Introduction

Hibiscus is the most diversified genus in the Malvaceae family which contains flowering plants that can be grown under variable climatic and soil conditions viz., extreme high temperature to low temperature. Geographically, this genus is well distributed in tropical, subtropical and temperate areas of the world. Until now, 250-300 Hibiscus species have been reported with diversified plant morphological and architectural characteristics (Akpan 2007; Bates 1965; Wise and Menzel 1971). H. syriacus (Rose of Sharon) national flower of Republic of Korea is prized for profuse flowering during summer when only a few shrubs produce flowers. This species can be used as an accent plant in gardens due to its strict, upright habit and large trumpet shaped flowers with conspicuous stamens (Shim 1994). The plant grows 2.5-3 m tall with open and loose branches, making this plant ideally suited for formal or informal gardening (Gilman and Watson 2014). This species grows well under partial shade or full sun in varying soil types with least maintenance practices. Commonly, the species produced solitary flowers in the axils with full range colors from white, red, pink, to blue and purple. Flower size varies from single to double according to cultivar and mostly blooming time ranged from Mid-July to Mid- October (Kim and Lee 1991).

The Malvaceae family is stenopalynous palynologically as pollen features are more or less similar (Fatanmi et al. 2013). Conversely, distinct pollen morphology was reported by Saad (1960) in Malvaceae and he observed distinguish morphological features between the genera. Also, this family was divided into six pollen categories; on the basis of aperture number and diameter, grain diameter and spine form (Culhane and Blackmore 1988). The pollens of most Hibiscus species are characterized as large spherical shape, having porporlate or colporate aperture with echinate sculpture. Despite this, pollens diverge greatly and these can be used in identification, classification and phylogeny analysis within Malvaceae family (Christensen 1986; El Naggar 2004; El Naggar and Sawady 2008).

H. syriacus is a hardy garden flowering shrub that can bears low temperature and prolonged dry spell and can be grown under diversified soil and climatic zones viz., arid conditions, farmlands, dry rocks, coastal regions, and along road-side (Lawton 2004; Yu et al. 1979). Since long this species has been cultivated in China and Korea and this flower symbolize as the national flower of Korea (Kim et al. 2009). In Korea, this species reveals diverse in the plant canopy and various other morphological traits viz., flower size, shape and color, leaf appearance and color, plant shape and height etc. Generally, trumpet shaped flowers are solitary borne in the axils having full range of flower colors with prominent stamens (Bean 1973; Kim and Lee 1991). Nonetheless, the taxonomic depiction, characterization and interrelationships of the species are challenging and complex (Wang et al. 1995).

The pollen morphology plays a significant role in plant systematics and taxonomy as demonstrated by many researchers (Adeonipekun and Ige 2007; Cranwell 1952; Erdtman 1969; Perveen and Qaiser 2009) as various pollen characters are subjective to strong selective forces responsible for different reproductive developments, viz. dispersal, pollination, fertilization, and germination (Stuessy 1990). Pollen and exine morphological characteristics are of investigative importance (Mbagwu and Edeoga, 2006; Mbagwu et al. 2008) and therefore can be used in plant identification during palynological analysis, plant taxonomy, evolution and environmental restoration activities (Ige 2009; Palazzesi et al. 2007). However, high plant species diversity makes a palynologist difficult to identify or differentiate some pollen types that might result in the neglecting some indicator species (Adekanmbi 2009). Also some times, traits subjected to dominant selection may be confusing if these reveal convergent evolution and exhibit same evolutionary response by dissimilar plant taxa under same ecological settings. Therefore, to use pollen morphological characteristics as taxonomic trait must be a challenging task and pollen morphology should be used for validation of evolutionary processes with additional features.

The objective of present research was to study the pollen morphological characteristics of H. syriacus using scanning electron microscope (SEM). The collected data could be used for in-depth insights of pollen features that lead to successful breeding in H. syriacus.

Material and Methods

Plant materials

During year, 2011-2014, twenty local and exotic cultivars of H. syriacus were collected from different nurseries of Korea. The collected cultivars were grown under field conditions at Kyungpook National University, Daegu, Korea (GPS coordinates 35.89, 128.612).

Pollen morphological analysis

Pollen morphological characterization of 24 cultivars was conducted. All plants were about 3 years old at the time of pollen collection. Data were collected from three samples per cultivar and then average was computed.

Observation using a scanning electron microscope (SEM)

In order to clearly examine the pollen morphology, scanning electron microscopy (SEM) was carried out. Fresh and mature pollen were collected from each cultivar at the start of anthesis and carried to the laboratory for different processes starting from fixation process. For this purpose, the Karnovsky’s fixation sol. containing paraformaldehyde (8%), glutaraldehyde (25%), and 0.2 M cacodylate buffer mixture was prepared, and pollens were placed in this solution for 24 hrs. After fixation process, pollen washing with 0.05M cacodylate buffer was done for 10 min for three times. For post fixation pollen were kept at 4℃ in osmic acid (1%) for 2 hrs. After post fixation, the pollens were washed with 0.05 M cacodylate buffer (3 times for 10 min.), ethanol (50% ethanol 2 times for 30 min.), and finally with amylacetate for 30 min. Then the pollens were kept in amylacetate prior to SEM examination. The pollens were dried through the critical point dryer (HCP-2, Hitachi, Tokyo, Japan) wherein the pollens were kept at 20 in L-CO2 (50-80%) for 20 mins. After drying, for white gold coating samples were placed in specimenholder fitted with ion sputter (E-1030, Hitachi, Tokyo, Japan). A scanning electron microscope (S-4300, Hitachi, Tokyo, Japan) was used for pollen morphological studies.

Results and Discussion

Pollen morphological analysis of 16 cultivars of H. syriacus collected locally from various parts of Korea was carried out. Present results showed that pollen diameter of 16 cultivars of H. syriacus ranges from 108.81 to 172.15 μm (Table 1). The pollen size of ‘Chongdansim’ was reported the maximum followed by cultivar ‘Paedal’. The pollen diameter was the minimum in cultivar ‘Yeonamsinjongsik’ which was 63.34 μm smaller than that of ‘Chongdansim’ (Table 1). The results are similar with the findings of Bibi et al. (2008), as they reported pollen diameter ranged from 159-186 μm in H. syriacus. There is variations controversy in pollen grain sizes for particular Hibiscus species. Pollen size of H. rosa-sinensis reported by Bibi et al. (2008) was ranged between 124-165 μm which was greater as measured by Lakshmi (2003) that is 72.80- 98.80 μm. Also, Fryxell and Hashmi (1971) also reported the pollen size ranged from 79 to 182 μm for most of Hibiscus species.

The present results confirmed EI Naggar (2004) findings that pollens in Malvaceae family had generally globular or spheroidal outline and had colporate or porate sculpture. Pollen size and other morphological features are reported to differ in different species due to a combination of different factors: genetic makeup, chromosome counting, pollen development, pollen maturity and also various environmental factors also contribute to variation in pollen size viz., temperature, humidity, nutrients, and pH etc. (Stanley and Linskins 1974).

It is also important to be cautious that pollen size also be affected while preparing pollens for SEM analysis during chemical treatments and fixation process (Stanley and Linskins 1974). Even though pollen size can be variable, this can be used in classification and characterization of different plant taxa. Closely related plant species sometimes showed a great difference in the pollen shape, size, and style length (Knight et al. 2010). A positive relationship between genome size and pollen size was reported by Beaulieu et al. (2008). To understand which factor controls the pollen features from a developmental perception can improve our knowledge of ecological and palynological significance of variations in the pollen size (Knight et al. 2010).

The pollens of H. syriacus are characterized by spiny tecta (Fig.1 and Fig. 2). In present study, the spines exhibited variations regarding its size (length), shape, number, and surface distribution (Table 1). From results it was observed that the maximum spine exine length was in ‘Jabae’ (25.29 μm) followed by ‘Collie mullens’ (23.25 μm), whereas, the minimum value was recorded in ‘Paedal’ (8.16 μm). The data regarding number of spiny exine showed that the maximum value in cultivar ‘Giant Red’ which had 84 number of spiny exine followed by ‘Hundang’ having 80 spines. The minimum number was counted in cultivar ‘Gyewolhyang’ with only 28 spines. Distance between spiny exine (μm) was also measured and presented in Table 1. The results showed that the maximum distance between spines was observed in cultivar ‘Gyewolhyang’ that had 45.03 μm distance. In cultivars ‘Freedom’ and ‘Arang’ 40.30 and 37.39 μm distance was recorded respectively. In ‘Seongchon’, the minimum distance 18.58 μm between spiny exine was found (Table 1). These differences are of taxonomic importance at various taxonomic levels, because these may recorded not only between species but also between cultivars of the same species. In literature, it was found that the spine length can be similar on pollen (monomorphic) as reported in most of species from tribe Abutilieae and Hibiscieae, but it can differ on the same pollen representing a dimorphic pattern as in the species of Malveae tribe.

Present study had emphasized on the morphological characteristics and differentiating features of the H. syriacus pollens. H. syriacus is a species in Malvaceae family that shared the same echinate features but peculiar in having isolated conspicuous spines. The echinate type of pollens was also reported in Malvaceae family (Perveen and Qaiser 2009). H. syriacus pollens were differentiated from H. rosasinensis in being greater in size and number of spines. Slight variations were however noted in pollen morphological features in other Hibiscus species reported by various researchers (Faegri and Iverson 1964; Shaheen et al. 2009). In stenopalynous plant families, external pollen features had been termed as the reliable, most constant and distinct trait through which pollens may be differentiated at various taxonomic levels (El Naggar 2004; Nair and Sharma 1965). It is apparent from this study that pollen morphology of various H. syriacus cultivars was fairly uniform, as reported by researchers From present study results, it could be concluded that pollen morphology could not be only used as the basis of taxonomic grouping of the Hibiscus. However, pollen morphology characteristics had spectacled evolutionary progression comparable to other organs. The pollen morphological analysis and cross-compatibility of different cultivars of same species, and also the cross-compatibility of other species is essential in further breeding research.