynological characters of this species. Poˇzegaˇca plum as well as the most of the species in Rosaceae has trizonocolpateobtus-triangular pollen. The pollen grains of P. domestica cv. Poˇzegaˇca are trizonocolpateobtus-triangular, as
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Fig. 3. Morphology of germinating pollen. (A) Pollen of Pdp4 genotype on medium with 15% PEG and 20% PEG (B).
Fig. 4. Pollen germination of Poˇzegaˇca plum after 24 h.
previously examined in species of this genus (P. bokhariensis, P. armeniaca, P. avium, P. persica, and P. cornuta) (Gilani et al., 2010). The equatorial axis, and the polar axis and diameter of the pollen grains of P. domestica are similar with four previously examined Prunus species (Gilani et al., 2010). SEM images of Poˇzegaˇca plum grains showed that these pollen grains had a well defined exine with specific architecture and ornamentation. The role of the exine is to protect the male spore and gametophyte from desiccation and other dangers of sub-aerial dispersal.
Fig. 5. Tube growth of Poˇzegaˇca plum pollen on different concentration PEG after 24 h.
Our results regarding pollen morphology and exine sculpturing are similar to the results Youshihiro and Hiroaki (1989), Arazani et al. (2005), Gilani et al. (2010) for pollen of the genus Prunus. In addition, the pollen morphology of different Rosaceae taxa studied by several researchers has reported that the exine features are for taxonomic and phylogenetic classification (Arazani et al., 2005; Gilani et al., 2010). Staining methods and fluorescence are very important because they allow for more complete information regarding pollen viability and fertilization potential (Atlagic´ et al., 2012). Pollen viability showed many differences between genotypes of Poˇzegaˇca plum. Genotype Pdp4 has pollen with the highest viability after acetocarmine and FDA staining. Our results indicate that genotypes have large differences in pollen viability, which is the results obtained for other species (Vaknin et al., 2003; Sakhanokho and Rajasekaran, 2010). Also, the pollen of the genotype Pdp4 had the greatest germination and length of pollen tubes. The results regarding the significant impact of genotype on Poˇzegaˇca plum pollen germination is in agreement with the results obtained in other Prunus species (Asma, 2008; Du et al., 2006; Hedhly et al., 2004; Koyuncu and Tosun, 2005; Sharafi, 2011). Previous research has studied the impact of temperature (Asma, 2008; Hedhly et al., 2005; Pirlak, 2002; Sorkheh et al., 2011; Suzuki et al., 1993), sucrose (Asma, 2008), polyamines (Sorkheh et al., 2011; Wolukau et al., 2004), plant growth regulators (Bolat and Pirlak, 2003), boric acid (Bolat and Pirlak, 2003) and fungicides (Zarrabi and Imani, 2011) on Prunus pollen germination with the exception of genotype effect. However, until now no one has studied the effect of PEG on pollen germination and growth of pollen tubes, which is presented in this manuscript. In general, the inclusion of PEG in the liquid medium improved both pollen germination and tube growth for examined species and varied according to PEG concentration. Regardless of the PEG concentration, genotype has an effect on germination and length of pollen tubes. Pollen morphology and ultrastructure have been shown to be correlated with growth habitat and pollination biology (Osborn et al., 2001). Our results show that binucleate pollen grains of Poˇzegaˇca plum retain viability 24 h after being exposed to dye. Binucleate pollen has weaker germination but longer survival than trinucleate pollen. It has been shown that plants which have binucleate pollen grains are pollinated via insects, which agrees with previously published results (Gottsberger, 1989).
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Sakhanokho and Rajasekaran (2010) reported that PEG functions as an osmoticum and it improves in vitro pollen germination frequency and tube growth by preventing tube bursting. Determining the viability and germination potential of pollen has a great importance for establishing fertilization potential when selecting genotypes. Conserving and cryopreserving pollen of the greatest viability and with the greatest germination potential is important for the conservation of this valuable plum cultivar. Acknowledgements This work is supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant No. 173015). References Acar, I., Kakani, V.G., 2010. The effects of temperature on in vitro pollen germination and pollen tube growth of Pistacia spp. Sci. Hortic. 125, 569–572. Alcaraz, M.L., Montserrat, M., Hormaza, J.I., 2011. In vitro pollen germination in avocado (Persea americana Mill.): optimization of the method and effect of temperature. Sci. Hortic. 130, 152–156. Arazani, K., Nejatian, M.A., Karimzadeh, G., 2005. Apricot (Prunus armeniaca) Pollen morphological characterization through SEM, using the multivariate analysis, New Zealand. J. Crop Hortic. Sci. 33, 381–388. Asma, B.M., 2008. Determination of pollen viability, germination ratios and morphology of eight apricot genotypes. Afr. J. Biotechnol. 7, 4269–4273. ´ J., Terzic, ´ S., Marjanovic-Jeromela, ´ Atlagic, A., 2012. Staining and fluorescent microscopy methods for pollen viability determination in sunflower and other plant species. Ind. Crop Prod. 35, 88–91. Bolat, I., Pirlak, L., 2003. Effects of three plant growth regulators and boric acid on pollen germination and tube growth in apricot (Prunus armeniaca L.). Bangl. J. Bot. 32, 53–56. Botu, M., Sarpe, C., Cosmulescus, S., Botu, I., 2002. The genetic control of pollen fertility, pollenizing and fruit set for the Prunus domestica L. plum cultivars. Acta Hortic. 577, 139–145. Coleman, A.W., Goff, L.J., 1985. Applications of fluorochromes to pollen biology. Mithramycin and 4’,6-diamidino-2-phenylindole (DAPI) as vital stains and for quantitation of nuclear DNA. Stain Technol. 60, 145–154. Dag, A., Eisenstein, D., Gazit, S., 2000. Effect of temperature regime on pollen and the effective pollination of ‘Kent’ mango in Israel. Sci. Hortic. 86, 1–11. Deng, Z., Harbaugh, B., 2004. Technique for in vitro pollen germination and short term pollen storage in Caladium. Hort. Sci. 39, 365–367. ´ c, ´ J., Tubic, ´ Lj., Zdravkovic-Kora ´ ´ S., Cingel, A., Cali ´ D., 2012. Devrnja, N., Milojevic, c, Pollen morphology, viability, and germination of Tanacetum vulgare L. Hort. Sci. 47, 440–442. Du, Y.H., Zhang, S.L., Jiang, T., Wu, J., 2006. Characteristics of pollen germination and pollen tube growth of Prunus mume in vitro. Acta Bot. 26, 1846–1852.
Dunez, J., Sutic, D., 1988. Plum pox. In: Smith, I.M., Dunez, J., Lelliot, R.A., Phillips, D.H., Archer, S.A. (Eds.), European Handbook of Plant Diseases. Blackwell Scientific Publications, London, pp. 44–46. Gilani, S.A., Qureshi, R.A., Khan, A.M., Potter, D., 2010. Morphological characterization of the pollens of the selected species of genus Prunus Linn. from Northern Pakistan. Afr. J. Biotechnol. 9, 2872–2879. Gottsberger, G., 1989. Comments on flower evolution and beetle pollination in the genera Annona and Rollina (Annonaceae). Plant Syst. Evol. 167, 89–94. Hedhly, Y.A., Hormaza, J., Herrero, I.M., 2004. Effect of temperature on pollen tube kinetics and dynamics in sweet cherry, Prunus avium (Rosaceae). Am. J. Bot. 91, 558–564. Hedhly, A., Hormaza, J.I., Herrero, M., 2005. The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach. Plant Biol. 7, 476–483. Herrero, M., Arbeloa, A., 1989. Influence of the pistil on pollen tube kinetics in peach (Prunus persica). Am. J. Bot. 76, 1441–1447. Heslop-Harison, J., Heslop-Harison, Y., 1970. Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Technol. 45, 115–120. Koyuncu, F., Tosun, F., 2005. Evaluation of pollen viability and germinating capacity of some sweet cherry cultivars grown in Isparta, Turkey. In: 5th International Cherry Symposium, 6–10 June, Bursa. Matsumoto, Y., Miyagi, M., Watanabe, N., Kuboyama, T., 2012. Temperaturedependent enhancement of pollen tube growth observed in interspecific crosses between wild Cucumis spp. and melon (C. melo L.). Sci. Hortic. 138, 144–150. ´ D., Milatovic, ´ D., 2010. Examining self-compatibility in plum (Prunus domesNikolic, tica L.) by fluorescence microscopy. Genetika-Belgrade 42, 387–396. Osborn, J.M., El-Ghazaly, G., Cooper, R.L., 2001. Development of the exineless pollen wall in Callitriche truncate (Callitrichaceae) and the evolution of underwater pollination. Plant Syst. Evol. 228, 81–87. Pirlak, L., 2002. The effects of temperature on pollen germination and pollen tube growth of apricot and sweet cherry. Gartenbauwissenschaft 67, 61–64. Sakhanokho, H.F., Rajasekaran, K., 2010. Pollen biology of ornamental ginger (Hedychium spp. J. Koenig). Sci. Hortic. 125, 129–135. Sharafi, Y., 2011. An investigation on the pollen germination and tube growth in some Prunus persica genotypes and cultivars. Afr. J. Microbiol. Res. 5, 2003–2007. Sorkheh, K., Shiran, B., Rouhi, V., Khodambashi, M., Wolukau, J.N., Ercisli, S., 2011. Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature, polyamines and polyamine synthesis inhibitor. Biochem. Syst. Ecol. 39, 749–757. Suzuki, N., Wang, X., Kataoka, I., Inoue, H., 1993. Effects of temperature on flowering and pollen germination in Japanese apricot cv. Nanko. J. Jpn. Soc. Hortic. Sci. 62, 539–542. Vaknin, Y., Mills, D., Benzioni, A., 2003. Pollen production and pollen viability in male jojoba plants. Ind. Crop Prod. 18, 117–123. Wolukau, J.N., Zhang, S.L., Xu, G.H., Chen, D., 2004. The effect of temperature, polyamines and polyamine synthesis inhibitor on in vitro pollen germination and pollen tube growth of Prunus mume. Sci. Hortic. 99, 289–299. Youshihiro, U., Hiroaki, T., 1989. Morphometric analysis of pollen exine patterns in roses. J. Jpn. Soc. Hort. Sci. 58, 211–220. Zarrabi, A., Imani, A., 2011. Effects of fungicides on in-vitro pollen germination, tube growth and morphology of almond (Prunus dulcis). Afr. J. Agric. Res. 6, 5645–5649.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Pollen morphology, viability, and germination of Prunus domestica cv. Poˇzegaˇca ´ c´ a,∗ , N. Devrnja a , I. Kostic´ b , M. Kostic´ c D. Cali a Department of Plant Physiology, Institute for biological Research “Siniˇsa Stankovi´c”, University of Belgrade, Despot Stefan Blvd. 142, 11 060 Belgrade, Serbia b Institute for Multidisciplinary Research, University of Belgrade, University of Belgrade, Kneza Viˇseslava 1,11 000, Belgrade, Serbia c Institute for Medicinal Plant Research “Dr Josif Panˇci´c”, Tadeuˇsa Koˇs´cuˇska 1, 11 000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 25 December 2012 Received in revised form 12 March 2013 Accepted 13 March 2013 Keywords: Pollen germination Pollen morphology Pollen nuclear status Pollen tube growth Poˇzegaˇca plum Viability
a b s t r a c t The pollen morphology and exine ornamentation of an important autochthon plum cultivar (Prunus domestica cv. Poˇzegaˇca L.), which has horticultural, medicinal and alimentary uses, was examined in detail using both light and scanning electron microscope (SEM). Acetocarmine and fluorescein diacetate were used to assess the pollen viability of four Poˇzegaˇca plum genotypes. Pollen viability varied from 67% in genotype Pdp1 to 99% in genotype Pdp4. Also, pollen nucleus status was determined, and binucleate mature pollens were detected. The effect of polyethylene glycol-PEG (10, 15 and 20%, w/v) on pollen germination and tube growth was evaluated. Overall, the inclusion of PEG in the medium improved both pollen germination and tube growth. Regardless of the PEG concentration genotype has an effect on germination and length of pollen tubes. Genotype has a pronounced effect on germination and length of pollen tubes, regardless of the PEG concentration used. Genotype Pdp4 had the highest germination percentage and the highest pollen tube length on all media when compared to the other three genotypes. The highest pollen germination (96%) and a tube length (822 m) of genotype PdP4 was attained in media with 20% PEG. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Poˇzegaˇca plum (Prunus domestica L., Rosaceae), is an important autochthon plum cultivar of Serbia, that is considered to be of high quality throughout the world. This cultivar has an ideal ratio of acids and sugars in it’s fruit, and is irreplaceable in brandy and jam production. However, this cultivar is extremely susceptible to Plum pox virus (PPV) which has caused its distribution to greatly diminish. Plum pox, also known as sharka, is the most devastating viral disease of stone fruit from the genus Prunus. Wild and ornamental species of Prunus may also become infected by some strains of the virus (Dunez and Sutic, 1988). The virus is transmitted by aphids and by the transfer of infected plant material to new locations. Plum pox poses no danger to consumers, but it can ruin the marketability of stone fruit by causing acidity and deformities. The only way to manage the disease is to destroy all infected trees, which has caused significant economic losses. Many studies have investigated the responses of pollen germination and pollen tube growth to a variety of factors such as
∗ Corresponding author. Tel.: +381 11 2078 366; fax: +381 11 2761 433. ´ c). ´ E-mail address: [email protected] (D. Cali 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.03.017
temperature (Dag et al., 2000; Wolukau et al., 2004; Acar and Kakani, 2010; Alcaraz et al., 2011; Matsumoto et al., 2012), sucrose (Vaknin et al., 2003; Alcaraz et al., 2011; Devrnja et al., 2012), polyethilenglycol (Sakhanokho and Rajasekaran, 2010). Many cultivars and genotypes with unfavorable pollen have been reported by breeders and researchers previously. For instance, some of the cultivars/genotypes have sterile pollen or pollen with a low germination percentage (Du et al., 2006; Koyuncu and ´ 2010). Therefore, studying Tosun, 2005; Nikolic´ and Milatovic, the characteristics of pollen from specific genotypes and cultivars is important for breeding programs (Sharafi, 2011). Many studies have investigated pollen viability and germination in peach, plum, sour cherry and other species and cultivars. Many studies have investigated pollen viability and germination in peach, plum, sour cherry and other species and cultivars, using different testing methods to determine the pollen viability (Botu et al., 2002; Herrero and Arbeloa, 1989). Results indicated that viability, germination and tube growth of pollens varied significantly according to species, cultivar, and even test that had been used Du et al. (2006), Hedhly et al. (2004), Koyuncu and Tosun (2005), and Sharafi (2011). The objective of this work was to determine viability, nuclear status, germination and tube growth capacity of pollens in four genotypes of Poˇzegaˇca plum.
´ ali´c et al. / Scientia Horticulturae 155 (2013) 118–122 D. C
2. Materials and methods
119
DAPI- treated microspores were examined under the Zeiss Axiovert fluorescent microscope equipped with a camera.
2.1. Plant materials 2.5. In vitro pollen germination and tube growth The study was conducted on four genotypes (Pdp1, Pdp2, Pdp3, Pdp4) of Poˇzegaˇca plum growing near Belgrade, Serbia. 2.2. Scanning electron microscopy The pollen samples were collected from fresh flowers, and processed for scanning microscopy without the usual fixation and dehydration procedures. In the SEM studies, the pollen grains taken from the anthers were placed directly on the stubs, and were covered with a thin layer of gold (ion sputtering coating) in a BALETECSCD 005 Sputtering Device, imaging at 15 kV, using a JSM-6390 LV (JEOL, Tokyo, Japan) scanning electron microscope. The detailed surface ornamentation and the aperture characteristics were examined under the SEM, and the microphotographs were taken. The measurements were made in micrometer (m). Scanning electron micrograph (SEM) images were taken on Poˇzegaˇca plum pollen grains at 190 × , 450 × , 1400 × , 1800 × and 10000 × (Fig. 1A-F). 2.3. Pollen viability test Anthers were longitudinal cutting and stained with 1% acetocarmine solution. Carmine solution was prepared in 45% acetic acid. Acetocarmine treated anthers were observed under DMRB microscope from Leica (Wetzlar, Germany). A rapid method with fluorescein diacetate -FDA (HeslopHarison and Heslop-Harison, 1970) was used to determine pollen viability after 1 and 24 h. FDA (2 mg L−1 ) dissolved in acetone was diluted by 0.5 M sucrose solution (1:1). Anthers were longitudinally rejected and free pollen grains were stained with 1–2 drops of FDA.
For the pollen germination test, the hanging drop technique was employed following published procedures (Deng and Harbaugh, 2004) with some modifications as described below. A “basic” liquid medium containing 1.2 M sucrose, 0.3 g L−1 calcium nitrate [Ca(NO3 )2 ], 0.10 g L−1 boric acid (H3 BO3 ), 0.1 g L−1 potassium nitrate (KNO3 ), and 0.2 g L−1 magnesium sulfate (MgSO4 ·7H2 O) was used. The effect of polyethylene glycol (PEG) concentrations (10, 15, and 20%, w/v) on pollen germination and tube growth was evaluated in four Poˇzegaˇca plum genotypes. PEG-free medium was used as a control. For the germination test with the PEGbased media, both pollen germination and pollen tube growth were recorded after a 24 h incubation period. Three replicates (slides) were used for each genotype. A pollen grain was considered to have germinated when pollen tube length equaled or exceeded the grain diameter. Germination percentage was determined by dividing the number of germinated pollen grains by the total number of pollen grains per field of view and multiplying by 100. To measure pollen tube growth, samples were prepared as described above. To measure pollen tube growth, samples were prepared as described above, and the slides were observed under the Zeiss Axiovert light microscope equipped with a camera. 2.6. Statistical analysis The measurements of the diameter, viability and nuclei status were taken on 600 pollen grains. The means were separated using Fisher’s LSD post hoc test for P ≤ 0.05. 3. Results 3.1. Scanning electron microscopy
2.4. Pollen nucleus status The number of pollen nuclei was determined by 4’, 6-diamidino2-phenylindole - DAPI (Coleman and Goff, 1985). The content of anthers was removed by squeezing and was stained with 1–2 drops of DAPI (1 g ml−1 ) solution, prepared in distilled water. FDA and
For the pollen morphological characterization different aspects of the pollens were studied including the shape, length, width, length and width ratio, number of colpi, presence or absence of spines and exine sculpturing in the polar and equatorial view. Shape of pollen is triangular-obtuse-convex in the polar view while
Fig. 1. Scanning electron micrographs of stamen, anters and pollen grains. (A) Stamen with mature anther and filament. Pollen emerging from mature anthers. (B) Cross section of anther. (C) Densely packed pollen grains. (D) Individual pollen grains in different views. (E). Equitorial view of pollen grain with a specific ornamental exine. (F) Striata detail of exine with pores.
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Fig. 2. Flower morphology, anthers and pollen photographed under a light and SEM microscope. Morphology of flower (A) and anthers (B). (C) Lengthwise dissection of acetocarmine stained anthers. Darker (viable) colored and lighter (nonviable, arrow) pollen grains. (D) Natural view of undyed pollen. (E) Viable and nonviable (arrow) pollen grains treated with FDA. (F) Binucleate pollen with a vegetative (V) and generative (G) nucleus staining by DAPI.
the shape of pollen is elliptic-acuminate-acute in the equatorial view. Pollen class is trizonocolpateobtus-triangular. The sculpturing of exine surface is striate. The number of colpi is three while the spines are absent. The horizontal distance (width) in the polar view is 54.37 ± 0.05 m, while the vertical distance (length) is 47.98 ± 0.11 m. The length - width ratio in the polar view is 1.13 m. The horizontal distance (width) in the equatorial view is 25.00 ± 0.37 m while the vertical distance (length) is 20.00 ± 0.43 m. The length - width ratio in the equatorial view is 0.80 m. Exine architecture of mature pollen grains taken at 10,000 × shown in Fig. 1F. Pori are elongated and also with a distinct margin. The porus latitude is smaller than the colpus latitude. One to three perforations were noticed on one square m of exine (Fig. 1F). 3.2. Pollen viability test Acetocarmine treated anthers originating from opened flowers (Fig. 2A, B) showed many viable pollen grains with different size, morphology and viability (Fig. 2C). Even pollen grains which are not acetocarmine treated showed different shape and size (Fig. 2D). The pollen grains are densely packed, therefore many more can fit inside of the anther, which was identified by cross sections of the anthers in both light and SEM microscopy. The pollen viability test was determined for 600 pollen grains of Poˇzegaˇca plum by FDA. Pollen viability was different between different genotype after a 1 and 24 h staining procedure. Viability is increased from genotype Pdp1, Pdp2, Pdp3 to genotype Pdp4 from 67, 73, 80 to 99% (Fig. 2E). Pollen grains have the same viability after both 1 and 24 hours. 3.3. Pollen nucleus status The number of pollen nuclei was determined with DAPI. All investigated pollen grains were binucleate (Fig. 2F).
3.4. In vitro pollen germination and tube growth The length of pollen tubes on PEG media was measured after 24 hours under a light microscope. The effect of PEG on pollen germination and tube growth varied according to concentration and time. Pollen grains from genotype Pdp 4 had the highest germination percentage on all media (Fig. 3A-B and 4). The highest germination percentage (96%) was obtained in a media with 20% PEG for genotype Pdp 4 (Fig. 3B). The germination percentage steadily improved with increasing PEG concentration for all genotype. The greatest influence of increased PEG concentrations was observed for genotype PdP1, where in comparison there was a very low germination rate for the control (1%). Overall in comparison to the control there was a 15 times greater germination potential for germination on 20% PEG (15%). Genotype Pdp4 had a greater germination percentage on PEG free medium than other three genotypes on the best medim with 20% PEG (Fig. 4). Genotype Pdp4 had 6.4 times greater germination on medium with 20% PEG compared to genotype Pdp1. Also, genotype Pdp4 had 2.13 times greater germination compared to genotype Pdp2 and 1.48 times in relation to genotype Pdp3 on same medium (Fig. 4). The influence of genotype and PEG was observed for longer pollen tubes, therefore pollen tube growth linearly increased from gen Pdp1 to gen Pdp4. A similar trend exists at higher PEG concentration. The highest pollen tube length (822 m) of genotype Pdp4 was obtained in medium containing 20% PEG (Fig. 4B; Fig. 5).
4. Discussion Since there are no reports on the pollen morphology, viability, and germination of Poˇzegaˇca plum, the present report gives an account of the palynological characters of this species. Poˇzegaˇca plum as well as the most of the species in Rosaceae has trizonocolpateobtus-triangular pollen. The pollen grains of P. domestica cv. Poˇzegaˇca are trizonocolpateobtus-triangular, as
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Fig. 3. Morphology of germinating pollen. (A) Pollen of Pdp4 genotype on medium with 15% PEG and 20% PEG (B).
Fig. 4. Pollen germination of Poˇzegaˇca plum after 24 h.
previously examined in species of this genus (P. bokhariensis, P. armeniaca, P. avium, P. persica, and P. cornuta) (Gilani et al., 2010). The equatorial axis, and the polar axis and diameter of the pollen grains of P. domestica are similar with four previously examined Prunus species (Gilani et al., 2010). SEM images of Poˇzegaˇca plum grains showed that these pollen grains had a well defined exine with specific architecture and ornamentation. The role of the exine is to protect the male spore and gametophyte from desiccation and other dangers of sub-aerial dispersal.
Fig. 5. Tube growth of Poˇzegaˇca plum pollen on different concentration PEG after 24 h.
Our results regarding pollen morphology and exine sculpturing are similar to the results Youshihiro and Hiroaki (1989), Arazani et al. (2005), Gilani et al. (2010) for pollen of the genus Prunus. In addition, the pollen morphology of different Rosaceae taxa studied by several researchers has reported that the exine features are for taxonomic and phylogenetic classification (Arazani et al., 2005; Gilani et al., 2010). Staining methods and fluorescence are very important because they allow for more complete information regarding pollen viability and fertilization potential (Atlagic´ et al., 2012). Pollen viability showed many differences between genotypes of Poˇzegaˇca plum. Genotype Pdp4 has pollen with the highest viability after acetocarmine and FDA staining. Our results indicate that genotypes have large differences in pollen viability, which is the results obtained for other species (Vaknin et al., 2003; Sakhanokho and Rajasekaran, 2010). Also, the pollen of the genotype Pdp4 had the greatest germination and length of pollen tubes. The results regarding the significant impact of genotype on Poˇzegaˇca plum pollen germination is in agreement with the results obtained in other Prunus species (Asma, 2008; Du et al., 2006; Hedhly et al., 2004; Koyuncu and Tosun, 2005; Sharafi, 2011). Previous research has studied the impact of temperature (Asma, 2008; Hedhly et al., 2005; Pirlak, 2002; Sorkheh et al., 2011; Suzuki et al., 1993), sucrose (Asma, 2008), polyamines (Sorkheh et al., 2011; Wolukau et al., 2004), plant growth regulators (Bolat and Pirlak, 2003), boric acid (Bolat and Pirlak, 2003) and fungicides (Zarrabi and Imani, 2011) on Prunus pollen germination with the exception of genotype effect. However, until now no one has studied the effect of PEG on pollen germination and growth of pollen tubes, which is presented in this manuscript. In general, the inclusion of PEG in the liquid medium improved both pollen germination and tube growth for examined species and varied according to PEG concentration. Regardless of the PEG concentration, genotype has an effect on germination and length of pollen tubes. Pollen morphology and ultrastructure have been shown to be correlated with growth habitat and pollination biology (Osborn et al., 2001). Our results show that binucleate pollen grains of Poˇzegaˇca plum retain viability 24 h after being exposed to dye. Binucleate pollen has weaker germination but longer survival than trinucleate pollen. It has been shown that plants which have binucleate pollen grains are pollinated via insects, which agrees with previously published results (Gottsberger, 1989).
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