II. Methods
1. Morphological Investigations
i. Whole Plant
Macromorphological attributes of the whole plant, inflorescence, flowers, fruits and seeds were described from the investigated specimens or compiled from text books such as Bailey (1949) and others. Macrophotographs for some of the studied taxa were made from living representatives and others were derived from specific sites.*
ii. Lamina Vein Architecture
Leaves were cleared in 5–20% KOH, rinsed in water, bleached in 25% NaHClO 2 , and placed in 250% chloral hydrate (CCl 3 CH(OH) 2 ) for 24 h.
Cleared and bleached leaves were then rinsed in water, dehydrated through an ethanol series, stained with 1% safranin, then placed onto glass slides and investigated by a microscope, photographs were presented.
Leaf architectural terminology generally follows Hickey (1973, 1979) and LAWG** (1999).
iii. Lamina Epidermal Morphology
Lamina epidermal characteristics were studied using fresh materials.
Leaf fragments of 5–10 mm2 were sampled from the median portion of each leaf.
The leaf fragments were then soaked in concentrated acetic acid and 9% hydrogen peroxide in Petri dishes for periods ranging from 1 to 3 days. The appearance of air bubbles on the surface of the leaf fragments indicated their suitability for separation.
They were transferred into water in a Petri dish with a pair of forceps.
Both epidermal surfaces were carefully separated by tearing up a corner of the leaf and pulling back the upper epidermis.
The epidermal surfaces were cleaned with a camel hair brush, rinsed several times in distilled water and later transferred into 50% alcohol to harden for about 2 min.
The epidermal srips were stained in safranine for about 3–5 minutes and excess stain washed off in water.
The ab- and adaxial epidermises were mounted in glycerine on a slide with the edges of the cover slip ringed with nail varnish to prevent dehydration.
The photomicrographs were taken using a Reichert Microstar IV microscope at the Plant Taxonomy Research Laboratory, Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
Descriptive terminology of epidermal characteristics based on Matcalfe & Chalk (1950), LAWG (1999) and Prabhakar (2004).
The stomatal index (SI) was calculated using the formula of Stace (1965): S/S+E×100, where "S" denotes the number of stomata per unit area and "E" the number of epidermal cells in the same unit area.
For SEM small pieces (7 mm2) of the leaf material were fixed on SEM stubs with double-sided tape, coated with gold in SPI-Module sputter coater, examined and photographed in Jeol JSM 5200 at different magnifications.
2. Molecular Investigations
i. RAPD-PCR
Genomic DNA extraction was performed as suggested by DNA extraction kit’s manufacturer Jena Biosciences, Plant DNA Preparation Kit, Genomic DNA purification from plant tissue.
a) Sample Collection: Fresh lamina tissue finely grounded with a mortar and pestle in liquid nitrogen prior to DNA isolation, tissue kept cold to minimize DNase activity.
b) Cell Lysis: The finely grounded tissue (10-30 mg) transferred to a 1.5 ml microtube, 300 µl Cell Lysis Solution added to the tissue, then incubated at 65°C for 60 min, the tube inverted occasionally during the incubation.
c) RNase Treatment: The RNase A Solution 1.5. µl added to the cell lysate, the sample mixed by inverting the tube 25 times and incubated at 37°C for 15-60 min.
d) Protein Precipitation: The sample cooled to room temperature and 100 µl of Protein Precipitation Solution added to the cell lysate, the solution mixed well by vortexing, centrifuged at 15,000 g for 3 min. (The precipitant form a tight, green pellet.
e) DNA Precipitation: DNA containing supernatant transferred to a clean 1.5 ml microtube containing 300 µl of Isopropanol >99%, The sample mixed by inverting gently 50 times, then Centrifuged at 15,000 g for 1 min, the supernatant discarded and tube drained briefly on clean absorbent paper, 300 µl Ethanol 80% added and tube inverted several times to wash the DNA Pellet, centrifuged at 15,000 g for 1 min, the ethanol discarded carefully, finally air dried at room temperature for 10-15 min.
f) DNA Hydration: DNA Hydration Solution 50-100 µl added to the dried DNA pellet, the DNA hydrated by incubating sample at 65°C for 60 minutes.
g) PCR analysis (Table 4): Polymerase chain reactions (PCR) were carried out in 50 µl volumes containing around 100 ng of sample DNA, nucleotides at 200 µM and 10-base oligonucleotide primers at 200 nM . each reaction containing 0.8units of Taq polymerase and 5 µl of Taq polymerase buffer was overlaid with 30 µl of light mineral oil. Reactions were carried out in a Techne PHC1 DRI block. The reaction parameters were 92C for 1 min; 35C for 1.5 min; 72C for 1.5 min for 45 cycles with a final extension step of 72C for 5 min. samples were then stored at room temperature.
Products from the PCR reaction were separated by agarose gel electrophoresis and visualized under UV light following ethidium bromide staining.
Table (4): Primers Used in RAPD Analysis
No. Primer Sequence No. Primer Sequence
1 SC10-5 TCGGAGTGGC 6 SC10-14 TCCCGACCTC
2 SC10-17 GTTAGCGGCG 7 SC10-18 GCCCTACGCG
3 SC10-22 CTAGGCGTCG 8 SC10-23 GGCTCGTACC
4 SC10-25 CGGAGAGTAC 9 SC10-30 CCGAAGCCCT
5 SC10-59 GCATGGAGCT 10 SC10-64 CCAGGCGCAA
ii. Isozyme Technique
Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted to identify isozyme variation among studied Caesalpinioideae using five isozyme systems.
Fresh and young leaf samples for each species were used separately for isozymes extraction.
The utilized isozymes are - and -esterase (- and - Est), acid phosphatase (Acph), alcohol dehydrogenase (Adh), and aldehyde oxidase (Alo).
These isozymes were separated in 10 % polyacrylamide gel electrophoresis according to Stegemann et al. (1985).
a) Stock Solutions
Extraction Buffer: 1M Tris HCl (pH 8.8): Solution was prepared by dissolving 12.11 g Tris in about 50 ml distilled water and pH was adjusted to 8.8 by conc. HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC.
Acrylamide Stock (30%): The solution was prepared by dissolving 30 g acrylamide and 0.8 g N, N, methylene bis–acrylamide in about 70 ml distilled water, then the volume was completed to 100 ml by distilled water. The stock solution was kept at 4oC.
Electrode Buffer (pH 8.65): Electrode buffer was prepared by dissolving 18.55 g boric acid and 2.5 g sodium hydroxide in 500 ml distilled water and mixed well with magnetic stirrer, then pH was adjusted into 8.6 by distilled water then kept at 4oC.
Gel Buffers: a. Separating gel buffer (1.5 M Tris – HCl, pH 8.8) (Tris 18.15 g, distilled water up to 100 ml). b. Stacking gel buffer (0.5 M Tris – HCl pH 6.8) (Tris 6.05 g, distilled water up to 100 ml). c. Ammonium persulfate solution (APS 10 %) (Amm. persulfate 0.1 g, distilled water up to 100 ml).
b) Extraction of Isozymes: Isozymes extraction from the different samples was performed separately for each taxa by homogenizing 0.5 g fresh leaves samples in 2 ml extraction buffer using a mortar and pestle. The extract was then transferred into clean eppendorf tubes and centrifuged at 10,000 rpm for 5 minutes. The supernatant was transferred to new clean eppendorf tubes and kept at –20 oC until use for electrophoretic analysis.
c) Gel preparation (Table 5): Vertical slab gel electrophoresis apparatus was used. All glass plates were washed with tap and distilled water, then surface sterilized with ethanol. Spacers of 1.5 mm were used. Separating gel was prepared by mixing the chemical components. The prepared gel solution was poured immediately between the two glass plates and overlaid with isopropanol and left to polymerize for at least one hour. After polymerization, isopropanol was removed. Stacking gel was similarly, prepared by mixing the chemical ingredients listed in the table and then poured over the separating gel. The comp was placed immediately. The gel was left to polymerize.
Table (5): Composition of Separating and Stacking Gels (- = absence)
Stock Solution 10% Separating Gel 4% Stacking Gel
Acrlyamide 8.3 ml 1.7 ml
Separating gel buffer 6.3 ml -
Stacking gel buffer - 1.25 ml
Distilled water 9.9 ml 6.8 ml
10 % Ammonium persulfate 250 μl 100 μl
TEMED 10 μl 10 μl
d) Application of Samples: A volume of 40 μl extract of each sample was mixed with 10 μl sucrose and bromophenol blue, then a volume of 50 μl from this mixture was applied to each well.
e) Electrophoresis Conditions: The gel glasses were fixed to both lower and upper tanks of the electrophoresis apparatus. The run (electrode) buffer was added to both lower and upper tanks. The apparatus was connected to the power supply. The run was performed at 100 volt until the bromophenol blue dye has reached the separating gel and then the voltage was increased to 125 volt. Electrophoresis apparatus was placed inside a refrigerator during running duration.
f) Enzymes Assay: After electrophoresis, the gels were stained according to their enzyme system with the appropriate substrate and chemical solutions then incubated at room temperature in dark for complete staining. In most cases incubation for about 1 to 2 hours is enough.
Acid Phosphatase (Acph): After electrophoresis, the gel was soaked in 100 ml of 50 mM Na-acetate buffer pH 5.0 containing 100 mg Fast blue BB salt, 100 mg -naphthyl phosphate, 100 mg MgCl2 and 100 mg MnCl2 (Wendel & Weeden, 1989). 50 mM Na-acetate buffer pH 5.0 was prepared by adding 5.15 ml glacial acetic acid and 2.85 g sodium hydroxide to 500 ml distilled water).
Alcohol Dehydrogenase (Adh): After electrophoresis, the gel was soaked in a solution of 4 ml isopropanol, 25 mg NAD, 20 mg NBT and 5 mg PMS in 100 ml of 0.05 M Tris HCl pH 8.5 (Jonathan & Wendel, 1990).
- and - Esterase Enzyme (- and - Est): After electrophoresis, the gel was soaked in 0.5 M borate buffer (pH 4.1) for 90 minutes at 4 oC. This procedure lowers the pH of the gel from 8.8 to about 7 at which the reaction proceeds readily. The low temperature minimizes diffusion of the protein within the gel. The gel then was rinsed rapidly in two changes of double distilled water. The gel was stained for esterase activity by incubation at 37 oC in a solution of 100 mg -naphthyl acetate or - naphthyl acetate (as a substrate) and 100 mg fast blue RR salt in 200 ml of 0.1 M phosphate buffer pH 6.5 (Scandalios, 1964).
Aldehyde Oxidase (Ao): After electrophoresis, the gel was soaked in 100 ml of aldehyde oxidase staining buffer pH 8.6 containing 20 mg NBT, 10 mg EDTA, 25 mg NAD, 100 mg KCl, 10 ml benzaldehyde and 5 mg PMS ((Jonathan & Wendel, 1990). Aldehyde oxidase staining buffer was prepared by dissolving 5.44 g Tris, 1.55 Boric, 0.37 g EDTA in 50 ml distilled water. After dissolving, the solution was completed to 100 ml by distilled water.
g) Gel Fixation: After the appearance of the enzyme bands, the reaction was stopped by washing the gel two or three times with tap water. This was followed by adding the fixative solution, which consists of ethanol and 20 % glacial acetic acid (9 : 11 v/v). The gel was kept in the fixative solution for 24 hours and then was photographed.
3. Numerical Analysis
a) Coding of Characters: Prior to analysis, the presence of character states (morphological and molecular) should be indicated as numerical values in order to make the comparison during similarity estimation feasible.
b) Standardization: The average data for each character was standardized using standardization program to get the linear transformation of the variables in the data matrix.
c) Similarity or Dissimilarity Assessment: This requires comparative recording of a set of characters for all studied taxa and is made by the use of a certain similarity coefficient. Then, average taxonomic distance was generated by using the similarity of interval data program.
d) Estimation the States of Characters: UPGMA (Unweighted Pair-Group Method using Arithmetic Averages) was used to estimate states of characters variation among the species, each taxa was considered as operational taxonomic unit (OTU) and states of characters analysed as binary characteristics.
e) Clustering: The formation of groups depending on the values of similarity. The sorting strategies are numerous and variable, but in erecting taxonomic relations it is usually hierarchical agglomerative i.e. forming groups or grouping the most similar OTU in groups, then these groups are linked in more larger groups and so on to the end of the analysis at which all OTU’s are in one group. Each step of the clustering is based on values of similarities between the groups. SAHN (Sequential, Agglomerative, Hierarchial and Nested clustering method) program was used as defined by Sneath & Sokal (1973) and Dunn & Everitt (1982).
f) Plotting the Tree: Phenogram was obtained by use of tree display graphics program.
All computations were carried out by the aid of the NTSYS-PC version 2.02 (Numerical Taxonomy and Multivariate Analysis System) software program (Rohlf, 1989).
Terminology
The terms used for lamina archirecture study.
Term Meaning
Admedially ramified Branching orinted toward the primary or midline.
Alternate percurrent Veins cross between secondaries wit an offset.
Brochidodromous Secondaries joined together in a series of prominent arches.
Campylodromous Several primary veins or their branches, originating at or near a single point and running in strongly recurved arches that converge apically.
Cladodromous Secondaries freely branching toward the margin.
Dichotomizing Veins branch freely.
Exmedially ramified Branching orinted toward the leaf margin.
Festooned brochidodromous Having one or more additional sets of loops outside of the main brochidodromous loop.
Fimbrial vein Higher vein orders fused into a vein running just inside the margin.
Opposite percurrent Veins cross between adjacent secondaries in parallel paths witout branching.
Pinnate Single primary vein
Random reticulate Tertiaries anastomose (rejoin) withother 3° veins or 2° veins at random angles.
Regular polygonal reticulate Veins anastomose with other 3° veins to form polygons of similar size and shape.
Semicraspedodromous Secondary veins branching just within the margin, one of the branches terminating at the margin and the other joining the superadjacent secondary.
The terms used for stomatographic study (LM).
Term Meaning
Anomocytic Five or more cells enclosing the guard cells, cells adjacent to the guard cells not differentiated in any way from the normal epidermal cells.
Cyclocytic Single ring of five or more small cells enclosing the guard cells.
Isotricytic Stomata completely surrounded by three subsidiaries, more or less equal in size.
Paracytic Two cells enclosing the guard cells with their long axis parallel to the long axis of the guard cells.
Tetracytic Stomata surrounded by only four subsidiaries of variable size and shape.
Terms used for Leaf Surface Sculpture (SEM)
Term Meaning
Colliculate With rounded broad elevations closely spaced.
Pusticulate With small broad slight elevations not so high or abundant as on colliculate and not having as abrubt elevations as a minutely tuberculate surface.
Reticulate With a rasied network of narrow and sharply angled line frequently presenting a geometric appearance, each area or depression outlined by the reticulum being an interspace.
Ruminate Penetrated by irregular channels giving an eroded appearance and running in different directions.
Tuberculate With small smooth rounded projections or knobs.
1. Morphological Investigations
i. Whole Plant
Macromorphological attributes of the whole plant, inflorescence, flowers, fruits and seeds were described from the investigated specimens or compiled from text books such as Bailey (1949) and others. Macrophotographs for some of the studied taxa were made from living representatives and others were derived from specific sites.*
ii. Lamina Vein Architecture
Leaves were cleared in 5–20% KOH, rinsed in water, bleached in 25% NaHClO 2 , and placed in 250% chloral hydrate (CCl 3 CH(OH) 2 ) for 24 h.
Cleared and bleached leaves were then rinsed in water, dehydrated through an ethanol series, stained with 1% safranin, then placed onto glass slides and investigated by a microscope, photographs were presented.
Leaf architectural terminology generally follows Hickey (1973, 1979) and LAWG** (1999).
iii. Lamina Epidermal Morphology
Lamina epidermal characteristics were studied using fresh materials.
Leaf fragments of 5–10 mm2 were sampled from the median portion of each leaf.
The leaf fragments were then soaked in concentrated acetic acid and 9% hydrogen peroxide in Petri dishes for periods ranging from 1 to 3 days. The appearance of air bubbles on the surface of the leaf fragments indicated their suitability for separation.
They were transferred into water in a Petri dish with a pair of forceps.
Both epidermal surfaces were carefully separated by tearing up a corner of the leaf and pulling back the upper epidermis.
The epidermal surfaces were cleaned with a camel hair brush, rinsed several times in distilled water and later transferred into 50% alcohol to harden for about 2 min.
The epidermal srips were stained in safranine for about 3–5 minutes and excess stain washed off in water.
The ab- and adaxial epidermises were mounted in glycerine on a slide with the edges of the cover slip ringed with nail varnish to prevent dehydration.
The photomicrographs were taken using a Reichert Microstar IV microscope at the Plant Taxonomy Research Laboratory, Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
Descriptive terminology of epidermal characteristics based on Matcalfe & Chalk (1950), LAWG (1999) and Prabhakar (2004).
The stomatal index (SI) was calculated using the formula of Stace (1965): S/S+E×100, where "S" denotes the number of stomata per unit area and "E" the number of epidermal cells in the same unit area.
For SEM small pieces (7 mm2) of the leaf material were fixed on SEM stubs with double-sided tape, coated with gold in SPI-Module sputter coater, examined and photographed in Jeol JSM 5200 at different magnifications.
2. Molecular Investigations
i. RAPD-PCR
Genomic DNA extraction was performed as suggested by DNA extraction kit’s manufacturer Jena Biosciences, Plant DNA Preparation Kit, Genomic DNA purification from plant tissue.
a) Sample Collection: Fresh lamina tissue finely grounded with a mortar and pestle in liquid nitrogen prior to DNA isolation, tissue kept cold to minimize DNase activity.
b) Cell Lysis: The finely grounded tissue (10-30 mg) transferred to a 1.5 ml microtube, 300 µl Cell Lysis Solution added to the tissue, then incubated at 65°C for 60 min, the tube inverted occasionally during the incubation.
c) RNase Treatment: The RNase A Solution 1.5. µl added to the cell lysate, the sample mixed by inverting the tube 25 times and incubated at 37°C for 15-60 min.
d) Protein Precipitation: The sample cooled to room temperature and 100 µl of Protein Precipitation Solution added to the cell lysate, the solution mixed well by vortexing, centrifuged at 15,000 g for 3 min. (The precipitant form a tight, green pellet.
e) DNA Precipitation: DNA containing supernatant transferred to a clean 1.5 ml microtube containing 300 µl of Isopropanol >99%, The sample mixed by inverting gently 50 times, then Centrifuged at 15,000 g for 1 min, the supernatant discarded and tube drained briefly on clean absorbent paper, 300 µl Ethanol 80% added and tube inverted several times to wash the DNA Pellet, centrifuged at 15,000 g for 1 min, the ethanol discarded carefully, finally air dried at room temperature for 10-15 min.
f) DNA Hydration: DNA Hydration Solution 50-100 µl added to the dried DNA pellet, the DNA hydrated by incubating sample at 65°C for 60 minutes.
g) PCR analysis (Table 4): Polymerase chain reactions (PCR) were carried out in 50 µl volumes containing around 100 ng of sample DNA, nucleotides at 200 µM and 10-base oligonucleotide primers at 200 nM . each reaction containing 0.8units of Taq polymerase and 5 µl of Taq polymerase buffer was overlaid with 30 µl of light mineral oil. Reactions were carried out in a Techne PHC1 DRI block. The reaction parameters were 92C for 1 min; 35C for 1.5 min; 72C for 1.5 min for 45 cycles with a final extension step of 72C for 5 min. samples were then stored at room temperature.
Products from the PCR reaction were separated by agarose gel electrophoresis and visualized under UV light following ethidium bromide staining.
Table (4): Primers Used in RAPD Analysis
No. Primer Sequence No. Primer Sequence
1 SC10-5 TCGGAGTGGC 6 SC10-14 TCCCGACCTC
2 SC10-17 GTTAGCGGCG 7 SC10-18 GCCCTACGCG
3 SC10-22 CTAGGCGTCG 8 SC10-23 GGCTCGTACC
4 SC10-25 CGGAGAGTAC 9 SC10-30 CCGAAGCCCT
5 SC10-59 GCATGGAGCT 10 SC10-64 CCAGGCGCAA
ii. Isozyme Technique
Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted to identify isozyme variation among studied Caesalpinioideae using five isozyme systems.
Fresh and young leaf samples for each species were used separately for isozymes extraction.
The utilized isozymes are - and -esterase (- and - Est), acid phosphatase (Acph), alcohol dehydrogenase (Adh), and aldehyde oxidase (Alo).
These isozymes were separated in 10 % polyacrylamide gel electrophoresis according to Stegemann et al. (1985).
a) Stock Solutions
Extraction Buffer: 1M Tris HCl (pH 8.8): Solution was prepared by dissolving 12.11 g Tris in about 50 ml distilled water and pH was adjusted to 8.8 by conc. HCl, then the volume was completed to 100 ml with distilled water then kept at 4oC.
Acrylamide Stock (30%): The solution was prepared by dissolving 30 g acrylamide and 0.8 g N, N, methylene bis–acrylamide in about 70 ml distilled water, then the volume was completed to 100 ml by distilled water. The stock solution was kept at 4oC.
Electrode Buffer (pH 8.65): Electrode buffer was prepared by dissolving 18.55 g boric acid and 2.5 g sodium hydroxide in 500 ml distilled water and mixed well with magnetic stirrer, then pH was adjusted into 8.6 by distilled water then kept at 4oC.
Gel Buffers: a. Separating gel buffer (1.5 M Tris – HCl, pH 8.8) (Tris 18.15 g, distilled water up to 100 ml). b. Stacking gel buffer (0.5 M Tris – HCl pH 6.8) (Tris 6.05 g, distilled water up to 100 ml). c. Ammonium persulfate solution (APS 10 %) (Amm. persulfate 0.1 g, distilled water up to 100 ml).
b) Extraction of Isozymes: Isozymes extraction from the different samples was performed separately for each taxa by homogenizing 0.5 g fresh leaves samples in 2 ml extraction buffer using a mortar and pestle. The extract was then transferred into clean eppendorf tubes and centrifuged at 10,000 rpm for 5 minutes. The supernatant was transferred to new clean eppendorf tubes and kept at –20 oC until use for electrophoretic analysis.
c) Gel preparation (Table 5): Vertical slab gel electrophoresis apparatus was used. All glass plates were washed with tap and distilled water, then surface sterilized with ethanol. Spacers of 1.5 mm were used. Separating gel was prepared by mixing the chemical components. The prepared gel solution was poured immediately between the two glass plates and overlaid with isopropanol and left to polymerize for at least one hour. After polymerization, isopropanol was removed. Stacking gel was similarly, prepared by mixing the chemical ingredients listed in the table and then poured over the separating gel. The comp was placed immediately. The gel was left to polymerize.
Table (5): Composition of Separating and Stacking Gels (- = absence)
Stock Solution 10% Separating Gel 4% Stacking Gel
Acrlyamide 8.3 ml 1.7 ml
Separating gel buffer 6.3 ml -
Stacking gel buffer - 1.25 ml
Distilled water 9.9 ml 6.8 ml
10 % Ammonium persulfate 250 μl 100 μl
TEMED 10 μl 10 μl
d) Application of Samples: A volume of 40 μl extract of each sample was mixed with 10 μl sucrose and bromophenol blue, then a volume of 50 μl from this mixture was applied to each well.
e) Electrophoresis Conditions: The gel glasses were fixed to both lower and upper tanks of the electrophoresis apparatus. The run (electrode) buffer was added to both lower and upper tanks. The apparatus was connected to the power supply. The run was performed at 100 volt until the bromophenol blue dye has reached the separating gel and then the voltage was increased to 125 volt. Electrophoresis apparatus was placed inside a refrigerator during running duration.
f) Enzymes Assay: After electrophoresis, the gels were stained according to their enzyme system with the appropriate substrate and chemical solutions then incubated at room temperature in dark for complete staining. In most cases incubation for about 1 to 2 hours is enough.
Acid Phosphatase (Acph): After electrophoresis, the gel was soaked in 100 ml of 50 mM Na-acetate buffer pH 5.0 containing 100 mg Fast blue BB salt, 100 mg -naphthyl phosphate, 100 mg MgCl2 and 100 mg MnCl2 (Wendel & Weeden, 1989). 50 mM Na-acetate buffer pH 5.0 was prepared by adding 5.15 ml glacial acetic acid and 2.85 g sodium hydroxide to 500 ml distilled water).
Alcohol Dehydrogenase (Adh): After electrophoresis, the gel was soaked in a solution of 4 ml isopropanol, 25 mg NAD, 20 mg NBT and 5 mg PMS in 100 ml of 0.05 M Tris HCl pH 8.5 (Jonathan & Wendel, 1990).
- and - Esterase Enzyme (- and - Est): After electrophoresis, the gel was soaked in 0.5 M borate buffer (pH 4.1) for 90 minutes at 4 oC. This procedure lowers the pH of the gel from 8.8 to about 7 at which the reaction proceeds readily. The low temperature minimizes diffusion of the protein within the gel. The gel then was rinsed rapidly in two changes of double distilled water. The gel was stained for esterase activity by incubation at 37 oC in a solution of 100 mg -naphthyl acetate or - naphthyl acetate (as a substrate) and 100 mg fast blue RR salt in 200 ml of 0.1 M phosphate buffer pH 6.5 (Scandalios, 1964).
Aldehyde Oxidase (Ao): After electrophoresis, the gel was soaked in 100 ml of aldehyde oxidase staining buffer pH 8.6 containing 20 mg NBT, 10 mg EDTA, 25 mg NAD, 100 mg KCl, 10 ml benzaldehyde and 5 mg PMS ((Jonathan & Wendel, 1990). Aldehyde oxidase staining buffer was prepared by dissolving 5.44 g Tris, 1.55 Boric, 0.37 g EDTA in 50 ml distilled water. After dissolving, the solution was completed to 100 ml by distilled water.
g) Gel Fixation: After the appearance of the enzyme bands, the reaction was stopped by washing the gel two or three times with tap water. This was followed by adding the fixative solution, which consists of ethanol and 20 % glacial acetic acid (9 : 11 v/v). The gel was kept in the fixative solution for 24 hours and then was photographed.
3. Numerical Analysis
a) Coding of Characters: Prior to analysis, the presence of character states (morphological and molecular) should be indicated as numerical values in order to make the comparison during similarity estimation feasible.
b) Standardization: The average data for each character was standardized using standardization program to get the linear transformation of the variables in the data matrix.
c) Similarity or Dissimilarity Assessment: This requires comparative recording of a set of characters for all studied taxa and is made by the use of a certain similarity coefficient. Then, average taxonomic distance was generated by using the similarity of interval data program.
d) Estimation the States of Characters: UPGMA (Unweighted Pair-Group Method using Arithmetic Averages) was used to estimate states of characters variation among the species, each taxa was considered as operational taxonomic unit (OTU) and states of characters analysed as binary characteristics.
e) Clustering: The formation of groups depending on the values of similarity. The sorting strategies are numerous and variable, but in erecting taxonomic relations it is usually hierarchical agglomerative i.e. forming groups or grouping the most similar OTU in groups, then these groups are linked in more larger groups and so on to the end of the analysis at which all OTU’s are in one group. Each step of the clustering is based on values of similarities between the groups. SAHN (Sequential, Agglomerative, Hierarchial and Nested clustering method) program was used as defined by Sneath & Sokal (1973) and Dunn & Everitt (1982).
f) Plotting the Tree: Phenogram was obtained by use of tree display graphics program.
All computations were carried out by the aid of the NTSYS-PC version 2.02 (Numerical Taxonomy and Multivariate Analysis System) software program (Rohlf, 1989).
Terminology
The terms used for lamina archirecture study.
Term Meaning
Admedially ramified Branching orinted toward the primary or midline.
Alternate percurrent Veins cross between secondaries wit an offset.
Brochidodromous Secondaries joined together in a series of prominent arches.
Campylodromous Several primary veins or their branches, originating at or near a single point and running in strongly recurved arches that converge apically.
Cladodromous Secondaries freely branching toward the margin.
Dichotomizing Veins branch freely.
Exmedially ramified Branching orinted toward the leaf margin.
Festooned brochidodromous Having one or more additional sets of loops outside of the main brochidodromous loop.
Fimbrial vein Higher vein orders fused into a vein running just inside the margin.
Opposite percurrent Veins cross between adjacent secondaries in parallel paths witout branching.
Pinnate Single primary vein
Random reticulate Tertiaries anastomose (rejoin) withother 3° veins or 2° veins at random angles.
Regular polygonal reticulate Veins anastomose with other 3° veins to form polygons of similar size and shape.
Semicraspedodromous Secondary veins branching just within the margin, one of the branches terminating at the margin and the other joining the superadjacent secondary.
The terms used for stomatographic study (LM).
Term Meaning
Anomocytic Five or more cells enclosing the guard cells, cells adjacent to the guard cells not differentiated in any way from the normal epidermal cells.
Cyclocytic Single ring of five or more small cells enclosing the guard cells.
Isotricytic Stomata completely surrounded by three subsidiaries, more or less equal in size.
Paracytic Two cells enclosing the guard cells with their long axis parallel to the long axis of the guard cells.
Tetracytic Stomata surrounded by only four subsidiaries of variable size and shape.
Terms used for Leaf Surface Sculpture (SEM)
Term Meaning
Colliculate With rounded broad elevations closely spaced.
Pusticulate With small broad slight elevations not so high or abundant as on colliculate and not having as abrubt elevations as a minutely tuberculate surface.
Reticulate With a rasied network of narrow and sharply angled line frequently presenting a geometric appearance, each area or depression outlined by the reticulum being an interspace.
Ruminate Penetrated by irregular channels giving an eroded appearance and running in different directions.
Tuberculate With small smooth rounded projections or knobs.
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