Trees (2011) 25:809–822 DOI 10.1007/s00468-011-0557-7
ORIGINAL PAPER
Influence of temperature on the in vitro pollen germination and pollen tube growth of various native Iranian almonds (Prunus L. spp.) species Karim Sorkheh • Behrouz Shiran • Vahid Rouhi Mahmood Khodambashi
•
Received: 18 November 2010 / Revised: 12 March 2011 / Accepted: 15 March 2011 / Published online: 31 March 2011 Ó Springer-Verlag 2011
Abstract Pollen germination and pollen tube growth was quantified among various native Iranian wild almonds (P. dulcis (Mill.) D. A. Webb, P. eleaegnifolia Mill., P. orientalis Mill., P. lycioides Spach, P. reuteri Bioss. et Bushe, P. arabica Olivier, P. glauca Browick and P. scoparia Spach in order to identify differences in the tolerance of pollen to temperature variations. Pollen germination and pollen tube growth were observed after incubation in darkness in a germination medium for 24 h at 10–50°C at 5°C intervals. Maximum pollen germination of the wild almond species and specify that 60% was obtained for P. orientalis pollen and 98% for P. scoparia. Pollen tube length ranged from 860 lm was obtained in P. lycioides and 1490 lm in P. scoparia. A modified bilinear model best described the response to temperature of pollen germination and pollen tube length. Almond species variation was found for cardinal temperatures (Tmin, Topt and Tmax) of pollen germination percentage and pollen tube growth. Mean cardinal temperatures averaged over eight almond species were 14.7, 24.2, and 43.7°C for maximum percentage pollen germination and 14.48, 25.3, and 44.4 °C for maximum pollen tube length. The principal component analysis (PCA) identified maximum percentage pollen germination and pollen tube length of the species, and Tmax for the two processes as the most important pollen parameters in describing a species tolerance to high temperature. PCA also classified Prunus L. spp. into four
Communicated by J. Carlson. K. Sorkheh (&) B. Shiran V. Rouhi M. Khodambashi Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahrekord University, Shahrekord, P.O. Box 115, Iran e-mail:
[email protected]
groups according to the tolerance of pollen to temperature variations. The Tmin and Topt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating species for high temperature tolerance. Keywords Prunus spp. Pollen germination Pollen tube growth Cell membrane thermostability
Introduction Temperature is the important factor controlling plant growth and development. Suitability of a crop to a given location depends not only on the threshold temperatures but also on the length of the growing season. Daily or seasonal temperatures above optimum and temperature extremes, should they coincide with critical stages of plant development, will become a major factor limiting crop production (Kakani et al. 2005; Acar and Kakani 2010). Extreme events such as warmer days with decrease in diurnal temperature range are projected to occur more frequently in the future climates (Dai et al. 2001; Kakani et al. 2005). Fruit set in many agronomic crops is sensitive to high temperature (Reddy et al. 1992; Peet et al. 1998). Fruit set was reduced on exposure to daytime temperatures of [30°C for about 13 h in Upland (Gossypium hirsutum L.) and Pima (G. barbadense L.) cottons (Reddy et al. 1992), 35°C for 4 h in Brassica napus L. (Young et al. 2004) and [28°C for 12 h during flowering in tomato (Lycopersicon esculentum Mill.) (Peet et al. 1998; Sato et al. 2002). Seed yield of wheat (Triticum aestivum L.) (Saini and Aspinall 1982), corn (Zea mays L.) (Mitchell and Petolino 1988) and rice (Oryza sativa L.) (Matsui et al. 1977) were reduced on exposure to daytime temperatures of 30°C for 16 h, 38°C for 16 h and [36°C for 6 h.
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Similarly, pod-set was reduced at day temperatures [28°C for 12 h in bean (Phaseolus vulgaris L.) (Prasad et al. 2002) and [28°C for 12 h in groundnut (Arachis hypogaea L.) (Prasad et al. 1999a; 2003). Iran, with a total land area of 1,648,195 square kilometres, lies between 25° and 39° N latitude and 44° and 63° E longitude and is primarily subtropical in the southern half of the country, temperate in the northern half part and mostly desert in the middle (Sorkheh et al. 2009). The resultant variability in environment and climate has made possible an extensive diversity of plant germplasm (Ghahreman and Attar 1999). Almond [Prunus dulcis (Mill.) D.A. Webb (syn. P. amygdalus (L.) Batsch)] production in Iran is based on locally adapted clones, with minimum to no inputs, and traditional management. Wild almond species (Prunus L. spp.) commonly grow in areas between 28° and 38° N and 41° and 54° E and from 1,100 m to 2,700 m altitudes (Komarov et al. 1941; Browicz 1969; Rickter 1972; Grasselly 1976; Denisov 1988; Kester et al. 1991). The limited gene pool in cultivated almond limits the cultivation to specific areas with Mediterranean climate. Related species demonstrate a greater resistance to abiotic and biotic stresses and so represent valuable germplasm sources for breeding. In addition, the wide adaptation of the related wild almond species indicates their potential as sources for resistance to abiotic and biotic stresses and represents valuable germplasm source for breeding. According to Barbosa et al. (1989), peach (Prunus persica L.) adapted to subtropical conditions can produce between 1,000 and 2,000 pollen grains per anther, and up to 80,000 per flower, with viability greater than 85% in the stone fruits. However, peach pollen viability can be influenced by diverse internal factors, such as the tree’s nutritional conditions (Willians 1970) and external factors, such as the hydratation level of the pollen and environmental temperature. High temperatures in the preblooming had a great influence on the number of pollen grains produced (Nava et al. 2009), and, depending on the intensity, can cause sterility in the male gametophytes. According to Sukhvibul et al. (2000), the effect of the temperature on the germination of pollen grains was inconsistent and varied between species and cultivars. In peach trees, Weinbaum et al. (1984) observed a germination peak of 20°C, but the same remained relatively high at 7 or 30°C. According to Kozai et al. (1999), the inhibition of the fertilization in peach trees at high temperature did not reduce the viability of pollen grains. The reproductive organs of plants are usually the most sensitive to chilling (Larcher and Bauer 1980). Low temperature during flower development has been found responsible for the occurrence of defective reproductive
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organs in different agricultural crops: almond (Prunus dulcis L.) (Egea and Burgos 1995), grape (Vitis venifera L.) (Ebadi et al. 1995), persimmon (Diospyros kaki Linn) (Fukui et al. 1990), white clover (Trifolium repens L.) (Pasumarty et al. 1995) and Arabidopsis thaliana (Boavida and McCormick 2007). Tropical and subtropical plants are prone to this damage when flower development occurs at suboptimal temperatures (Polowick and Sawhney 1985; Mercado et al. 1997). Temperature requirement for the pollen germination and pollen tube growth in fruit species and cultivars may differ in early or late flowering period. For example, the desired temperature to optimal pollen germination of walnut (Juglans regia L.) was lower in early-flowering varieties than late-flowering walnut counterparts (Luza et al. 1987). Furthermore, almond pollens could germinate even at 5°C but the rate of pollen germination was considerably low (Godini et al. 1987). However, even a germination temperature of 10°C was not enough for pear (Pyrus communis L.) and pollen (Mellenthin et al. 1972). Additionally, Egea et al. (1992) observed that pollen germination and tube growth speed was low at 5°C for apricot (Prunus armeniaca L.) varieties and the germination rate increased with increasing temperature. Extreme temperatures during the flowering period not only impede pollen germination and tube growth but also reduce bee (Vasilakakis and Porlingis 1985) and other insect activities, which is of an important role in pollination. Pollen performance, which includes pollen germination, pollen tube growth rate and pollen competition, is an important component of fertilization success in seed-producing plants. Pollen performance is clearly affected by the genotype of the pollen (Snow and Spira 1991; Sharafi et al. 2010). Temperature is also one of the most important environmental factors that could affect pollen performance during the progamic phase (Hedhly et al. 2005). It has been shown that temperature affects pollen germination (Elgersma et al. 1989; Shivanna et al. 1991) and pollen tube kinetics in the style (Jefferies et al. 1982; Elgersma et al. 1989). Temperature ranges and optimum temperature values for pollen germination and pollen tube growth were studied for different fruit species, including jojoba (Simmodsia chinensis (Link) Schneider) (Lee et al. 1985), pears (Pyrus communis L.) (Mellenthin et al. 1972; Vasilakakis and Porlingis 1985), papaya (Carica papaya L.) (Cohen et al. 1989), cherimoya (Annona cherimmola Mill.) (Rosell et al. 1999), mango (Mangifera indica L.) (Sukhvibul et al. 2000; Dag et al. 2000), Prunus mume L. (Wolukau et al. 2004), almond (Godini et al. 1987), apricot (Vachun 1981; Egea et al. 1992; Austin et al. 1998; Pirlak 2002), sour cherry (Cerovic and Ruzic 1992), sweet cherry (Pirlak 2002) and Pistacia L. spp. (Acar and
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Kakani 2010). The optimum temperature required for pollen germination was about 15 and 20°C for apricot, sour cherry and sweet cherry (Vachun 1981; Egea et al. 1992; Cerovic and Ruzic 1992). Cell membrane leaf thermostability is a vegetative physiological parameter widely used in studying plant tolerance to temperature. It was successfully used to screen crops for high-temperature tolerance (ur Rahman et al. 2004; Acar and Kakani 2010). The crops showing high membrane thermostability gave higher seed yield under high-temperature conditions during flowering and bollfilling period (Malik et al. 1999; ur Rahman et al. 2004). Recent studies in peanuts showed that cell membrane thermostability was not highly correlated with yield loss or pollen germination under high-temperature conditions (Kakani et al. 2002; Craufurd et al. 2003; Kakani et al. 2005). Thus, it is essential to understand the relationships between responses of pollen to high temperature and leaf membrane thermostability in wild almond (Prunus L. spp.) species. Although optimum temperature for pollen germination and pollen tube growth is known to vary among and within species, there is no extensive study, within the literature, on the response of pollen germination and pollen tube growth to germination temperature ranges in Prunus species of almond. The cardinal temperatures (Tmin, Topt and Tmax) for maximum percentage pollen germination and maximum pollen tube length have not been reported for native Iranian almonds species. This is the first study of the effect of temperature on pollen germination and pollen tube growth of native Iranian almonds species. The objectives of this study were to (a) quantify the effect of temperature on pollen germination and pollen tube growth of different almond species, (b) determine cardinal temperatures for pollen germination and pollen tube growth and (c) compare pollen (total germination and maximum pollen tube growth) response to temperature with leaf membrane thermostability.
Materials and methods Wild almond species Wild almond species studied included in the genus Prunus, subgenus Amygdalus were P. dulcis (Mill.) D. A. Webb, P. eleaegnifolia Mill. and P. orientalis Mill. [syn. P. argentea Lam] in section Euamygdalus Spach; P. lycioides Spach and P. reuteri Bioss et. Bushe [syn. P. hordia Spach] in sect. Lycioides Spach; and P. arabica Olivier, P. glauca Browick and P. scoparia Spach in sect. Spartioides Spach (Kester et al. 1991; Browicz and Zohary 1996) (Table 1). Pollen collection and growth medium Flowers were collected from ten plants per species at the time of anther dehiscence, between 7 and 8 a.m. according to Kakani et al. (2002) and Acar and Kakani (2010), and immediately placed in Petri dishes lined with moistened filter paper to avoid pollen desiccation. Pollen from ten randomly selected flowers was put on a slide and mixed using a nylon hairbrush. The pollen was collected either by pressing the keel petal or by removing pollen from the anthers using a needle. After mixing, the pollen was transferred within 30 min of picking the flowers on to the growth medium. The germinating media consisted of 0.1% sucrose, 0.1% H3BO3, 0.25% Ca (NO3)2, 0.2% MgSO4 and 0.1% KNO3 in deionized water (Niles and Quesenberry 1992). The media was solidified with 2% agar. Pollen germination and pollen tube growth were determined by placing 2 mL of germinating media on a glass slide and inoculating it with a sample of pollen. Slides with media and pollen were then placed in Petri dishes lined with moist filter paper thus serving as germination chambers. The growth media on slides were placed in the incubator 15 min prior to pollen inoculation. An even spread of pollen on the surface of the growth medium was achieved by gently tapping the nylon hairbrush loaded with pollen grains.
Table 1 Evaluation of agronomical traits in the wild almond species studied Section
Species
Flowering date
Self-compatibility
Ripening date
Kernel taste
Euamygdalus
P. dulcis
Middle
Self-incompatible
Middle
Bitter
Euamygdalus
P. eleagnifolia
Very late
Self-incompatible
Late
Bitter
Euamygdalus
P. orientalis
Middle
Self-incompatible
Late
Bitter
Lycioides
P. lycioides
Middle
Self-incompatible
Middle
Bitter
Lycioides
P. reuteri
Late
Self-incompatible
Late
Bitter
Spartioides
P. arabica
Late
Self-incompatible
Late
Slightly bitter
Spartioides
P. glauca
Very late
Self-incompatible
Late
Slightly bitter
Spartioides
P. scoparia
Very late
Self-incompatible
Late
Slightly bitter
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Temperature treatments Petri dishes with media slides containing pollen were maintained at temperatures between 5 and 50°C, at 5°C intervals, in the incubator under dark conditions. The temperature of the medium in three Petri dishes in each of the incubators was measured using copper–constantan microthermocouples inserted into the growth medium. The temperatures were measured at 10-s intervals according to Kakani et al. (2002), and averaged and stored every 10 min using a data logger (Campbell Scientific, Shepshed, UK). The average temperature of the growth medium during the period of germination was used in all analyses.
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diameter (Rodriguez-Riano and Dafni 2000; Kakani et al. 2002). Germination percentage was determined by dividing the number of germinated pollen grains per field of view by the total number of pollen per field of view and expressed as percentage. Measurements of pollen tube length were recorded directly by an ocular micrometer fitted to the eyepiece of the microscope. Mean pollen tube length was calculated as the average length of 20 pollen tubes measured from each Petri dish after 24 h. The replicated values on maximum pollen germination and tube length were analysed using the one-way ANOVA procedure (SAS Institute 1979). Curve fitting procedures and analysis
Pollen viability in vitro To study pollen viability in vitro, anthers were sampled in the field, at late balloon stage, from eight Prunus species during 2 years (2007–2008). The anthers were dried in an incubator at 20–22°C until dehiscence, which usually took about 24 h. The viability tests were then immediately performed. The optimum experimental condition to study pollen viability in vitro had previously been determined (Sorkheh et al., unpublished results). The germination substrate consisted of 1% agar and 15% sucrose dissolved in boiling water at pH 5.6. The substrate was poured into 90 mm Petri dishes, and pollen was distributed on the surface of the cooled, but still somewhat fluid, substrate. The Petri dishes were then kept at 25°C. A pollen grain was considered germinated when the tube had grown to a length of approximately twice the diameter of the pollen grain. Counts of germinated pollen grains were made under a light microscope (Olympus, CX31, Japan) after 3 h. Counts were postponed till 6 h for poorly germinating material. For each Prunus species, three replicates with approximately 100 pollen grains were counted. In addition viable, semi-viable and dead pollen numbers and their percentages were determined on 1% 2, 3, 5-triphenyl tetrazolium chloride (TTC) according to Mert (2009). One or two drops of TTC solution was put on a clean microslide and pollen grains were sprinkled on these drops with a brush. Then, the drop was carefully covered by a cover glass without trapping air and kept for 4 h at ambient conditions. Viable pollen was dyed in red, semi-viable pollen dyed in light red-pink and dead pollen not dyed at all.
Maximum pollen germination percentage and pollen tube length recorded after 24 h of incubation, at each temperature, were analysed using linear and nonlinear regression techniques to quantify developmental responses to temperature. Quadratic (Yan and Wallace 1998), cubic or higher order polynomial (Tollenaar et al. 1979) and modified broken-stick or bilinear (Omanga et al. 1995) equations were applied to data and examined to determine the best fit model according to Kakani et al. (2005). The modified bilinear equation (Eq. 1) provided the greatest R2 value and smallest root mean squared deviation for both pollen germination and pollen tube length and was used to estimate cardinal temperatures, minimum (Tmin), optimum (Topt) and maximum (Tmax), for pollen germination and pollen tube length of all wild almond species (Kakani et al. 2002, 2005; Acar and Kakani 2010). The PROC NLIN procedure in SAS (1979) was used to estimate parameters in the modified bilinear equation. A modified Newton–Gauss iterative method was used to determine Topt based on the lowest root mean squared deviation values between observed and predicted values. Values of Tmin and Tmax were estimated using parameters derived from the modified bilinear equations (Eqs. 2, 3). Replicated values of cardinal temperatures were then analysed using the one-way ANOVA procedure in SAS (1979). Pollen germination ð%Þ or Pollen tube length ¼ a þ b1 T Topt þ b2 ABS Topt T
ð1Þ
Tmin ¼
a þ ðb2 b1 Þ Topt b1 b2
ð2Þ
Pollen germination and pollen tube measurements
Tmax ¼
a ðb2 þ b1 Þ Topt b1 þ b2
ð3Þ
Observations on pollen germination were determined by direct microscopic observation (Olympus, CX31, Japan). A pollen grain was considered germinated when pollen tube length was at least equal to or greater than the grain
where a, b1 and b2 are equation constants, T the various temperatures at which germination and tube growth were studied, and Topt the optimum temperature for germination or pollen tube growth.
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Cell membrane thermostability measurements At the time of pollen sampling, cell membrane thermostability of leaves was measured using the procedure described by Martineau et al. (1979) and Kakani et al. (2002), (2005). Each sample assay consisted of two sets of five leaf discs cut with a 1.2-cm-diameter punch from five fully expanded leaves on the main stem. Samples were replicated three times each. Before each assay, the two paired sets of leaf discs were placed into two separate test tubes with 20 mL of deionized water after washing them thoroughly with at least four changes of deionized water to remove electrolytes released from cut cells at the periphery of the discs. To avoid evaporation and leakage of contents, test tubes were sealed with aluminum foil. One set of test tubes was incubated for 20 min at 55°C in a temperaturecontrolled water bath, whilst the other set was left at room temperature of approx. 25°C. Test tubes were then immediately incubated at 10°C for 12 h and inverted several times to mix the contents. After incubation, the initial measurement of conductance was measured by an electrical conductivity meter (Corning Checkmate II; Corning Inc., New York, USA), after which tubes were sealed with aluminum foil and autoclaved at 120°C and 0.15 MPa for 20 min to kill leaf tissues. Autoclaved tubes were cooled to 25°C, contents mixed thoroughly and final conductance was recorded. Relative injury (RI) to cell membranes resulting from the temperature treatments was calculated using Eq. (4) according to Kakani et al. (2002) and Acar and Kakani (2010). RI% ¼ f 1 ½1 ðTi =Tf Þ=½1 ðCi =Cf Þ g 100
ð4Þ
where T and C refer to the conductance of the treatment (55°C) and control (25°C) solution, respectively, and the subscripts i and f indicate initial and final conductance, respectively. The ratio of the initial to the final conductance (Ti/Tf) is a relative measure of electrolyte leakage caused by elevated temperature and consequently a measure of the extent of damage to cellular membranes. One-way ANOVA in SAS (1979) was carried out to identify Prunus species differences. Principal component analysis A principal component analysis (PCA) using PROC PRINCOMP of SAS (1979) was applied to pollen germination and pollen tube growth parameters to identify the parameters that best describe Prunus L. spp. tolerance to temperature. Values of maximum pollen germination percentage and pollen tube length, cardinal temperatures (Tmin, Topt and Tmax) for pollen germination and pollen tube length and relative injury of eight Prunus species were included in the PCA. Eigenvectors generated by PCA were
used to identify parameters that best differentiated Prunus L. spp. for temperature tolerance. The first two PC scores, PC1 and PC2 that accounted for maximum variability of the parameters tested, were used to group the wild almond species. The Prunus species which had ?PC1 and ?PC2 scores were classified as tolerant, ?PC1 and -PC2 scores as moderately tolerant, -PC1 and ?PC2 as moderately susceptible and finally -PC1 and -PC2 as susceptible (Kakani et al. 2005).
Results In vitro pollen viability Pollen viability of eight Prunus species during 2 years, Prunus species, year and Prunus species 9 year interaction were all highly significant (p = 0.0001) with corresponding F-values of the same order. Thus, different Prunus species reacted differently to environmental changes. However, some Prunus species were less variable than others and thus should be preferred as pollen donors in rootstock breeding. Examples of viable, semi-viable and dead of pollen P. dulcis (Fig. 1a), P. scoparia (Fig. 1b) and P. lycioides (Fig. 1c) with variation for pollen viability are presented in Fig. 1, for clarity. The pollen could be stored in desiccators at 4–6°C for at least 1 year with only a slight loss of viability as tested in vitro. Pollen germination Pollen germinated rapidly and reached their maximum percentage germination within 50 min of contact with the agar medium. The modified bilinear equation provided best-fit to predict the Prunus species pollen germination response to temperature. The average R2 value for all eight species tested was 88%. There was no pollen germination observed for the Prunus species at a temperature of 50°C (Fig. 2a); only the pollen grains of some species germinated at 5°C, and the germination rates were below 1% (Fig. 2b). In addition, massive pollen germination was observed at the control temperature (25°C) (Fig. 2c). The observed values and fitted lines for the response to temperature of pollen germination of Prunus dulcis and P. orientalis are shown in Fig. 3. The effect of ten constant temperature regimes (from 5 to 50°C at 5°C intervals) on the in vitro pollen germination of eight Prunus species was evaluated and expressed as the percentage of germinated pollen grains (Table 2). Figure 3 shows the variation for pollen germination in response to temperature of Prunus dulcis and P. orientalis. There was considerable variability in the cardinal temperatures for pollen germination among the almond species.
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Fig. 1 Pollens stained with TTC (left) and percentage of pollen status (viable, semiviable and dead pollen grains) in P. dulcis (a), P. scoparia (b), and P. lycioides (c). Error bars indicate ± SD. Bare indicate 100 lm
Maximum germination percentage ranged from 60.8% (P. orientalis) to 98.2% (P. scoparia), with a mean of 86.7% (Table 2). Cardinal temperatures for pollen germination differed among Prunus species. Values of Tmin ranged from 11.2°C (P. eleagnifolia) to 18.4°C (P. scoparia) with an average of 14.7°C. Optimum temperature (Topt) ranged from 22.0°C for P. glauca to 26.5°C for P. reuteri with an average of 24.2°C. The Tmax values ranged from 41°C for P. dulcis to 46.3°C for P. glauca with an average of 43.7°C (Table 2). P. eleagnifolia had the lowest Tmin value of 11.2°C and the pollen germination was 70% at Topt 23°C. P. dulcis had the highest pollen germination of about 98% and had a Tmax of 41°C. P. scoparia had the highest Tmin (18.4°C) and the highest Tmax (46°C), and its pollen germination was 98%. The average cardinal temperatures for pollen germination were 14.7°C (Tmin), 24.2°C (Topt) and 43.7°C (Tmax) (Table 2). The pollen tube length was between 860 and 1,490 lm with an average of 1,212 lm (Table 3).
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Pollen tube growth Some various wild species of almond (Prunus L. spp.) differed significantly in pollen tube length at optimum temperatures (Table 3). Similar to pollen germination, the variation in pollen tube length of P. lycioides and P. scoparia in response to temperature is shown for clarity (Fig. 4). The modified bilinear model fit is shown for Prunus L. spp. that had high variation in pollen tube length and cardinal temperatures for pollen tube growth (Table 3). The Tmin ranged from 11.9°C for P. eleagnifolia to 18.9°C for P. arabica with an average of 14.48°C. The Topt ranged from 22.0°C for P. scoparia to 30.2°C for P. reuteri with an average of 25.3°C. Values of Tmax ranged from 42.6°C for P. dulcis to 46.3°C for P. scoparia with an average of 44.4°C (Table 3). P. eleagnifolia had the lowest Tmin (11.9°C) and had a maximum pollen tube length (1,200 lm), while P. scoparia had the highest Tmax (about 46.3°C) and had 1,490 lm for pollen tube length. The wide-spread species, P. arabica,
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Fig. 3 Response of pollen germination of Prunus dulcis and P. orientalis to temperature. Prunus species with variation for maximum pollen germination are presented for clarity. Error bars indicate ± SD
Principal component analysis
Fig. 2 Pollen germination in P. scoparia after incubation at 50°C (a), 5°C (b) and 25°C (c) for 24 h. Bare indicate 100 lm
also had the highest Tmin of 18.9°C and a high Tmax of over 45.3°C and maximum pollen tube length was 1,090 lm. The average cardinal temperatures for pollen tube growth were 14.48°C (Tmin), 25.3°C (Topt) and 44.4°C (Tmax) (Table 3). Cell membrane thermostability The leaf cell membrane thermostability expressed as percentage relative injury (RI %) differed significantly among Prunus species of almond and ranged from 33% for P. scoparia including in sect. Spartioides to 80.2% for P. lycioides that in sect. Lycioides with an average of 58% (Table 3). Relative injury had poor correlation with pollen germination and pollen tube length (Fig. 5).
The PCA is a multivariate technique for examining relationships among several quantitative variables and is especially a valuable analytical technique in exploratory data analysis (Johnson 1998; Sorkheh and Amini 2010). The PCA identified the pollen parameters that best separated Prunus species of almond for their tolerance to temperature. The first three PCs accounted for 38, 22 and 15% of the total variation among the wild species (Table 4). The vectors of PC1 contrasted species for percentage germination, and Topt and Tmax for pollen germination and tube growth (all negative loadings) with Tmin for germination and tube growth. Prunus species with negative scores for PC1 had higher values of Topt and Tmax for pollen germination and pollen tube length than those with positive scores, as well as having higher percentage pollen germination. The second PC contrasted species with high values of Tmin for germination and pollen tube length (positive loadings) with low values for pollen tube length (negative loading). The Prunus species with short pollen tube lengths and high values for Tmin therefore had positive scores. The third PC contrasted species having long pollen tubes with low values of Tmax for pollen germination and pollen tube length. The wild species of almond were divided into four groups based on the scores of the first two principal components: group 1 includes Prunus species as tolerant with positive scores for PC1 and PC2, group 2 as moderately tolerant with positive PC1 and negative PC2 scores, group 3 as moderately susceptible with negative PC1 and positive PC2 and finally group 4 as susceptible with negative PC1 and PC2 scores (Table 4; Fig. 6).
Discussion Temperature is one among the most important environmental factors affecting plant reproductive processes such
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Table 2 Relative injury, maximum pollen germination percentage and cardinal temperatures for pollen germination of eight almond species (Prunus L. spp.) of in response to temperature Section
Species
Relative injury (%)
Maximum pollen germination (%)
Cardinal temperatures (°C) Tmin
Topt
Tmax
Euamygdalus
P. dulcis
70.0
98.7
12.8
25.7
41.0
Euamygdalus
P. eleagnifolia
72.6
70.5
11.2
23.4
41.9
Euamygdalus Lycioides
P. orientalis P. lycioides
50.0 80.2
60.8 90.2
13.2 15.6
23.0 25.5
42.1 42.8
Lycioides
P. reuteri
75.0
96.4
14.1
26.5
44.8
Spartioides
P. arabica
42.0
88.4
17.4
23.7
45.2
Spartioides
P. glauca
34.0
90.7
15.6
22.0
46.3
Spartioides
P. scoparia
33.0
98.2
18.4
24.0
46.0
86.7 ± 4.18b
14.7 ± 0.30a
24.2 ± 0.23a
43.7 ± 0.37b
Mean ± SD a,b
Significant at p \ 0.05 and p \ 0.01 level, respectively
Table 3 Maximum pollen tube length (lm) and cardinal temperatures for pollen tube length of eight almond species (Prunus L. spp.) in response to temperature Section
Species
Maximum pollen tube length (lm)
Cardinal temperatures (°C) Tmin
Topt
Tmax
Euamygdalus
P. dulcis
1,430
12.7
26.4
42.6
Euamygdalus
P. eleagnifolia
1,200
11.9
25.4
42.9
Euamygdalus Lycioides
P. orientalis P. lycioides
1,100 860
13.4 14.3
22.3 27.9
43.1 44.6
Lycioides
P. reuteri
1,240
14.2
30.2
45.1
Spartioides
P. arabica
1,090
18.9
25.4
45.3
Spartioides
P. glauca
1,289
15.6
23.0
45.6
Spartioides
P. scoparia
1,490
14.9
22.0
46.3
12.12 ± 80.32a
14.48 ± 0.78b
25.3 ± 1.65b
44.4 ± 0.65b
Mean ± SD a,b
Significant at p \ 0.05 and p \ 0.01 level, respectively
Fig. 4 Response of pollen tube length of Prunus lycoides and P. scoparia to temperature. Prunus species with variation for maximum pollen germination are presented for clarity. Error bars indicate ± SD
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as pollen germination, pollen tube growth and fruit set (Kakani et al. 2005; Acar and Kakani 2010). Within each year, no significant differences were found among the pollen viability rates of the Prunus L. spp. except viable pollen rate in the year 2007. Su¨tyemez (2007) reported that the pollen viability rates of 32 different walnut cultivars varied between 81 and 94%. In another study pollen viability ratio of selected 19 walnut cultivars varied between 77 and 92% (Su¨tyemez 2007). In general, in some previous studies, similar results for pollen viability were reported in hazelnut (49–97%) by Beyhan and Odabas¸ (1995), in chestnut (8.8–35.8%) by Beyhan and Serdar (2009), in apricot (76–86%), sweet cherry (67–81%) and sour cherry (71%) by Bolat and Pırlak (1999) and in walnut (75–89%) by Mert (2009). Our
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Fig. 5 Relationship between cell membrane thermostability (expressed as relative injury, %) and maximum pollen germination percentage (a), and pollen tube length (b) recorded at optimum temperature of eight researched Prunus species Table 4 Principal component analysis eigenvectors of eight almonds (Prunus L. spp.) species for maximum percentage pollen germination, maximum pollen tube length and their respective cardinal temperatures (Tmin, Topt and Tmax), relative injury (%) and the variation accounted for by each eigenvector Parameter
PC1
Pollen germination
-0.42
0.02
0.26
Pollen tube length
-0.18
-0.48
0.56
0.04
0.53
0.10
Tmin pollen germination
PC2
PC3
Topt pollen germination
-0.30
0.25
0.37
Tmax pollen germination
-0.50
0.12
-0.45
Tmin pollen tube length
0.02
0.58
0.42
Topt pollen tube length
-0.47
-0.23
0.18
Tmax pollen tube length
-0.49
0.16
-0.40
Relative injury (%)
-0.19
-0.43
0.15
% variation
38.2
22.34
15.25
results are in agreement with the previous studies in various species. In the present study, the average of germinated pollen (36–52%) for various wild Prunus L. spp. is similar to percentages previously reported for diploid apples (Stott 1972; Kaufmane and Rumpunen 2002), also with clear fluctuations between years. The highest percentage noticed
in our study for Prunus L. spp. including to sect. Spartoidies (98%) is compared to the very high value reported for some apple varieties (over 95%) (Stott 1972). Addition of boron to the substrate would perhaps have improved germination percentages as has previously been reported for many rosaceaous species (Visser 1955; Voyiatzi 1995; Calzoni et al. 1979). No disintegrating pollen grains were noticed, and high germination percentages were obtained for some wild Prunus species in sect. Spartioidies. Our results in this regard accord with that of Kaufmanea and Rumpunen (2002). The current study, which examined pollen response to temperature from 10 to 50°C, shows very clearly that pollen germination and pollen tube growth processes can also be described in terms of temperature. All eight of wild Prunus species of almond had clear temperature optima, above and below which pollen germination and maximum pollen tube length were reduced, and this response was well described by a modified bilinear regression model. Pollen from the different Prunus species tested in this work responded differently. In vitro pollen germination and pollen tube growth of species were severely reduced under both high- and low-temperature conditions (Fig. 2a, b). The optimum value for both pollen germination and pollen tube length was 24.9°C for the Prunus species used in the study. The effects of temperature on pollen germination and pollen tube growth have been reported by several researchers (Vachun 1981; Egea et al. 1992; Kakani et al. 2002, 2005; Kremer and Jemric´ 2006; Acar and Kakani 2010). The Prunus species pollen germination was slow at low temperature and increased linearly, and reaching its maximum value at 25°C; beyond that degree, germination percentage decreased (Sorkheh et al., unpublished results). Low temperature did not inhibit in vivo pollen germination of apricot at 10°C (Austin et al. 1998) and that of sour cherry at 15°C (Cerovic and Ruzic 1992). The optimum temperature required for in vitro pollen germination of apricot was between 15 and 20°C, and degrees above 25°C caused a decrease in pollen germination rates (Vachun 1981; Kakani et al. 2002). The lowest pollen germination and pollen tube length of apricot and sweet cherry were obtained at 5°C, and the best results were observed between 15 and 20°C (Pirlak 2002). It was also reported that degrees lower than 15°C reduced pollen germination in pears (Mellenthin et al. 1972; Vasilakakis and Porlingis 1985). The validity of the in vitro evaluation of pollen germination is a predictor of in vivo behaviour (Hormaza and Herrero 1999). There were significant differences among the in vitro pollen performances of different various wild species of almond at each temperature. The Prunus species pollen germination percentage was between 60 and 98% with a mean of 86% (Table 2), which was much higher
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818
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Fig. 6 First, second and third principal component analysis (PCA) scores for the identification of Prunus species response to temperature. The latent vectors are indicated by green lines showing the direction (angle) and magnitude (length). Maximum pollen germination percentage (1), and maximum pollen tube length (2) Tmin, Topt and Tmax are cardinal temperatures for percentage pollen germination (3, 4, 5) and pollen tube length (6, 7, 8) according to Kakani et al. (2002, 2005)
than that observed in Pistacia L. spp. by Therios et al. (1985), Atli et al. (1995), Caglar and Kaska (1995), Kuru (1995), Acar and Ak (1998), Acar (2004) and Kamiab et al. (2006). The values of pollen germination and pollen tube growth obtained from the Prunus species were similar to those reported for jojoba (Lee et al. 1985), pear (Vasilakakis and Porlingis 1985), papaya (Cohen 1989), cherimoya (Rosell et al. 1999), mango (Sukhvibul et al. 2000), Prunus mume Siebold et Zucc (Wolukau et al. 2004) and Pistacia L. spp. (Acar and Kakani 2010). According to Sukhvibul et al. (2000), the effects of temperature on pollen germination are inconsistent and seem to be cultivar or species dependent. Therefore, the differences in pollen germination and pollen tube length observed in the present study were a reflection of genotype variability. Pollen with heat-stress tolerance has been successfully used to transfer the tolerance into sensitive genotypes. Such studies in other crops have shown that pollen could be used to screen genotypes for tolerance to high temperature. In cotton plants, pollen of the tolerant breeding line (7,546 of Gossypium barbadense L.) exposed to temperatures of [35°C were used to cross the emasculated flowers of a sensitive genotype (Paymaster 404) resulting in tolerance in further generations (F1 and further backcross populations) of this otherwise heat-sensitive genotype (Garay and Barrow 1988). Currently, studies are being conducted at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) using the identified tolerant cultivars to evaluate whether the tolerance can be simply inherited. Studies along similar lines to differentiate the response of male and female tomato organs (Lycopersicon esculentum Mill.) to high temperatures by Peet et al. (1997) indicated that viable
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pollen supply alone is not sufficient for fruit set under high temperatures. Further studies are needed to identify the role of megagametophytes in heat tolerance. Further studies are required in field conditions for screening wild almond species with variable water status and high temperature during fruit set. From the results, it is inferred that a modified bi-linear equation can be used to describe the percentage pollen germination and pollen tube length response to temperature. In a study conducted by Prasad et al. (1999b) warmer night temperatures (28 versus 22°C) reduced fruit set due to reduced pollen production and pollen viability in peanut (Arachis hypogaea L.). The reduced pollen production and viability would also affect the total percentage of pollen germinated. Similarly, high temperature prior to anthesis was also known to reduce microsporogenesis in peanut (Arachis hypogaea L.) (Prasad et al. 2001), tomatoes (Garay and Barrow 1988) and cranberry (Vaccinium macrocarpon Aiton) (Cane 2009). Further studies are required to evaluate the temperature response of pollen grains developed during pre-anthesis high-temperature treatments. In this study, the membrane thermostability expressed as relative injury ranged between 35 and 82%, but had a poor correlation with pollen parameters (Fig. 5). Recently, ur Rahman et al. (2004) also concluded that membrane thermostability is not a useful parameter for discriminating high-temperature tolerance of cotton (Gossypium hirsutum L. cv Deltapine 50) cultivars under ambient temperatures. In cotton, heat tolerance does not correlate with degree of cell membrane lipid saturation (Rikin et al. 1993), suggesting factors other than membrane stability may be limiting reproductive growth and development at high temperature. However, the Prunus species differences for
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pollen germination and pollen tube growth identified in this study could be due to the variation in their pollen carbohydrate concentration. Studies have shown that carbohydrates are responsible for pollen development and, especially, pollen cytoplasmic carbohydrates and sucrose are involved in protecting pollen viability during exposure and dispersal (Pacini 1996) and for pollen germination, simple sugars are the primary substrates (Stanley 1971). In pepper (Capsicum annuum L. cv. Mazurka) plants, exposure to high temperature (32/26°C) for 8 days resulted in pollen germination of 6% and shorter pollen tubes compared with maximum pollen germination of 25% obtained at normal temperature (28/22°C) (Aloni et al. 2001). This was attributed to a decrease in sucrose utilization by pollen grains under high temperature, even though the pollen grains accumulated more starch and sugars than under normal temperature conditions. In contrast, a decrease in starch and sugar concentration was recorded in tomato pollen grown under high-temperature (32/26°C) conditions (Pressman et al. 2002). Therefore, under-utilization or unavailability of carbohydrates hinders pollen germination on exposure to high temperatures. Future studies need to study the genotypic differences or pollen carbohydrate concentration and its role in determining the temperature tolerance of Prunus spp. pollen. There was significant variation among Prunus species in Topt and Tmax for germination and pollen tube length. Temperatures warmer than optimum temperature for germination and pollen tube length greatly reduced the number of pollen germinating and their tube length. The PCA is perhaps the most useful statistical tool for screening multivariate data with significantly high correlations (Sorkheh and Amini 2010). The first three principal components, PC1, PC2 and PC3 from PCA explained about 75% of the total Prunus species pollen variability in response to temperature. The cluster analysis applied to the principal components divided the cultivars into four distinct groups (Table 4). On the basis of the PCA of these responses, various wild species of almonds were classed as tolerant, intermediate or susceptible. According to Kakani et al. (2002, 2005), Prunus species with a negative PC1 and PC2 score (\1) had high optimum and maximum temperatures for pollen germination and tube growth, which should contribute to greater heat tolerance. Within this group, PC2 discriminated Prunus L. spp. with the longest pollen tube length (negative score). In contrast, Prunus species with positive PC1 scores had lower optimum and maximum temperatures, which should equate to greater sensitivity to high temperature. Thus, the species in sect. Lycioides (P. lycioides, P. reuteri) and P. dulcis are the most heat-tolerant species, the species in sect. Euamygdalus (P. eleagnifolia, P. orientalis) is intermediate and the
819
species from sect. Spartioidies (P. arabica, P. glauca, P. scoparia) is the most heat-susceptible. A significant relation between Tmin and Tmax for pollen germination and pollen tube growth (Fig. 7) is indicative of the temperature limits for these processes in almond species that is according to the results by Kakani et al. (2002) that exposed on groundnuts with different temperature regimes. The variability in Topt across the wild species spectrum reveals the adaptive nature of wild almond species to temperature conditions at a given location.
Fig. 7 Relationships between (a) Tmin, (b) Topt and (c) Tmax temperatures for pollen germination and pollen tube growth for various native Iranian Prunus species
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820
Conclusion In conclusion, in vitro pollen germination and pollen tube growth of different Prunus species varied with temperature. The maximum percentage pollen germination and pollen tube length of species, and Tmin and Tmax were the most important parameters describing species tolerance to low and high temperatures. In addition, in all investigated Prunus species, the modified bi-linear model best described the response of pollen to temperature. The maximum percentage pollen germination and pollen tube length of genotypes and Tmax were the most important parameters describing genotypic tolerance to high temperature, whereas Tmin and Topt for pollen germination and tube growth and also the pollen tube growth rate were the least important. Future studies will also be required to validate the performance of high temperaturetolerant cultivars identified by these in vitro methods in hightemperature environments. Acknowledgments The authors offer grateful thanks to Shahrekord University for financial assistance, as well as to the Agriculture and Natural Resources Research Center of Shahrekord for access of various wild species of almond trees. We are grateful to Ms. Kh. chenaneh-Hanoni for her kind help in undertaking this study.
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