AgroLife Scientific Journal - Volume 5, Number 1, 2016 ISSN 2285-5718; ISSN CD-ROM 2285-5726; ISSN ONLINE 2286-0126; ISSN-L 2285-5718 POLLEN MORPHOLOGY AND GERMINATION OF INDIGENOUS GRAPEVINE CULTIVARS ŽILAVKA AND BLATINA (Vitis vinifera L.) Tatjana JOVANOVIC-CVETKOVIC 1, Nikola MICIC 1,2, Gordana DJURIC 1,2, Miljan CVETKOVIC 1 1 University of Banja Luka, Faculty of Agriculture, Bosnia and Herzegovina 2 University of Banja Luka, Genetic Resources Institute, Bosnia and Herzegovina Abstract Corresponding author: miljan.cvetkovic@agrofabl.org The indigenous cultivars Žilavka and Blatina of V. vinifera are of the greatest economic importance for wine production in Bosnia and Herzegovina. In vineyards, the grape productivity of Žilavka is normal while that of Blatina is extremely low. The objective of this work was to study the pollen morphology of V. vinifera cvs. Žilavka and Blatina using scanning electron microscopy. The pollen grain of Žilavka showed three furrows, but acolporated (without furrows or pores) pollen grains were observed in Blatina. Pollen germination in Žilavka was satisfactory, whereas Blatina pollen lacked the capacity to germinate. The present research is a contribution to amore detailed analysis of major grapevine cultivars in the Balkans. Key words: indigenous cultivars, scanning microscopy, pollen. INTRODUCTION Although there is a range of indigenous grapevine cultivars available in Herzegovina region (southeastern part of Bosnia and Herzegovina) for many years the greatest enological and economic importance has been placed on cvs. Žilavka and Blatina. As opposed to excellent reproductive characteristics and satisfactory yields of Žilavka (Mijatović, 1988; Kurtović et al., 1989; Tarailo, 1991) Blatina exhibits low productivity which is typically associated with the characteristics of the functionally female flower and fertilization problems, which have not been systematically examined and explained so far. Pollen grains of V. vinifera are normally 3-zonocolporate, spheroidal to prolate, with very long, narrow, slit-like, slightly, but distinctly sunken ectoaperture (colpus). The endoaperture is a circular pore, with narrow, but distinct costae (Galardo et al., 2009). However, a considerable body of previous research (Lombardo et al., 1978; Cargnello et al., 1980; Cabello Saenz Maria et al., 1994; Slimane-Harbi et al., 2004; Marasali et al., 2005; Abreu et al., 2006) on some economically important cultivars (hybrids) of grapevine (and their clones) has demonstrated the presence of pollen roundish 105 in shape, without furrows and germination pores. In the wild grapevine V. vinifera subsp. sylvestris (Gmelin) Hegi, pollen dimorphism has also been observed (Inceoglu et al., 2000; Galardo et al., 2009) and attributed to differences between pollen grains of male and female plants. Regardless of the report (Lombardo et al., 1978) that the presence of pollen with pores and germination apertures is responsible for high grapevine productivity, Gargnelo et al. (1980) found that the absence of germination pores can be major cause, although not the only cause of low yields in some grapevine cultivars and populations. The objective of this research was to analyse the pollen morphology and germination of indigenous cvs. Žilavka and Blatina to determine their morphological specificities. MATERIALS AND METHODS Research was conducted in the 2010/2012 flowering season using two main indigenous cultivars in Bosnia and Herzegovina - 'Blatina' with functionally female flowers and 'Žilavka' with hermaphrodite flower type. Samples were taken from commercial vineyards in Mostar region (characteristics of the Mediterranean climate, average temperature in January 2.3 o C,
in July 22.5 o C to 25.7 o C). At different stages of flowering, inflorescences were removed from the vines in early morning and kept in a sealed vial in an insulated container for transport to the laboratory, where they were refrigerated at 4 o C. Pollen samples were coated with 0.02 μmgold alloys in a BAL-TEC SCD 005 sputtercoater and monitored at 15 kv with a JEOL JSM-6390LV SEM. Pollen grains were observed and photographed at 500, 1,500 and 2,000 (whole grain) and 10,000 15,000 magnification (for exine pattern characteristics). Pollen grains were measured directly on the screen of the electron microscope and pictures of pollen grains were taken for each of the tested cultivars. Both dry and hydrated pollen grains were examined. For each pollen grain (30 per cultivar) the following parameters were monitored: pollen shape and size (length, width and length/width - L/W ratio) and exine ornamentation characteristics (Punt et al., 2007). Pollen separation for germination analysis was performed at the initial flowering stage. Pollen viability was assessed by the hanging drop method under in vitro conditions using 12%, 15%, 18% sucrose solutions. The germination of pollen grains was examined under a light microscope, with readings taken in microscopic fields of view. In each field of view (5 per cultivar), total number of pollen grains and number of germinating pollen grains were counted. Germination was photodocumented using an Olympus/DP/SOFT light microscope and an Olympus/DP camera, and the resulting images were edited by the Image Analyzer software. Statistical analysis was carried out via the statistical software package SPSS 22 (IBM 2013). Figure 1. Pollen grain - distribution in anthers ('Žilavka') Figure 2. Pollen grain - equatorial view ('Žilavka') RESULTS AND DISCUSSIONS The morphological analysis of the dry pollen grain of 'Žilavka' shows that it hasthe morphology of a wheat grain as caused by the closed colpi due to the morphological and physiological preparation for viability at the dissemination stage (Figure 1 and 2). Once the pollen grain reaches the stigma, the colpi open, thus giving the hydrated pollen grain of 'Žilavka' the shape of a slightly flattened rotating ellipsoid (Figure 3). Figure 3. Pollen grain - polar view ('Žilavka') In 'Blatina', the dry pollen grains are visually collapsed, with a depression in the interior of the grain itself (Figure 4 and 5). 106
reticulate-foveolatein 'Blatina' and foveolate in 'Žilavka'. Figure 4. Pollen grain - distribution in anthers ('Blatina') Figure 7. Pollen grain-exine pattern ('Žilavka') Figure 5. Pollen grain - equatorial view ('Blatina') During hydration the pollen grain of 'Blatina' absorbs the fluid and obtains the shape of a flattened rotating ellipsoid with undifferentiated colpi - furrows and without germination apertures (Figure 6). Figure 8. Pollen grain - exine pattern ('Blatina') The detailed analysis of the collapsed pollen grains of 'Blatina' shows interesting details that suggest the effort of the cytoplasm to invaginate the external membrane and morphologically prepare the pollen grain for release by the anther (Figure 9 and 10). Figure 6. Pollen grain - polar view ('Blatina') In both cultivars, the exinetakes the form of a reticulate pattern in the equatorial zone (Figure 7 and 8). In the polar region, the exine is 107 Figure 9. Sporadic occurrence of an initial furrow, suggesting signs of colpus positioning
Typically two aperture types were observed. Tricolporate pollen grains were observed in the pollen of 'Žilavka', the tricolporate form being characteristic of regular-bearing cultivars and clones (Cabello Saenz Maria et al., 1994; Slimane-Harbi et al., 2004; Marasali et al., 2005; Abreu et al., 2006) as well as of male plants of the wild grapevine V. vinifera subsp. sylvestris (Gmelin) Hegi (Inceoglu et al., 2000; Galardo et al., 2009). Figure 10. Sporadic initial invagination of the external membrane, suggesting colpus formation efforts The shape of a pollen grain (Table 1) in 'Žilavka' is prolate (26.86 15.41 μm), and that in 'Blatina' subprolate (23.12 21.90 μm). Table 1. Pollen grain diameters in the tested cultivars (average for 2010-2012) Pollen grain diameters Length Width (μm) (μm) L/W ratio cultivar ± ± 'Blatina' 23.448 ± 0.86 18.164 ± 0.74 1.290 'Žilavka' 26.861 ± 0.87 15.416 ± 0.73 1.742 During the research, 'Blatina' showed no pollen germination in any experimental year (Figure 11), whereas the germination of 'Žilavka' ranged from 18.21 0.237 to 2.52 0.404 depending on the sucrose content in the medium and experimental year (Figure 12 and Table 2). Figure 12. In vitro germination of 'Žilavka' tricolporated pollen Table 2. pollen germination of 'Žilavka' in 12%, 15% and 18% sucrose solution year sucrose solution % V k 2010 12 36.25 0.562 17.32 15 31.68 0.599 20.56 18 32.06 0.486 14.31 2011 12 34.64 0.603 18.26 15 42.52 0.404 11.36 18 29.26 0.436 14.59 2012 12 38.41 0.636 19.32 15 28.77 0.680 24.56 18 18.21 0.237 12.88 Figure 11. In vitro germination of 'Blatina' acolporated pollen Upon hydration, the pollen grains of 'Žilavka' become hexagonal, due to the preparation for germination, and do not take a different form of pollen of the same plant (Cabello Saenz Maria et al., 1994). Pollen germination of this cultivar was satisfactory and in agreement with previous results (Kurtovićet al., 1989), ranging between 29.0 and 31.5% depending on the germination medium. In 'Blatina', the presence of inaperturatepollen grains was observed, as also found in certain cultivars and clones (Cabello Saenz Maria et al., 1994) of plants of economically important cultivars (Lombardo et al., 1978)and of female 108
plants of the wild grapevine V. vinifera subsp. sylvestris (Gmelin) Hegi (Inceoglu et al., 2000; Galardo et al., 2009). In analysis of the pollen of female plants of V. vinifera ssp. silvestris, Caporali et al., (2003) found a high percentage of collapsed pollen grains with a typical depression towards the interior, which are identical to the collapsed pollen grains in 'Blatina'. Such pollen grains do not have the capacity to germinate, although the present research confirmed that the pollen grains of 'Blatina' show a normal cytogenetic constitution which enables them toperform fertilization (results not presented in this paper). Pollen grain size in 'Žilavka' and 'Blatina' is correlated with the results of Marasali et al. (2005) and Galardo (2009), whereas somewhat larger grains were observed by Cabello Saenz Maria et al. (1994). Differences in exineornamentation in the polar region and that in the equatorial view were also confirmed by Abreu et al. (2006). CONCLUSIONS 'Žilavka' has morphologically functional pollen grains that exhibit a high germination rate, thus ensuring high regular yields regardless of the presence of a pollinator in the vineyard. In 'Blatina', reproductive success is inhibited not only by functionally female flowers but also by the aporate pollen, which is not morphologically predisposed to germination. ACKNOWLEDGEMENTS This work has been supported by the Ministry of Science and Technology of the Republic of Srpskaunder the projects 'Anatomorphological and cytohistological evaluation of reproductive organs of indigenous grape cultivars in Bosnia and Herzegovina' (Contract No. 06/6-020/961-119/08) and 'Genotype characterisation of cv. 'Blatina' male sterility' (Contract No. 19/6-020/961-177/09). REFERNCES Abreu I., Costa I., Oliveria M., Cunha M., De Castro R., 2006. Ultrastructure and germation of Vitis vinifera L. cv Loureiropollen. Protoplasma 228: p. 131-135. Cabello-Saennz S.M., Luis-Villota F., Tortosa-Tortola P., De-Luis-Villota M.E., 1994. Palynological study of the pollen grain of Vitis vinifera L. cultivars: some aspects of sculpturing and pollination. Vitis 33: p. 57-61. Caporali E., Spada A., Marziani G., Failla O., Scienza A., 2003. The arrest of development of abortive reproductive organs in the unisexual flower of Vitis vinifera ssp. silvestris. Sexual Plant Reproduction 15: p. 291-300. Cargnello G., Carraro L., Lombardo G., Gerola F., 1980. Pollen morphology of Picolit grown in different Italian regions. Vitis 19: p. 201-206. Gallardo A., Ocete R., Angeles Lopez M., Lara M., Rivera D., 2009.Assessment of pollen dimorphisim in populations of Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi in Spain.Vitis 48 (2): p. 59-62. Inceoglu Ö., Pinar N.M., Oybak-Donmez E., 2000. Pollen morphology of Wild Vitis sylvestris Gmelin (Vitaceae). Turk J. Bot. 24: p. 147-150. Kurtović M.. Mijatović D., Blesić M., Tarailo R., 1989. Klijavost polena vinskih i stonih sorti vinove loze (Vitis vinifera L.). Poljoprivrednipregled 4,5 i 6: p. 23-27. Lombardo G., Cargnello G., Bassi M., Gerola F.M., Carraro L., 1978. Pollen ultrastructure in different vine cultivars with low productivity.vitis 17: p. 221-228. Marasali B., Münevver P., Büyükkartal N., 2005. Palynological Study on the Pollen Grains of Selected Turkish Grape (Vitis vinifera L.) Cultivars. Turk J. Agric. For 29: p. 75-81. Mijatović D., 1988. Ispitivanje karakteristika rodnosti i kvalitet agrožđa sorte Žilavka u interakciji važnijih agroekolo škihčinilaca. Doktorska disertacija Poljoprivredni fakultet Univerziteta u Sarajevu. Punt W., Hoen P.P., Blackmore S., Nilsson S., Le Thomas A., 2007. Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology, 143: p. 1-81. Slimane-Harbi M.B., Chabbouh N., Snoussi H., Bessis R., Gazzah M., 2004. Etude du germoplasme de vignes autochtones de Tunisie. Precisions sur I' origine du millerandage du "Razzegui". Bulletin O.I.V, Vol. 77, No. 881-882: p. 487-501. Tarailo R., 1991. Proučavanje poulacije sorte Žilavka u cilju izdvajanja odlika sa najpovoljnijim biološkim i privredno-tehnološkim karakteristikama. Doktorska disertacija. Poljoprivredni fakultet Univerziteta u Sarajevo. 109