Pollen grains and seed morphology as related to biochemical patterns in five species of genus Ocimum L . ( Lamiaceae Juss . ) of Saudi Arabia

Genus Ocimum L. (tribe Ocimeae) is one of the largest genera Lamiaceae Juss; it comprise about 150 species mainly in tropical and warm temperate countries. Six species of Ocimum L. are recorded in Saudi Arabia (Al-Farhan et al., 2005; Masrahi, 2012). Palynology has been used considerably in the angiosperms taxonomy and helped in tracing the history of plant groups and species (Moore and Webb, 1978). Patel and Datta (1958) and Sowunmi (1973) are some of the researchers who have worked on the pollen grains morphology and emphasized their significance architecture in phylogeny. Bentham (1832) divided the Ocimum into three sections; Ocimum (Ocymodon Benth.), Hierocymum Benth and Gymnocymum Benth. Pollen grains of three species and a variety of Ocimum occurring in South-western Nigeria by light microscopy (LM) have been reported by Arogundade and Adedeji (2009). Morphological characteristics of family Lamiaceae of seeds of Al-Taif in Saudi Arabia are focused by Hassan and Altobatti (2015) and formerly by El-Gazzar and Watson (1970). The protein electrophoretic separations now have an established place in modern chemotaxonomic practice (Harborne and Turner, 1984). Genetic diversity among Ocimum L. populations in Egypt as reflected by morphological and electrophoretic techniques are carried out by Abd El-Zaher et al. (2006). Current work aims to describe in details the pollen and nutlet morphology (macro and micro characters) of five Ocimum L. species in Saudi Arabian by light microscopy (LM) and scanning electron microscopy (SEM) and their correlations to the biochemical analyses to give modern data may can support to evaluate the systematic relationships of this species in genus Ocimum.


INTRODUCTION
Genus Ocimum L. (tribe Ocimeae) is one of the largest genera Lamiaceae Juss; it comprise about 150 species mainly in tropical and warm temperate countries.Six species of Ocimum L. are recorded in Saudi Arabia (Al-Farhan et al., 2005;Masrahi, 2012).Palynology has been used considerably in the angiosperms taxonomy and helped in tracing the history of plant groups and species (Moore and Webb, 1978).Patel and Datta (1958) and Sowunmi (1973) are some of the researchers who have worked on the pollen grains morphology and emphasized their significance architecture in phylogeny.Bentham (1832) divided the Ocimum into three sections; Ocimum (Ocymodon Benth.),Hierocymum Benth and Gymnocymum Benth.Pollen grains of three species and a variety of Ocimum occurring in South-western Nigeria by light microscopy (LM) have been reported by Arogundade and Adedeji (2009).Morphological characteristics of family Lamiaceae of seeds of Al-Taif in Saudi Arabia are focused by Hassan and Altobatti (2015) and formerly by El-Gazzar and Watson (1970).The protein electrophoretic separations now have an established place in modern chemotaxonomic practice (Harborne and Turner, 1984).Genetic diversity among Ocimum L. populations in Egypt as reflected by morphological and electrophoretic techniques are carried out by Abd El-Zaher et al. (2006).Current work aims to describe in details the pollen and nutlet morphology (macro and micro characters) of five Ocimum L. species in Saudi Arabian by light microscopy (LM) and scanning electron microscopy (SEM) and their correlations to the biochemical analyses to give modern data may can support to evaluate the systematic relationships of this species in genus Ocimum.

MATERIALS AND METHODS
Five species of Ocimum L., were collected from different localities in Jazan area of Saudi Arabia as follows: Ocimum americanum L., Jabal Fayfa; Ocimum basilicum, Al-Arda; Ocimum filamentosum Forssk., Jabal Fayfa; Ocimum forsskalii Benth, near Bani Malik and Ocimum tenuiflorum L., Pollen grains and seed morphology as related to biochemical patterns in five species of genus Ocimum L. (Lamiaceae Juss.) of Saudi Arabia Wael Taha Kasem 1,2 * Al-Arda.Specimen identification was identified according to Chaudhary (2001) and Al-Farhan et al., 2005, the voucher specimens are deposited at the Jazan University Herbarium, KSA (JAZUH).

Pollen Morphology
Pollen grains of the taxa were studied by LM and SEM, for LM, pollen grains were first treated with 70% alcohol to remove oily substances.For LM, the pollen grains were observed and photographed by a Nikon E1100 microscope.The measurements are based on 20 readings from each slide.The polar axis (P), equatorial diameter (E), and P/E ratio were calculated.For SEM, acetylation was according to the Erdtman technique (Erdtman, 1952).Pollen grains were dehydrated in ethanol sequences and mounted on a metallic stub in few drops of ethanol.The specimens were coated with gold in Apolaron E1100 ion sputtering device, then viewed at 20 KV in a JOEL JSM 5300 SEM of the Central Laboratory, Faculty of Science, Alexandria University, Egypt.The terminology based on Barthlott (1981;1984).

Seed Morphology
Mature seeds of the five species were collected from their natural habitats.10 seeds of each species were examined for size, shape, and color.During SEM, mature seeds (2-3) from each species were selected and mounted onto stubs with double-sided adhesive tape and coated with gold.The seed surface pattern was examined on the lateral surfaces of the seeds.For each sample, photographs of seeds were taken using a 20 KV in a JOEL JSM 5300 at different magnifications powers and photomicrography.The terminology used here follows authors such as Stearn (1992), Barthlott (1981), and Koul et al. (2000).

Electrophoretic Techniques
Species Seeds were collected, washed in distilled water, dried and ground to fine powder and used for protein and isoenzyme determination.Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was conducted as the method outlined by Stegemann et al. (1988).Bands were determined and scanned using Hoefer scanning densitometer GS 300.Protein gel bands scanned and photographed.Homogeneous PAGE was conducted for esterase isoenzyme measurements as outlined by Stegemann et al. (1988).After electrophoretic process; the specific staining solution used according to Graham et al. (1964) and Jonathan and Wendel (1990).The esterase gel is stained by adding 1 ml of 1% alpha naphthyl acetate in 60% acetone to 25 ml phosphate buffer (pH 6.5) and 10 mg of fast blue RR were added to 50 ml of the same buffer.Gel scanned using also Hoefer scanning densitometer GS 300.

Data Analysis
A statistical analysis of the identified data was carried out by multivariate cluster analysis using using Minitab 13.1 release-PC computer program (Minitab 2000).A table illustrating the means of all parameters was prepared, cluster diagram of the total values was constructed.

O. americanum L.
The pollen types were subprolate, with concave form.Polar length ranged in between 25.3 and 27.7 µm, and the equatorial one was 18.3-19.2µm with an average P/E ratio of 1.42 µm.Pollen wall thickness ranged from 5.93 to 6.24 µm.Doubly reticulate (bi-reticulate) tectum has a distinctive regular primary and secondary stratum.Coarsely perforate structure with enlarge pores were noticed in secondary lumina (substratum) (Figure 2 -1a and b).

O. basilicum L.
Possessed prolate, with convex-shaped pollen grains.The polar axis was 21-23 µm while the equatorial one was 13-17 µm, and the average P/E ratio was 1.5 µm.Thick pollen walls of 7.93-8.42µm were noticed.The tectum examination by SEM showed bireticulate patterns and deep with regular rough granular particles in the inner part of substratum (Figure 2 -2a and b).

O. filamentosum Forssk.
The observed pollen type was subprolate (Figure 1 -3a and b), with concave shape.Six apertures were situated in both the polar and equatorial regions.The polar and equatorial lengths were 26.4-27.3µm and 21.7-22.5 µm, respectively, with mean P/E ratio of 1.22 µm, wall thickness in between 7.93 and 8.42 µm.SEM revealed a regular tectum; primary lumina has a smooth wall texture, and regular reticulate structure was common in the secondary lumina (Figure 2 -3a and b).

O. forsskalii Benth.
Hexacolpate pollen type was observed, subprolate, ovoidal to spherical shape, concave.Fairly large pollen grains found.The polar length was 32.1-34.3µm while equatorial length was 26.3-27.4µm, and P/E ratio was 1.31 µm.Thick pollen wall (8.6-9.2 µm) was estimated.Exine structure explained a fairly deep tectum with rough stratum; the length of 19.1-22.8µm were found.P/E ratio was 1.19.The thickness of the pollen wall was 7.8-8.3µm.Pollen tectum was angular and deep, stratum has a smooth texture whereas the substratum filled with irregular perforate patterns (Figure 3 -5a and b).O. tenuiflorum L.

Seed protein profiles and esterase (EC.3.1.1.1) isoenzymes
Protein and esterase electrophoretic patterns for the five species of Ocimum are presented in Table 1 and illustrated in Figure 6 a and b.From gel scan, ten protein groups obtained with migration distances ranged from 0.80 to 4.78 mm.Protein bands of 1, 3, 5, 7, and 10 (monomorphic) found in all the studied species.The highest number of nine protein profile found in O. basilicum whereas the lowest one was noticed in O. forsskalii (Figure 6a).On the other hand, eight esterase patterns obtained scanning the gel with migration distances in between 0.78 and 5.43 mm.These profiles revealed that Est 2, Est 5, and Est 8 pattern were found in all species.The highest number of eight esterase isoenzyme bands found in O. basilicum whereas the lowest one of three bands noticed in O. tenuiflorum.
39 different characters (Table 1) included pollen grains, seed morphology and electrophoretic polymorphism of seed protein and esterase isoenzymes used in cluster analysis (Figure 7).From the phenogram, O. basilicum separated in a single level than the remainders which gathered at the different similarity level.In the second The seed micro-and macromorphological parameters showed that nutlet is fairly variable in shape and size.Ellipsoid shape was observed in all the studied species such variability in seed shapes existed a within a given species agree with Mayer and Mayber (1975) and Hassan and Altobatti (2016).Seed color ranged from dark brown, gray or black; Husain et al. (1990) and Hussein (2000) considered the seed color as having a very limited taxonomic value in this genus because it is fairly similar color.In dimensions' measurements, obtained results showed fairly range of variations in nutlets size, so, the color, shape, and size can be of little taxonomic important.Similar conclusions have also been given by Karakish (1993), Hamed and Mourad (1994), Shaheen (2002), Kaya and Dirmencl (2008), Budantsev (1993), andKasem (2016).
Key to the studied species based on pollen microsculpture A1 -Pollen grains are prolate O. basilicum A2 -Pollen grains are subprolate B1 -Substratum is rugose patterns O. filamentosum B2 -Substratum is perforate shaped C1 -Rough perforation with regular pattern O. americanum C2 -Coarsely perforate with irregular pattern O. tenuiflorum C3 -Finely perforate with semi-solid structure O. forsskalii Seed Morphology and Seed Coat Sculpturing Macro-and micro-morphological seed parameters of the five species as shown by LM and SEM are illustrated and photographed.Ocimum americanum L. Slightly large seeds (1.5-1.6 × 0.77-0.79mm) with smooth texture.Ellipsoid-oblong in shape with upper and basal rounded ends with short projection.Seed color is gray to black.Nutlet coat sculpture revealed thin anticlinal walls, undulate with regular polygonal fork form and raised boundaries.periclinal cell walls are smooth fairly concave form (Figure 4 -1a and b).
30-1.35 × 0.4-0.50mm) with smooth texture, gray color, ellipsoid-ovate with upper rounded end in which a short projection appeared while basal end is oval.The examined anticlinal cell wall boundaries, demonstrate a fairly finely raised, quirky polygonal cells.Periclinal cell walls are smooth and fairly convex.(Figure 4 -2a and b).
Seed size, 1.1-1.5 × 0.3-0.4mm, rough texture, black nutlet.Ellipsoid oblong-oval shape with oval upper end and short projection while seed basal end is rounded.Seed scan by SEM showed a thin anticlinal cell walls with undulated polygonal depressed deformation boundary cells.Smooth irregular periclinal cells, convex with compactly reticulated patterns.(Figure 4 -3a and b).O.forsskalii Benth.Seed diameter, 1.4-1.45× 0.77-0.78mm.Black nutlet, rough texture, oblong-ovoid.Seed upper end is oval with short projection whereas the lower end is circular.secondary lumina filled with very finely perforation form and semi-solid structure (Figure 3 -4a and b).

Figure 3 :Figure 4 :Figure 5 :
Figure 3: Scanning electron microscope micrographs of pollen grains.(a) Polar view; (b) enlargement part exine; (4 a & b) O. forsskalii, (5 a & b) O. tenuifl orum Periclinal and anticlinal wall discriminated in certain species such as undulate in Americanum and O. filamentosum, quirky in O. basilicum, rounded in O. forsskalii and straight in O. tenuiflorum.On the other hand, molecular patterns in seed protein and esterase isoenzymes differed in band numbers and migration distance.The highest protein bands found in O. americanum whereas minimum bands found in O.