Pollen Morphology and Pollen Fertility Estimation of Three Medicinal Plant Species of Hypericum L . from Kaghan Valley , Northern Pakistan

Pollen morphology & pollen fertility estimation of three medicinal species belonging to genus Hypericum L. from flora of Kaghan valley, Northern Pakistan was investigated by light and scanning electron microscopy for the first time. The species are Hypericum elodeoides Choisy in, Hypericum oblongifolium Choisy, and Hypericum perforatum L. Pollen morphological characteristics with considerable variation are provided for these taxa. Among the studied taxa, the pollen grains were 3-4 colporate type and the largest pollen size was found in H. oblongifolium in polar view 23.0 μm and in equatorial view was 25.35 μm. The smallest pollen was of H. elodeoides in polar view which was 19.91 μm and in equatorial view was observed 12.53 μm in H. perforatum. The shape of the all pollen grain was circular to semi-angular in polar view, however in equatorial view the shape of the grains was oblate-spheroidal, in H. elodeoides, and H. oblongifolium while prolate in H. perforatum. Exine sculpturing was observed by scanning electron microscopy which showed foveolate sculpturing in H. elodeoides, reticulate in H. oblongifolium and scabrate in H. perforatum. Pollen fertility estimation ranged from 82.66% to 90.90% which shows that flora of selected species is well established.


Introduction
The flowering plant genus; Hypericum L. is one of the 100 large angiosperm genera and the largest genus of nine genera of the family Hypericaceae with about P. Mazari  Hypericum [1] [2] [3].Family Hypericaceae belongs to order Malpighiales and contains well-known medicinally valuable species [2] [4].The genus Hypericum is known worldwide for its traditional and modern uses.There are variety of bioactive compounds found in many species of Hypericum, which are used pharmacologically as well as to treat many diseases like tumors, cancer, AIDS, mild depression and kidney failure etc. [2] [5] [6].The most well-known species of the genus Hypericum is St. John's wort Hypericum perforatum which is economically important crop plant with antiviral, anticancer, antibacterial and antidepressant properties [7] [8].Flowers of this plant are known to use for relieving body pains and tea preparation in Kaghan Valley [9].
Genus Hypericum L has been studied by various researchers for its taxonomic, chemotaxonomic, pharmacological, karyological, ethnobotanical aspects.
However a recent phylogenetic study based on morphological characters was carried out (Nurk and Blattner, [10] while based on phylogenetic characters was studied by (Nurk [11] and Rhufel et al. [12].[6] [13]- [24] but the pollen morphology of Hypericum from Pakistan is still not known.However it has been largely studied for its medicinal properties, ethnobotanical uses, traditional knowledge and antioxidant capacity [9] [25] [26].
The knowledge of pollen morphology and taxonomy can be used as an instrument of multiple scientific researches in systematic botany, paleobotany, paleoecology, pollen analysis, aeropalynology, criminology, allergy, stratigraphic correlation of oil-bearing rocks and coal fields, drugs in the field of medicopalynology and improvement of honey in the field of mellittopalynology and copropalynology [27].Application of pollen morphology in plant taxonomy is best evidence in the flowering plants, especially in the angiosperms.The largest variety of pollen morphology occurs among the angiospermic plants [28].Like other disciplines pollen grains have an important part in modern issue of plant taxonomy [29].
The present investigation is based on pollen morphology and pollen fertility estimation of three medicinally important plant species of genus Hypericum studied for the first time from Pakistan.

Materials & Methods
Study Area-The Kaghan valley covering an area of 19790.4hectare spreads on either bank of river Kunhar, situated in the district Mansehra, Hazara division, Northern Pakistan (Figure 1).

Light Microscopy (LM)
For light microscopy slides were prepared by using glycerin jelly and safranine according to modified method of Mazari et al. [32].The slides were prepared and observed at different magnification.The micro-graphs of these mounted materials were taken using a digital camera (Meiji CCD Model 00179048, Canada) fitted on the Lieca light microscope (DM 1000, Germany).All photographs were taken on different object lenses by using oil emulsion.

Scanning Electron Microscopy (SEM)
The pollen grains were prepared for scanning electron microscopy (SEM).Pollen grains suspended in a drop of water were directly transferred with a fine pipette to a metallic stub using double sided cello tape and coated with gold in a sputtering chamber (Ion-sputter JFC-1100).Coating was restricted to150A.The S.E.M examination was carried out on a Jeol microscope JSM-2.

Pollen Fertility Estimation
Pollen fertility percentage was calculated on the basis of staining by Glycerin jelly.The prepared slides were observed at low magnification.Glycerin jelly was used in pollen staining and prepared according to modified method of Mazari et al. [33].Percentage of pollen fertility was calculated on the basis of staining by Glycerin jelly.The number of stained and unstained pollen grains was counted.
Fully stained pollens were considered fertile while the lightly stained or un-

Results
The pollen morphology and pollen fertility estimation of the three selected species of Hypericum was carried out by using light and scanning electron microscopy.Micrographs of pollen grains are presented in Figure 2 and Figure 3.
Findings of each species are represented in an order of local name, English name, phytogeography, flower color and season and palynomorph features.
These results are summarized in Table
English Name: Marsh Stewart John's wort.
Phytogeography: It is found from Kashmir to Yunnan and Burma.
In Pakistan it is found in Basaoli, Budrawur, Gulmarg, Kashmir & Northern areas.
Phytogeography: Europe and N. Africa to W.China and N.W., India.Introduced to East Asia, America.Australasia and South Africa.Pollen fertility: Pollen fertility is valuable for the taxonomists in attempting to distinguish putative hybrids from the parent plants and is also useful to determine the degree of fertility in those plants that are grown under unfavorable conditions [40].The pollen fertility studies have been proved helpful for recognizing wide range of variation existing within plant species and differentiating plant species within genera.Many workers have tried to correlate in vitro pollen fertility with a survival of a plant under its natural habitat [41].Matin et al. [42] studied pollen fertility of 50 medicinal plants of Shogran Valley.Fertility ratio of the plants ranged from 98% -100%, which indicated that flora of Shogran was well established and stable.In the present study among the 3 species of Hypericum; pollen fertility percentage ranged from 82.66% to 90.90%.It indicates that Hypericum species from Kaghan valley is well established and the species are stable in the area.

Discussion
In recent studies on palynology of Hyperium by Otaghvari et al., Martonfi et al. and Ocak, et al. [6] [22] [24] the regular and irregular pollen grains have been discussed.In the present study pollen examination of selected Hypericum taxa was conducted from Pakistan for the first time.Interesting findings were found regarding pollen characters of selected species.The pollen fertility estimation of the species was also investigated.Generally pollen grains were small, monad and isopolar.The type of pollen grains 3 -4 colporate in H. elodeoides while 3-colporate in other two species.In polar view in all species pollen shape was circular to semi-angular (Figure 2 .The present study showed palynological variation among these three species of Hypericum especially in exine sculpturing of pollen grain which was foveolate sculpturing in Hypericum elodeoides, reticulate in H. oblongifolium and scabrate in H. perforatum.Some of our findings regarding shapes and small to medium size of pollen grains are in accordance with [6] pollen grains but in contrast with regard of exine sculpturing which is different in all species studied.
Similarly palynologiacal analysis of seven Hypericum taxa were studied by [31], by light and scanning electron microscopy.According to them there were no essential differences in the exine structure of seven Hypericum species.The present showed much variation in regard of pollen shape in equatorial view and sculpturing pattern of exine which was different in all species studied.Pollen grains are mainly classified on the basis of their shape, size, aperture, polarity, symmetry and exine sculpturing.Along with these characters; exine sculpturing pattern is of great importance from phylogenetic and evolutionary point of view [39].The present showed that the studied palynological characters would be more useful for further palynological, karyological and taxonomic studies of Hyperium species.

Figure 1 .
Figure 1.Map of the study area.
stained and deformed pollens were considered sterile.Percentage of pollen was determined by the formula: No. of total pollen/ No. of fertile pollen = Percentage Pollen fertility.The descriptive terms for pollen morphology are used according to terminology of Erdtman), Kremp, Faegri & Iversen, Punt et al., and Walker & Doyle [34]-[39].

P.
Mazari et al.DOI: 10.4236/ajps.2017.8122073081 American Journal of Plant Sciences et al.DOI: 10.4236/ajps.2017.8122073074 American Journal of Plant Sciences 469 species occurring naturally and have been introduced to, every continent except Antarctica.Approximately 80% of the diversity of the family is within Choisy It is found in Western Himalayas from Kurram to Nepal.In Pakistan it is found in Kurram, lower Swat, Hazara, Malakandi, Kaghan, Nathiagali, Muree, Kashmir, Poonch.Flower color & period: Yellow & March-August.