biosystematics of four species of euphorbia l. grown in...

Republic of Iraq Ministry of Higher Education And Scientific Research University of Baghdad College of Science Biology Department

biosystematics of four species of

euphorbia l. grown in baghdad

university campus- jadiriyah

A thesis Submitted to the College of Science

University of Baghdad As partial fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Biology By

silva a. yakoub zokian

M.Sc. Biology-College of Science University of Baghdad

2006

Supervised By

Prof. Al-Musawi Ali H.,Ph.DAssist.Prof. AlJibouri Abidaljasim M., Ph.D

2011A.C. 1432 H.

جمهورية العراق وزارة التعليم العالي والبحث العلمي

كلية العلوم/جامعة بغداد قسم علوم الحياة

التصنيف الحياتي الربعة انواع من الجنس

Euphorbia L. -النامية في مجمع جامعة بغداد الجادرية

اطروحة جامعة بغداد - كلية العلوم مقدمة إلى

وهي جزء من متطلبات نيل درجة دكتوراه فلسفة في علوم الحياة/ نبات

من قبل

سيلفا انرتانيك يعقوب زوكيان

جامعة بغداد- كلية العلوم -ماجستيرفي علوم الحياة 2006

شرافءبا

علي حسين الموسوي أ.م.د. عبد الجاسم محيسن الجبوري أ. د.

هـ1432 م2011

Chapter One Introduction & Literature Review

1

chapter one

introduction & literature review 1- introduction

The history of plant systematic-the biological classification of plants-

stretches from the work of ancient Greek to modern evolutionary biologists.

As a field of science, plant systematic came into being only slowly, early

plant lore usually being treated as part of the study of medicine. Later,

classification and description were driven by natural history and natural

theology. Until the advent of the theory of evolution, nearly all classification

was based on the scala naturae. The professionalization of botany in the 18th

and 19th century marked a shift toward more holistic classification methods,

eventually based on evolutionary relationships. A major influence on plant

systematic was the theory of evolution (Charles Darwin published origin of

species in 1859), resulting in the aim to group plants by their phylogenetic

relationships. To this added the interest in plant anatomy, aided by the use of

the light microscope and the rise of chemistry, allowing the analysis of

secondary metabolites ( www.en.wikipedia.org).

The Arabs in the early times gave much attention to the study of

plants of the Arabian Peninsula, North Africa and Spain from different

angles especially for their uses in medicine. Mention needs to be made in

this connection of Eben Sina or Avicenna (980-1037), Ghafiqy (1160), and

Eben Al-Baithar (1248).

The study of the plants of Iraq as a modern science may be started by later

part of eighteenth century and after A. Michaux(1782); Oliver Bruguiers


2

(1795); Aucher-Eloy(1835); Col.Chenseney(1836); Theodore Kotschy

(1841); Noe(1851) and Haussknecht(1865-1867) worked on the plants of

Arabia which have been incorporated in Boissier’s Flora Orientalis(1876).

Further J. Bornmu ller(1892-1893); Oppenheimer(1893); F. Nbe’lek(1909);

H.F.V. Handel-Mazzetti(1910); C.Pau and C.Vicioso(1910); E.Guest

(1929); A.Eig(1929-1946), Z.Zohary(1933) and Col.Meinertzhagen (1934)

amongst others made extensive collections of Iraqi plants. But it is worth

mentioning that most of their collections are not deposited In Iraqi herbaria

but remain scattered in different herbaria of the continent and elsewhere.

E.Guest began to establish the nucleus of a herbarium of Iraq-plants in the

year 1929. Further collections have been added to the herbarium by many

workers later (Al-Rawi, 1964).

Systematics is important in providing a foundation of information

about the tremendous diversity of life. Virtually all fields of biology are

depend on the correct taxonomic determination of a given study organism,

which relies on formal description, Identification, naming, and classification.

Systematics is also an integrative and unifying science. One of the fun

aspects of systematic is that it may utilize data from all fields of Biology:

Morphology, Anatomy, Embryology/Development, Ultra structure,

Paleontology, Ecology, Geography, Chemistry, Physiology, Genetics,

Karyology, and cell/ Molecular biology (Simpson, 2006). While scientists

have agreed for some time that a functional and objective classification

system must reflect actual evolutionary processes and genetic relationships,

the technological means for creating such a system did not exist until

recently. In the 1990s DNA technology saw immense progress, resulting in


3

unprecedented accumulation of DNA sequence data from various genes

present in compartments of plant cells. The genus Euphorbia L. is one of the largest and most complex genera of

flowering plants. High morphological plasticity and diversity of this genus

make taxonomical studies on Euphorbia attractive for botanists.

The species of Euphorbia have their own economic value and hence

contribute to the floristic wealth of tropical and subtropical countries of the

world. This genus is also well reputed for the production of valuable

secondary metabolites like alkaloids, flavonoids and terpenes in nature.

Local Euphorbia species are quite rich, and have not been studied yet.

There are about 44 species of Euphorbia in Iraqi flora (Radcliff-Smith,

1980), and more than four species just in University of Baghdad Campus in

Jadiriyah.

The aims of this study: 1- Capable of providing a comprehensive monograph of genus

Euphorbia through appropriate field exploration, study of herbarium

and living collections from fields and gardens of University of

Baghdad Campus -Jadiriyah.

2- Identify the morphological characters.

3- Identify the anatomical characters

4- Study the habitat and geographic distribution in Iraq.

5- Identify the species of Euphorbia by investigation for proteins by

using the technique of protein electrophoresis.

6- Identify the species of Euphorbia by using molecular analysis tool

(the technique of RAPID- PCR).


4

7- Investigation for the response of the species of Euphorbia for callus

induction by using the technique of tissue culture under different

parameters in vitro.


5

2- literature reviews

2.1 systematic position of the family

Euphorbiaceae family is one of the largest families of plants .The

Euphorbiaceae was first adequately delimited as a natural group of plants by

A.L. de Jussieu in 1789 (Bruce and Perry, 1943, Simpson, 2006, Takhtajan,

2009). Since this time many contributions have been made to the

classification, phylogeny, morphology, and anatomy of the group. Though

the family has been known for many years, considerable differences of

opinion still exist as to the number of genera and species involved. Estimates

of the number of species in the family vary from 3000 to 8000 (Bruce and

Perry, 1943); reach’s to 8910 species in flora of China (Bingtao et al., 2008).

The family is closely related to the Geraniales by structure of the

gynoecium, although widely separated from other families in the order by

the amount of reduction in the most of its flowers. Small combines the

Euphorbiaceae and the Callitrichaceae into a separate order - Euphorbiales.

This order placed between the Polygalales and the Sapindales, with the

Geraniales immediately preceding this group (Heywood, 1978; Radcliff-

Smith, 1980; Webster, 1994; Judd et al., 1999).

The family is characterized as follows: Flowers hypogynous,

actinomorphic, mostly unisexual; perianth rarely double, usually simple or

wanting; androecium 1-∞; ovary of 3 carpels, trilocular, with 1 or 2

suspended ovules in each cell; micropyle directed upwards and outwards,

and covered with a fleshy outgrowth (caruncle). Fruit almost invariably a

schizocarp-capsule, splitting into carpels, often elastically (Heywood, 1978;

Judd et al., 1999; Singh, 2006; Takhtajan, 2009).


6

As we said the Euphorbiaceae are recognized as one of the largest

families of the dicotyledons. They are relatively natural group, although

showing many lines of evolution. The impression exists among many plants

mention that the Euphorbiaceae have a general preference for semi-desert or

desert regions, but a survey of the geographic distribution of the family

proves this to be erroneous . Its members live in the varied habitats, in many

different areas of the tropical and temperate world, and exhibit considerable

diversity in growth types. Because of this great diversity of form and habitat,

the family is of special interest. The Euphorbiaceae is of further interest due

to the great diversity of chromosome numbers and chromosome sizes both

between and within so called natural groups. Certain tribes, or other

taxonomic groups, appear cytologically to be natural groups in that one

basic chromosome number is found in each, while in other group nearly all

the basic numbers present in the family are found, suggesting that the unit is

merely descriptive and artificial and not phyletic (Bruce and Perry, 1943).

The Euphorbiaceae consists of trees, shrubs, herbs but are rarely

woody climbers. The larger genera are: Euphorbia (about 2,000 species),

Croton (700 species), Phyllanthus (500 species), Acalypha (430 species),

Jatropha (175 species), Manihot (170 species) (Aworinde et al.,2009).

Additionally Judd et al. (1999) stated more major genera like

Glochidion (300 species), Macaranga (250 species), Antidesma (150

species), and Tragia (150 species).

According to the most recent molecular research Euphorbiaceae is a

complex family previously comprising five subfamilies: the Acalyphoideae,

the Crotonodeae, the Euphorbiodeae, the Phyllanthoideae and the

Oldfieldioideae. The first three are uni-ovulate sub-families while the two


7

last one are bi-ovulate (Heywood, 1978; Radcliff-Smith, 1980; Takhtajan,

2009). The Euphorbiaceae has been split into five families: the three uni-

ovulate subfamilies have become the Euphorbiaceae in the strict sense, with

the tribe Galearieae in the Acalyphoideae forming the most of the family

Pandaceae. The bi-ovulate subfamily Phyllanthoideae has become the family

Phyllanthaceae, with the tribe Drypeteae as family Putranjivaceae and, the

tribe Centroplaceae part of the Pandaceae. The other bi-ovulate subfamily

Oldfieldioideae has become the Picrodendraceae (Heywood, 1978; Webster,

1994; Tokuoka and Tobe, 1995; Takhtajan, 2009). Euphorbioideae divided

to five tribes:

Tribe Euphorbieae

Tribe Hippomaneae

Tribe Hureae

Tribe Pachystomateae

Tribe Stomatocalyceae

(Heywood, 1978; Webster, 1994; Llamas, 2003; Mwine and Van Damme,

2011).

Webster suggested that “Phllanthoideae” are the primitive group from

which the other subfamilies are derived. They are almost surely

paraphyletic, and are characterized by having two ovules per locule, usually

alternate leaves, nonspiny pollen, and nonarillate seeds. Oldfieldioideae also

have two ovules per locule, and may be monophyletic on the basis of their

spiny pollen. Members of Euphorbiaceae having only one ovule per locule

probably form clade, which has been divided into three subfamilies by

Webster above: the Acalyphoideae (Acalypha, Alchornea, Tragia, Ricinus,

and relatives), which lack latex; and Crorotonoideae (Croton, Manihot,


8

Jatropha, Codiaeum, Aleurites, Cnidoscolus, etc.) and Euphorbiodeae

(Hippomane, Hura, Euphorbia, Gymnanthes, Stillingia, Sapium, etc.), both

of which have latex. The Contonoideae have distinctive polyporate pollen,

often stellate, peltate, or branched hairs, and colored to white, noncaustic

sap, while Euphorbiodeae have tricolporate pollen, simple hairs, and white,

often caustic sap. Euphorbioideae contain the large tribe Euphorbieae

(mainly Euphorbia, which includes Poinsettia, Chamaesyce, etc.), which are

considered monophyletic on the basis of their inflorescences, which are

cyathia. The carpellate flower is surrounded by numerous staminate flowers

(each of which is reduced to a single stamen) within a cuplike structure

formed from a highly reduced cymose inflorescence and associated bracts.

One to five nectar glands, sometimes with petal-like appendages, are

associated with the cuplike axis of each cyathium (Willis, 1973; Judd et al.,

1999; Prenner and Rudall, 2007). Most Euphorbiaceae are insect-pollinated

(flies, bees, wasps, and butterflies), with nectar providing the floral

attractant, yet some are probably pollinated by birds, bats, or other amimals.

Acalypha, Ricinus, and Alchornea, among others, are wind-pollinated.

Outcrossing is promoted by maturation of carpellate flowers before

staminate ones. Most have elastic schizocarps. The large fruits of hura (Hura

crepitans) or hevea (Hevea brasiliensis) are able to explosively eject their

seeds, shooting them several meters. Some are secondarily water-dispersed,

while those with oily arils are sometimes secondarily dispersed by ants.

Some taxa have fleshy arils (or indehiscent fleshy fruits) and are dispersed

by birds (Judd et al., 1999).


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2.2 systematic position of the genus

The genus Euphorbia is one of the largest, most complex and diverse

groups of flowering plants on earth. It contains at least 2000 species

(Heywood, 1978; Radcliff-Smith, 1980; Judd et al., 1999; Pritchard, 2003).

Many of the species are known as "spurges." They all produce a mostly

white latex which they exude when cut, and this sap is often toxic. There are

many herbaceous spurges, especially in temperate zones worldwide, but the

genus is best known for its many succulent species, some of which appear

very similar to cacti. Succulent euphorbias are most diverse in southern and

eastern Africa and Madagascar, but they also occur in tropical Asia and the

Americas (Pritchard, 2003; Heywood, 1978; Takhtajan, 2009). All flowers

in the Euphorbiaceae are unisexual (either male or female only), and they are

often very small in size. In Euphorbia, the flowers are reduced even more

and then aggregated into an inflorescence or cluster of flowers known as a

"cyathium" (plural cyathia). This feature is present in every species of the

genus but nowhere else in the plant kingdom. Whereas most other large

genera of plants differ in features of the flowers themselves, Euphorbia

varies instead in features of the cyathium, which can show amazing

modifications in different groups within the genus (Pritchard, 2003;

Heywood, 1978).


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FIGURE (1): Typical Flower of Euphorbia

(Diagram from www.Euphorbiaceae.Org (PBI))

The main defining feature of the cyathium is the floral envelope or

involucre that surrounds each group of flowers. The involucre almost always

has one or more special glands attached to it, most often on the upper rim,

and these glands and their appendages vary greatly in size and shape. There

may be specialized leaves called cyathophylls or cyathial leaves that

surround the cyathium and give an overall flower-like appearance to the

whole complex inflorescence. Inside the involucre are the flowers, usually

with a number of extremely simplified male flowers consisting of a single

anther, filament, joint and pedicel. Generally there is a single female flower

in the center consisting of a pedicel, a three-lobed ovary, and no petals or

sepals associated with it (Radcliff-Smith, 1980; Simpson, 2006; Takhtajan,

2009) Figure (1).


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FIGURE (2): Cyathium of Euphorbia

(Diagram from www.Euphorbiaceae.Org (PBI))

With this basic model of the cyathium, many modifications upon it have

evolved in the genus, as well as in the aggregation of cyathia into higher

order units (Heywood, 1978; Radcliff-Smith, 1980; Webster, 1994; Judd et

al., 1999) Figure (2).

2.3 fruits and seeds

Fruits of Euphorbia are capsules that typically split open explosively

when ripe. There are potentially three seeds per capsule, and there is a wide

variety of size, shape, and surface features of the seeds and capsules. Seeds

of some species have a fleshy appendage called the caruncle above the point

of attachment to the central column of the fruit (Mangaly et al., 1979).


12

Structure of the seed coat is characteristic for the family and does not

provide evidence for a polyphylectic origin of the family (Webster, 1994).

2.4 diversity of life forms

The variety of habits or life forms is one of the most salient features of

Euphorbia. There are many annual or perennial herbs, and these tend to

retain leaves through their active growing periods. At its simplest, in a

number of species in the Chamaescyce lineage, the plant will germinate,

dichotomously branch, flower, fruit, and die in a matter of weeks. There are

also leafy shrubs and trees as members of species that can reach 20 meters

high. A large portion of Euphorbias, however, are succulent, with thickened,

photosynthetic stems and very ephemeral leaves if present at all. Many

succulents are in turn thorny, and some have well developed underground

tubers (Radcliff-Smith, 1980, Pritchard, 2003).

2.5 systematics and classification

The understanding of the relationships of Euphorbia has been bolstered

by comparative DNA sequence data from many species, and these results

support a broad view of the genus that includes a number of groups that

were formerly recognized as different genera, such as Chamaesyce,

Monadenium, Pedilanthus, and Poinsettia. The most current information

places Euphorbia species into four distinct monophyletic groups or clades .

Steinmann & Porter (2002) has examined different regions from the three

plant genomes (nuclear, chloroplast, and mitochondrial), and this shows

clear support for the following relationships among the four main clades of


13

Euphorbia: clade B (subgenus Esula) is the sister group to clade A

(subgenus Rhizanthium), and that is in turn sister to both clades C and D

(subgenus Euphorbia and subgenus Chamaesyce) Figure (3).

FIGURE (3) Major Clades of Euphorbiaceae

Diagram from www.Euphorbiaceae.Org (PBI))

2.6 origin of the botanical (latin) name of

euphorbia

King Juba of Mauritania (today’s Morocco) is credited as the first person

to discover a succulent Euphorbia and give the genus its name. Somewhere

between 25 BC and 18 AC, he discovered a plant in the Atlas mountains: it

was most likely E.officinarum or E.resinifera, King Juba named the plant

after his doctor whose name was Euphorbus, the meaning of this word being

“well fed”, the king comparing his fat flashy doctor with the plant

(Pritchard, 2003; Gledhill, 2008). Non- succulent species had been known


14

back in ancient Greek times as “Titbymalus”. The two names existed side by

side until 1583 when a botanical link was made by Andrea Cesalpino, then

in 1753 Linnaeus listed both under one name “Euphorbia”. Today

“Titbymalas” still exists as section or subgroup of Euphorbia (Radcliff-

Smith, 1980; Pritchard, 2003; Simpson, 2006)

2.7 meaning of the common name "spurge"

Many of the herbaceous, leafy species of Euphorbia are commonly called

"spurges". This word derives from the old French word espurgier (Latin

expurgare), which means "to purge." The sap of many herbaceous

Euphorbia species have traditionally been used as a purgative, or laxative

(www.Euphorbiaceae.Org). Radcliff-Smith (1980) stated that Bailey (1939)

points out that Euphorbia is sometimes improperly referred to as Milkweed

and that the name spurge, though often applied to the genus as a whole,

cover more correctly the smaller herbaceous species. In our own territory

several general name, covering a number of different and unrelated plants

with milky juice, have frequently been noted for the weedy vegetal and ruder

species of Euphorbia which occur in fields, gardens and waste places in

lower Iraq (E. puples, E. granulata, E. chaemaesyse, E. prostrate, E.

petiolata, E. helioscopia): UM AL-HALIB ام الحليب, HALIB حليب and

ALBAINA البينة or LUBAINA لبينة (all colloquial names denoting “milk”).

Similarly, in the north, names embodying SHIR شير (‘milk”, kurd.) have

been noted for several species in the mountains - SHIR KITIK شير كتك

(“cat‘s milk”, Kurd.) for E.macroclada in Rowanduz district, SHIR-I MAR

or SHIR MAR شير مار (“snake‘s milk”, Kurd. ) at Amadiya. Another


15

common name in the north for plants with bitter milky juice is KUJILK

sometimes KHUZHILKA, SHIR KHUZHILK) خريلكةor KHURHILKA كجيلك

etc., Kurd). Ibn Al-Bitar indeed mentions the name FORBIYUN فربيون

(Euphorbium) and quotes an extract from Ibn Radwan who speaks of

LABAN AS-SUDA as a gum exported from (”negress‘s milk“) لبن الصودة

Morocco which resolves tumors and has other medicinal properties. This he

says is obtained from the plant known as RAQIB ASH-SHAMS راقب الشمس

(“sun observer,” in Greek “helioscopia”), suggesting that the Sun Spurge

(E.helioscopia) which contains euphorbium has long been used medicinally

(Radcliffe-Smith, 1980). Additionally the common names of E.helioscopia

are: SUN SPURGE, EUPHORBE REVEILLE-MATIN فربيون الشمس

(Chemaly and Chemaly, 2007). Also it is known as KHANNAIQ-AD-

DIJDJ ,Al-Rawi and Chakravarty) عص الكلبة US-AL-KALBA ,خنيق الدجاج

1964).

While E.peplus known as: PETTY SPERG, EUPHORBE DES VIGNES

.(Chemaly and Chemaly, 2007) فرفخ

E.hirta known as the AUSTRALIAN ASTHEMA HERB or

QUEENSLAND ASTHEMA WEED, CAT‘S HAIR, HAIRY SPURGE

(Ekpo and Pretorius, 2007).

There are many local names for particular species of Euphorbia.

"POINSETTIA" is the much-used common name for Euphorbia

pulcherrima cultivars BINT AL-QINSIL بنت القنصل , harking back to when

the genus Poinsettia was used for this and related species. "CROWN OF

THORNE" is the English common name for Euphrobia milii cultivars.


16

"MILKBUSH" or "PENCIL CACTUS"are both used for the much-cultivated

Euphorbia tirucalli.

Some other names used for different species of Euphorbia include

"SNOW-ON-THE-MOUNTAIN", "MEDUSA‘S HEAD", "MEXICAN

FIRE PLANT", and "SCARLET PLUME" (www.Euphorbiaceae.Org ).

2.8 previous work on euphorbia

The last complete monograph of Euphorbia was the treatment by

Boissier (1862) in de Candolle's Prodromus, in which 740 species were

recognized (www.Euphorbiaceae.Org).

Handel Mazzetti (1910) had described 4 species of Euphorbia with their

geographical distribution in Iraq and Syria. Guest (1933) described 4 species

with mentioning to their cultivating time and their colloquial names in Iraq

(our studied species E.helioscopia was one of them). In the same year (1933)

Post reported 44 species of Euphorbia for flora of Syria, Palestine, and Sinai

(our studied species E.helioscopia, E.granulata and E. peplus were among

them). Zohary in (1950) mentioned (25) species (E.helioscopia was among

them). Rechinger in (1959) stated about 36 species in a description for flora

of Syria and Lebanon. But in (1964) Rechinger described 25 species and

their geographical distribution in Iraq. Rechinger and Schiman-Czeika (1964) described the family of Euphorbiaceae in flora of Iran and reported

98 species, 29 species of them are distributed in Iraq. Additionally Al- Rawi

(1964) reported about 39 species distributed in Iraq (E.helioscopia,

E.granulata and E.peplus were among them). Migahid (1978) reported 14

species in flora of Saudi Aarabia (E.helioscopia, and E.granulata were


17

among them). Radcliff-Smith (1980) gave a detailed description for the

family Euphorbiaceae of Iraq and recorded 44 species. Ridda and Daood

(1982) published the geographical distribution of 32 species grown in Iraq.

Daoud (1985) reported 3 species of Euphorbia In flora of Kuwait (E.

granulata were one of them). Chemaly and Chemaly (2007) in Illustrated

flora of Lebanon reported 38 species of Euphorbia with their descriptions

and the local names.

Recently the most previous works in Iraq had involved investigation of

medicinal properties of some Euphorbia species, for instance: Antibacterial

activity of E.granulata extracts against some pathogenic bacteria (Al-

Zubaidy et al., 2005); The effect of phenols, alkaloids and terpenoids of

Euphorbia granulata Forssk. on the reproductive performance of Albino

male mice (Al-Kemisie, 2006); as well as Yousif (2008) studied the effect of

extracted phenols, alkaloids and terpenoids of Euphorbia helioscopia L. on

the biological performance of mosquito Culex molestus Forsskal (Diptera:

Culicidae); (Al-Jaryian, 2008) studied the effect of extracted alkaloids and

terpenoids of Euphorbia peplus L. on the biological performance of house

fly Musca domestica L. (Diptera: Muscaidae). Al-Haidari (2010) studied the evaluation of some locally grown plant extracts in control of algal growth.

Also, Al-Dubaisy (2008) studied the Morphology of pollen grain of

Euphorbia grown in Baghdad University Campus/ Jadiriyah.

As far as we know there is no complete systematic study for the genus

Euphorbia available in Iraq. There is lack in information deal with

morphology, anatomy, molecular study, and the response for tissue culture

technique of this genus.


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2.9 chemical contents and medicinal uses

Euphorbia has a reputation for being incredibly toxic plants. Some of the

Euphorbia are used in folk medicine to cure skin diseases, gonorrhea,

migraines, intestinal parasites, and warts. The genus Euphorbia has been the

source of large number of biological active compounds. Tannins, flavonoids,

unsaturated sterols/triterpenes, carbohydrates, lactones and proteins/amino

acids were reported as major active constituents of some Euphorbia species.

A variety of diterpenoids with antibacterial, anticancer, prostaglandin E2-

inhibitory, antifeedant, anti-HIV, and analgesic activity have also been

isolated from different Euphorbia species. They include jatrophane, ingenol,

and myrsinane diterpenoids . These diterpenoids have been reported to act in

diverse ways; they have been found to be skin- irritants, tumor-promoters. In

addition to anti-tumor activity, several species of this genus have been

investigated for their immunomodulatory activity and some immunotoxic,

immunosuppressive and immunostimulatory effects have been reported.

These broad range and diversity of biological activities in the Euphorbia

genus, perhaps due to the presence of various components with different

modes of action in the plants (Sayed, 1980 ; Amirghofran et al., 2008;

Gyuris et al., 2009; Noori et al., 2009; Kumar et al., 2010).

On the other hand Ressler (1985) reported that the latex of

Euphorbiaceae cause keratoconjunctivitis. Phorbol esters are considered to

be responsible for the toxicity of the latex e.g. in the case of E.helioscopia.

Over seventy jatrophane, modified jatrophane, segetane, pepluane, and

paraliane diterpenoids, fifty of them reported for the first time, were

extracted, purified and characterized from E.dendroides, E.characias,


19

E.peplus, E.helioscopia, and E.papalias. These compounds showed

interesting pharmacological activities (Corea et al., 2008).

Chemical investigation of E.peplus revealed almost identical profiles of

secondary metabolites affording ingenanes, jatrophanes, and tetracyclic

diterpene with new carbon skeleton for the name pepluane is proposed.

(Jakupovic et al., 1998). In a study for Mucsi et al. (2001) cytotoxicities and

anti-herpes simplex activities of nine diterpenes isolated from Euphorbia

species were determined. From a pro-inflammatory active extract of

E.peplus two new diterpine polyester based on the pepluane and

jatrophanes skeletons were isolated. Ingenol 3-angelate, which was obtained

for the first time from this plant, is an irritant toxin with high activity

(Hohmann et al., 2000). Non- polar component of the latex of E.peplus was

found to contain nobtusifoliol, cycloartenol, 24-methylenecycloartaenol,

lanosterol, and 24-methylenelanosterol in the free and esterified triterpene

alcohol fractions (Giner et al., 2000). As well as Williams (2005) stated that

inginol 3-angelate (PEP005) is diterpene ester isolated from E.peplus used as

a home remedy for skin cancer. PEP005 is a novel anticancer agent was

previously shown to modulate protein kinase C (PKC), resulting in

antiproliferative and proapoptotic effects in several human cancer cell lines

(Serova et al., 2008). Additionally, Weedon and Chick (1976) reported that

the sap of the E.peplus used as a home treatment for warts and basal cell

carcinomas and documented its successful use on a biopsy-proven basal cell

carcinoma. Also, Al-Haidari (2010) stated that the growth of algae

Anabaena cylindrica, Nostoc commune, and Microcystis aeruginosa were

stimulated by phenolic compounds extracted only from flower and leaf of

local E.peplus, while alkaloid extracts had no effect against studied algae.


20

Sharawy and AL-Shammari (2009) stated the toxic principles of

E.granulata include terpenes, triterpenols, glycosides, and phenols present in

the milky juice, all parts of these plants or the milky latex can cause an

inflammation or irritation when in contact with the skin. If eaten by animals,

the plants may cause vomiting and severe purgation, the same as for

E.peplus. On the other hand the feeding of lactating goats on usual green

fodder, contaminated with E.helioscopia or E.nubica, results in poisoning of

the dams as well as their kids. General sings of toxicity were emaciation,

depression, shedding body hair, arching of back, and possible death. The

goat also suffered from deterioration of renal function. Necrosis of epithelial

cells of kidney tubules were noticed. Considerable degenerative changes

were also observed in heart and lung. The pathophysiological appearances

indicate that by feeding on the E.peplus reported previously. Such

intoxication most likely is done to irritant and hyperphysiogenic diterpene

ester (DTE) toxins. Usually present in the aerial part of Euphoeria species

and well known as tumor promoters in mouse skin. As discussed previously

in lactating goat feed on fodder contaminated with E.peplus, tumor

promoters of the DTE type may enter the human food chain via this source

of milk (Nawito et al., 2000).

Leaf extract of E. granulata is effective against the pathogenic bacteria

(Klebsiella pnemoniae, Salmonella typhi, and Staphylococcus epidermidis);

there is little reference to the use of plant in medicine but its latex is locally

applied against poisonous bites (Salamah et al., 1989).

Pohl et al., (1975) had isolated flavonol glycoside from

E.helioscopia. Irritant diterpine ester toxins were isolated from E.helioscopia

and E.nubica which contaminate the green fodder of livestock in Egypt. Six


21

short-to medium-chain polyfunctional diterpene esters of the ingenane type,

generally containing unsaturated acids were obtained. These compounds

considered to be more or less active tumor promoters, i.e., conditional (non-

genotoxic) cancerogens (Zayed et al., 2001).

Methanol and chloroform extracts of E.helioscopia and other Saudi

Arabian Euphorbiales have molluscicidal activity against the snail

Biomphalaria pfeifferi (Al-Zanbagi et al., 2000). While Salamah et al.,

(1989) further stated that the leaf extract of E.helioscopia is specifically

lethal to the bacteria Salmonella typhi.

E. hirta is used in the treatment of gastrointestinal disorders, bronchial

and respiratory diseases and in conjunctivitis. Hypotensive and tonic

properties are also reported in E.hirta. The aqueous extract exhibits

anxiolytic, analgesic, antipyretic, and anti-inflammatory activities. The stem

sap is used in the treatment of eyelid styles and a leaf poultice is used on

swelling and boils. Extracts of E.hirta have been found to show anticancer

activity. The aqueous extract of the herb strongly reduced the release of

prostaglandins. The aqueous extract also inhibits aflatoxin contamination in

rice, wheat, maize, and mustard crops. Methanolic extract of leaves have

antifungal and antibacterial activities (Rao et al., 2010; Kumar et al., 2010).

Decoction of dry herbs is used for skin diseases. Decoction of fresh herbs is

used as gargle for the treatment of thrush. Root decoction also beneficial for

nursing mothers deficient in milk. Roots are also used for snake bites. The

polyphenolic extract of E. hirta has antiamoebic and antispasmodic activity.

Quercitrin, a flavanoid glycoside, isolated from the herb showed an

antidiarrheal activity. It is reported to have of whole plant shows


22

hypoglycemic activity in rats. It has a sedative effect on the genito-urinary

tract (Lanhers et al.,1993; Kumar et al.,2010). The study of Adedapo et al.

(2005) has clearly explained that the aqueous crude extracts of E.hirta after

its administration into dogs produced a significant increase in PCV (Packed

Cell Volume), RBC(total erythrocytes count), Hb(Hemoglobin

concentration, Total WBC (Leucocyte count) and lymphocyte counts. The

fecal egg counts also showed a remarkable and significant reduction in the

levels of the identified helminths. Thus E. hirta could serve as antihelmintic

agent. Liu et al. (2007) explained that bioassay-guided fractionation of

methanolic extracts of E.hirta aerial parts led to the isolation of flavanol

glycosides afzelin, myricitrin, the two compounds showed proliferation

inhibition of Plasmodium falciparum, on the other hand they exhibited little

cytotoxic property against human epidermoid carcinoma KB cells. Johnson

et al. (1999) proved the diuretic effect of the E.hirta leaf extract in rats using

diuretic drugs. The positive results validate the traditional use of the plant as

diuretic agent. Ogueke et al.(2007) stated that the ethanolic extract of E.hirta

is hematologically not toxic to rats, as well as the antimicrobial activities

were believed to be due to the tannins, alkaloids, and flavonoids which were

identified in the extract. The results are significant in the health care delivery

system and justifies the use of the plant in the treatment of sores-boils,

wounds and control of dysentery and diarrhea.

Chapter Two Morphological studies

23

Chapter two

Morphological studies

1- Introduction

Morphological characters are features of external form or

appearance. They currently provide most of the characters used for

practical plant identification and many for hypothesizing phylogenetic

relationships. These features have been used for a longer time than

anatomic or molecular evidence and have constituted the primary

source of taxonomic evidence since the beginnings of plant

systematic. Morphological characters are easily observed and find

practical use in keys and descriptions. Phylogenetically informative

characters may be found in all parts of the plant, both vegetative and

reproductive (Judd et al., 1999; Simpson, 2006).

Euphorbiaceae is one of the largest families of flowering plants.

It is widespread throughout the globe, strongly represented in tropics.

There are seven genera in Iraq (4 native, 2 naturalized and now

subspontaneous, and 1 quite recently introduced). Euphorbia is the

largest genus cosmopolitan with some 2000 species (Radcliff-Smith,

1980).

There are 44 species in Iraq most of them native, but including

a few naturalized species and 2-3 were introduced as an ornamental

species (Radcliff-Smith, 1980). High morphological plasticity and

diversity of this genus in Iraq, as well as few studies on the genus


24

make taxonomical studies on Euphorbia attractive for many botanists.

Taxonomic and phylogenic significance of morphological features of

Euphorbia are emphasized by many (Rechinger, 1959; Rechinger,

1964; Rechinger and Czeika, 1964; Bentham and Hooker, 1965;

Mangaly et al., 1979; Radcliff-Smith, 1980; Pahlevani, 2007;

Aworinde et al., 2009).

In the present study an attempt has been made to collect as

much information as possible about four species of Euphorbia. With

the aim of providing useful taxonomic data that would give further

insight into proper classification and identification.

2- Materials and Methods

Plant materials of E.helioscopia, E.peplus, E.granulata and

E.hirta, used for this investigation were obtained from Baghdad

University Herbarium (BUH), and newly collected specimens from

different parts of University of Baghdad were studied and identified

using corresponding scientific papers and the flora of Iraq (Radcliff-

Smith, 1980). Field collection were conducted between the flowering

and fructifying throughout the years (2008-2010). Photographs were

taken from fields by using digital camera (model Sony Cyber-Shot T

700) as well as fitted on dissecting microscope. These photographs

were useful for identification and differentiation of morphological

features.


25

2.1 Observation of Characters

The macromorphological characters were assessed on stems,

mature leaves, trichomes, cyathia and seeds at comparative position.

Morphological characters of stems include stem length, width,

shape, color and stem types (the mode of branching).

Morphological characters were assessed on mature leaves

include leaf apex, margin, shape, leaf surface, and leaf base, as well

as leaf length, width at the widest point, petiole length, petiole width

and blade. As well as the presence of the trichomes.

The species have been studied for cyathial characteristics

including number of type of cyathia, involucres shapes and

diameters, lobe shapes, numbers, colors and shapes of glands as

well as the apexes of glands.

Mature seeds of Euphorbia were collected from plants growing

in the University of Baghdad campus and studied to understand their

general morphology, which included seed shape, diameter, color and

surface configuration in addition to the presence of curuncle.


26

3- Results and Discussion

3.1 Habit and Duration

Euphorbia species are herbs in Iraq, unarmed or spinous, with

a milky juice. Our native species are all herbaceous, small herbs on

the plaines and in the mountains shrub-like herbs with woody

rootstocks. Many species of Euphorbia have a xerophytic habit and

grow in very dry places (and are usually cactus like in some cultivated

species). However they are readily distinguishable from cactus, even

when not in flower by the presence of latex, figure (4).

The species E.helioscopia, E.peplus and E.hirta are annual

herbs, where as E.granulata is perennial herb, with erect or

ascending or prostrate, simple or branched stem, the whole life cycle

from seed germination to seed production requires (2-6) months,

plate (1).

E.helioscopia, E.peplus and E.hirta grow in disturbed habitats,

while E.granulata is thermophilous plant with desert habitat.


27

FIGURE (4) Latex of Euphorbia.

Euphorbia helioscopia

Euphorbia peplus

PLATE (1-a): Species of Euphorbia in nature.


28

Euphorbia granulata

Euphorbia hirta


29

PLATE (1-b): Species of Euphorbia in nature.

3.2 Vegetative parts Morphology

3.2.1 Stems

The stems of Euphorbia species have taxonomic importance. It

was found in this study that the mode of branching, color, length,

thickness, presence of stipules and trichomes are characters of

taxonomic values as shown in table (2-1).

Stems are erect, ascending or prostrate, simple or ramified,

single or with ascending branches near base. Stems of E.helioscopia

have scars at the base where the leaves have fallen, also

E.helioscopia and E.peplus are glabrous (sometimes E.helioscopia

may has unicellular hairs in upper part of stem) characterized by

absence of stipules. Stems of E.granulata are un-branched,

occasionally branched at the end, usually woody at base, many from

base, ascending or prostrate, internodes conspicuous (has distinct

swollen nodes), pilose on one side of the stem and characterized by

presence of stipules. E.hirta stems branched from the middle or

above (usually few branched), ascending to erect or prostrate, with

mixture of long yellow – brown multicellular hairs and much shorter

white hairs, in addition to presence of distinct hairy stipules as shown

in plate (2).

Our field observations in University of Baghdad campus-

Jadiriyah revealed occurrence of two ecotypes for E.helioscopia and


30

E.hirta, one erect and the other prostrate in relation to the moisture

level of the soil and the degree of biotic factors.


31

E.hirta is erect and grows in more shady areas, has more

active growth of one or two branches is more useful, since the plant

has to struggle for the maximum availability of sun light, and therefore

all or most of the extra-axillary branches disappear.

Mangaly et al. (1979) reported that the erect ecotype of E.hirta

predominates during the rainy season, whereas the prostrate one

predominates during winter months and exhibits higher reproductive

potentialities under extreme biotic disturbances.


32


33

3.2.2 Leaves

Leaves are simple, alternate and opposite, stipules absent or

membranous, petiole absent or present ( short or long ), obovate to

spatulate or subelliptic or lanceolate-oblong, apex rounded, obtuse or

acute, margin entire or dentate, base cuneate or oblique or obliquely

rounded, color pale to yellowish green or green to red, glabrous or

hairy as shown in table ( 2-2 ). The leaf size shows considerable

variation within the studied species. The largest size recorded in

E.helioscopia and the smallest in E.granulata. Also, we observed in

E.hirta leaves that hairs of abaxial side is more abundant and longer

than the hairs of adaxial side.

Inflorescence of E.peplus is a terminal pseudoumbel, rays

irregulary branched, total branches few; primary involucral leaves 3 or

4, short; cyathophylls 2, similar to normal leaves. While E.helioscopia

Inflorescence a compound pseudumbel, usually rather compound;

primary invollucral leaves 5, yellowish green, obovate-oblong 3-4 ×

0.8-1.4 cm, progressively shorter; cyathophylls 2, obovate, base

rounded, margin dentate, apex rounded.


34


35

3.2.3 Trichomes

E.peplus is glabrous; while E.helioscopia is glabrous or

sparsely hairy in the upper part (unicellular hairs). E.granulata has

multicellular hairs on one side of the stem as seen in plate (2);

however E.hirta is with mixture long yellow-brown multicellular hairs

and much shorter white hairs as shown in plate (2) and figure (5).

.

FIGURE (5) Multicellular gland-like trichomes in E.hirta

(100X).


36

E.helioscopia E.peplus

E.hirta E.granulata

PLATE (2): Stems and trichomes in species of Euphorbia.

a- Trichomes in E.helioscopia, one side trichomes in E.granulata, mixture of long yellow and shorter white hairs in E.hirta.

b- Hairy stipules.

a

b

a

b

a


37

3.3 reproductive parts Morphology

3.3.1 Cyathia

The cyathial characteristics have shown well differences.

Cyathia of the four species were subsessile, axillary or dense. The

shape of involucres were cuplike, campanulate or turbinate. Lobes

were rounded, subtruncate or triangular- ovate, glabrous or pilose. All

the species studied had four glands, with appendages - such as

E.granulata and E.hirta - or without appendages -such as

E.helioscopia and E.peplus which varied from the rest of the species

by having two horned glands, as well as the color of glands differ

from green, pale brown to red, also the shape of glands varies

between crescent shape, disk like shape, irregular and rounded to

transversely elliptic as shown in table (2-3) and plate (3). Also E.hirta

differs in having multicellular uniseriate rugose hairs on all parts of

the cyathium except style and stigma, in the meantime E.granulata

has hairs only on ledges of cyathium. We believe that this is an

important characteristic for these species.


38


39

-A- -B- -C-

E.helioscopia

E.peplus


40

E.granulata

E.hirta

PLATE (3): The Cyathia of Euphorbia Species.

A: Cyathium B: female flower C: C.S. of immature fruit (Bars ═ 1.0 mm)

3.3.2 The Male and Female Flowers

The study of male and female flowers aid to the differentiation of


41

the four species of Euphorbia as shown in table (2-4), Figure (6). The

pedicel of the stamen is longer than the filament, despite the different

dimensions and forms of the anthers and the variation in numbers of

male flowers, splitting of anthers is horizontal in all studied species. In

floras there are no real numbers for male flowers of studied species.

According to flora of China, numbers of male flowers are many in

E.helioscopia, E.peplus and E.granulata, whilst numbers for male

flowers in E.hirta are 4 or 5 (Bingtao et al., 2008); this result differs

from our finding recorded in table (2-4).

FIGURE (6): Cyathia of Euphorbia. A. Cyathium in E.peplus , B. Mature opened cyathium showing sex organs,

C. Cyathium in E.hirta a- male flower b- 2-horned gland

(Bars ═ 1.0 mm)

A B

a b

C

a


42

3.3.3 Seeds.

The taxonomic value of the seeds of Euphorbiaceae had long

been recognized (Mangaly et al., 1979; Webster, 1994; Tokuoka and


43

Tobe, 1995). The morphological characters of seeds for the four

species of Euphorbia shown in table (2-5). Seeds shapes were

ovoid-angulate, ovoid, tetragonal or subglobose-tetragonal. The

seeds size shows considerable variation within the species studied.

The largest diameter recorded in E.helioscopia (1.3-0.8) mm and the

smallest in E.hirta (0.8-0.5) mm. As well as seeds had different colors

and surface configurations such as gray - micropores, brown -

reticulated, brown - adaxially grooved and brown to reddish smooth

seeds. Besides the seed often shows a fleshy outgrowth from the

integuments-known as caruncle-that often functions as an

inducement for animal (usually ants) dispersal. The presence and

type of caruncle had taxonomically importance, which separated the

species studied for into groups, one related to E.helioscopia and

E.peplus that had white sessile caruncle, and the other belongs to

E.granulata and E.hirta which were ecarunculate (without caruncle),

plate (4). Webster, (1994) as well as Tokuoka and Tobe (1995)

explained that seeds are useful for comparison between and within

subfamilies.


44


45

E.helioscopia E.peplus E.granulata E.hirta

PLATE (4) Seed of species of Euphorbia.

A - Seed in ventral view B - Seed in dorsal view

(Bars ═ 1.0 mm)

These morphological studies have revealed important stable

taxonomic characteristics that can be used as a key in Euphorbia

species distinction, such as the presence of hairy stipules,

Multicellular gland-like trichomes in both species E.granulata and

E.hirta; Also the presence of swollen nodules and the distribution of

hairs on one side of stem in E.granulata. In addition to the variation

of cyathia, male and female flowers in Euphorbia species that were

studied for the first time in addition to the characteristics of seeds.

-A-

-B-

Chapter three Anatomical studies

44

chapter three

anatomical studies

1- introduction

The role of anatomical data in traditional taxonomy has been long

recognized since the variations within the species, genera or family are

usually reflection of anatomical features as well. The anatomical features of

roots, stems, leaf epidermis, stomata, trichomes and other characters are

useful anatomical tools (Judd et al., 1999; Simpson, 2006; Ahmad et al.,

2010). Anatomical features are of a particular value to scientists who need to

identify small scraps of plant material. There has been a remarkable

evolution in the past 50 years or so in the investigation of vascular plant

anatomy and its uses in classification (Ahmad et al., 2010).

The anatomical structure of Euphorbiaceae exhibits a wide range of

variations in correlation with the diversity of habit, and no important

character occurs throughout the numerous tribes into which the family is

divided (Metcalfe and Chalk, 1950).

The variable literatures of the anatomy of the Euphorbia species for

vegetative organs far as we know is quite rich (Metcalfe and Chalk, 1950;

Kakkar and Paliwal, 1972; Gales and Toma, 2006; Gales et al., 2008b; Jafari

and Nasseh, 2009; Ahmad et al., 2010). But the local literatures of the field

is poor and includes no study exclusively on the structure of the Euphorbia

species.


45

So comparative internal structure study on Euphorbia was carried

out for the first time. In the present study, vegetative organs (stem and leaf)

anatomy in cross section were investigated; so as epidermis, stomata,

trichomes were studied.

2- materials and methods

Fresh samples of E.helioscopia, E.peplus, E.granulate and E.hirta

used for this investigation were collected throughout the years (2008-2010),

from different parts of University of Baghdad campus fields. The sample

materials were subjected to analysis (stem and leaf) have been cross-

sectioned by hand, so slides of stems and slides of both abaxial and adaxial

sides of leaves were prepared and observed under light microscope.

Microphotographs were taken by using digital camera (model Sony Cyber-

Shot T 700) fitted on light microscope. These micrographs were useful for

identification and differentiation of cell of vegetative organs on the base of

microscopic features.


46

3- results and discussion

3.1 the stem

Cross section of the stem variable in shape in Euphorbia species, and

generally has a circular shape as seen in figure (7) and plate (5).

a- Epidermis

Epidermis of Euphorbia stem is uniseriate, but it is biseriate in E.peplus

and with red color because of Anthocyanin presence. Epidermis thickness in

E.helioscopia and in E.peplus were about 20-25 µm, while in E.granulata

15-20 µm and in E.hirta were about 45-50 µm. The epidermal cells seem to

be isodiametric. The cuticle layer is distinct over the epidermis reached

about 2.5 µm.

b- Cortex

Cortex of Euphorbia stems is distinctly formed in the species studied,

and with 8-10 rows of cells in E.helioscopia and E.peplus, but with about 5-

6 rows in E.granulata and E.hirta. These cells are rich in chloroplasts,

therefore it is chlorenchyma. Chlorenchyma of E.granulata and E.hirta

constitute the whole cortex, but with three outer rows in E.peplus , in time

that the cortex of E.helioscopia lack chloroplast. The chloroplast in E.hirta

present in the cortex, phloem, xylem and within 1-2 layers of the pith cells,

were as in E.granulata chloroplasts presence continue deeply within the

pith centre. Thickness of the cortex in E.helioscopia and E.peplus were


47

about 200-225 µm, while in E.granulata 90-100 µm and in E.hirta was

about 300-320 µm.

C- Central Cylinder (Stele)

Euphorbia has got wavy central cylinder in the species E.granulata

because of the bundle cap which prolongated and projected along the

phloem positions. This waving may be initiated by two ways:

1- Variations of the vascular bundles positions, so that there are internal

vascular bundles and external ones which are alternated.

2- Variations among bundle cap diameters at each two following bundle

caps so that one is projected out and the other in.

The central cylinder in the other Euphorbia species is slightly wavy,

wide and with peripheral position because of their similar size and regular

positions of the bundle caps.

Tracheary elements of wood resembled by vessels and trachieds, which

are in radial rows in the species studied. Xylem parenchyma is distinct

within the wood. Wood arms projected clearly toward the pith, and they are

about 22-25 in E.helioscopia, 20-25 in E.peplus15-18 in E.granulata and

about 24-30 in E.hirta. Wood arms (xylary arms) may be single, double or

triples. The mean thickness of wood is about 100-150µm in E.helioscopia

and E.peplus, 50-65µm in E.granulata and about 250-300 µm in E.hirta

The phloem is in external position and usually surrounded by thick

fibrous tissue which resemble the bundle caps. The sieve elements and other

cells of phloem distributed regularly between the bundle caps and xylem,


48

and there are few fibers among them as in E.granulata and E.hirta, or these

cell elements present as an islands between the bundle cap fibers, and

segregated from each other as in E.helioscopia and E.peplus. The mean of

phloem thickness in species studied was about 15-20 µm.

All Euphorbia stems have wide pith except E.granulata which has got

pith moderate in size. The pith of most species studied has a distinct gap or

central cavity at maturation stage. The pith cells are big, with thin walls and

distinct intercellular spaces. The pith may reach in diameter up to 300 µm

as in E.helioscopia and E.peplus , 150-200 µm E.granulata and reached

900-1000 µm in E.hirta. Pith cells increase in size toward the centre, and

these cells are similar in shape (usually spherical) and polyhedrals. Pith

often becomes hollow in mature stage. Also laticiferes are distinct within

the pith and variable in size.


49

FIGURE (7): Cross Section in E.helioscopia Stem (100X). a- Epidermis, b-Cortex, c-Phloem, d-Xylem, e-Pith

a

b

c

d

e


50

E.helioscopia (100X) E.peplus (100X)

E.granulata (100X) E.hirta (100X)

PLATE (5) Cross Section in Stems of Species of Euphorbia.


51

3.2 the leaf

a- Epidermis

The epidermis is uniseriate, regular, thin walled, usually similar

in diameters and covered with thin cuticle layer in all the species

studied.

b- Mesophyll

Mesophyll is differentiated into palisade layer and spongy layer

in E.granulata and E.hirta, but it is undifferentiated in E.helioscopia

and E.peplus, so in the latter case, mesophyll is composed of

parenchyma cells which are variable in size. The palisade layer in

E.granulata and E.hirta is on the adaxial side of the leaf and

composed of 2-rows of (elongated at right angles to the epidermis)

cells. Because of that, these plants are xerophytes, the palisade layer

is increased into two rows so that obtaining of the energy can be

suitable for the photosynthesis. The spongy layer is in a good growth

in general, and their cells are spherical and subspherical with big and

small intercellular spaces. The spongy layer thickness is different

around the midrib region, compared with other parts, so it has 2-6

rows of cells. Laticifers are present in the middle part of the

mesophyll and usually between palisade layer and spongy layer, as it

is clear in leaf vertical sections, figure (8) and plate (6-a,b).


52

C- Vascular bundles

Vascular bundle shape can be seen in cross section as a sub-

circular, and these bundles occupied third part of the section in the

species studied. The xylem elements initiated perfectly and composed

of many straight rows of mainly vessels. The xylem elements

surrounded internally by variable parenchyma cells. These

parenchyma cells are different in size, so that vascular bundle may be

enclosed completely by parenchyma cells. Vascular bundle phloem

elements are abundant and occupied a good part of the vascular

bundle as a semicircle shape.

FIGURE (8) Vertical Section in Leaf Blade of E.hirta (100X). a- Upper epidermis, b- Palisade parenchyma, c- Bundle- sheat, d- Xylem, e- Phloem, f- Laticifer, g- Spongy parenchyma, h- Lower epidermis

c

b a

d e

f

g

c

h


53

E.helioscopia (100X)

E.peplus (100X)

PLATE (6-a): Vertical Section in Leaves of the Species of Euphorbia.


54

E.granulata (100X)

E.hirta (100X)

PLATE (6-b): Vertical Section in Leaves of the Species of Euphorbia.


55

3.3 the foliar lamina

The epidermal cells on adaxial side may be circular, rectangular or

polygonal in outline. Cells with feebly undulated walls seen in

E.helioscopia, straight to feebly sinuous walls in E.peplus, undulated in E.

granulata and highly undulated E hirta on abaxial side as shown in plate (7-

a,b).

Raju and Rao (2008) studied the variation in the structure and

development of foliar stomata in the Euphorbiaceae and found that the

epidermal cells are polygonal, trapezoidal or variously elongated in different

direction and diffusely arranged. The epidermal anticlinal walls are straight,

arched or sinuous. The occurrence of curved walls in species studied agreed

with the suggestion of Stace (1965) that curved wall is a mesomorphic

character and that environmental conditions such as humidity play a

significant role in determining the pattern of anticlinal cell walls (Aworinde

et al., 2009).

a. Stomatal Complex

The studied species of Euphorbia possess amphistomatic leaves bearing

anomocytic and anisocytic stomatal complexes, as well as paracytic stomatal

complexes seen on adaxial side as shown in plate (7-a,b and 8).

Generally stomatal complexes occur only on the lower surface

(hypostomatic leaves). But in studied species they are located in both abaxial

and adaxial surfaces. Metcalfe and Chalk (1950) stated the presence of

amphistomatic leaves in some species of Euphorbia.


56

Freire et al., (2005) found anomocytic stomatal complexes in E.peplus.

Whereas, Kakkar and Paliwal (1972) stated that various species of

Euphorbia have been found to possess anomo-, aniso-, para-, and cyclocytic

types of stomatal complexes or they may have even a single subsidiary cell,

as well as some species may show combinations of two or three different

types of stomatal complexes on the same leaf surface.


57

-A- -B-

E.helioscopia (100X)

E.peplus (100X)

PLATE (7-a) Stomatal Complex of Species of Euphorbia. A- adaxial side, B- abaxial side


58

-A- -B-

E.granulata (100X)

E.hirta (100X)

PLATE (7-b) Stomatal Complex of Species of Euphorbia. A- adaxial side, B- abaxial side


59

E.granulata (100X)

E.hirta (100X)

E.hirta (100X) PLATE (8) Adaxial Side of E.granulata and E.hirta Leaf.

a-Stellated epidermal cells b-Convex epidermal cells

a b

a

a


60

b- Trichomes

Unicellular and multicellular, uniseriate trichomes occur in the four

species investigated. E.granulata and E.hirta differ from the others in having

multicellular uniseriate rugose hairs on all vegetative parts and on the

cyathium except style and stigma, figure (9). Further distinction can also be

made on the basis of multicellular gland-like trichomes. Tichomes that

present on leaves of E.granulata and E.hirta are surrounded by stellated

arranged epidermal cells ranged between 12-14 cells around the base of each

hair in E.hirta, and around 7-8 cells inserted at the base of each hair in

E.granulata, as shown in plate (8). The epidermal cells appear convex in

surface appearance, so each epidermal cell can be distinguished from those

of neighboring as shown in plate (8). Metcalfe and Chalk (1950) hold that

the trichome frequency and size are environmentally controlled, while Stace

(1965) believes that hairs are constant in a species when present and showed

a constant range of form and distribution useful in diagnosis.

Trichome of E.granulata (400X) Trichome of E.hirta (400X)

FIGURE (9): Thrichomes of Euphorbia.


61

c- Laticifers

Anatomical investigation pointed out that the laticifers are present in all

the vegetative organs of all species studied. Laticifers present constant

characters (non-articulated, ramified, polygonal shape in transverse section,

cellulosic wall thicker than of adjacent cells).

In the aerial stem, laticifers are present in the internal zone, in the phloem

periphery, usually between or in the thickness of the cordons of

sclerenchymatous fibers and in the primary phloem, as shown in figure (10).

In the leaf, laticifers are localized in the medium nervure (generally, in close

proximity of the phloem of median vascular bundle) as well as in the

mesophyll, figure (11).

These findings agreed with Gales and Toma (2007), as well as Jafari and

Nasseh (2009) results. Also Metcalfe and Chalk (1950) reported that

laticifers vessels occur chiefly in the phloem of both stem and leaf, at the

margin of the pith and the primary cortex. As well as they sometimes extend

into the mesophyll. Laticiferous cells, not always clearly distinguish from

laticiferous vessels as it mentioned in most literatures. In mature plants

laticifers occur in the pith and primary cortex of the axis as well as in the

veins and sometimes free in the mesophyll of the leaf.

Laticifers generally contains milky latex in living material, but becoming

brown or grey in herbarium specimens.

In present study we found that latex of species of Euphorbia is containing

rod-or bone-shaped starch grains as shown in figure (11), these results

agreed with Metcalfe and Chalk (1950), and Evert (2006) statements.


62

FIGURE (10): Laticifers in Species of Euphorbia (100X). The arrows showing Laticifer above phloem and in pith

FIGURE (11): Rod-Shaped Starch Grains in Latex of Euphorbia

(400X).

Chapter four Ecology & Geographical Distribution

63

chapter four

ecology and geographical

distribution

1. introduction

The data on geographical distribution have always been important to

determine whether taxa are sympatric or allopatric when assessing their

status or the barriers to gene - exchange which exist between them.

The identification of areas of diversity can help in understanding the

course of evolution within a genus, while, information on the habitat

occupied by the species can provide added insights, since there is evidence,

from several groups, of progressive evolution from stable, mesic

environments to disturbed and more xeric conditions (Al-Musawi, 1979).

It is obviously known that the plant morphology is influenced by

geographical and environmental agents therefore the members of Euphorbia

species show a high variation in their appearance under different

environmental conditions.


64

2. materials and methods

The information on the geographical distribution of Euphorbia species

has been assembled from the herbarium specimens that have been examined,

as well as from literature citations and personal field observations.

Every specimen examined had its geographical and habitat data recorded

on an index card. Specimens that were collected from Jadiriyah campus,

dried and labeled with information: Scientific name, Date of collection,

Locality, Altitude, Soil, Habitat, Abundance, etc..

Also the data based on the lists of Iraqi plants were published by: Handel-

Mazzetti, 1910; Nabelek, 1929; Zohary, 1950; Al-Rawi, 1964; Ridda and

Daoud, 1982; and on some Floras such as: Flora of Syria, Pal., Sin. (Post,

1933); Flora of Lowland Iraq (Rechinger, 1964); Flora Iranica (Rechinger

and Schiman-Czeika, 1964); (Radcliffe-Smith, 1980).

The Map of Physiographic regions and Districts of Iraq printed from

Flora of Iraq (Guest, 1966) with some modifications figure (12).

3. results and discussion

3.1 ecology and geographical distribution

This part of the investigation was allocated to study the geographical

distribution of the studied Euphorbia and recording some ecological notes

which are helpful to isolate the species.

E. helioscopia is common in the lower forest zone and steppe region of

Iraq, and on the alluvial plain region: (DWD), Haditha, Baghdadi highway

on the road side in cultivated fields, Khan Baghdadi, Ramadi province


65

grown in cultivation; (MJS), Jabal Sinjar, Kursi, grown in limestone, rocky

soil. grooves; (FUJ), around Mosul city; (MAM), Aqra city on the side of

water fall in rocky grooves; (FAR), Arbil province on roads, limestone

ledges and on stream side slopes; (MRO), Shaglawa and Gendian,

Rawandus on slopes in rocky soil; (MSU), North side of Darbandikhan mt.,

Deyala province in open oak forest, Sulaimaniya distributed in heavy clay

soil of hillsides; (FKI) in several places as weeds; (FPF), North of Jaloula in

sandy gravel on mountain sides. Distribution continuous to the middle of

Iraq (LEA)on road sides and fields; (LCA), Khan Baghdadi, Ramadi

province and in north of Baghdad, in orchards near river side, also we found

a big robust specimen which had been collected from Jadiriya and Karada in

a field near the river, from Baghdad University campus, near water stream,

Kut Al-Hai. To the south (LSM) grown on roads and fields; (SDS), southern

desert, Basra province grown under palmetto, Abu- Alkhassib.

The results of our field observations in Baghdad University Campus

revealed that E. helioscopia is well grown on road sides, watered fields and

it recorded the highest length near water pipes.

General distribution of E.helioscopia : Almost throughout Europe,

Cyprus, Aegean isles, Syria, Lebanon, Palestine, Jordan, Egypt, Saudi

Arabia, Turkey, Caucasus, Iran, W. Pakistan, Afghanistan, N. India, China,

C. Asia, N. Africa (Morocco, Algeria, Libya). Introduced in to N. America

and other parts of the world .

Habitat of E. helioscopia : In the mountains and upland plains, on a cliff,

on grassland, in waste places, gardens and fields, a common weed in

gardens, orchards, fields and wasteland on the irrigated alluvial plain.

Flowering in January, March, April, fructifying in April and May.


66

E.peplus is quite common on the irrigated alluvial plain : (DWD), Ana,

Ramadi province, near Euphrates river, 50 Km. North of Rutba grown on

rocks ,outcrops fields, hill side, Habaniyh and Diyala, as a weed among

vegetables; (DLJ); (FNI); (MSU), Darbandikhan (2000)Km; (FPF) in fields

as weeds; (LEA), Baquba; (LCA), Khan Bani Saad, Abu-Ghraib west of

Baghdad, Jadiriyah in University of Baghdad distributed in fields; (LSM) on

roads and wastes areas ; (SDS), Shatt Hamdan, Basra Liwa grown under

palmetto.

This species has very good abundance and well distribution in Baghdad

University Campus, it is grown everywhere on roads, fields, wastes areas,

near water pipes and it is often companied by E.helioscopia.

General distribution of E.peplus : Widespread in Europe, Aegean Isles,

Cyprus, Syria, Lebanon, Palestine, Sinia, Egypt, Arabia, Kuwait, Turkey,

Iran, W. Pakistan, Asia, N. Africa (Morocco, Algeria, Libya). Introduced

into China, Japan, N. and tropical America and most other parts of the

world.

Habitat of E. peplus : Ruderal weed in date grooves, orchards, gardens,

generally on irrigated alluvial soil and near urban centers; flowering in

January - March, fructifying in February - May.

E. granulata has occasional distribution in the northern sectors of the

desert region of Iraq : (DWD)Western Desert, 55Km West of Ramadi,

depression in sandy gravel desert, South east of Rutba, in Haswa desert;

(DLJ), about 10 Km South of Beiji , on the top of sandy hill; (MJS), North

of Jabal Sinjar grown on road sides; (MSU), North East of Dawana, grown

in very saline plain; (FKI) in many places; (FPF) in fields and open areas;


67

(LEA) in some fields as a weeds; (LCA), about 20 Km West Falluja on

sandy planes and in Baghdad grown in several places.

E.granulata has well distribution in University of Baghdad Campus in dry

and watered fields, as well as on rock grooves, rarely seen on roads, and it

may stay all over the year if there is enough water and nutrition. Field

observations revealed that this species may companied by E.chamaesyce

which has similar appearance, hence sometimes cultivators confuse between

them.

General distribution of E.granulata : Syria, Palestine, Jordan, Sinai,

Egypt, Kuwait, Bahrain, Caucasus, Iran, W. Pakistan, Afghanistan, N. India,

C. Asia, N. Africa (Morocco, Algeria, Libya), tropical Africa.

Habitat of E.granulata: In the desert, on sandy or gravel soil,

gypsiferous slopes, roadside depressions, stony canal banks, irrigated

alluvium, fields and gardens, sometimes slightly saline depressions;

flowering and fructifying throughout the year.

E.hirta is rare in Iraq only found in one locality on the alluvial plain in the

desert region: (LCA), Jadiriyah and Karada in Baghdad as a weed in

irrigated gardens and cultivation (LEA). As well as this species has limited

distribution in Baghdad University campus.

General distribution of E.hirta : Lebanon, Palestine, Arabia, India,

China, Japan, Malaya. Originally a native of Mexico and other parts of

tropical America, now found throughout most of the tropics and subtropics

of both hemispheres.

Habitat of E. hirta : Weed in irrigated areas and cultivation; flowering in

October, fructifying in October and November.


68

M- MOUNTAIN REGION MAM Amadiya District

MRO Rowanduz District

MSU Sulaimaniya District

MJS Jabal Sinjar District

F- UPPER PLAINS AND FOOTHILLS REGION FUJ Upper Jaziera District

FNI Nieneveh District

FAR Arbil District

FKI Kirkuk District

FPF Persian Foothills District

D- LOWER PLATEU REGION DLJ Lower Jaziera District

DGA Ghurfa-Adhaim District

DWD Westren Desert District

DSD Southren Desert District

L-LOWER MESOPOTAMIAN REGION LEA Eastern Alluvial Plain District

LCA Central Alluvial Plain District

LSM Southern Marsh District

LBA Basra Estuarine District

Physiographic Regions and Districts of Iraq (Guest, 1966).


69

: E.helioscopia, : E.peplus, : E. granulata, : E.hirta

FIGURE (12) Physiographic regions and Districts Map of Iraq.

(From Guest, 1966, with some modifications)

w

N

FUJ

GORDAN

0


70

It appears from above that the species E.helioscopia, E.peplus and

E.hirta are annual herbs, where as E.granulata is perennial herb and they

mostly distributed in desert and alluvial plain region of Iraq.

Rabia, et al., 2008 mentioned that E.granulata is perennial or annual herb,

while Rechinger, 1964; Radcliffe-Smith, 1980; Jafari and Nasseh, 2009

recorded it as an annual.

The most annual species were forming dominance on area and spreading

in the large area. The possible reason could be the availability of plentiful

moisture (as that in studied area of Baghdad University campus-Jadiriyah).

Furthermore, the common species found were more competitive due to rapid

growth than the rest of species (Rabia et al., 2008).

In Iraq E.helioscopia and E.peplus are wider distributed than E.granulata

and E.hirta.

In Baghdad University campus the most distributed species were E.peplus,

E.helioscopia, and E. granulata, whereas E.hirta was seen just in few areas.

Rechinger and Schiman-Czeika (1964) had listed E.helioscopia, E.peplus,

E.granulata in flora Iranica without any mention about their distribution in

Iraq.

Euphorbia species have morphological plasticity and diversity in Iraq and

neighboring countries. The chief interest centers on those species that inhabit

very dry places and have consequently a xerophytic habit (Willis, 1973).

This study revealed that E.peplus is quite common on the irrigated alluvial

plain, in moisture and shaded places; this result disagrees with Radcliffe-

Smith (1980) statement that E.peplus distributed in the desert region of Iraq.


71

From herbarium investigation we noticed that in recent years E.granulata

has well distribution in watered areas, gardens and fields as a weed in the

middle region of Iraq in addition to the northern sectors of the desert region.

E.granulata has allelopathic effects of other weeds (such as with Cynodon

dactylon). Besides weeds are important in cultivated fields since they not

only compete with the crops, but may exhibit allelopathy. Farmers generally

leave litter to organic matter and thereby releasing minerals during its

decaying. On the other hand providing habitats for microorganisms, the litter

enhances the porosity and water-holding of soil (Hussain, 1980).

The active principles in the seeds and foliage of E.helioscopia are not

affected by drying, as well as it accumulates boron and may enrich compost

to which the weed is added (Salisbury, 1961).

In many parts E.helioscopia litters irrigation fields of grain crops, less

often vegetable crops. It occurs frequently, but with low abundance. The

plant also litters tilled crops and spring grain in all areas. This plant did not

name as a main weed. The species is also found in anthropogenic

landscapes, i.e., in garbage places, along roads, railways, and on fallow

lands (Nikitin, 1983).

E. hirta is a very common plant of the wild that can grow in almost all

types of habitat. It can grow in various types of positions in sandy soil and

even in pure sand mixed with little nutrients. It grows abundantly on rubble,

old building sites, at the margins of streams etc. where water logging does

not occur. Euphorbia hirta cannot grow properly in polluted area. So it can

also be taken as a pollution indicator (www.Ecosensorium.org.).

Pollination is principally brought about by insects or especially by ants

Metcalfe and Chalk (1950). Seeds get dispersed through simple mechanical

process. Though a number of seeds get destroyed and eaten up by birds and


72

insects, it does not cause any impact on the general population density of the

plant due to abundance of its seeds, figure (13).

FIGURE (13) Member of Aphidae Insect Live and Feed on

E.helioscopia (Distinct pollen on insect leg).

There is lack of precise geographical distribution for the species E. hirta

in flora of Iraq and in Baghdad University Herbarium (BUH) specimens.

There is just two local specimens for E. hirta in BUH (No.99126 and

No.49093) collected from University of Baghdad campus-Jadiriyah fields by

Al-Dubaisy; Actually after examination we found that specimen has

incorrect naming E.densa in time it is E.hirta not E.densa.

In recent years E.hirta may have well distribution in many regions of Iraq

because it has active growth in Baghdad as well as in neighboring countries;

and because of its abundance pollination.

Depending on our observation species has appeared in Iraqi area within

the three past decades.


73

From the results of herbarium and field observations we noticed that the

species of Euphorbia exhibit variations -ecotypes- when grown under

different conditions of light intensity and soil moisture and these results

agree with Metcalfe and Chalk (1950); Mangaly et al. (1979) findings.

The plant abundance, leaves and stems size as well as plant height varies

from year to year in relation with rainy seasons and the presence of water

sources like rivers, streams, water pipes, sewages, etc.

Euphorbia species assume striking adaptation to dry situations. Many

species are horticultural interest due to their ornamental nature. Some have

been found useful for reclamation of waste lands for their excellent

adaptability to extreme conditions of life. Their decomposed body also

enriches the soil by supplying organic matter (Chakravarty, 1976; Hussain,

1980).

Chapter five Protein analysis

74

chapter five

protein analysis

1-introduction

Many plants and cultivars can be distinguished morphologically.

Vegetative and floral morphology does not always provide a clear basis for

identification. Numerous biochemical and chemical analytical procedures

have been used in chemotaxonomic studies, often these procedures are used

in conjunction with traditional taxonomic interpretations for comparisons at,

or above the species level. Electrophoretic separation of proteins and

isozymes has been a widely used and valuable technique in the field of

biochemical systematics. Many chemotaxonomic studies have used gel

electrophoresis of proteins to study the relationships between species,

hybrids and interspecific hybridsens (Werner and Sink, 1977; Al-Jibouri and

Dham, 1989; Jensen et al., 1994; De Freitas et al., 2010). Proteins and

isozymes are under genetic control, being the consequence of nucleotide

sequence at the gene level. Thus, morphological differences between

cultivars not environmentally manifested nor caused by histogenic

rearrangement should have a biochemical basis reflected as differences in

protein structure (Werner and Sink, 1977).

Electrophoresis is a technique for measuring the rate and direction of

movement of organic molecules (in this case, proteins) in response to an

electric field. The rate and direction of protein movement in a starch or an

agar gel will depend on the protein's net surface charge, size, and shape.

Proteins can then be stained, resulting in a series of bands in the gel. Those


75

proteins that migrate to the same place in a slab of agar gel and yield similar

banding patterns when stained are considered to represent homologous

proteins. The banding patterns resulting after electrophoresis of seed or

pollen proteins (or of specific types of enzymes) can be compared, and the

presence or absence of particular bands used as taxonomic characters (Judd

et al., 1999).

In this study there was an investigation for proteins by using the

technique of electrophoresis to identify the species of Euphorbia.


76

2-materials and methods

2.1 materials

2.1.1 equipments and devices

Table (5-1) illustrates all equipments and devices used in this study.

TABLE (5-1): The apparatus used in the study.

Apparatus Names Company and Origin Autoclave Express –Japan Cold centrifuge Eppendorf-Germany Deep freeze Sanyo-Japan Distiller Controls-England Polyacrylamide Gel Electrophoresis apparatus

Consort-Belgium

pH-Meter Bio Red – Italy Power supply Consort- German Refrigerator Ishtar-Iraq Sensitive electronic Balance Sartorius-Germany Magnetic stirrer Scientific Industries-USA Water bath Memmert – Germany


77

2.1.2 chemicals and biological materials

TABLE (5-2): illustrates all Chemicals and Biological Materials used in

this study.

TABLE (5-2): Chemicals and Biological Materials.

Chemicals and Biological Materials Company and origin

Tris-Hcl BDH – England

Tris-Base BDH – England Ammonium persulfate BDH – England N,N,N,N Tetra methyl ethylene diamine (TEMED)

BDH – England

Acrylamide powder BDH – England Bis acrylamide BDH – England Glycine BDH – England Hydrochloric acid BDH – England Phosphoric acid BDH – England Methanol Alcohol BUH ــ England Perchloric acid BUH ــ England Trichloroacetic acid (TCA) BUH ــ England Acetic acid Fluka ــ Germany Sodium dedocyl sulfate (SDS) Fluka ــ Germany Bromophenol Blue powder Sigma ــ USA Coomassi brilliant blue R-250 Riedel de Haën Germany


78

2.1.3 extraction buffer

This solution prepared by dissolving 0.985 gm of Tris-HCl, 2gm of Sodium dodecyl sulfate (SDS), 10 ml Glycerol, and 10 ml α-

mercaptoethanol in distilled water. The pH was adjusted to 8.0 and the

volume was completed to 100 ml by distilled water (Dzayee, 2002).

2.1.4 gel electrophoresis

Solutions were prepared according to Garfin (1990), as described below.

2.1.4.1 Resolving gel buffer solution:

This solution prepared by dissolving 18.2 gm of Tris-Base in 80 ml

distilled water. The pH was adjusted to 8.8 by 1M HCl solution and the

volume was completed to 100 ml by distilled water.

2.1.4.2 Stacking gel buffer:

This solution prepared by dissolving 6 gm of Tris-Base in a volume of

distilled water. The pH was adjusted to 6.8 by using 1M HCl, and the

volume was completed to 100 ml by distilled water.

2.1.4.3 Sodium dodecyl sulfate solution (10% SDS):

prepared by dissolving 10 gm of SDS in a volume of distilled water,

and the volume was completed to 100 ml by distilled water, to give 10%

SDS solution.


79

2.1.4.4 Reservoir buffer solution:

This solution prepared by dissolving 3 gm of Tris-Base with 14.4 gm

glycine in 800 ml distilled water. The pH was adjusted to 8.3 by 1M HCl,

then 10 ml of 10% SDS solution was added and the volume was completed

to 1000 ml by distilled water.

2.1.4.5 Acrylamide bisacrylamide solution:

prepared by dissolving 30 gm of Acrylamide and 0.8 gm

bisacrylamide in 80 ml distilled water, and the volume was completed to 100

ml by distilled water, kept in dark bottle.

2.1.4.6 Ammonium persulfate solution:

Prepared fresh by dissolving 1 gm of ammonium persulfate in 10 ml

distilled water, to gave 10% ammonium persulfate solution.

2.1.4.7 N,N,N,N Tetra Methyl Ethylene Diamine (TEMED)

This solution prepared fresh. It composed of 40% methanol and 10%

trichloroacetic acid (TCA). It was prepared by dissolving 100 gm of TCA in

500 ml distilled water, 400 ml of methanol was added, and the volume was

completed to 1000 ml by distilled water.

2.1.4.8 Bromophenol blue:

A dye prepared by dissolving 0.5 gm of bromophenol blue in 100 ml

distilled water, mixed until completely dissolved.


80

2.1.4.9 Coomassie Brilliant Blue R-250:

This stain prepared as follows: 10 ml 70% perchloric acid (PCA)

diluted to 200 ml by distilled water. 0.8 gm of dye was dissolved in it; the

solution was mixed for 1 hour then filtered and kept in dark bottle.

2.1.4.10 Fixing Solution:

This solution prepared by mixing 10 ml of trichloroacetic acid (TCA)

with 40 ml of methanol, and volume was completed to 100 ml by distilled

water.

2.1.4.11 De-staining solution:

This solution composed of 40% methanol and 10% acetic acid. It was

prepared by mixing acetic acid, methanol and distilled water in a ratio of

1:4:5, respectively.

2.1.4.12 Preparation of the Resolving Gel:

The (10%) resolving (separating) gel prepared by mixing 5 ml of 30%

acrylamide bisacrylamide, 6.3 ml distilled water, 3.75 ml resolving gel

buffer, 0.1 ml SDS, 150 µl of 10% Ammonium persulfste and 15 µl of

TEMED were added to solution and mixed gently. Using a Pasteur pipette,

the resolving gel was transferred to polyacrylamide gel electrophoresis slab.

Using another pipette, the top of the gel was covered with isobutyl alcohol.

The gel was then allowed to polymerize for 1 hour at room temperature

(25ᵒ C).


81

2.1.4.13 Preparation of the stacking gel:

Stacking gel was prepared by mixing 0.65 ml of 30% Acrylamide

solution, 1.6 ml of stacking gel buffer, 4.2 ml of distilled water, and 0.1 ml

of 10% SDS solution. 67µl Ammonium persulfate solution and 6.7 µl of

TEMED were added then mixed gently. The stacking gel was transferred

slowly over the resolving; the top of the gel was covered with isobutyl

alcohol. The gel was then allowed to polymerize for 1 hour at room

temperature (25ᵒ C).

2.1.4.14 Standard Protein Solution (Markers)

The protein markers Tris-Glycine 2-4 % markers (10-180 KDa) were

prepared according to the manufacturer instructions.

2.2 methods

2.2.1 protein extraction buffer from dry seeds of

the species of euphorbia

Seed samples 0.2 gm were homogenized with 3 ml of extraction

buffer in a chilled pestle and mortar at 4ᵒC. the homogenate was

centrifuged in a refrigerated centrifuge at 14,000×g for 10 minute. The

supernatants were stored in small aliquots at -85ᵒC for SDS-PAGE.

Supernatant samples were mixed with equal volumes of solublizing buffer

62.5 mM Tris-HCl, pH 6.8,20% (W/V) glycerol, 2%(W/V)SDS, 5% (V/V)

2-mercaptoethanol and 0.01%bromophenol blue and heated for 4 minute at

95ᵒC, cooled on ice before loading on 10% polyacrylamide slab gels.


82

2.2.2 preparation of samples of protein

The samples were prepared by adding 15µl of extracted proteins to 5

µl of loading dye, then was transferred to water bath 65ᵒ C for 10 minute

before loading to the slab gel.

2.2.3 polyacrylamide gel electrophoresis of

species of euphorbia

In order to determine the molecular weight of the proteins obtained

from Euphorbia species, protein polyacrylamide gel electrophoresis was

performed (Garfin, 1990) for total protein as follows:

The slab gel was submerged in the reservoir buffer and 20µl of

sample protein solution of each species was loaded on the gel surface

respectively. The power supply was connected and run. Current was 25 Am.,

Voltage was 125 Volt, Power was 300 W and the total time was 120

minutes. Then the polyacrylamide gels were removed from the slab gel and

placed separately in plate. Gels were immersed in fixing solution for 30

minutes, fixing solution was then poured off and the gels were immersed in

the staining solution (Coomassie Brilliant Blue R-250) for 3 hours. Then

staining solution poured off and the gel was immersed in the de-staining

solution to remove the unbound stain. The de-staining process continued

until blue band of protein were obtained. The gel was stored in 7% acetic

acid solution.


83

To determine the molecular weight of the appearing protein bands, the

distance of the bromophenol blue dye transfer from the top of the gel to the

center of the dye band was measured. Also the distance from the top of the

gel to the center of the separated standard protein bands were measured.

The relative mobility (Rm) was calculated for each protein according to the

following equation:

Relative mobility (Rm) = Distance of Protein / Distance of Bromophenol

Blue Dye

The relation between Rm and log molecular weight of the standard

proteins was plotted.

The molecular weight of the appearing protein bands were determined

by projecting the Rm values of the corresponding bands on the standard

curve of the standard proteins.


84

3- results and discussion

In this investigation we tried to make a comparative study for protein

profile. The banding pattern of proteins were different for the four studied

species of Euphorbia, such differences included the number of bands,

relative mobility value (Rm), molecular weight and intensity on the gel as

shown in figure (14).

FIGURE (14) Polyacrylamide Gel (SDS/PAGE) patterns of extractable seed proteins of the four studied species of Euphorbia. Bands were fractionated by electrophoresis on Polyacrylamide Gel (SDS/PAGE) (Current 25 Amp.,125 V, 300 W, 120 min.) and visualized by Coomasi Brilliant Blue R-250 staining

From left M: Markers, 1: E.granulata 2: E.hirta 3: E.helioscopia 4: E.peplus

The total number of protein bands ranged between (9-10) bands, with

molecular weight ranging from (10.00 to 93.33) KDa. These differences are

obvious in table (5-3).

M

1 2 3 4 KDa 180

130

75

48

35

28

10


85

TABLE (5-3): The total number of protein bands with molecular

weight of proteins of the four species of Euphorbia.

Species E.granulata E.hirta E.helioscopia E.peplus

The Bands

number with

Molecular

Weight in Kilo

Daltons

2 - - 10 10 3 10 12.58 10.71 10.71 4 11.75 16.98 19.95 12.58 5 14.13 24.54 23.98 19.95 6 27.54 35.48 31.62 21.87 7 36.30 54.95 36.30 27.54 8 54.95 69.18 41.68 41.68 9 63.11 91.20 54.95 70.79 10 - - 83.17 93.32

Total No. of Bands 9 9 10 10 *Colored cells indicate for bands with similar molecular weights

The total number of protein bands of E.granulata were (7) bands

with molecular weight ranged between (10.00-63.11)KDa, and (2) bands

with molecular weight less than the minimum molecular weight of marker

protein. As well as E.hirta has (8) bands as a total number with molecular

weight ranged between (10.00-91.20)KDa, and (1) band with molecular

weight less than the minimum molecular weight of marker protein. The total

number of protein bands for E.helioscopia were (9) bands with molecular

weight ranged between (10.00-83.18)KDa. Also E.peplus has (9) bands with

molecular weight ranged between (10.00-93.33)KDa. Both species have (1)

band with molecular weight less than the minimum molecular weight of

marker protein.

The banding pattern revealed the presence of bands with similar molecular

weight among the species studied. The molecular weight of the second

bands of E.helioscopia, E.peplus and E.granulata are equal 10KDa; the


86

third band of E.hirta and the forth band of E.peplus have the same

molecular weight 12.58 KDa; the sixth band of E.granulata and the seventh

band of E.peplus have the same molecular weight 27.54 KDa; Also the

seventh bands of E.granulata and E.helioscopia, in addition to the eighth

bands of both E.helioscopia and E.peplus have similar molecular weight

reached to 36.30 KDa and 41.68 KDa, respectively. Finally the eighth band

of E.granulata and the ninth band of E.helioscopia have the same molecular

weight 54.95 KDa.

The banding patterns of the same protein (a protein with the same

function) may be different in different species; because over evolutionary

time, the DNA of the two species accumulates differences due to mutation.

The proteins in the two types of organisms (which are encoded by the DNA)

may have slightly different sequences, which accounts for different banding

patterns on the gels, but still retain the same or similar functions.

(Barraclough et al., 2004).

Each band represents specific protein of known molecular weight.

Since the protein is gene product (Scandalios, 1969; Al-Jibouri, 1988;

Hussain et al., 1989; DeFreitas et al., 2010), we can conclude, that there are

genetic differences between the studied species, these genetic variations are

confirmed by the phenotypic differences in plants as well as on the

molecular level (DNA). However the similar or close bands may show the

genetic convergence between these species.


87

This study can be significant in the relation to classification,

comparative studies between species, cultivars and determination of the

purity of mutant genotypes (Al-Jibouri, 1988; Al-Jibouri and Dham, 1989;

Hussain et al., 1989; Judd et al., 1999; DeFreitas et al., 2010).

Chapter six Molecular systematic

88

chapter six

molecular systematic study

1-introduction

One of the most exciting developments in the past decade has been the

application of nucleic acid data to problems in systematics.

The term molecular systematic is used to mean macromolecular

systematic- the use of DNA and RNA to infer relationships among

organisms. Molecular data have revolutionized our view of phylogenetic

relationships, although not for the reasons initially suggested. Early

proponents of molecular systemaics claimed that molecular data were more

likely to reflect the true phylogeny than morphological data, ostensibly

because they reflected gene-level changes, which were thought to be less

subject to convergence and parallelism than were morphological traits. This

early assurance appears to be wrong, and molecular data are in fact subject

to most of the same problems that morphological data are. The big

difference is that there are simply many more molecular characters available,

and their interpretation is generally easier. In many cases, molecular data

have supported the morphology of groups that were recognized on

morphological grounds. More importantly, molecular data often have

allowed systematists to choose among competing hypotheses of

relationships (Judd et al., 1999).


89

As we said genetic identification can be performed by examining

morphological or phenotypical characteristics but such characteristics are

affected by environmental conditions. However, DNA based techniques

allow scanning the genome directly without being environmental affected.

Random amplified polymorphic DNA (RAPD), which is polymerase

chain reaction (PCR) based, was developed by Williams, et al. (1990) and

Welsh and McClelland (1990). Today genetic variety or similarity can be

revealed in short time and easily, and the population can be examined

rapidly through RAPD- PCR (Sesli and Yegenoglu, 2010).

In this study there was an attempt to identify the species of Euphorbia by

the technique of RAPD- PCR.


90

2-materials and methods

2.1 materials


Table (6-1) illustrates all equipments and devices used in this study.

TABLE (6-1): The apparatus used in the study.

Apparatus Names Company and Origin

Autoclave Express -Japan Cold microfuge Eppendorf-Germany Deep freeze Sanyo-Japan Distiller Controls-England Electrophoresis unit Consort-Belgium Gel documentation system Consort-Belgium Germination Cabinet Hoffman-USA Hot plate magnetic stirrer IKA-USA Laminar air flow hood Techne- UK Magnetic stirrer Scientific Industries-USA Master cycler personal Eppendorf-Germany Microcentrifuge Eppendorf- Germany Microwave Oven LG -Korea pH-Meter Bio Red – Italy Power supply Consort- German Refrigerator Ishtar-Iraq Sensitive electronic Balance Sartorius-Germany Spectrophotometer Shimadzu- Japan Ultra violet transilluminator Consort-Germany Vortex Stuart Scientific-UK Water bath Memmert – Germany


91

2.1.2 chemicals and biological materials

Table (6-2) illustrates all Chemicals and Biological Materials used in this

study.

TABLE (6-2): Chemicals and Biological Materials.

Chemicals and Biological

Materials Company and origin

Absolute ethyl alcohol BDH-England Agarose Promega-USA Ammonium Acetate (CH3COONH4) Thomas Bakar-India Bench top PCR markers (50-1000bp) Promega- USA Boric acid Fluka – Switzerland Bromophenol Blue Sigma-USA Chloroform BDH-England CTAB(cetyltrimethyl ammonium bromide) Riedel-de Haën Ethanol Alcohol Hazard-UK Ethidium bromide Promega- USA EDTA(ethylene diaminetetra acetate) BDH-England Go Taq®Green master mix Promega – USA Glycerol Fluka – Switzerland Hydrochloric acid BDH – England High Pure GMO Sample Preparation Kit Roche – Germany Isoamyl Alcohol Thomas Bakar-India Isoprobanol BDH-England

(1) Kb DNA Ladder (250-10000)bp Promega – USA

Na2EDTA Riedel-de Haën Sodium chloride (NaCL) BDH-England Sodium hydroxide (NaOH) Fluka – Switzerland Tris-Base Thomas Bakar-India TBE-buffer (10x) Promega – USA


92

2.1.3 solutions used in agarose gel electro-

phoresis

2.1.3.1 Tris-Borate (TBE) Buffer

(0.89M Tris-Base; 0.88M Boric acid; 20 mM EDTA, pH 8.0)

To prepare 10 X TBE solutions, the components used as following:

108 gm of Tris-Base, 55gm of Boric acid, 40ml of 0.5 M EDTA (pH=8.0)

in an appropriate amount of D.W, pH was adjusted to 7.8 and volume

completed to 1 liter with D.W. The solution was sterilized by autoclave and

stored at room temperature (Sambrook et al., 1989).

2.1.3.2 Loading Buffer It was prepared by dissolving 0.25gm Bromophenol blue dye in 50ml

D.W, 30ml glycerol was added, volume completed with D.W to 100ml

(Sambrook et al., 1989).

2.1.3.3 Ethidium Bromide Dye (10 mg/ml)

Prepared by dissolving 1gm of Ethidium Bromide in 100ml of a sterile

D.W, and kept in dark bottle (Maniatis et al., 1982). Ethidium is a powerful

mutagen; gloves and mask were worn during weighing and through all steps

of handling.

2.1.3.4 Molecular Weight Markers The DNA markers (Bench top PCR markers 50-1000bp and 1 Kb DNA

Ladder 250-10000bp were prepared according to the manufacturer

instructions.


93

2.2 methods

2.2.1 dna extraction from dry seeds of euphorbia

The DNA was extracted from dry seeds by using commercial kit, High

Pure GMO Sample Preparation Kit, that was provided by (Roche –

Germany).

2.2.2 estimation of the dna concentration by the

spectrophotometer

Five microliters (µl) of each sample were added to 495µl of D.W and

mixed well to determine the DNA concentration and its purity by using the

Spectrophotometer.

A spectrophotometer was used to measure the optical density (O.D.) at

wave length of 260nm and 280nm. An O.D of 1 corresponds to

approximately 50µg/ml for double stranded DNA (Sambrook et al., 1989).

The concentration of DNA was calculated according to the formula:-

DNA concentration (µg/ml) = O.D 260 nm ´ 50 ´ Dilution factor

The spectrophotometer was used also to estimate the DNA purity ratio

according to this formula:-

DNA purity ratio = O.D 260 nm / O.D 280 nm

This ratio was used to detect nucleic acid contamination in protein

preparations. DNA quality can be also assessed by simply analyzing the

DNA by agarose gel electrophoresis (Maniatis et al., 1982).


94

2.2.3 agarose gel electrophoresis

Agarose gels in different concentrations were used 0.8% for extracted

DNA, and 1.2% for visual checking to separate DNA fragments, of RAPD

product. Gels were run horizontally in 0.5X TBE buffer.

Electrophoresis buffer was added to cover the gel and run for 2 hours at

5V/cm. Agarose gels were stained with ethidium bromide 0.5mg/ml for 20-

30 minutes. DNA bands were visualized by U.V transilluminator at 365 nm

wavelength (Maniatis et al., 1982). A gel documentation system was used to

document the observed bands.

2.2.4 rapd-pcr analysis of genomic dna of

euphorbia species

1- Randomly primers:

Nine random sequence decamer primers were used, synthesis by (Alpha

DNA-Canada) from different series (A, C, D, P and R) in a lyophilized form

and were dissolved in sterile distilled water to give a final concentration of

10 pmol/µl as recommended by provider. The primers used and their

sequences are listed in table (6-3).


95

2- Go Taq®Green Master Mix (2X):

Go Taq®Green Master Mix is a ready to use mixture that contains Taq

DNA polymerase, MgCl2, pure deoxynucleotides (dNTPs), reaction buffer

and two dyes (blue and yellow) that allow monitoring of progress during

electrophoresis, with concentration 2X. Go Taq®Green Master Mix was

provided by (Promega-USA).

Amplification was performed on ice in aseptic conditions in laminar air

flow using 0.2 ml tight cap Eppendorf tubes.

A negative control reaction in each PCR experiment was set up

containing all components of the reaction without template DNA so that any

contaminating DNA present in the reaction would be amplified and detected

on agarose gel.

2.2.4.1 The Protocol of RAPD-PCR

PCR was performed with a protocol includes the following:

v PCR Primers: The random PCR primers as indicated in table (6-3).

v PCR mix: About 12.5 µl of the PCR ready mix (Go Taq®Green

Master Mix) was added when the final reaction volume was 25µl to

obtain a final concentration 1X as recommended by provider and

sterile distilled water was used to achieve a total volume of 25 µl after

added each of primers and DNA template.


96

TABLE (6-3): The names of the random primers used in the study

and their sequences(Ahmadikhah and Alvi, 2009).

(Ahmadikhah and Alvi, 2009)

v Amplification reaction Amplification of random fragments of genomic DNA was preformed

with the following master amplification reaction.

RAPD-PCR master mix (final reaction volume = 25 µl)

Genomic DNA 2µl (50ng/µl) + mix 23µl.

No. Primer'sname Sequence 5'------ 3' 1 A07 GAAACGGGTG 2 A08 GTGACGTAGG 3 A13 CAGCACCCAC 4 C05 GATGACCGCC 5 D20 ACCCGGTCAC 6 P06 TCGGCGGTTC 7 P07 CTGCATCGTG 8 R02 GTCCTCGTGT 9 R03 ACGGTTCCAC

Material's concentration and manufacturer

Final concentration

Volume for (1) tube

D.W ______ 9.5ml Promega Green Mix ( 2X)

1X 12.5ml

Primer (10 pmol/ml) 10 pmol/ml

1ml Total reaction volume

23ml


97

v PCR Program

The amplification program was run as follow:

Time: 5 min Temp.: 94°C Initial denaturation

No. of cycles = 45 cycles

Time: 1 min Temp.: 94°C Denaturation

Time: 1 min Temp.: 36°C Annealing

Time: 2 min Temp.: 72°C Extension

Time: 10 min Temp.: 72°C Final extension

Approximately 20µl of PCR amplified products were separated by

electrophoresis in 1.2 % agarose gels (2 hr, 5V/cm, 0.5X Tris-borate buffer).

Gels stained with ethidium bromide, PCR products were visualized by U.V

transilluminator and then were imaged by gel documentation system

(Hashemi et al., 2009).

The amplified products usually consist of 1-10 discrete bands and may

reach to 15 bands, the size of RAPD-PCR products estimated by comparing

with the marker 1Kb DNA ladder 250-10,000 bp.


98


3.1 dna extraction from dry seeds of euphorbia

species

The extraction of genomic DNA from dry seeds of Euphorbia spp. using

commercial kit produced good quality and high purity of intact DNA to use

in the RAPD-PCR analysis. The DNA yield of E.peplus, E.helioscopia, E.

granulata and E.hirta were (11.5, 18.5, 9.5, 14.0) μg per mg, respectively, of

dry seeds powder, while the purity of the extracted DNA were (1.3, 1.6, 1.3,

1.4), respectively. The integrity of the extracted DNA checked by agarose

gel 0.8 %.

Molecular biological studies of plants, such as the PCR techniques,

require pure DNA (Kang and Yang, 2004; Ahmadikhah and Alvi, 2009).

One of the advantages of the PCR techniques is the rapid DNA analysis of

many plant samples using small quantities of DNA.

The DNA samples extracted from seeds were very stable and could be

stored in (4 to -80) °C for a long time without degradation, so it could be

used in further studies (Ahmadikhah and Alvi, 2009).

3.2 rapd-pcr analysis

RAPD-PCR technique was used to reveal DNA polymorphism in DNA

of the studied Euphorbia spp. in order to search for the sources of

differences that could be used as a DNA marker represent the Euphorbia

spp.


99

The primers used in this study were selected randomly. Nine primers had

been tested with same DNA samples under optimum conditions.

The primers were classified into three groups according to results

obtained. The first group, gave no amplified products and this group include

(P07). Similar results were reported in different studies and a number of

random primers were scored as non amplification producing primers (Al-

Judy, 2004; Sujatha, et al, 2005; Younan, 2010; Sesli and Yegenoglu, 2010).

The second group, that gave results in term of amplification and

polymorphism, including (A13, C05 and D20).

The third group which include (A08, A07, R02, R03 and P06) primers

gave amplification and polymorphism of the genomic DNA for some

species, while no amplification was detected with other species.

The reasons of failure of these primers to amplify genomic DNA may be

absence of suitable priming site for these primers on template DNA (Devos

and Gale, 1992).

The analysis of PCR amplified DNA fragments relies on several bases

including the absence or presence of bands, differences in molecular weight;

also, there were distinct divergence in intensity of the bands, but in this

study, it was not taken into account because the presence of obvious

differences in total number of bands and their molecular weight among

species studied.

Levels of polymorphisms were generated in this study among the four

species of Euphorbia and also some primers generate unique bands that

could be used as a DNA marker to distinguish between the local species of

Euphorbia. In some instances, the reasons behind DNA polymorphism

among samples may be due to a single base changes in genomic DNA.

Other sources of polymorphisms may include deletions of a priming site,


100

insertions that render priming sites too distant to support amplification, or

insertions that change the size of a DNA segment without preventing its

amplification (Williams et al., 1990). Furthermore it had been reported that

single nucleotide changes in a primer sequence caused a complete change in

the pattern of amplified DNA segments.

v Primer A13 PCR results of primer A13, that amplified genomic DNA of species

studied of Euphorbia shown (33) bands as a total number of bands. These

total number of bands were distributed into (19) main bands, there were

polymorphic bands. The range of bands between (3-12) bands, E.granulata

produced the lower number of bands only 3 bands, while E.helioscopia

produced the highest number of bands (12) bands as shown in figure (15).

Primer A13 generated eight unique bands, the second, third, tenth and

nineteenth bands with molecular weight of about (2712)bp, (2402)bp,

(1283)bp and (527)bp respectively, which distinguished E. peplus. The other

unique bands were the first , fourth, eighth and eighteenth bands with

molecular weight of about (2955)bp, (2213)bp, (1500)bp and (618)bp ,

respectively which differentiated E.helioscopia from other species of

Euphorbia as shown in table (6-4).


101

FIGURE (15): Agarose gel electrophoresis of RAPD-PCR reaction for random primer A13 for DNA samples of Euphorbia species. Bands were fractionated by electrophoresis on a 1.2% agarose gel (2hr, 5V/cm, 0.5X Tris-borate buffer) and visualized under UV light by ethidium bromide staining.

M: 1 Kb ladder. NC: negative control. Lanes: 1. E.peplus, 2. E.helioscopia, 3. E.granulata, 4. E.hirta

NC

1

4 2 3 M

pb

2500

1000

750

500

250

1500

2000

3000


102

TABLE (6-4): The polymorphic, monomorphic and unique bands with

their molecular weight for primer A13.

1: present of band 0: absent of band

: Unique bands : Polymorphic bands

Lanes: 1. E.peplus, 2. E.helioscopia, 3. E.granulata, 4. E.hirta

v Primer C05

Euphorbia four spp. Band M.wt in bp No.

4 3 2 1 0 0 1 0 2955 1 0 0 0 1 2712 2 0 0 0 1 2402 3 0 0 1 0 2213 4 1 0 1 0 2650 5 1 0 0 1 1810 6 0 0 1 1 16450 7 0 0 1 0 1500 8 1 1 0 1 1400 9 0 0 0 1 1283 10 0 0 1 1 1090 11 1 0 0 1 970 12 0 1 1 1 865 13 0 1 1 0 814 14 0 0 1 1 775 15 1 0 1 0 750 16 1 0 1 0 690 17 0 0 1 0 618 18 0 0 0 1 527 19 6 3 12 11 Total number of band


103

Genomic DNA of Euphorbia spp. was amplified by using primer C05 and

the results included, a total number of bands were (30) distributed into (17)

main bands. Out of (30) bands, (8) bands were polymorphic, ranging in

molecular weight of (439-1569)bp, and one band were monomorphic with

molecular weight a bout of (690)bp (Table 6-5). The present monomorphic

band in result of PCR reaction means there was share DNA fragment in

genomic of all four species of Euphorbia. Primer C05 produced bands in

range (6-9) bands, E.peplus produced the lowest number of bands (6) bands,

while E.hirta had the highest number of bands that produced (9) bands as in

figure (16).

Primer C05 generated eight unique bands as shown in table (6-5),

E.peplus had one unique band which was the third band with molecular

weight of about (1225)bp, as well as E.helioscopia distinguished by one

unique band, the fourteenth band with molecular weight (492)bp, while

E.granulata was differentiated by three unique bands, the fourth, ninth and

sixteenth bands, a molecular weight of about (1169)bp,(871)bp and (401)bp,

respectively. Also E.hirta distinguished by three unique bands, the second,

tenth and seventeenth bands a molecular weight of approximately (1321)bp,

(825)bp and (376)bp, respectively.


104

FIGURE (16): Agarose gel electrophoresis of RAPD-PCR reaction for random primer C05 for DNA samples of Euphorbia species. Bands were fractionated by electrophoresis on a 1.2% agarose gel (2hr, 5V/cm, 0.5X Tris-borate buffer) and visualized under UV light by ethidium bromide staining.


250

3000

500

750

1000

1500

2000 2500

M NC 2 3 1 4

pb


105

TABLE (6-5): The polymorphic, monomorphic and unique bands with their molecular weight for primer C05.

Euphorbia species Bands M.Wt in bp No.

4 3 2 1 1 0 1 1 1569 1 1 0 0 0 1321 2 0 0 0 1 1225 3 0 1 0 0 1169 4 1 0 0 1 1090 5 1 0 1 0 1031 6 0 1 0 1 960 7 1 0 1 0 910 8 0 1 0 0 871 9 1 0 0 0 825 10 0 1 1 0 560 11 1 1 1 1 690 12 1 1 0 0 605 13 0 0 1 0 492 14 0 1 1 1 439 15 0 1 0 0 401 16 1 0 0 0 376 17

9 8 7 6 Total number of bands


: Unique bands : Monomorphic bands : Polymorphic bands



106

v Primer D20 The results of PCR reaction of primer D20, that reacted with genomic

DNA of the species of Euphorbia, appeared (31) bands as a total number of

bands, distributed into (16) main bands, of which (21) bands were

polymorphic bands, the range of their molecular weight were between (241-

2476)bp (Table 6-6), and there were one monomorphic band with size of

about (431)bp. E.helioscopia produced the highest number of bands (13)

bands compared with E. granulata that produced (3) bands.

This primer generated six unique bands (Table 6-6). The first, sixth

thirteenth bands with molecular weight of approximately (2476)bp, (980)bp,

and (391)bp, respectively, distinguished E.helioscopia .The other unique

bands were the second, fourteenth and sixteenth bands with molecular

weight of about (2240)bp, (339)bp, and (250)bp, respectively ,that

distinguished E.peplus as in figure (17).


107

FIGURE (17): Agarose gel electrophoresis of RAPD-PCR reaction for random primer D20 for DNA samples of Euphorbia species. Bands were fractionated by electrophoresis on a 1.2% agarose gel (2hr, 5V/cm, 0.5X Tris-borate buffer) and visualized under UV light by ethidium bromide staining.


250

500

1500

2000 2500 3000

1000

750

M 1 NC 2 3 4

pb


108

TABLE (6-6): The polymorphic, monomorphic and unique bands with their molecular weight for primer D20.


: Unique bands : Monomorphic bands : Polymorphic bands


Euphorbia species Bands M.Wt in bp

No. 4 3 2 1 0 0 1 0 2476 1 0 0 0 1 2240 2 1 0 1 0 1500 3 0 0 1 1 1300 4 0 0 1 1 1100 5 0 0 1 0 980 6 1 0 1 0 880 7 1 0 1 0 790 8 1 0 1 1 678 9 1 0 1 0 590 10 0 1 1 1 520 11 1 1 1 1 431 12 0 0 1 0 391 13 0 0 0 1 339 14 1 1 1 0 275 15 0 0 0 1 250 16

7 3 13 8 Total number of bands


109

v Primer A08 PCR results of primer A08, shown (9) bands as a total number of bands

for the two species E.helioscopia and E.peplus. All bands were unique, the

size of these bands ranged from (500-2000)bp,

This primer produced amplification products with range (5) bands of the

first species and (4) bands of the second as shown in figure (18) which

include the molecular weight of amplified bands, their presence or absence,

and the unique bands.

FIGURE (18): Agarose gel electrophoresis of RAPD-PCR reaction for random

primer A08 for DNA samples of Euphorbia species. Bands were fractionated by electrophoresis on a 1.2% agarose gel (2hr, 5V/cm, 0.5X Tris-borate buffer) and visualized under UV light by ethidium bromide staining.

M. 1 Kb ladder. NC: negative control. Lanes: 1. (E.peplus), 2. (E.helioscopia), 3. (E. granulata), 4. (E.hirta),

2500

250

750

500

1000

1500

3000

2000

3

pb 2

M 1

NC 4


110

v Primers A07, R02, R03 and P06 The genomic DNA of the two species of E.granulata and E.helioscopia

were amplified by using the primers A07, R02, R03 and P06, while the

genomic DNA of E.peplus and E.hirta had no amplification.

The results of primer A07 were (8) bands as a total number of bands, the

size of these bands had ranged between (500-2100) bp. This primer

produced amplification products with range (3) bands for E.granulata and

(5) bands for E.helioscopia.

The genomic DNA of these two species was amplified by using primer

R02, the results that appeared were (7) bands as a total number of bands,

size of bands were ranged between (545-1400) bp. This primer gave

amplification products with range (4) bands for E.granulata and (3) bands

for E.helioscopia.

While the results of primer R03 included, total number of bands for the

two spp., were (12) bands, size of bands were ranged between (350-2200)

bp. This primer gave amplification products with range of (9) bands for E.

granulata and (3) bands for E.helioscopia .

The primer P06 amplified genomic DNA of E.granulata and E.

helioscopia. The total number were (2) bands, the size of the first band were

about (2400) bp for E.granulata and the second band were about (1000) bp

for E. helioscopia.

The figure (19- A, B, C, D) below for the four primers include the molecular

weight of amplified bands, their presence or absence, and the unique bands.


111

(A) Primer A07 (B) Primer R02

(A) Primer A07 (B) Primer R02 (C) Primer R03 (D) Primer P06 FIGURE (19): Agarose gel electrophoresis of RAPD-PCR reaction for random

primers A07, R02, R03 and P07 for DNA samples of Euphorbia species. Bands were fractionated by electrophoresis on a 1.2% agarose gel (2hr, 5V/cm, 0.5X Tris-borate buffer) and visualized under UV light by ethidium bromide staining.

M. 1 Kb ladder. NC: negative control. Lanes: 1. E.peplus, 2. E.helioscopia, 3. E.granulata, 4. E.hirta

1 2 3 4 NC M

1 4 NC 3 2 M

1 NC 2 M 4 3 1 2 3 4 M NC 2 1 3 4 NC M


112

RAPD-PCR analyses based on the second group of primers (A13, C05

and D20) because it gave results in term of amplification and polymorphism

for the species of Euphorbia as seen in table (6-7).

The second group of primers produced a total of 52 main bands across the

four species, table (6-8). Of these 52 PCR products generated 3.85% (2

bands) were monomorphic across the studied species. The remaining 50

bands (96.2% of the total products scored) were polymorphic among the

species studied, this means that there is high difference among the genotypes

of the species of Euphorbia.

A total of 50 (96.2%) polymorphic bands were observed ranging from 8

to 11 bands. The primer A13 gave the highest number of polymorphic bands

(11), while the minimum number of polymorphic bands (8) by using C05

primer. The average number of polymorphic bands per primer among the

species was (9.3).

Polymorphism of each primer was calculated as the percentage of

polymorphic bands to the number of total main bands produced by the

designated primer. The obtained high polymorphism rate indicates a high

genetic diversity.

The number of bands generated by each primer varied, A13 generated

maximum number of bands (33) while R02 amplified minimum number of

bands (2). The variation in the number of bands amplified by different

primers influenced by variable factors such as primer structure, template

quantity and less number of annealing sites in the genome (Kernodle et al.,

1993).

Visual examination of electrophoresis gels and analysis of banding

patterns confirmed that E.peplus and E.helioscopia had high degree of


113

similarity appeared in the pattern of DNA with most of primers compared

with other species, but there were clear differences among them specially in

term of unique bands. While E.hirta and E.granulata had less similarity

pattern of DNA.

TABLE (6-7): The species of Euphorbia and the primers that appeared the unique bands, the number and molecular weight of these bands.

No. Species name Primer Molecular weight

of unique bands Unique bands

number

1 E. peplus

A13

2712 2nd 2402 3rd 1283 10th 527 19th

C05 1225 3rd

D20 2240 2nd 339 14th 241 16th

2 E. helioscopia

A13

2955 1st 2213 4th 1500 8th 618 18th

C05 492 14th

D20 2476 1st 980 6th 391 13th

3 E. granulata C05

1169 4th 871 9th 401 16th

4 E.hirta C05

1321 2nd

825 10th

376 17th


114

TABLE (6-8): Distinct characteristic of random primers included in the study: primer's name, total number of bands, number of polymorphic bands and percentage of polymorphism in species of Euphorbia.

No. Primer Total number of main bands

Number of polymorphic bands

Polymorphism %

1 A13 19 11 84%

2 C05 17 8 47%

3 D20 16 9 56%

Total 52 28 ___

The RAPD assay generated specific products in all of the species studied.

These may be used as DNA fingerprints for species identification. It would

be of immense use for the establishment of proprietary rights and the

determination of species purity. On the other hand, RAPD markers had been

useful as the first step to produce a genetic map in plants with unknown or

much or less known genetic series (Sesli and Yegenoglu, 2010). On the

other hand these results confirm the isolation of the four species of

Euphorbia from each other obviously; as well as distinction of E.peplus and

E.helioscopia from the other two species E.hirta and E.granulata, and this

true corresponds to the morphological features of these species. These

results may be applied in isolation of similar species that could not be

isolated by using other qualities and characteristic features.

This is one of the goals of this biosystematic study which can be applied

such methods in the diagnosis of the rest of Euphorbia species existing in

Iraq.

Chapter seven Plant tissue culture

115

chapter seven

plant tissue culture

1-introduction

Plant tissue culture techniques are essential to many types of academic

inquiry, as well as to many applied aspects of plant science. In the past, plant

tissue culture techniques have been used in academic investigations of

totipotency and the roles of hormones in cytodifferentiation and

organogenesis. Also tissue cultures are required in the formation of somatic

haploid embryos from which homogyous plants can be generated. Thus,

tissue culture techniques have been, and still are, prominent in academic and

applied plant science (Mineo, 1990). Researchers are constantly exploring

new evidence for natural resources. A lot of economical factors are based on

our resources which make a transition from one element to another very

difficult.

` The members of Euphorbiaceae are valuable source of different kinds of

useful products like dyes, edible tubers, oil crops, furniture, agricultural

implements, ornamental plants, pharmacological products, rubber, timber

and aesthetic items. Micropropagation is an alternative mean of propagation

that can be employed in conservation of the flora in relatively shorter time.

Tissue culture is useful for multiplying and conserving the species, which

are difficult to regenerate by conservation methods and save them from

extinction. Cryopreservation of germplasm would help in maintaining the

genetic diversity of the endangered population. Improved cell and tissue

culture technologies would help in producing the active compounds in vitro


116

with better productivities without cutting down the natural resources. There is sufficient progress at research level to suggest that the tissue

culture of Euphorbiaceae can and should be further developed (Rajesh et al.,

2009). In this study we have made an investigation for the response of the

species of Euphorbia for callus induction by using nodule and leaf explants

grown in the dark and in the light condition to reveal the reflection of the

genotypes in vitro and to state a biotechnical sort to preserve the rare and

endangered plants.

2- materials and methods

2.1 materials


The following equipments and device were used throughout the

experimental work:

Apparatus Company and Origin Autoclave Karl ــGermany Distiller GFL ــ Germany Electric balance Mettler ــ Switzerland Hot plate with magnetic stirrer Gallenkamp ــ England Laminar air flow cabinet ESCO ــ Singapore Micropipettes Slamid ــ Germany Oven Gallenkamp ــ English pH-meter Meter Gmbh-teledo ــ England Refrigerator Ishtar ــ Iraq Sensitive balance Delta range ــ Switzerland Vortex Buchi ــ Switzerland


117

2.1.2 plant material

The plant material (mature leaves and stems) of the four species of

Euphorbia was obtained from Baghdad University gardens for developing

tissue culture protocol for study the callus growth in response to different

concentrations of plant growth regulators, light and dark , then selecting the

best conditions for callus induction.

2.2. methods

2.2.1 sterilization of explants

Mature leaf and stem explants were excised, rinsed with tap water for 30

min, then transferred to a laminar air flow-cabinet where rinsed with sodium

hypochlorite 2% NaOCl for 15 min (Yang et al., 2009, Al-Naqshabandy,

2010), followed by three rinses in sterile distilled water. The nodal explants

were trimmed 1cm at the base and cultured with the cut surface in contact

with medium surface. Leaves were cut into 1cm2 segments and placed with

the basal side in contact with the medium.

2.2.2 preparation of culture medium

Culture medium consisted of MS (Murashige and Skoog, 1962)

(Table 7-1), salts and vitamins, 30 g/l sucrose and 9 g/l agar. For callus

initiation nodal and leaf explants were cultured on medium supplemented

with 1mg/l Cytokinin BA (Benzyl adinine) and Auxin 2,4-

Diclorophenoxyacetic acid at different concentrations individually. The pH

was adjusted to (5.8) using NaOH or HCl (1 N), then 9 g/l of the agar type

(agar-agar) was added to the medium, placed on a hot plate magnetic stirrer

until boiling, then aliquots of 20 ml were dispensed into (8×6) cm culture


118

vessels. Then autoclaved at 121 ºC under 1.04 Kg/cm² pressure, for 15 min.

The medium was left at room temperature to cool and become ready to

culture explants.

TABLE (7-1): Composition of Murashige and Skoog medium (1962).

Weight (mg/l) Chemical formula Components Stock (1) Macronutrients

1650 NH4NO3 Ammonium nitrate

1900 KNO3 Potassium nitrate

440 CaCl2.2H2O Calcium chloride.2H2O

370 MgSO4.7H2O Magnesium sulphate.7H2O

170 KH2PO4 Potassium phosphate monobasic

Stock (2) Micronutrients 6.20 H3BO3 Boric acid

0.83 KI Potassium iodide

22.30 MnSO4.4H2O Manganese sulphate.4H2O

8.60 ZnSO4.7H2O Zinc sulphate.7H2O

0.25 Na2MoO4.2H2O Molybdic acid (sodium salt).2H2O

0.025 CuSO4.5H2O Cupric sulphate.5H2O

0.025 CoCl2.6H2O Cobalt chloride.6H2O

Stock (3) Chelated Iron

37.3 Na2-EDTA.2H2O Sodium ethylene diamine tetra acetate

27.8 FeSO4.7H2O Ferrous sulfate.7H2O

Stock (4) Vitamins and organics

0.1 Cl2H17ClN4OS.HCl Thiamine.HCl (B1)

0.5 C8H11NO3.HCl Nicotinic acid (free acid) (B3)

0.5 C6H5NO2 Pyridoxine.HCl (B6)

2.0 C2H5NO2 Glycine (free base)

100 C6H6(OH)6 Myoinositol

30000 C12H22O11 Sucrose


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To prepare one liter of MS medium we need:

Stock (1) Macro 100 ml

Stock (2) Micro 10 ml Stock (3) Fe-EDTA 10 ml Stock (4) Vitamins 1 ml Inositol 100 mg Sucrose 30 gm Agar 9 gm

2.2.3 plant growth regulators

Different concentrations of the Auxin 2,4-D (0,0.5, 1, 1.5, 2) mg/l and

the Cytokinin BA 1 mg/l were prepared and added to the culture medium as

required before autoclaving.

2.2.4 incubation of cultures

Surface sterilized leaf and stem explants 1cm in diameter were

inoculated into the culture vessels under aseptic conditions. Cultures were

divided for two groups .the first group cultures were incubated at 25± 2 ºC

under 16 hr photoperiod, white fluorescent lights 40 µmolQ/ m2 s. The

second group were incubated at 25± 2ºC under dark. Each treatment

consisted of three replicates.

2.2.5 initiation of callus cultures

Observation on number of sprouted initiated callus were recorded

after 2, 4, 6 and 8 weeks of culture on the primary medium.


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2.2.6 maintenance of callus cultures

Eight weeks callus was removed from the explants using forceps and

scalpel, then pieces weighing about 50 mg were subcultured onto fresh

medium supplemented with different concentrations of the 2,4-D (0, 0.4, 0.8,

1.2, 1.6 ) mg/l and the BA 1mg/l. The data were recorded after four weeks.

Callus fresh weight was determined using sensitive balance, and then oven

dried at 40 ºC for 24 hrs for callus dry weight measurements.

2.2.7 statistical analysis The observation of experiment were analyzed by statistical analysis

system – SAS (2004) was used to study the effect of Concentration of 2,4-D

and Euphorbia species (factorial experiment) in study traits. The least

significant difference (LSD) test was used to significant compare between

means.


121


The most important factors contributing the induction of callus and

plant regeneration are: the explants type, medium formulation and growth

regulators (Buyukalaca and Mairtuna 1996). Callus induction in different

plants have been achieved using a variety of media ranging from relatively

dilute - White's medium (1963) to a more concentrated formulations of

Gamborg et al. (1968), Schenk and Hildebrandt (1972), as well as

Murashige and Skoog (MS) medium (1962); However, over 70% successful

cases have used MS salts or its derivatives (Evans et al. 1981). Among the

plant growth regulators, generally auxin is known to be essential for the

induction of somatic embryogenesis and 2,4-D is the most commonly used

auxin (Ammirato 1983). The results of the present study revealed the

different response of the species of Euphorbia for tissue culture technique

under different parameters.

3.1 the response for callus induction

a- Callus induction from nodule explants of Euphorbia species

cultured on MS medium supplemented with different

concentrations of 2,4-D incubated in dark and light conditions Results of table (7-2) show different Callus induction among Euphorbia

spp. in control treatment and MS medium supplemented with different

concentrations of 2,4-D incubated in dark and light conditions.

The control treatment (without 2,4-D) of E.helioscopia was induced 25%

in third and fourth two weeks in light condition only, while the other species


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failed to produce callus from nodule explants on MS medium incubated in

dark and light conditions.

In the first two weeks, nodules incubated in dark condition showed

responses for callus induction in E.peplus reached 25% and 75 % on MS

supplemented with 1.5 and 2 mg/l 2,4-D, respectively. While in light

condition the response reached 25%, 50% and 50 % on MS medium with 1,

1.5 and 2 mg/l of 2,4-D, respectively. As well as in E.hirta reached 50 % on

MS with 1.5 mg/l 2,4-D.

The second two weeks showed responses for callus induction in all

species of Euphorbia. The maximum callus induction of nodules incubated

in dark was recorded in E.peplus reached 100% on MS supplemented with 2

mg/ml 2,4-D, and in E.hirta reached 75% on MS with 1and 2 mg/l 2,4-D.

As well as in E.granulata and E.helioscopia reached 75% on MS with 1.5

mg/l 2,4-D. Whereas in light condition the response of E.hirta reached

100% on MS supplemented with 2 mg/l. In E.peplus reached 75% on MS

with 1, 1.5 and 2 mg/l. While in E.granulata reached 75% on MS with 1.5

mg/l and in E.helioscopia reached 50% on MS supplemented with 1 mg/l

2,4-D.

The third two weeks the maximum callus induction of nodules incubated

in dark was recorded in E.peplus 100% on MS with 2 mg/l 2,4-D. While in

E.hirta reached 75% on MS supplemented with all concentrations of 2,4-D.

In E.granulata reached 75% on MS with 1.5 mg/l 2,4-D, and in

E.helioscopia reached 75% on MS with 0.5 mg/l. However the maximum

callus induction of nodules in light were recorded in E.peplus 100% on MS

supplemented with 1, 1.5 and 2 mg/l 2,4-D. In the meantime E.hirta reached

75% on MS supplemented with all concentrations of 2,4-D. In E.granulata


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reached 75% on MS with 1.5 and 2 mg/l 2,4-D and in E.helioscopia reached

75% on MS with 1 mg/l 2,4-D.

The final two weeks the response for callus induction in dark condition in

E.hirta reached 75% in all concentrations of 2,4-D. In E.peplus reached to

100% on MS with 0.5 and 2 mg/l 2,4-D. In E.granulata recorded 75%

callus induction on MS with 1, 1.5and 2 mg/l 2,4-D. While E.helioscopia

recorded 75% on MS with 0.5 mg/l 2,4-D. However the light condition was

better for the response of E.hirta reached 100% in all concentrations of 2,4-

D. Also, in E.peplus reached 100% on MS supplemented with 1, 1.5 and 2

mg/l 2,4-D. In E.granulata reached 75% on MS with 1.5 and 2 mg/l 2,4-D

and in E.helioscopia reached 75% on MS with 1 mg/l 2,4-D.

From above we can conclude that E.hirta prefers light condition while

the other species prefer dark condition for best callus induction.


124


125

b- Callus induction on MS medium supplemented with 1mg/l

BA and different concentrations of 2,4-D from leaf explants of

Euphorbia species incubated in dark and light conditions The results in table (7-3) explain the response of leaf explants of

Euphorbia spp. for callus induction. All studied species failed to produce callus from leaf explants on MS

medium incubated in dark and light conditions in control treatment (without

2,4-D) on period of eight weeks..

In the first two weeks, E.peplus showed first response for callus

induction compared with other species reached 75% on MS supplemented

with 0.5 mg/ml 2,4-D in leaf explants incubated in dark condition only,

while other species failed to produce callus.

The second two weeks showed maximum callus induction on leaves

incubated in dark in E.peplus reached 100% on MS supplemented with 0.5

mg/l 2,4-D. While E.hirta recoded 25% on MS with 1 and 2 mg/l 2,4-D.

The maximum responses for callus induction incubated in light were

recorded in E.peplus 75% on MS supplemented with 1 and 1.5 mg/l 2,4-D.

While E.hirta recorded 75% on MS with 1.5 mg/l 2,4-D. In E.granulata

reached 50% on MS with 2 mg/l 2,4-D and in E.helioscopia reached 50% on

MS with 1 mg/l 2,4-D.

The third two weeks the maximum callus induction of leaf explants

incubated in dark was recorded in E.peplus 100% on MS with 0.5,and 2

mg/l 2,4-D.While in E.hirta reached 75% on MS with 1.5 and 2 mg/ml 2,4-

D. In E.granulata reached 75% on MS with 1.5 and 2 mg/l 2,4-D, and in

E.helioscopia reached 25% on MS with 0.5, 1.5 and 2 mg/l 2,4-D.


126

However the maximum responses for callus induction incubated in light

were recorded in E.peplus and E.hirta 100% on MS with 1.5 mg/l 2,4-D.

In E.granulata reached 75% on MS with 1.5 and 2 mg/l 2,4-D and in

E.helioscopia reached 50% on MS with 1 and 1.5 mg/l 2,4-D.

The final two weeks the maximum response for callus induction of leaves

incubated in dark reached in E.hirta 100% on MS supplemented with 1.5

and 2 mg/l 2,4-D. As well as in E.peplus recorded 100% on MS with 0.5,

1.5 and 2 mg/l of 2,4-D. While E.granulata recorded 75% callus induction

on MS with 1.5 and 2 mg/l 2,4-D. E.helioscopia recorded 25% on MS with

0.5, 1.5 and 2 mg/l 2,4-D. In light condition the response of leaves of

E.hirta reached 100% on MS with 1 and 1.5 mg/l of 2,4-D. As well as in

E.peplus reached 100% on MS with 1.5 and mg/l 2,4-D.

In E.granulata reached 75% on MS with 1.5 and 2 mg/l 2,4-D. However

E.helioscopia recorded 50% on MS with 1 and 1.5 mg/l 2,4-D.

From the results above we can conclude that leaf explants of E.hirta and

E.peplus have highest responses for callus induction among the other species

in dark and light conditions, while E.helioscopia has the lowest response for

callus induction. As well as the nodule explants were better than leaf

explants in the base of response for callus induction in dark and light

condition.

A semi-compact and pale-yellow callus was seen on nodule and leaf

explants of studied species incubated in dark conditions after eight weeks

(Figure 20). In the meantime a semi-compact pale-green callus was seen on

nodule and leaf explants of studied species incubated in light conditions after

eight weeks (Figure 21).


127


128

FIFURE (20) Callus induction on MS medium supplemented with

2,4-D in nodule and leaf explants of E.hirta incubated in dark

after 60 days. a- Nodule explants b- Leaf explants

FIFURE (21) Callus induction on MS medium supplemented with

2,4-D in nodule and leaf explants of E.peplus incubated in light

after 60 days. a- Nodule explants b- Leaf explants

a b

a b


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3.2 callus production

3.2.1 Callus production and the fresh weight from nodule and

leaf explants of Euphorbia species incubated in dark Calli were produced on nodule explants taken from Euphorbia species

and cultured on MS medium with different concentrations of 2,4-D.

Results in Table (7-4) explain the response of nodule explants of the species

of Euphorbia for callus production incubated in dark. The mean of fresh

weight on nodule explants of E.hirta and E.peplus were significantly

increased compared with the other species, reached 1.174 mg and 1.156 mg

respectively. While E.helioscopia recorded the minimum mean of fresh

weight on nodule explants reached 0.496 mg and showed significant

differences from other species.

The concentrations of 2,4-D also showed significant differences in the

maximum mean of callus fresh weight on nodule explants reached 1.200 mg

and 1.060 at the concentration of 0.4 mg/l and 0.8 mg/l, respectively. While

the minimum mean of callus fresh weight on nodule explants were recorded

in control treatment reached 0.230 mg and showed significant differences

from other concentrations of 2,4-D.

The interaction between the species of Euphorbia and 2,4-D

concentration recorded the maximum callus fresh weight in E.peplus

reached 1.77 mg callus fresh weight with 0.4 mg/l 2,4-D.


130

Table (7-4): Effect of 2,4-D and species of Euphorbia on callus fresh weight

produced from nodule explants cultured on MS medium supplemented with

different concentrations of 2,4-D incubated in dark condition.

Conc. of 2,4-D mg/l

Callus fresh weight (mg)

E.helioscopia E. peplus E.granulata E.hirta Mean

0 0.000 0.000 0.000 0.920 0.230 0.4 0.800 1.770 1.260 0.970 1.200 0.8 1.680 0.850 0.580 1.140 1.060 1.2 0.000 1.630 1.000 1.210 0.960 1.6 0.000 1.530 0.640 1.630 0.950

Mean 0.496 1.156 0.696 1.174 --

LSD (P≤0.05)

2,4-D : 0.136 * Euphorbia species: 0.121 * 2,4-D x Euphorbia species: 0.272 *

*Significant

On the other hand, different results were observed with leaf explants.

Table (7-5) explains the response of leaf explants of the species of

Euphorbia for callus production incubated in dark. The mean of fresh weight

significantly increased in E.hirta compared with the other species, reached

1.03 mg of mean callus fresh weight on leaf explants. While E.peplus

recorded the minimum mean of fresh weight on leaf explants reached 0.370

mg and showed significant differences from the other species.

The concentrations of 2,4-D also showed significant differences which

recorded maximum mean of callus fresh weight on leaf explants reached

0.92 mg at the concentration 0.8 mg/l and showed significant differences

from all concentrations except 1.6 mg/l 2,4-D. While the minimum mean of

callus fresh weight on leaf explants was recorded on control treatment

reached to 0.220 mg and showed significant differences from other

concentrations.


131


concentration was significant, the maximum callus fresh weight on leaf

explants in E.peplus and E.hirta reached 1.550, 1.510 mg callus fresh weight

with 1.6 and 0.6 mg/l 2,4-D, respectively and showed significant differences

from all combinations.


produced from Leaf explants cultured on MS medium supplemented with different

concentrations of 2,4-D incubated in dark condition

Conc. of 2,4-D mg/l

Callus fresh weight (mg) E.helioscopia E. peplus E.granulata E.hirta Mean

0 0.000 0.000 0.000 0.870 0.220 0.4 0.450 0.000 1.010 1.510 0.740 0.8 1.290 0.000 1.240 1.150 0.920 1.2 0.630 0.300 1.090 0.770 0.700 1.6 0.000 1.550 0.890 0.860 0.830

Mean 0.470 0.370 0.850 1.030 -- LSD

(P≤0.05) 2,4-D : 0.116 * Euphorbia species: 0.l04 *

2,4.D x Euphorbia species: 0.233 * *Significant 3.2.2 Callus production and the fresh weight from nodal and

leaf explants of Euphorbia species incubated in light

The results of callus production and the fresh weight of Euphorbia species

from nodule explants in light were revealed in table (7-6). The mean of fresh

weight significantly increased in E.hirta compared with the other species,

reached 1.64 mg of mean callus fresh weight on nodule explants. While

E.peplus recorded the minimum mean of fresh weight on nodule explants


132

reached 0.390 mg and showed significant differences from other species

except E.helioscopia.

The concentrations of 2,4-D showed significant differences, reached

1.14 mg of mean callus fresh weight on nodule explants at the concentration

of 1.6 mg/l. While the minimum mean of callus fresh weight on nodule

explants were recorded in control treatment and showed significant

differences from other concentrations of 2,4-D.


concentration recorded the maximum callus fresh weight on nodule explants

in E.hirta reached 2.70 mg and 2.530 mg callus fresh weight with 1.6 mg/l

2,4-D and 1.2 mg/l 2,4-D, respectively, and showed significant differences

from all other combinations.


produced from nodule explants cultured on MS medium supplemented with

different concentrations of 2,4-D incubated in light condition.

Conc. of 2,4-D mg/l


0 0.000 0.000 0.000 0.000 0.000 0.4 0.730 0.520 0.820 1.550 0.905 0.8 0.650 0.410 1.020 1.450 0.882 1.2 0.570 0.550 0.650 2.530 1.075 1.6 0.580 0.470 0.810 2.700 1.140

Mean 0.506 0.390 0.660 1.646 -- LSD

(P≤0.05) 2,4-D : 0.188 * Euphorbia species: 0.168 *

2,4-D x Euphorbia species: 0.376 *

*Significant


133

While table (7-7) Show the response of leaf explants of the species of

Euphorbia for callus production incubated in light. The mean of fresh

weight significantly increased in E.hirta compared with the other species,

reached 0.890 mg of mean callus fresh weight on leaf explants.

Whereas, E.peplus recorded the minimum mean of fresh weight on

leaf explants reached 0.360 mg and showed significant differences from

other species.

The concentrations of 2,4-D also showed significant differences,

reached 1.00 mg of mean callus fresh weight on leaf explants at the

concentration of 0.8 mg/l. While the minimum mean of callus fresh weight

on leaf explants were recorded in control treatment and showed significant

differences from other concentrations of 2,4-D.


concentration recorded the maximum callus fresh weight on leaf explants in

E.helioscopia reached 1.750 mg with 0.8 mg/l 2,4-D and showed significant

differences from all other combinations.


134


produced from Leaf explants cultured on MS medium supplemented with different

concentrations of 2,4-D incubated in light condition.

Conc. of 2,4-D mg/l


0 0.00 0.000 0.000 0.000 0.000 0.4 0.770 0.280 1.150 0.980 0.790 0.8 1.750 0.490 0.790 0.960 1.000 1.2 0.700 0.390 0.910 1.180 0.800 1.6 0.000 0.660 0.880 1.310 0.710

Mean 0.640 0.360 0.750 0.890 -- LSD



*Significant 3.2.3 The dry weight of callus from nodule and leaf explants of

Euphorbia species incubated in dark The species of Euphorbia showed significant differences in nodules

explants callus dry weight incubated in dark as seen in Table (7-8)

The maximum mean of dry weight showed significant differences in

E.peplus compared with E.helioscopia and E.granulata species, which

reached 0.044 mg. While E.granulata recorded the minimum mean of dry

weight on nodule explants reached 0.021 mg and showed significant

differences from E.peplus and E.hirta.

The concentrations of 2,4-D showed significant differences, reached

0.047 mg of mean callus dry weight on nodule explants at the concentration

of 0.4 mg/l. While the minimum mean was recorded in control treatment

0.008 mg and showed significant differences from the other concentrations.


135


concentration recorded the maximum callus dry weight in E.peplus reached

0.083 mg with 1.2 mg/l 2,4-D and showed significant differences from most

combinations.

Table (7-8): Effect of 2,4-D and species of Euphorbia on callus dry weight produced

from nodule explants cultured on MS medium supplemented with different

concentrations of incubated in dark condition.

Conc. of 2,4-D mg/l

Callus dry weight (mg) E.helioscopia E. peplus E.granulata E.hirta Mean

0 0.000 0.000 0.000 0.034 0.008 0.4 0.060 0.045 0.032 0.050 0.047 0.8 0.073 0.038 0.024 0.032 0.042 1.2 0.000 0.083 0.023 0.026 0.033 1.6 0.000 0.052 0.027 0.054 0.033

Mean 0.026 0.044 0.021 0.039 -- LSD

(P≤0.05) 2,4-D : 0.009 Euphorbia species: 0.008 *

2,4-D x Euphorbia species: 0.019 * *Significant

Table (7-9) explains the dry weight of callus from leaf explants incubated

in dark. The species of Euphorbia showed significant differences in leaf

callus dry weight which significantly increased in E.helioscopia and

E.granulata compared with the other species, reached in both 0.028 mg of

mean callus dry weight of leaf explants and showed significant differences

from the other species except E.peplus which recorded the minimum mean

of dry weight on leaf explants reached 0.014 mg and showed significant

differences from other species.

The mean of concentrations of 2,4-D showed significant differences,

the maximum mean of callus dry weight on leaf explants reached 0.034 mg


136

at the concentration of 0.8 mg/l. While the minimum mean was recorded in

control treatment 0.007 mg and showed no significant differences from the

other concentrations.


concentration recorded the maximum callus fresh weight in E.hileoscopia

reached 0.075 mg with 0.8 mg/l 2,4-D and showed significant differences

from the other combination.


from Leaf explants cultured on MS medium supplemented with different

concentrations of 2,4-D incubated in dark condition.

Conc. of 2,4-D mg/l

Callus dry weight (mg) E.helioscopia E. peplus E.granulata E.hirta Mean

0 0.000 0.000 0.000 0.031 0.007 0.4 0.033 0.000 0.039 0.043 0.028 0.8 0.075 0.000 0.036 0.025 0.034 1.2 0.035 0.027 0.036 0.017 0.029 1.6 0.000 0.046 0.030 0.018 0.023

Mean 0.028 0.014 0.028 0.027 -- LSD



*Significant 3.2.4 The dry weight of callus from nodule and leaf explants of

Euphorbia species incubated in light The measurements of nodule explants callus dry weight revealed high

differences among the species as shown in table (7-10).

The maximum mean of callus dry weight was significantly increased in

E.hirta compared with the other species, reached 0.052 mg of mean callus

dry weight of nodule explants and showed significant differences from other


137

species. While the minimum mean was recorded in E.peplus reached 0.020

mg and showed significant differences from other species.

The concentrations of 2,4-D showed significant differences , reached

0.041 mg of mean dry weight at the concentration of 1.6 mg/l and differs

significantly from all other combinations. While the minimum mean was

recorded in control treatment 0.011 mg and also showed significant

differences from the other concentrations. The interaction between the

species of Euphorbia and 2,4-D concentration recorded the maximum callus

dry weight in E.hirta reached 0.074 mg with 1.2 mg/l 2,4-D and showed

significant differences from most combinations.


from nodule explants cultured on MS medium supplemented with different


Conc. of 2,4-D mg/l

Callus dry weight (mg) E.helioscopia E.peplus E.granulata E. hirta Mean

0 0.000 0.000 0.000 0.043 0.011 0.4 0.047 0.032 0.023 0.033 0.034 0.8 0.038 0.017 0.033 0.043 0.033 1.2 0.031 0.021 0.023 0.074 0.037 1.6 0.055 0.021 0.021 0.068 0.041

Mean 0.034 0.018 0.020 0.052 -- LSD


2,4-D x Euphorbia species: 0.011 * *Significant


138

However Table (7-11) explains the mean of leaf explants callus dry

weight which significantly increased in E.helioscopia, reached 0.040 mg and

showed significant differences from other species. While the minimum mean

was recorded in E.peplus and E.hirta reached in both 0.021 mg and showed

significant differences from other species.

The concentrations of 2,4-D also showed significant differences

reached 0.043 mg of mean callus dry weight on leaf explants at the

concentration of 0.8mg/l. whereas the minimum mean was recorded in

control treatment and showed significant differences from the other

concentrations.

The interaction between the species of Euphorbia and 2,4-D concentration

recorded the maximum callus dry weight in E.helioscopia reached 0.090 mg

with 0.8 mg/l 2,4-D and showed significant differences from other

combinations.


from Leaf explants cultured on MS medium supplemented with different


*Significant

Conc. of 2,4-D Mg/ml

Callus fresh dry (mg/ml) E.helioscopia E. peplus E.granulata E.hirta Mean

0 0.000 0.000 0.000 0.000 0.000 0.4 0.065 0.029 0.042 0.022 0.039 0.8 0.090 0.033 0.030 0.017 0.043 1.2 0.047 0.015 0.033 0.022 0.029 1.6 0.000 0.029 0.028 0.045 0.025

Mean 0.0400 0.021 0.026 0.021 -- LSD (P≤0.05)

2,4-D: 0.027 Euphorbia species : 0.005* 2,4-D x Euphorbia species: 0.0114 *


139

A loose granuliform and yellow to pale green callus was seen on nodule and

leaf explants of E.helioscopia, E.peplus and E.granulata incubated in light

and dark conditions after four weeks, plate (9). The cullus texture and color

of E.helioscopia agreed with Yang et al. (2009) that further stated “callus

turn brown when the concatenation of 2,4-D rose to 4.0 mg/l”.

While a loose granuliform and pale yellow to pale brown callus was

seen on nodule and leaf explants of E.hirta incubated in light and dark

conditions after four weeks (Plate 10). Also, it is obvious that the calli

incubated in dark have more humidity in all species studies.


140

PLATE (9) Callus production on MS medium supplemented with 0.4 mg/l 2,4-D in nodule and leaf explants of E.peplus incubated in light

and dark conditions after 30 days. a- Callus of nodule explant incubated in light

b- Callus of leaf explant incubated in light

c- Callus of nodule explant incubated in dark

d- Callus of leaf explant incubated in dark

a b

c d


141

PLATE (10) Callus production on MS medium supplemented with 0.4 mg/l 2,4-D in nodule and leaf explants of E.hirta incubated in

light and dark conditions after 30 days. a- Callus of nodule explant incubated in light

b- Callus of leaf explant incubated in light

c- Callus of nodule explant incubated in dark

d- Callus of leaf explant incubated in dark

a b

c d


142

On the other hand, the results of diagram (1) indicated to the presence of

differences between the mean of fresh weight of vegetative parts (nodule and

leaf) of the species of Euphorbia initiated on MS medium for callus

production, incubated in dark and in light conditions. In addition to

presence of differences among the four studied species of Euphorbia in the

base of vegetative parts.

E.helioscopia showed no significant differences in callus dry weight

from nodule and leaf explants either those incubated in dark or those

incubated in light conditions. The maximum mean of callus fresh weight

was 0.644 mg on leaf explants incubated in light condition. While the

response of E.peplus for callus production showed significant differences

between vegetative parts and incubation conditions, the maximum mean of

fresh weight reached 1.156 mg on nodule incubated in dark conditions and

significantly differed from other vegetative parts which recorded no

significant differences. E.granulata just like E.helioscopia showed no

significant differences in callus production, the maximum mean of callus

fresh weight reached 0.856 mg on leaf explants incubated in dark condition.

But E.hirta showed significant differences in callus induction. The

maximum mean of callus fresh weight recorded 1.646 mg on nodule

explants incubated in light condition and showed significant differences

from other vegetative parts incubated in dark or light conditions.

From diagram (1) we can reveal that E.hirta has the highest response for

callus production, while E.helioscopia has the lowest response for callus

production among the studied species. As well as the highest callus

production achieved with E.peplus nodule explants incubated in dark

conditions, and E.hirta nodule explants incubated in light conditions that

recorded 1.156 mg and 1.646 mg, respectively.


143

While, the results of diagram (2) showed presence of differences between

the mean of dry weight of vegetative parts (nodule and leaf) of the species of

Euphorbia initiated on MS medium for callus induction, incubated in dark

and light conditions. In addition to presence differences among studied

species of Euphorbia in the base of vegetative parts.

E.helioscopia showed no significant differences in callus induction from

nodule and leaf explants either those incubated in dark or those incubated in

light. The maximum mean of dry weight for callus production reached 0.040

mg on leaf explants incubated in light condition. While the response of

E.peplus for callus production showed high significant differences between

vegetative parts and among incubation conditions, the maximum mean of

dry weight reached 0.043 mg on nodule incubated in dark conditions and

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8


Cal

lus f

resh

wei

ght (

mg)

Species of Euphorbia

Diagram (1) Fresh weight of callus produced from nodule and leaf explants of species of Euphorbia cultured on MS medium supplemented

with different concentrations of 2,4-D incubated in dark and light conditions.

Nodule explants grown in dark

Leaf explants grown in dark

Nodule explants grown in light

Leaf explants grown in light

1.646

1.156

E.hirta LSD (P≤0.05): 0.367*

E.peplus LSD (P≤0.05): 0.452*


144

significantly differed from other vegetative parts which recorded no

significant differences. E.granulata showed no significant differences in

callus dry weight, the maximum mean of dry weight for callus production

reached 0.028 mg on leaf explants incubated in dark condition. But E.hirta

showed high significant differences in callus dry weight. The maximum

mean of dry weight for callus production recorded 0.052 mg on nodule

explants incubated in light condition and showed significant differences

from other vegetative parts incubated in dark or light conditions.

In addition the diagram (2) showed that E.hirta has the highest response

for callus induction, while E.granulata has the lowest response for callus

induction among studied species. As well as the highest callus production

achieved with E.peplus nodule explants incubated in dark conditions, and

E.hirta nodule explants incubated in light conditions that recorded 0.043 mg

and 0.052 mg, respectively.


145

According to the results of tissue culture stated above, we can conclude

that there are significant differences within studied species on the base of

response to callus induction. E.hirta achieved the highest response for callus

induction reached (75-100) % in period eight weeks according to the

concentration of 2,4-D supplemented to MS medium. While E.helioscopia

achieved the lowest response for callus induction reached (25-50) % in the

period eight weeks according to the concentration of 2,4-D supplemented to

MS medium. This response revealed on the fresh and dry weight of callus

production of the studied species. Also these results revealed on callus

production in the second experiment specially on E.peplus nodule explants

incubated in dark and E.hirta nodule explants incubated in light that

achieved highest callus production compared with the other species.

0

0.01

0.02

0.03

0.04

0.05

0.06


Cal

lus d

ry w

eigh

t (m

g)

Species of Euphorbia

Diagram (2) Dry weight of callus produced from nodule and leaf explants of species of Euphorbia cultured on MS medium

supplemented with different concentrations of 2,4-D incubated in dark and light conditions.

Nodul explants grown in dark

Leaf explants grown in dark

Nodule explants grown in light

Leaf explants grown in light

E.peplus LSD (P≤ 0.05): 0.014*

E.hirta LSD (P≤ 0.05): 0.011*

0.052

0.043


146

Yang et al. (2009) stated that the highest frequencies of callus induction

in E.helioscopia observed on MS media supplemented with 3.0 mg/l 2,4-D.

The differences of these results may be due to the differences in

genotypes of studied species in the base of the response to callus induction

and production, as well as due to endogenous concentration of growth

hormones.

Similar results were reported in different studies (Hameed, 2001;

Aljibouri et al., 2005; Yang et al., 2009; Aljibouri et al., 2010).

Previous results reveal the significant differences in the response of the

four species for callus induction in different conditions. These differences

can be used as secondary supported characteristics among the species

studied, in addition to the other characteristics used in distinction and

identification. Also we can use these results in this section in segregation

and identification of species which have very close or similar characteristics,

in all species of plant within this genus of this family or any other species

taxa. Furthermore, in this study we stated a technique that may be used by

any taxonomist to preserve and regenerate plants (unavailable in studied area

or preserve important rare or endangered plants), as well as this technique

may be used in studies of ontogeny.

Chapter eight General Discussion

147

chapter eight

general discusson

In the preceding chapters of this thesis detailed information has been

provided on the variation in morphology, anatomy, ecology and distribution

of the four species of the genus Euphorbia grown in University of Baghdad

campus. In addition, data of protein electrophoresis on polyacrylamide gel

and RAPD-PCR analysis, as well as investigation of the response of species

studied to callus induction as a new taxonomic tool. These results permit a

clear indication of the systematic position of the four species of Euphorbia.

Latex production is a major feature of the family Euphorbiaceae and a

useful field character. Latex usually signals to toxicity and this is true for this

family. Two floral features provide good recognition characters for the

family; unisexual flowers and tricarpellate gynoecium with each carpel

containing a single seed. The latter produces a three-lobed ovary and fruit

that, when present on pistillate flower, is a very key character. In addition to

the seed type (carunculate or ecarunculate) and configuration which are good

characteristics for the intergeneric and interspecfic taxa of the family to be

able to recognize by sight (www.Euphorbiaceae.org).

According to the flora of China (Bingtao et al., 2008) and the

molecular phylogenetic of Frajman and Scho nswetter (2011) E.helioscopia

and E.peplus belong to subgenus Esula, a group of annual to perennial herbs

and shrubs. Stems often little branched, often hollow, often dying after

flowering, leaves usually alternate; stipules absent; leaf blade symmetrical,


148

usually persistent. Inflorescence usually a terminal pseudoumbel, sometimes

compound, and also with axillary cymes from uppermost axils forming

cylindric thyrse. Cyathia subtended by cyathophylls loger than cyathia,

mostly green, occasionally colored; glands usually 4, sometimes 5, simple

with 2 horns. Seeds with or without caruncle.

However E.granulata and E.hirta belong to subgenus Chamaesyce a

group of herbs or shrubs. Main stem branched, lateral stem usually many.

Leaves opposite; stipules membranous; leaf blade oblique. Cyathia lateral at

nodes, sometimes gathered in to terminal inflorescence by reduction of

subtending leaves, in cymes or solitary; cyathophylls inconspicuous; glands

with pink or white petal like appendages. Seeds not carunculate.

In this first attempt to assess the overall variations in the studied

species of Euphorbia the following characters have been found to be of

greatest value in delimiting the species recognized in the systematic

treatment: 1- Leaf morphology (shape, size, color, presence of trichomes,

presence of petiole and leaf arrangement); 2- Stem morphology (shape, size,

color, presence of stipules, presence of trichomes, type of stem and type of

branching); 3- morphology of cyathium (shape, size, appendages, shape and

color of glands); 4- morphology of stamens; 5- morphology of stigma; 6-

Seed morphology (shape, size, color, configuration and presence of

caruncle). All these morphological characteristics confirm the segregation of

the species studied to the subgenus Esula and Chamaesyce as explained in

flora of China.


149

The stem of E.granulata is pilose on one side, this characteristic found in

local species and as far as we know it is reported for the first time in this

work.

The anatomical characters of vegetative parts of Euphorbia species have

a taxonomic importance. The variation of epidermis and cortex thickness of

the stem; the presence of chlorenchyma in whole cortex of E.granulata and

E.hirta; the presence of wavy central cylinder in the species E.granulata, and

the width of the pith, are characters of taxonomic values, according to our

knowledge it is studied for first time. Metcalfe and Chalk (1950) pointed out

to the presence of terminal tracheids in species of Euphorbia; as well as

sclerenchyma said to be absent from around the veins. The present study has

not noticed or investigated such characters.

In addition, the variations in leaf texture of the species studied have a

taxonomic importance. The mesophyll is differentiated into palisade layer

and spongy layer in E.granulata and E.hirta, but it is undifferentiated in

E.helioscopia and E.peplus (i.e. dorsi-ventral mesophyll). This confirming

the results of Jafari and Nasseh (2009) about E.helioscopia, but disagreed

about E.granulata. Also, they stated in their study that isolateral and dorsi-

ventral mesophyll were in mesophytic and xerophytic species. It sounds the

variation in anatomy characters of studied species is related to ecologic

factors. Cutler et al. (2007) explained, because some leaves lack distinction

of layers and others have very marked layers, the mesophyll can be used as

an aid to identification. It cannot often be used as a guide to taxonomic

position of a plant, but within a group of related plants there may be close

similarities of arrangement. Furthermore, the environmental variations will


150

not alter arrangements that are rigidly controlled by the genome. Palisade

cells can be present next to the upper or lower surface, or both. There are,

however, striking changes that can occur to the layers themselves. Also,

because in some plants the leaves growing in bright light may be thicker and

have more layers of palisade cells than those leaves that have developed in

the shade, this is not sound diagnostic character and is clearly an effect of the

environment (Cutler et al.,2007). This explanation confirms our anatomical

results in relation to habitat of the species studied.

The anatomical study of leaf epidermis revealed several interesting

epidermal features some which have not previously been studied. The surface

of the blade presents several types. Cells with feebly undulated walls seen in

E.helioscopia and E. peplus, undulated in E. granulata and highly undulated

in E.hirta. The undulating pattern has an important taxonomic value

(Metclafe and Chalk, 1950; Evert, 2006; Cutler et al., 2007). Ahmad et al.

(2010) had studied the taxonomic diversity in epidermal cells of some sub-

tropical plant species, and stated that the type of epidermal cells in

E.helioscopia are tetra to penta and hexagonal only on adaxial side and its

walls are wavy on abaxial side. Also, Raju et al. (2008) find that the

epidermal cells in Euphorbiaceae are polygonal or elongated in different

directions and diffusely arranged. The epidermal anticlinal walls are straight,

arched or sinuous.

Leaves are amphistomatic bearing anomocytic, anisocytic and diacytic

stomatal complexes in all the species studied. The mode of stomatal

development has been found to be identical in a given species. Kakkar and

Paliwal (1972) had made a study of epidermis in 150 species of Euphorbia.


151

Stomata of anomo-, para-, anisocytic types have been recorded; anomocytic

type being most common. Often, more than one type of stomata may occur

on the same leaf surface. Besides, they stated that E.peplus has anomocytic

stomatal type, E.granulata has anomocytic, anisocytic and paracytic stomatal

type whereas E.hirta has anomocytic, para-, anomo- and dia-cytic stomatal

type.

In the mean time, Kandalkar et al. (2009) and Aworinde et al. (2009) pointed

out to the presence of anomocytic type of stomatal complex in E.hirta, which

agreed with our findings. Aworinde et al.(2009) stated that leaf epidermal

characters such as pattern of epidermal cells, types of stomata and presence

of trichomes are constant in some species of Euphorbia and variable in

others, and thus of great significant in understanding the relation between and

within species. Moreover, the stomatal types found on the adaxial surface

may differ from those on the abaxial surface of the same species. In spite of

fact that, vegetative and floral characters are markedly modified in relation to

the habitat and pollination mechanism (Kapil and Bhatnagar, 1994).

There is another valuable character, E.granulata and E.hirta differs from

the others in having multicellular uniseriate rugose hairs (trichomes) on all

parts of the cyathium except style and stigma, as well as on leaves and stems,

with special ornamental epidermis. The hair base is surrounded by stellated

arranged epidermal cells ranged between 12-14 cells around the base of each

hair in E.hirta and 7-8 cells around the base of each hair in E.granulata. The

epidermal cells appear convex in surface appearance. As we know, these

results are stated for first time in our work. Although trichomes vary widely

in structure within families and smaller groups of plants, they are sometimes


152

remarkably uniform in a given taxon and have long been used for taxonomic

purposes (Evert, 2006; Cutler et al., 2007).

Evert (2006) explained that plants growing in arid habitats tend to have

hairier leaves than similar plants from more mesic habitats. Studies of arid-

land plants indicate that increase in leaf hairiness reduces the transpiration

rate by (1) increasing the reflection of solar radiation, which lowers leaf

temperatures, and (2) increasing the boundary layer (the layer of still air

through which water vapor must diffuse). Moreover the basal or stalk cells of

the trichomes of at least some xeromorphic leaves are completely cutinized,

precluding apoplastic water flow into the trichomes. Many air plants utilize

foliar trichomes for the absorption of water and minerals (Evert, 2006). The

greatest value of hairs is in identification, that is, they have high diagnostic

value. They are constant in a species when present, or show a constant range

of form (Metclafe and Chalk, 1950). Consequently, small fragments of leaf

with hairs can often be matched with known material. Examination of hair

types can help in quality control, for example of dried herbs, like mint

(Mentha) where cheaper substitutes may have been added. Also, in some

families individual can be defined on the form of their hairs alone (Cutler et

al., 2007).

Laticifers present constant characters and has important taxonomic value

for Euphorbiaceae (Metcalfe and Chalk, 1950; Webster, 1994; Evert, 2006;

Cutler et al., 2007; Biesboer and Mahlberg, 2008). Laticifers are present in

all vegetative organs of all the species studied. They are undoubtedly served

as systems to sequester toxic secondary metabolites, which may function as

protection against herbivores (Raven et al., 2005). Systematic comparative


153

studies of laticifers are scarce, and possible phylogenetic significance of the

variation in the degree of their specialization has not yet been revealed, in

spite of laticifers have been object of intensive study since the early days of

plant anatomy for instance De Bary (1884) and Sperlich (1939) studies

(Evert, 2006). Starch grains occur in laticifers of some genera of

Euphorbiaceae (Metcalfe and Chalk, 1950, Mahlberg and Assi, 2002; Evert,

2006; Biesboer and Mahlberg, 2008) and assume various forms -rod, needle-

like, osteoid, discoid, and intermediate forms- and may become very large.

Metcalfe and Chalk (1950) pointed out to the presence of rod-or bone shaped

in species of Euphorbia, Hippomane, Hura, and Pedilanthus. This agreed

with our results.

Gales et al. (2008a) had studied the morphology and anatomy of the fruit and

seed development of E.helioscopia which agreed with our findings in

morphological aspects of the ovary. Additionally they explained that the one

layered epidermis of the ovary wall shows xeromorphic characters. But it is

worth to mention that the morphological view of seeds is incorrect, they

presented the seed view of E.peplus instead of E.helioscopia.

Despite the diversity of Euphorbia species, their inflorescence has been a

homogeneous organization, whose rendering during the time have launched

to contradictory hypothesis. Tournefort (1700), Linnaeus (1753), Payer

(1857) and Baillon (1858) concluded that the true status is that of a single

hermaphroditic flower. Le Maout (1842) adduces a new interpretation of this

curious “flower”, named cyathium by Warming (1912), the notion being a

long time discussed (Gales et al., 2008).


154

Mishra and Sahu (1983) have studied the cyathial characteristics and hair

types of some prostrate Euphorbias common in India including E.hirta. The

morphological results of current study agreed with their findings in the base

of cyathial characteristics and hair types of E.hirta, also they reported that the

study of trichome types and their organographic distribution on the cyathium

provide and aid to the differentiation of the species.

Also the species of Euphorbia exhibit variations -ecotypes- when grown

under different conditions of light intensity and soil moisture as mentioned in

several literatures (Metcalfe and Chalk, 1950; Mangaly et al., 1979). We

found the occurrence of two ecotypes in species E.helioscopia and E.hirta,

one erect and the other prostrate in agreement with Ramakrishan (1960) and

Mangaly et al.(1979).

Scientists have agreed for some time that a functional and objective

classification system must reflect actual evolutionary processes and genetic

relationships, the technological means for creating such a system did not exist

until recently (www.en.Wikipedia.org).

The banding pattern that forms in an experiment like protein

polyacrylamide gel electrophoresis is distinctive for each set of proteins of

species studied. The total number of protein bands of the studied species of

Euphorbia ranged between (9-10) bands, with molecular weight ranging from

(10.00 to 93.33) KDa. These results confirm the segregation of the four

species clearly, as well as indicates to the similarity between E.helioscopia

and E.peplus that belong to subgenus Esula, and the similarity between

E.granulata and E.hirta that belong to the subgenus Chamaesyce.


155

In fact, comparison of the same protein in different species can show

different banding patterns in the different species (Werner and Sink, 1977;

Al-Jibouri and Dham, 1989; Jensen et al., 1994). Using this sort of

distinguishing information, scientists can be helped to identify to what plant

species an individual plant belongs. Protein is gene product, so this explain

the genetic differences between the studied species. These genetic variations

are confirmed by the phenotypic results among the differences of species; as

well as explain similarity in certain features. Jensen et al. (1994) reported that

the classification dilemma illustrates the need for additional taxonomic and

phylogenetic criteria to improve our understanding of the systematic of

Euphorbiaceae; it is worth to mention that several posteriori statements have

been supported by the evidence seed proteins have provided in a variety of

systematic studies such as, the studies of Cristofolini, 1980; Fairbrothers,

1983; Jensen and Fairbrothers, 1983.

The genetic identification can be performed by examining morphological

or phenotypical characteristics but such characteristics are affected by

environmental conditions. However, DNA based techniques allow scanning

the genome directly without being environmental affected. Today genetic

variety or similarity can be revealed in short time and easily, and the

population can be examined rapidly through RAPD- PCR technique. In

present study, the results of RAPD- PCR confirm the isolation of the four

species of Euphorbia from each other obviously. Because even closely

related individuals may show some sequence variation that may determine

potential primer sites, these different individuals will show different

amplification products (Simpson, 2006).


156

It was concluded that RAPD markers could be used to gain information

about genetic similarities or differences that are not evident from pedigree

information (Demeke and Adams, 1994). In many cases, molecular data have

supported the monophyly of groups that were recognized on morphological

ground. More importantly, molecular data often have allowed systematists to

choose among competing hypotheses of relationships. In other cases,

molecular data have allowed the placement of taxa whose relationships were

known to be problematic (Judd et al., 1999). Zimmermann et al. (2010)

summarize the molecular evidence about the phylogenetic relationships

within Euphorbia, their results support the hypothesis that Euphorbia

evolved in Africa from progenitors of subgenus Esula. In the meantime

Frajman and Scho nswetter (2011) stated that the majority of Euphorbia

belongs to subgenus Esula, including about 500 taxa, and the phylogenetic

relationships among its constituents remain poorly understood; they have

sampled DNA sequences from about 100 European taxa of subgenus Esula in

order to infer its phylogenetic history. The data support monophyly of

subgenus Esula. As well as, character state reconstruction illustrates that the

annual life form developed independently several times in different clades of

subgenus Esula from perennial ancestors.

Scientific research in the field of purely taxonomic research through to the

technical applications aims to conservation for all plants groups, and their

pragmatic applications in the floriculture, herbal and medicinal plant

industries. In present investigation the response for callus induction in the

studied species of Euphorbia showed significant differences according to

different parameters: (1)The species, (2) concentrations of the auxin 2,4-D

(3) type of explants and (4) incubation conditions. The results of this


157

investigation pointed to the segregation of the studied species on the base of

genotypes. Vardja and Vardja (2001) inferred that all cells of the plants

normally carry the same genetic information, but the morphogenic responses

vary according to the spatial and temporal distribution of the cells and their

physiological and developmental stages. As well as, the genetic make-up,

varied endogenous concentrations of growth hormones and response of the

genotype to different concentrations of growth hormones play a key role.

We believe that the response for callus induction is a reflection of species

genotype; so this behavior may be used as one of secondary distinction tools

among the species studied. As well as micropropagation can be used

to conserve rare or endangered plant species, in addition to the other

applications. Kondamudi et al. (2009) explained the importance of

Euphorbiaceae and their economic value and hence contribute to the floristic

wealth of tropical and subtropical countries of the world. The family

comprises a number of endemic and endangered taxa. However the in vitro

tissue culture studies are confined only to few genera of aesthetic, medicinal,

timber yielding, rubber yielding, dye yielding, cottage industries, ornamental

and food crops.

It is obvious from the above and referring to data reported further

studies are necessary, including both field and laboratory work, but it is

hoped that this work has provided information leading to above scenario

which gives a firm basis for future investigations.

Chapter nine Conclusions & Recommendations

158

conclusions

1- Morphological study of the vegetative parts revealed several interesting

taxonomic characteristics. The Presence of petiole, stipules and trichomes

are important taxonomic characteristics for stems and leaves. Also the

cyathium reveals obvious characteristics in all the species studied. The

taxonomic value of the seeds are clearly recognized. The largest seed

diameter recorded in E.helioscopia. Seeds have different colors and

distinct configurations. The presence and type of caruncle have

taxonomic importance too.

2- Anatomical study reveals constant taxonomical characteristics such as:

* Presence of constitute chlorenchyma in whole cortex of E.granulata

and E.hirta stems, three outer rows in E.peplus, in time E.helioscopia

lack chlorenchyma.

* Presence of distinct wavy central cylinder in E.granulata.

* Presence of differentiated mesophyll into palisade layer and spongy

layer in E.granulata and E.hirta; and undifferentiated in E.helioscopia

and E.peplus.

* Presence of distinct leaf pattern of epidermal cells. Presence of

anomocytic, anisocytic and paracytic stomatal complexes, as well as

presence of trichomes which are significant constant characteristics in

species studied.


159

3- From an environmental perspective E.helioscopia showed a wider range

of distribution, while E.hirta showed such confined distribution in Iraq

and at the University of Baghdad Campus, with xerophytic and

mesophytic habitat. E.helioscopia, E.peplus and E.hirta are nnual herbs,

whereas E.granulata is perennial.

4- The banding pattern of protein electrophoresis reveals the isolation of the

four species of Euphorbia, in addition to converge E.helioscopia with

E.peplus, and E.hirta with E.granulata.

5- RAPD-PCR technique confirms the isolation of the four species of

Euphorbia obviously. The analyses based on three primers A13, C05 and

D20 that gave results in term of amplification and polymorphism. Primer

A13 produced the highest percent of genetic polymorphism compared

with primer C05. These primers could be used as markers distinguishing

the studied Euphorbia species.

6- E.hirta achieved the highest response for callus induction reached (75-

100)%, whereas E.helioscopia achieved the lowest response for callus

induction reached (25-50) %. Also, E.peplus nodule explants incubated in

dark and E.hirta nodule explants incubated in light achieved the highest

callus production compared with the other species studied. These

differences give more confirm to morphological, anatomical and

molecular characteristics.


160

recommendations

1- Investigation of ecology and geographical distribution for all species of

Euphorbia in Iraq and try to find out new taxa.

2- Comparative studies are needed for all taxa of the genus Euphorbia

grown in Iraq including all parts of plant.

3- Expansion in study of detailed morphological, anatomical and pollen

grain features of species of Euphorbia by using electron microscope.

4- Conduct chemical investigation by using the technique of HPLC or GC

for all Iraqi species.

5- Using wider range of universal primers to find if there is any genetic

relationship among the studied species as well as among the other

species of Euphorbia.

6- Adoption of DNA markers and molecular techniques in futuristic

taxonomical studies.

7- Adoption of the technique of tissue culture as a part of biosystematical

studies that can be used as secondary supported characteristics.

Additionally its importance in studies of ontogeny and conserve rare

or endangered plant species.

Refferences

161

references

a

· Adedapo, A., Shabi, O. and Adedokun, O. (2005). Anthelmintic Efficacy of The

Aqueous Crude Extract of Euphorbia hirta Linn. In Nigerian Dogs. Veterinarski Arhiv.

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Euph. Planetary Biodiversity Inventory Project (PBI).

· http://www.en.Wikipedia.org

History of plant systematic.

31

Table: ( 2-1 ) Morphological characters of stems of studied species of Euphorbia

species Stem Length (cm)

Stem Width (cm)

Stem Shape

Stem Color

Stem Type Type of branching

E.helioscopia (15-70) 28 (0.6-1.8) 0.8 cylindrical green to red

erect or ascending

single or branched at base

E.peplus (10-35) 20 (0.3-0.5) 0.35 cylindrical pale green erect or ascending

single or branched at base

E.granulata (6-20) 12 (0.1-0.2) 0.15 cylindrical pale brown

ascending or prostrate

usually un-branched, occasionally branched

at end

E.hirta (30-75) 40 (0.3-0.5) 0.33 cylindrical green to red

ascending to erect, or prostrate

branched from middle or both

33

Table : ( 2-2 ) Morphological characters of the leaves of studied species of Euphorbia

spp. Apex Margin shape Base Trichome color Arrange-ment

leaf length (cm)

leaf width (cm)

petiole length (cm)

petiole width (cm)

E. helioscopia rounded dentate obovate to spatulate cuneate _ yellowish

green alternate (1.5-4.5) 3.0

(0.7-1.8) 1.5

absent absent

E. peplus rounded entire obovate to spatulate

cuneate below _ pale

green alternate (1.0-2) 1.2

(0.5-1.0) 0.7

absent absent

E. granulata obovate

or oblong

entire subelliptic obliquely rounded

₊ green to red opposite (0.3-0.6)

0.45 (0.2-0.4)

0.25 0.1 0.09

E. hirta acute

entire or few

serrate below middle

lanceolate-oblong oblique ₊₊ green to

red opposite (2.5-3.5) 2.8

(0.3-1.6) 0.8

0.35 0.2

37

Table: (2-3) Morphological characters of the cyathia of studied species of Euphorbia

SPP. type of cyathium

shape of involucre

shape of lobes

no. of glands appendages color of

gland shape of

gland apex of gland

E.helioscopia Sub-sessile campanulate 2.5×2 mm

rounded smooth and

glabrous pilose at margin

4 no appendages bright green

Disk-like shortly concave

no horned

E.peplus Sub-sessile cuplike 1×1 mm

rounded, ciliate 4 no

appendages bright green crescent-shaped

2- horned

E.granulata

single, axillary

peduncle exist

turbinate 1.5×1.5 -2

mm

Sub-truncate white pilose,

marginal lobes 5

4

white & unequal narrow

adaxially

pale brown irregular

concave or pepand

no horned

E.hirta

dens head-like

pedunclate cymes at

upper nodes

campanulate 1×1 mm

triangular-ovate, pilose,

marginal lobes 5

4

white to reddish

margin entire to slightly undulate

red

rounded to transversely

elliptic, center

slightly sunken

no horned

40

Table: (2-4) Morphological characters of male and female flowers of studied species of Euphorbia

species

no. of male

flower

diameter of

anther (mm

)

diameter of

filament (m

m)

color of filam

ent

length of pedicel (m

m)

ovary shape &

diameter (m

m)

length of ovary pedical (m

m)

no. of style

length of style (m

m)

branches of style

no. of stigma

stigma shape &

color

E.helioscopia (5-15) 9 0.2×0.1 0.2×0.01 white 0.8 3 lobed (3×3) 1 3 0.4 2 6

round, pale

yellow

E.peplus (5-15) 9 0.2×0.1 0.2×0.01 white 1 3 lobed (1.5×1.6) 2.8 3 0.2 2 6

round, pale

yellow

E.granulata ( 4-10) 6 0.03×0.01 0.02×0.01 white 0.06 3 lobed (1.2×1.4) 3.5 3 0.02 2 6 discoid,

red

E.hirta ( 9-12) 10 0.025×0.01 0.04×0.01 white 0.5 3 lobed (1×1) 1.5 3 0.35 2 6 discoid,

red

42

Table: ( 2-5) Morphological characters of seeds of studied species of Euphorbia

species Seed shape Seed diameter (mm) Seed color & configuration Seed caruncle

E.helioscopia ovoid 2.0×1.5 dark brown,reticulately wrinkled compressed, white, sessile

E.peplus ovoid-angulate 1.3×0.8 gray or gray white, each surface with 3 -5 micropores

peltate, yellow - white, sessile

E.granulata Subglobose-tetragonal 0.8×0.5 browen to reddish, wavy wrinkled absent

E.hirta tetragonal 1.5×1.0 gray, adaxially grooved, smooth absent

Summary

The present study has dealt with many biosystematic aspects

for four species of Euphorbia L. grown in University of Baghdad

campus-Jadiriyah. The species were compared to the adoption of

field and herbarium specimens.

A detailed morphological study of the stems, leaves, cyathia

and seeds is presented, and revealed several interesting taxonomic

characteristics, some of which have not previously been studied in

Iraq. The Presence of petiole, stipules and trichomes are important

taxonomic characteristics for stems and leaves. The largest leaf size

recorded in E.helioscopia L. and the smallest in E.granulata Forssk..

Also the cyathium reveals obvious characteristics in all the species

studied. The taxonomic value of the seeds are clearly recognized.

The largest seed diameter recorded in E.helioscopia and the

smallest in E.hirta L.. Also, Seeds have different colors and distinct

configurations. The presence and type of caruncle have taxonomic

importance too, E.helioscopia and E.peplus L. have white sessile

caruncle, while E.granulata and E.hirta are ecarunculate.

Anatomical studies reveals constant taxonomical

characteristics such as in the stems, the chlorenchyma of E.granulata

and E.hirta constitute the whole cortex of the stems, but with three

outer rows in E.peplus, in time that the cortex of E.helioscopia lack

chloroplast and presence of distinct wavy central cylinder in

E.granulata. Leaf anatomy was strongly supported in species

segregation, so that mesophyll is differentiated into palisade layer

and spongy layer in E.granulata and E.hirta, but it is undifferentiated

in E.helioscopia and E.peplus. Leaf pattern of epidermal cells and

presence of trichomes are significant constant characteristics in

species studied. From an environmental perspective has been

studying the habitat and the distribution of the herbarium species in

Iraq and at the University of Baghdad Campus, and a map was

prepared for distributions of the four species on the provinces.

E.helioscopia showed a wider range of distribution, while E.hirta

showed such confined distribution. The chief interest focus in those

species that inhabit very dry places and have consequently a

xerophytic habit. E.helioscopia, E.peplus and E.hirta are annual

herbs, whereas E.granulata is perennial herbs and they are mostly

distributed in desert and alluvial plane region of Iraq.

From chemical and genetic perspective there were studies to

perform a protein electrophoresis using polyacrylamide gel

electrophoresis for the total protein extracted from dry seeds of

species studied. The total number of protein bands were (9 and 10)

bands, with molecular weight ranged between (10.00-93.32)KDa. The

banding pattern reveals great significance in understanding the

relationships among species. Also there was an attempt to identify

the four species of Euphorbia and find the genetic polymorphism

among them by using DNA markers in Polymerase Chain Reaction

(PCR) technique. Total genomic DNA of species studied was

extracted from dry seeds by using commercial kit. Molecular analysis

was performed by using nine random markers in Random Amplified

Polymorphic DNA (RAPD-PCR) technique. RAPD-PCR analyses

based on three primers A13, C05 and D20) that gave results in term

of amplification and polymorphisim for the four species studied. The

genetic polymorphisms value of each primer was determined and

ranged between (47-84%), primer A13 produced the highest percent

of genetic polymorphism compared with primer C05. (RAPD-PCR)

technique confirm the isolation of the four species of Euphorbia

obviously.

To complete the molecular studies on the species there was an

attempt to investigate the response of the species for callus induction

on Murashige and Skoog's (MS) medium supplemented with different

concentrations of auxin hormone 2,4-Dichlorophenoxy acetic acid

(2,4-D) by using nodule and leaf explants incubated in dark and light

conditions. E.hirta achieved the highest response for callus induction

reached (75-100) % , whereas E.helioscopia achieved the lowest

response for callus induction reached (25-50) %. Additionally

E.peplus nodule explants incubated in dark and E.hirta nodule

explants incubated in light achieved the highest callus production

compared with the other species studied. These differences give

more confirm to morphological, anatomical and molecular

characteristics, and can be used as a secondary supported

characteristics used in distinction and identification of Euphorbia

species.

الخالصة

أنواع من الجنس تعاملت الدراسة الحالية مع العديد من الجوانب التصنيفية الحياتية الربعة

Euphorbia النامية في مجمع جامعة بغداد-الجادرية، حيث جرت مقارنة االنواع باالعتماد على

العينات الحقلية و المعشبية.

قدمت دراسة مظهرية مفصلة للسيقان و االوراق والنورة الكاسية و البذور، بعضها لم

تدرس مسبقا في العراق. وجدت الدراسة ان للسويقات و االذينات و الشعيرات خصائص هامة

اكبر حجم للورقة، والنوع .E.helioscopia L بالنسبة للسيقان واالوراق. سجل النوع

E.hirta L. اصغرها. كما بينت النورة الكاسية صفات مميزة في جميع االنواع التي شملتها

الدراسة. وكانت القيمة التصنيفية للبذور واضحة تماما، اذ كان لها الوان وزخارف مميزة. وكانت

اصغرها، وكان .E.granulata Forsskاكبرها حجما، وبذور E.helioscopia بذور

و L. E.peplus اهمية تصنيفية مميزة ايضا للنوعينCaruncleللجسم االسفنجي

E.helioscopia حيث تكون جالسة sessile بيضاء اللون، بينما تكون مفقودة في النوعين

E.granulata و E.hirta .

اظهرت الدراسة التشريحية خصائص تصنيفية ثابتة في السيقان اذ تشكل الكلورنكيما في

E.peplus القشرة باكملها، لكنها تشكل ثالثة صفوف في E.hirta و E.granulataالنوعين

فضال عن وجود اسطوانة مركزية E.helioscopia.، وتكون عديمة البالستيدات في النوع

. اعطى تشريح الورقة دعما قويا لعزل االنواع اذ تكون E.granulataمتموجة واضحة في

E.granulata طبقة الميزوفيل متمايزة الى طبقة عمادية و طبقة اسفنجية في النوعين

. ووجد ان انماط E.peplus و E.helioscopia ، وغير متمايزة في النوعين E.hirtaو

البشرة ووجود الشعيرات هي خصائص ثابتة مهمة في االنواع التي شملتها الدراسة.

من الناحية البيئية تم مسح للعينات المعشبية ودراسة انتشارها في العراق و في حرم جامعة

بغداد، وأعدت خارطة لتوزيع االنواع على المقاطعات . وتركز االهتمام على االنواع التي تستوطن

هي E.hirta وE.peplus و E.helioscopiaالمناطق الجافة جدا خاصة الصحراوية. االنواع

هي اعشاب معمرة، تنتشر على االغلب في المنطقة E.granulataاعشاب حولية في حين

الصحراوية و منطقة السهل الرسوبي في العراق.

من الناحية الكيميائية والجينية تم اجراء الترحيل الكهربائي للبروتين باستخدام هالم البولي

بالنسبة للبروتين الكلي المستخلص من polyacrylamide gel electrophoresis اكريالميد

حزم عند 10 و9البذور الجافة لالنواع التي شملتها الدراسة. وكان العدد االجمالي لحزم البروتين

) كيلو دالتون. كما يظهر نمط توزيع الحزم اهمية 10.00-39.32 ( وزن جزيئي يتراوح ما بين

، Euphorbiaكبيرة في فهم العالقة بين االنواع فضال عن محاولة تشخيص االنواع االربعة لل

المعتمدة على تقنية انزيم DNA markersوايجاد التباينات الوراثية باستخدام مؤشرات الدنا

وتم استخالص الدنا Polymerase Chain Reaction (PCR) . بلمرة الدنا المتسلسل

. Kit من البذور الجافة باستخدام العدة الخاصة بالعزل Total genomic DNAالمجيني الكلي

Random Amplified تقنية اجري التحليل الجزيئي باستخدام تسع بادئات عشوائية باستخدام

Polymorphic DNA(RAPD-PCR) اعتمد التحليل الجزيئي العشوائي على ثالث بادئات .

التي اعطت بدورها نتائجا للتضاعفات وتباينات وراثية لالنواع االربعة D20 وC05 و A13هي

التي شملتها الدراسة. وتم تحديد قيمة التباين الوراثي التي انتجتها البادئات الوراثية وتراوحت بين

. اكدت C05 اكبر عدد من الحزم المتباينة مقارنة مع البادئ A13 % . وقد حقق البادئ 47-84

بشكل واضح تماما. Euphorbia انعزال االنواع االربعة للجنسRAPDتقنية ال

ألستكمال الدراسات الجزيئية لالنواع كانت هناك محاولة للتحري عن استجابة االنواع

المجهز بتراكيز Murashige and Skoog (MS)على وسط Callus الستحداث الكالس

ستخدام نبيتات با2,4-Dichlorophenoxy acetic acid (D-2,4)مختلفة من منظم النمو

حقق المحضنة في ظروف الظالم والضوء. Stem and leaf explantsالساق والورقة

E.hirtaفي حين حقق %،100-75 اعلى استجابة للكالس E.helioscopia اقل استجابة

المحضنة في ظروف الظالم، ونبيتات E.peplus%. كما اعطت نبيتات الساق للنوع 25-50

المحضنة في ظروف الضوء اعلى انتاجية للكالس مقارنة مع االنواع E.hirtaالساق للنوع

االخرى. هذه النتائج تعطي مزيدا من التاكيد على الصفات المظهرية والتشريحية والجزيئية ويمكن

. Euphorbiaاستخدامها بوصفها صفات ثانوية ساندة في عزل وتشخيص انواع ال

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