POLLINATION, FERTILIZATION AND FRUIT FORMATION IN EGGPLANT (SOLANUM MELONGENA L.)

The aim of the investigations was to determine the regularity of pollination and fertilization as dependent on the phenotype of fl ower in eggplant. The experiment was conducted in 2005 and 2006 at the Agricultural University in Kraków, Poland. The object of the investigations was eggplant (Solanum melongena L.) ‘Epic F1’, grown in the open fi eld. Flower phenotype (long-, mediumor short-styled), fruit setting and the number of seeds per fruit were recorded on experimental plants during the fl owering period. The number of pollen tubes in the middle of the style and the number on fertilized ovules in ovaries were evaluated for all types of fl owers. The course of pollination was different in particular types of eggplant fl owers. Differences concerned only the number of pollen tubes and fertilized ovules of short, mediumand long-styled fl owers and the number of seeds in the fruit. There was not observed any incompatibility in the growth on pollen tubes in the styles of all types of eggplant fl owers. Stereomicroscopy observations of cross sections of the ovaries do not show differences in the formation and position of the ovules on the placenta. Short-styled fl owers were characterized by signifi cantly lower pollination and fruit set effi ciency and they produced fruits with a signifi cantly lower number of seeds. The low number of pollen tubes, fertilized ovules and seeds can be a straight consequence of the morphology of short-styled fl owers: small-sized stigmas and spatial separation of anther pores and stigmas, which makes pollination diffi cult.


INTRODUCTION
Flowering and fruit set are two most important factors determining the yield of cultivated eggplants.Genotype and environmental factors infl uence the course of fl owering and the fl ower morphology, especially the style length which determines the effectiveness of fruit setting.Eggplant is a self-pollinated plant -pollen does not need a pollinator to transfer it in the fi eld.It is a partly self-incompatible species and needs cross-fertilization for greater fruit set (A m o a k o and Ye b o a h -G y a n , 1991).In hot humid climate, cross-pollination may occur up to 20%, mainly due to heterostyly, even though it is treated as a self-pollinated crop (S w a r u p , 1995).Q u a g l i o t t i (1979) estimated the amount of cross pollination in eggplant at between 0 and 46%, but cross pollination at distances of 50 m or more was nonexistent.The fl ower remains open for 2-5 days without closing at night.Anthesis and pollen release occur in the morning, but these phenomena are infl uenced by the daylight, temperature, and humidity.Pollen viability is retained for 8-10 days at a temperature of 20-22°C with a relative humidity of 50-55% (C h e n , 2001).In eggplant, fertilization is completed within 3 days after pollination and gene expression 3 days after anthesis is induced by stimuli of both pollen tube elongation and fertilization.Ovaries of unpollinated fl owers began to senesce 3 days after anthesis, although half of them had the potential to set fruit (N a g a s a w a et al. 2001).
The variation in style length and its position towards stamens was widely described in eggplant.Longstyled fl owers (the stigma is above the stamen or on the same level as the stamen) are always more appreciable in the number than short-styled fl owers (the stigma is below the stamen).Sometimes fl owers with the stigma on the same level as the stamen are defi ned as mediumstyled ones.K r i s h n a m u r t h i and S u b r a m an i a m (1954) described four types of fl owers: 1) long styled with large ovary, 2) medium styled with medium sized ovary, 3) pseudoshort-styled with rudimentary ovary, and 4) true-short-styled with very rudimentary ovary.Pseudo-and short-styled fl owers fail to set fruits, whereas long-and medium-styled fl owers produce fruits.Fruit set rates are higher for long-styled fl owers compared with short-styled fl owers, so short-styled ones are not totally sterile (C h e n , 2001).In long-styled fl owers, the stigma may project beyond the anthers, what favours cross-pollination.N o t h m a n n et al. (1983) stressed that stylar heteromorphism affected fruit set more than did the position of the fl ower in the cluster.According to P a s s a m and B o l m a t i s (1997).the proximity of the stigma to the anther pores at anthesis infl uences fruit set, fruit size and seed content, but not seed quality of eggplant.Fruit weight and seed formation were maximal in fl owers with the stigmata at maturity situated close to the anther pores.There are some investigations designed to determine the infl uence of agronomical and environmental factors on the occurrence of different types of fl owers in eggplant.P a ss a m and B o l m a t i s (1997) showed that the style length in eggplant is a varietal characteristic.B o b a d i and van D a m m e (2003) stated that nitrogen failed to infl uence style length and type of fl owers of eggplant.According to K o w a l s k a (2003), eggplant formed the highest number of fl owers with a long pistil and much less fl owers with medium and short pistils regardless of the pollination method (self pollination, using bumble bees) and fl ower hormonization.
The production of short-styled fl owers with a small and highly reduced gynoecium, often infertile or with small possibilities of setting fruits, seems to be the fi rst step to functional andromonoecy -the production of staminate and hermaphroditic fl owers.Andromonoecy is widespread in the genus Solanum (D u l b e rg e r et al. 1981; S o l o m o n , 1986; D i g g l e and M i l l e r , 2004).The aim of the present investigations was to discuss the reasons and benefi ts of eggplant stylar heteromorphism and to determine the regularity of pollination and fertilization as dependent on the type of fl ower.

MATERIALS AND METHODS
The experiment was conducted in 2005 and 2006 at the Agricultural University in Kraków, Poland.The object of investigations was eggplant (Solanum melongena L.) 'Epic F 1 ', grown in the open fi eld, in the Experimental Station in Mydlniki, near Kraków, South Poland.Seeds were sown on 4th March 1995 and 3rd March 2006 in seed-boxes fi lled with peat substrate and placed in a controlled environment: temperature 27 ± 1°C and RH 80%.After emergences, temperature was maintained at 26 ± 2°C (day) and 21 ± 1°C (night), RH at 80%.When the fi rst true leaf was expanding, uniform seedlings were pricked out into plastic containers Ø 8 cm, fi lled with peat based substrate.Uniform transplants with 5-6 leaves were fi nally transplanted into open fi eld at the beginning of May, 70 and 62 days after sowing (in 2005 and 2006, respectively), in a spacing of 0.75 × 0.60 m.Eggplant was grown in standard agronomic conditions.The observations were conducted on 20 plants during the whole fl owering period.Single fl owers were numbered according to the order of appearance on each plant.Flower phenotype (long-, medium-or shortstyled), fruit setting and the number of seeds per fruit were recorded for all plants.Results were evaluated by ANOVA, Tukey's test, at p = 0.05.At full fl owering (17.07.2005 and 26.07.2006), 60 fl owers of each type (long-, medium-and short-styled) were collected.Only pollinated fl owers were taken into consideration.In the fi rst part of the investigations, 30 styles of each above mentioned type were isolated and fi xed immediately in FAA (formalin-acetic-alcohol), according to Martin's method (M a r t i n , 1959).Germination of pollen on stigmas, growth of pollen tubes and fertilization of ovules were examined under fl uorescence microscopy.The number of pollen tubes in the half of the style and the number of fertilized ovules were evaluated.In the second part of observations, the cross sections of 30 sty-les of each abovementioned type were observed under stereomicroscopy to evaluate the regularity of ovule formation.

RESULTS
Stylar heteromorphism was a distinct phenomenon of eggplant 'Epic F 1 ' in the conditions of the present experiment.The plants produced three types of fl owers: long-styled, medium-styled and short-styled (Figs 1, I-III).Stereomicroscopy observations of cross sections of the ovaries do not show differences in the formation and position of the ovules on the placenta (Figs 2, I-III).Bumble bees (Bombus terrestris) were widely observed on turned down fl owers (Fig. 3).There were no morphological differences in the forming of anthers among the long-, medium-and short-styled fl owers.The stamens formed a cone like a tube around the style and dehisced at the terminal pores (Fig. 4).Stamen pores of the me-dium-styled fl owers were localized closest to the stigma of the style.
Pollination effi ciency of the long-and mediumstyled fl owers was the greatest and was contained in the range of 38.8-50.8%(Tab.1).Pollination effi ciency of the short-styled fl owers was signifi cantly lower (17.3 and 30.2%, in subsequent years of the experiment).In 2005, fruit set effi ciency of the long-and medium-styled fl owers was much higher as compared to the short-styled ones.In 2006, signifi cant differences in fruit setting between the investigated types of fl owers were not observed.Fruits set from the long-and medium-styled fl owers contained a comparable number of seeds (371.0 seeds per fruit, on average).Fruits set from the short-styled fl owers contained about two times less of seeds (179.4 seeds per fruit).

Percent of obserwations
There was not observed any incompatibility in the growth on pollen tubes in the styles of all types of eggplant fl owers.Pollen tubes grew into the style with seven ducts.Long-, medium-, and short-styled fl owers were differentiated only in relation to the number of pollen tubes observed in the styles.There was found 10-300 pollen tubes in the middle of the style in the short-styled fl owers, 150-900 in medium-styled, and 200-1000 in long-styled ones (Fig. 5).The ovaries of the short-styled fl owers contained 0-70 fertilized ovules.Many more fertilized ovules were found in the remaining types of fl owers: 40-400 in medium-styled and 100-400 in longstyled ones (Fig. 6)., 1989).Differences between medium-and longstyled fl owers concerned only the pistil morphology; the pollination and fruit set effi ciency as well as the number of seeds per fruit of the mentioned types of fl owers were

Percent of obserwations
Percent of obserwations similar.In the conditions of the present experiment, the short-styled hermaphroditic fl owers of eggplant were not functionally pistillae.The short-styled fl owers were fertile and able to set fruits but fruit set rates were greater for the medium-and long-styled fl owers, as it was reported by C h e n (2001) and K o w a l s k a (2003).The high frequency of short-styled fl owers was coincident with the peak fl owering period of the investigated eggplant cultivar (data not shown).It may be an adaptation in preventing excessive fruit set.P a s s a m et al. (2000) found that the transitory reduction in style length, due to the presence of the fruit in the stage of growth, may lead to a temporary reduction in fertility of eggplant.Authors suggested that fruit load and development affect fl ower mass and style length of eggplant, whereas auxins infl uence the number of fl owers.The fl owers of plants that did not set fruits showed only minor fl uctuations in fl ower and pistil mass and style and anther length.
S a m b a n d a m (1964) stated that 30-40% of the eggplant fruit set is attributed to pollination by contact, gravity and wind, the rest is dependent on insects.C h e n (2001) stated that the cone-like formation of eggplant anthers favors self-pollination; but since the stigma ultimately projects beyond the anthers, there is ample opportunity for cross-pollination.Such kind of the androecium indicates the adaptation to the buzz-pollination mechanism.In buzz-pollination, insects grasp the anther cone and transmit vibrations through their bodies to the anthers, thus producing pollen expulsion from the anther apices (B u c h m a n , 1983).This feature restricts pollination to a group of insects which are able to vibrate the anthers, i.e. bumble bees (B e z e r r a and M a c h a d o , 2003).Bumble bees were common on the fl owers of eggplant in the conditions of the present experiment (open fi eld plantation).The buzz-pollination mechanism enabled bumble bees to pollinate all types of eggplant fl owers, whereas self-pollination was prevented in short-styled fl owers because of the spatial separation of terminal pores and the stigma in turned down fl owers (Fig. 1, III; Fig. 4).It was possible that the short-styled fl owers were maintained by the plant to attract insects with pollen as a reward.The short-styled fl owers were fertile, despite of the reduced size of the pistil, but pollination was hampered.If it had occurred (mainly by insects), fl owers set fruits.C o n n o l l y and A n d e r s o n (2004) investigated the role of the androecium in male and hermaphroditc fl owers of andromonoecious Solanum carolinense.The anthers in these two types of fl owers had the same size and they bear the same quantity of pollen, but their differed in their role in pollination.Male organs of hermaphroditic fl owers were the source of pollinator reward (pollen), at least as near distance attractive elements, and as landing platforms.The stamens of the male fl owers functioned primarily as possible near-distance attractors and as the source of pollen for reproduction.In Solanum palinacanthum, the yellow anthers were shown to be more important attractants than was the corolla (C o l e m a n and C o l e m a n , 1982).
The incompatibility in the growth on pollen tubes in the short styles was not observed, so a smaller number of pollen tubes, noticed in short-styled fl owers, may have a morphological substratum: the above mentioned pollination diffi culties and small sized stigmas of shortstyled fl owers which absorb less pollen.A low number of pollen tubes, fertilized ovules and seeds can be a straight consequence of short-styled pistil morphology and a way of pollination.R y l s k i et al. (1984) showed that pistils with a style length of less than 0.5 cm tend to be smaller than medium-or long-styled ones and carry a relatively small stigma with undeveloped papillae and a low sugar content that precludes pollen germination and thus results in pollen drop.P a s s a m and B o lm a t i s (1997) found that fl owers of style length <0.5 cm had signifi cantly smaller ovaries and failed to set fruits.In fl owers with style length greater than 0.51 cm, the ovary size was quite similar.

Fig. 5 .
Fig. 5. Number of pollen tubes in the styles of diff erent types of eggplant fl owers.

Fig. 6 .
Fig. 6.Number of fertilized ovules in the pistils of different types of eggplant fl owers.

Table 1
The effectiveness of fertilization and fruit setting as dependent on the type of fl ower in eggplant.