Viability and in vitro germinability of pollen grains of olive cultivars grown in different environments

Yield of olive orchard depends on many factors; among them potential male fertility plays a primary role in both self-fertile and self-incompatible cultivars. Genetic and environmental factors have been indicated as important factors affecting pollen grain viability and germinability. In this study these two parameters were investigated on fresh pollen grains collected from plants of the cultivars ‘Arauco’, ‘Arbequina’, ‘Coratina’, ‘Farga’, ‘Frantoio’, ‘Leccino’, ‘Leccio del Corno’, ‘Morchiaio’, ‘Morettini’, ‘Nevadillo’, ‘Nocellara’, ‘Sevillana Fina’, ‘Urano’. The cultivars were grown in Mendoza (Argentina), different localities of Buenos Aires (Argentina) and Tuscany (Italy). Viability was assessed by the fluorochromatic reaction with double coloration with fluorescein diacetate and propidium iodide. In vitro germinability was evaluated on pollen grains cultivated in droplets of H3BO3, Ca(NO3)2 and sucrose contained in petri dishes. Pollen grains showed different rates of viability and germinability in relation to cultivar and place of cultivation. The percentage of viable pollen grains consistently resulted higher than the rate of in vitro germinability. No linear correlation was found between the viability and germination rates. INTRODUCTION Selfand inter-incompatibility, and morphological aberrations are relevant factors of sterility in olive (Olea europaea L.); furthermore low productivity, associated to reduced fecundation, is very often due to insufficient viability and germinability of pollen grains (Bini, 1984; Tombesi, 2003; Romano et al., 2006). Pollen viability can be studied through different techniques, among which the lactophenol aniline blue staining (Hayman, 1956); the redox staining test with 2,3,5triphenyltetrazolium chloride – Triphenyl (TTC) (Lakon, 1942; Stanley and Linskens, 1974); the triphenilmetane “Aniline blue” (Smith-Huerta and Vasek, 1984); acetocarmine jelly (Radford et al., 1974; Raamsdonk, 1985); fluorescein-diacetate/propidium-iodide (FDA-PI) test (Heslop-Harrison and Heslop-Harrison, 1970; Greissl, 1989) are mostly adopted. The in vitro growth of pollen tube is adopted to evaluate pollen grain germinability. Olive pollen germinability is very sensitive to the composition of in vitro growing media (Ferri et al., 2008). Actually the knowledge about the compounds, their presence and concentration on the media, are referred to water, sucrose, agar, boric acid, tetracycline and citric acid (Morettini, 1972; Lee et al., 1985; Shivanna and Johri, 1985; Pinney and Polito, 1990; Al-Dehadheh et al., 2004). Factors that affect pollen viability and germinability are length of time of conservation, temperature (usually low values extend time of viability) and air humidity (very dry or very wet are deleterious, being 28-33% of relative humidity (RH) optimal levels) (Heslop-Harrison et al., 1981; Lee et al., 1985; Pinney and Polito, 1990; Barnabas and Kovacs, 1997; Ferri et al., 2008). The aim of this work was to evaluate the male fertility of olive pollen grains on a broad range of cultivars including oil and table cultivars, grown in Italy and Argentina, in Proc. VII IS on Olive Growing Eds.: F. Vita Serman et al. Acta Hort. 1057, ISHS 2014 66 order to supply information on their suitability to be used as pollinizers in new orchards and possibly to reduce cases of low productivity in specific olive groves. MATERIALS AND METHODS Viability and germinability were assessed on pollen grains of trees grown in Italy and three sites of Argentina. The olive trees in Italy are located in an experimental field belonging to the Faculty of Agronomy of the University of Florence in the “Azienda Montepaldi”, S. Casciano Vai di Pesa (Florence Province) (43°N, 11°E; 230 m a.s.l.). The plants were 5 years old, not irrigated and they were grown in typical Tuscany landscape of olive orchards. In Argentina, pollen samples were collected from the olive germplasm collection of the EEA INTA Junín, Mendoza province (33°S, 68°W, 653 m a.s.l.). The trees were planted in 1956 to 12×12 m distance with irrigation through basin, according to the scheduling typically used in the area. While, the other two Argentine populations are located at Dorrego (37°S, 62°W) and Puan (38°S, 60°W), in the southern of Buenos Aires province, olive trees were 4-6 years old and were not irrigated. Randomly distributed flowers were collected from the trees at the E stage (i.e., corolla changes colour from green to white) and F stage (i.e., 10% of flower open) (Loussert and Brousse, 1978) and stored in petri dishes for 48 h at room temperature with Silica Gel; pollen grains, extracted from dehiscent anthers, were stored at +4°C (RH=29%) until the assessment tests. Assessment of Viability Pollen grain viability was assessed by the Greissl test (1989). Before staining, 1-2 drops of a 0.3 M sucrose solution were used to reactivate membrane functions on pollen grains; 1-2 drops of fluorescine diacetate solution (FDA) and 1 drop of propidium iodate solution where added after 15 and 25 min, respectively. Pollen grains were then incubated in dark for 24 hours at 24°C. A Leica DM 1000 microscope with a highpressure mercury lamp of 50 W at blue excitation (450-490 nm) and ultraviolet excitation (340-380 nm) was used for pollen grains observations. Pollen grains showed bright green color were considered viables (V), the opaque green, yellow, orange and red were considered not viables (NV) (Fig. 1). Viability values (percentage of viable on total observed pollen grains) were classified as high (>50%), medium (from 25 to 50%) and low (<25%) (Rovira and Tous, 2005). The percentage of aborted pollen grains was determined as pollen grains which showed some kind of developmental anomaly. Assessment of In Vitro Germination Petri dishes containing distilled water in the base and with nutrient solution droplets deposited on the inner face of the petri dish lid were used as moist chambers for the germination of pollen grains. The nutrient solution employed was 30% sucrose solution with added 2×10 M H3BO3 and 6×10 M Ca(NO3)2. Pollen grains were sown in five or six droplets during 24 h at 24°C. The pollen grains were considered germinated when the length of pollen tube was 3-fold the grain diameter (Fig. 2) (Morettini, 1972; Pinney and Polito, 1990). Data on viable/non viable and germinated/non germinated pollen grains were analyzed by means of Chi-squared test (p=0.05) and average and standard errors are reported. The coefficient of correlation between viability and germinability results was calculated. RESULTS AND DISCUSSION Viability The general average percentage of viability observed on pollen grains of cultivars independently of collection localities was 58.52±3.13, with 32.45% the lowest value (‘Leccio del Corno’ cultivated in Mendoza, Argentina) and 87.79% the highest (‘Arbequina’ grown in Mendoza, Argentina). The values observed in all the studied cases