Abstract: The objective of this study was to determine the phenological stages and viability, germination capability of pollen grains in vitro and fertility level of Érdi bõtermõ, Debreceni bõtermõ, Kántorjánosi, Újfehértói fürtös, Éva, Petri, Oblacsinszka, Pándy279 and Csengõdi sour cherry (Prunus avium L.) cultivars grown in Hungary. It was determined that active pollen levels significantly varied between 12.4 and 93.7% in TTC (2, 3, 5-triphenyl-tetrazolium-chloride) tests. Anther of Kántorjánosi showed the highest (148) number of pollens, whereas the lowest number (27 was obtained from Oblacsinszka. The highest germination was obtained from Oblacsinszka by 64.5% and the lowest obtained by Pándy 279 on 28.8% after 48 h. In orchard trials the mean fruit set of self-pollination were 6.3% with max. 18.6% in the case of Oblacsinszka and min. 0% in the case of Pandy279, while in open pollination trials it was 15.2% max. 28.5% in Oblacsinszka and min. of 7.5% in Pándy279.
INTRODUCTION
Determining of the phenological stages and viability, germination capability of pollen grains in vitro and fertility level of new cultivars are very important for breeders and growers. The majority of commercially grown sour cherry cultivars are self-fertile and for the purpose of finding adequate polleniser cultivar for auto-incompatible cultivar, have to consider not only the number of anthers per flower, but also the quantity of pollen per anther and the germination viability of pollen grains. According to Thompson (2004), first requirement for economical fruit production is the availability of an adequate source of viable and compatible pollen.
The maximum fruit set by self fertilisation may be more than 50% in sour cherry (Kozma et al., 2003). Nyéki et al. (1999) reported that high pollen production and pollen viability are important for fertilization. The environmental factors and genetic characteristics are effective in the case of cultivation of fruit trees; an abundant crop of cherries is dependent upon a successful completion of a sequence of reproductive events. Failure or deficiency of any aspect of this process can result in a crop reduction or total loss. According to Nyéki et al. (2008) the three selected species examined prove the tendency that self-fertile varieties produce about 2.5 times more pollen than the auto-incompatible ones. The partially self-fertile varieties produced 1,688, i.e., more than the auto-incompatible cultivars (Nyéki et al., 2002). Some sour cherry cultivars under favorable condition approach the self-fertile types, whereas under unfavorable conditions they are almost self-incompatible. The types of Pándy sour cherries are auto-incompatible (Kozma et al., 2003). Pollen performance includes pollen produced in a flower, pollen morphological homogeneity, pollen germination, pollen tube growth and pollen competition; it is an important component of fertilization success in fruit trees (Thompson, 2004; Hedly et al., 2004). Applying enough pollen does not guarantee a good fruit set unless the pollen used is viable with a high germination percentage. The use of selected pollen with a high degree of viability will ensure a better fruit set and consequently an acceptable yield (Bots and Mariani, 2005).
The literature on pollen viability that appeared until 1974 has been reviewed by Stanley and Linskens (1974). The accumulated data indicated that pollen viability was influenced by relative humidity, cold and hot temperature, atmospheric composition and oxygen pressure. Elevated CO2 levels protect pollen viability against heat stress during development (Aloni et al., 2001).
According to Bots and Mariani (2005), pollen grains of many species may be sensitive to excessive dehydration caused by low relative humidity, but adaptations to prevent such dehydration are species specific. In addition, drought stress during pollen development in the anther can also strongly affect pollen viability.
Pollen viability could be assessed by different methods like staining with nuclear or non-vital dyes or in vitro germination tests (Parfitt and Ganeshan, 1989; Heslop-Harrison and Shivanna, 1984; Voyiatsiz and Paraskevopoulou, 2002).
Therefore, knowledge of pollen viability and germination are the most important properties in cherry tree fertilization and selection of suitable polliniser while the orchard is being established (Brown et al., 1996; Nyéki et al., 2008). Also, important research materials for morphological, physiological and the way it is affected by environmental factors may help predicting hybrid formation.
The objectives of this study were to investigate the viability and germination rate of pollen collected from some pollen donor sour cherry cultivars that will be used for the cultivar combinations program in the future. Additionally, the weather conditions of last year and its effect on pollen production level. For this purpose, pollen viability, pollen production, pollen germination rate were assessed under in vitro and orchard conditions.
MATERIALS AND METHODS
The study was carried out on a sour cherry collection at the fruit Research and Extension Center for Fruit Growing, Újfehértó, located in the northeastern part of Hungary. The latitude is 38°10´, the longitude is 30°37´ and the altitude is 1,050 m. The mean annual temperature is 9.5 °C and the annual rainfall over 50 years was 583 mm. In general, the weather conditions during flowering time of sour cherry were partially rainy and the pollination period occurs in rainy weather, with the exception of May 7, which it was sunny. Winds are predominantly blowing from the east-northeast.
Pollen collection and viability: At the full bloom time of sour cherry (April 2008) some late balloon staged (not opened) flowers were collected from the Érdi bõtermõ, Debreceni bõtermõ, Kántorjánosi, Újfehértói fürtös, Éva, Petri, Pándy279, Oblacsinszka and Csengõdi sour cherry cultivars. The flowers were transferred to the laboratory immediately. Anthers were removed and placed into dark-colored bottles to promote dehiscence at room temperature. In the stain tests, pollen viability was estimated by using TTC (2, 3, 5-triphenyl-tetrazolium-chloride) 1% stains. Pollens were scattered onto TTC solutions and stained pollens were counted after 2 h. To determine pollen viability, pollens of each cultivar were observed on three slides under a light microscope (x120 magnifications). The stained pollens were considered as viable in these tests.
The pollen productions of flowers: The pollen number of each individual flower of sour cherry cultivars was calculated over 7 years` observations (1973-1974, 1988-1991 and 2008). Pollens from each individual flower of sour cherry cultivars were calculated following the method described by Cruden (1977). Fifteen mature undehisced anthers were washed in distillated water, mounted in 1:1 solution of 1% acetocarmine and 50% glycerine and shook for 2 min. Each anther was gently squashed to release the pollen. Five slides of samples were observed under a microscope and the total number was counted. This number was multiplied with the total number of anthers to calculate pollen production per flower.
Pollen germination: For this reason, pollen grains were sowed in the medium containing 0.5% agar + 15% sucrose + 5 ppm boric acid medium and incubated at the constant temperature of 25 °C. The percentage of pollen germination was determined after a 24 h incubation period. The pollen tube growth was evaluated in the laboratory and 6 slides of samples were observed, using a Ziss microscope.
Statistical analysis: The percentage data were first transformed to arcsine square root transformation and an analysis of variance was performed. The differences among means were analyzed using the Duncan`s multiple range test at p<0.05 significance.
RESULTS AND DISCUSSION
Phenological stages: The bud burst appeared during the second
week of March. The balloon stage started in the first half of April. The
flowering period took approximately 8-19 days. The genotypes have different
flowering times: Érdi bõtermõ flowers in 13-April,
which was the early blooming cultivar, while the Kántorjánosi
and Petri flowers in 20-April which were the late blooming cultivars.
This is related to the different chilling requirements needed to flower
that are lower for Érdi bõtermõ than for Kántorjánosi
and Petri. The fruits set were completed in the last week of April.
Fruits turned to red color in the first and second week of June. Harvest
time was at the end of June except for Érdi bõtermõ
and Oblacsinszka cultivars which were harvested 10 days earlier
(Table 1). The earliest cultivars were Érdi
bõtermõ and Oblacsinszka while Kántorjánosi
cultivar was the latest. The phenological dates can change with temperature.
Nyeki et al. (2002) studied three main phenological stages in different
sour cherry cultivars: the duration between bud burst and beginning of
flowering was 32-33 days, the flowering period was 10-11 days and duration
between the end of flowering and fruit ripening was 63-66 days. With respect
to weather conditions, flowering time can show alteration. An early flowering
cultivar as Érdi bõtermõ cannot be recommended
as a pollinator for the latest cultivar and it might not be able to pollinate
other late blooming cultivars.
| Table 1: | Phenological stages of sour cherry cultivars under climatic conditions of Újfehértó, Hungary (2008) |
| Table 2: | The characteristic of flowers of sour cherry cultivars, Hungary (2008) |
| Values within a row followed by different letter(s) are significantly different (p<0.05) | |
Pollen production: Anther of Kántorjánosi cultivar showed the highest (148) number of pollens, whereas the lowest numbers (27) were obtained from Oblacsinszka and Debreceni bõtermõ (29) cultivars (Table 2). Similar observations were made by Nyéki et al. (2008) as for the weak pollen production of Pándy clones. Redalen (1984) scored 35 sour cherry varieties as for their pollen production. According to Nyéki et al. ( 2008 ) sour cherry cultivars could be grouped on the basis of their pollen production per anther: 1. Small (0-600); 2. Intermediate (600-1,200); 3. Large (>1,200).
In this experiment, all the examined cultivars belonged to the first
group. The pollen production of Pándy 279 is very low. Substantial
differences were produced by different seasons regarding the means of
cultivars. The results of over 6 years of observations (1973-1974, 1988-1991)
of 20 sour cherry cultivars shows that very low number of pollen grains
(345-865) were found in the 5 Pándy meggy clones, whereas most
(1976 per anther) were typical for the variety Meteor.
| Table 3: | The quantity of pollen per anther of 20 sour cherry cultivars (1973-1974, 1988-1991) |
Substantial differences were produced by different seasons regarding the means of varieties. In 1991, the number was 518, whereas in 1974 it was 1,468 (Table 3).
Pollen germination tests: The pollen viability values and germination rates of sour cherry cultivars are given in Table 4. According to Davarynejad et al. (1993) high pollen viability and germinating as well as pollen production capacity are important to decide on a suitable pollinator, since not all pollen grains germinated on stigma can reach to the ovule. On the basis of TTC examinations, pollen viability values vary between 12.4 of Pandy 279 and 93% Oblacsinszka. Tosun and Koyuncu (2007) studied sour cherry pollens using TTC and reported that pollen viability ratios reached 80 to 93%.
The highest germination was obtained from Oblacsinszka (64.59%) while the lowest was from Petri (29.12%) after 24 h of incubation (Table 4). Results of incubation duration experiments were similar to the findings of Tosun and Koyuncu (2007), who reported that the pollen germination rate of sour cherry cultivar after 24 h were between 57-67%.
With regard to meteorological characteristics of 2007, it can be seen
that the precipitation in the time induction and differentiation of flowers
was very low. It could be reduce the number of pollen grains per anther
on some cultivars. For example in Újfehértó, precipitation
from June 21, 2007 to July 4 was only 20 mm and from July 6 to August
1 was only 15.1 mm (data not shown), which was non effective irrigation
for trees. Present results were congruent with the results obtained by
Bots and Mariani (2005).
| Table 4: | The number of pollen grains per anther in sour cherry cultivars of different fertility relations |
| Auto-incompatible (>1%)*, Partially self-fertile (1-10%)*, Self-fertile (<10%)* fruit set after self-pollination. Values within a row followed by different letter(s) are significantly different (p<0.05) | |
Investigating the self-fertility of the cultivars in orchard: The highest fruit set was obtained from free pollination with 58.5% fruit set. Fruit set exceeding 5% can be considered as acceptable degree of self-fertility (Table 4). Nyéki et al. (2000) classified the cultivars into three groups: adequate (above 10%), acceptable (5-10%) and weak (below 5%). The fruit set of self pollination was highest in case of cv. Újfehértói fürtös and lowest in case of cv. Pándy272. The other cultivars were intermediate. Surpasses the desired level of 10% required for safe fruit set. In case of other cultivars, the average degree of self-fertility is between 3 and 6% (Nyéki et al., 2008).
However, self-fertile cultivars of sour cherries produced more pollen than auto-incompatible ones. The partially self-fertile varieties displayed less pollen than the latter ones; we cannot state a general rule concerning the correlation between fertility relations and the number of pollen grains per anther. Present observations match the statement of Kozma et al. (2003), which the auto-incompatible sour cherry cultivars produce less pollen per anther than the self-fertile cultivars.
CONCLUSION
Testing the viability and germination of pollen grains of sour cherry cultivars are the most important properties in cherry tree fertilization for a fruitful cultivation, especially when the new cultivars are used as pollen donor cultivars purpose, especially in terms of selecting which cultivars should be used by growers. Érdi bõtermõ, Debreceni bõtermõ, Kántorjánosi, Újfehértói fürtös, Éva, Petri, sour cherry cultivars selected in Hungary and similar to the old Pandymeggy. The low percentage of viability of Újfehértói fürtös and Debreceni bõtermõ explain the high variation of self pollination in different years. In this context, this study may provide useful information for facilitating the evaluation of sour cherry cultivars based on their pollen performance. All the examined cultivars no produce enough pollen except of Kántorjánosi and Oblacsinszka which could be used as a good pollen donor for others.