9.agri sci - ijasr - applications full

7/29/2019 9.Agri Sci - IJASR - Applications Full

1/22

APPLICATIONS OF BIOTECHNOLOGY ON EGYPTIAN CLOVER

[(BERSEEM)( TRIFOLIUM ALEXANDRINUML.)]

EHAB MOHAMED ZAYED

Forage Crops Research Department, Field Crops Research Institute, Agriculture Research Center, Giza-12619, Egypt

ABSTRACT

Berseem is the added value in Egyptian economic world. The cycle of berseem capital equal tens B.US$

Berseem is the main forage crops for livestock and have produce milk and /or meat production in Egypt. So too berseem is

the guard on Egyptian soil fertility. Berseem clover was planted since 6000 years BC. Egyptian clover have many

scientific problems that need to integrate highly technical studies of bio-and what led him to provide solutions to the

breeders of Egyptian clover. The purpose of this study: review of some the results of research in Egypt and the world

areas: biotechnology and some branches of biotechnology such as tissue culture, genetic transformation. As well as, was

an also signed result of molecular genetics of protein and isozymes, and molecular markers of RAPD, ISSR, SSR-PCRs

and AFLP. Studies have been mentioned Cytogenetics to distinguish between the two types of a monocut berseem Fahl

and multicut berseem clover. Models of tolerant berseem the harsh conditions were explored, such as tolerating heat and

cold. And ultimately was presented a map of the berseem wild type, as well as wild relative species. Also, it included that

the origin of berseem has not yet been decided. It is clear that, the origin of berseem needs of many of the efforts between

the different countries to approve it; but likely to be the origin of berseem in Egypt.

KEYWORDS: RAPD, ISSR, SSR-PCRs and AFLP, Berseem clover, Cytogenetics

INTRODUCTION

Berseem are the add value in Egyptian economic. The cycle of berseem capital equal tens billions American

dollar. If the disarmament of berseem Egyptian agriculture map there will be no livestock or produce milk or meat. So too

is the guard on Egyptian soil fertility. Berseem is the Arabic and Coptic word. .And it grows in Egypt since 6000 years

BC. Egyptian clover, (Trifolium alexandrinum L.), is the traditional forage crop in Egypt. It is grown as an annual crop in

the winter and spring. After several cuttings for animal feeding and hay use, a few fields are left for an extra month to

harvest seeds, while the rest of the crop is plowed as a preparation way for the summer crops cultivation. Egyptian clover

is part of a mandated crop rotation in the Nile delta where cotton, rice and mays are grown,( Zayed et al, 2011, Zayed et al,

2010 and Tarrad and Zayed, 2009). Breeding Egyptian clover has many scientific problems that need to be highly studied

through bio-and molecular basis, which could provide solutions such problems. There is a need needs to define the genes

responsible for drought, salinity, diseases and insects resistance in berseem, and explain the mechanical injury of these

abiotic and biotic stresses at the molecular level. It needs also to draw a genetic map, and the relationship of commercial

varieties and wild type, and whether we can offer the genetically engineered Egyptian clover. Also, berseem need a strong

push at the increasing number of studies in this field which we hope it will improve the clover cultivation future in Egypt

and the countries that grows it.

In providing this historical account of the science as well as the personalities of plant biotechnology, and the many

debates and controversies, the review highlights the value of historical studies in science education and research (Indra K.

Vasil 2008). The concept of biotechnology application has raised early eighties of the last century. January 1983, is

International Journal of AgriculturalScience and Research (IJASR)

ISSN 2250-0057

Vol. 3, Issue 1, Mar 2013, 99-120

TJPRC Pvt. Ltd.


2/22

100 Ehab Mohamed Zayedarguably one of the most important dates in the history of plant biotechnology, where genetic engineering has generated, at

the Miami Winter Symposium. In this congress, three independent groups describedAgrobacterium tumefaciens-mediated

genetic transformation, leading to the production of normal, fertile transgenic plants. The global acreage of biotech crops

increased rapidlyat an annual rate in excess of 10%so that in 2007 more than 282 million acres (nearly 8% of total

world crop acreage) were planted in 23 countries, for a cumulative (19962007) total acreage of over 1.7 billion acres(James 2007).

There are two phases in biotechnology on Egyptian clover, the first phase was the establishment of the techniques

at the beginning eighties. The second phase is application of biotechnology techniques which started in mid-nineties and

continued to current time. Unfortunately, a little work of biotechnology on Egyptian clover was published so far. The

total number of publication in berseem from nineties to 2012 is 1872 (ENAL 2012),amongst only 199 paper published on

the field of biotechnology representing only 10.6% (ENAL 2012). More information of biotechnology statistics on

berseem are in the following site: (http://srv5.eulc.edu.eg/eulc_v5/libraries/start.aspx?fn=StuffPapersView&ScopeID=1.)

Biotechnology is a tool to help plant breeder to solve problems, which are unable to be solved by conventional

methods. We are trying to implement molecular breeding concept in our breeding program. The biotechnological

approaches include molecular techniques for understanding the genetic structure of the plants, inserting foreign genes

directly into the plant genome, in-vitro regeneration of plants from any plant part. A number of techniques such as:

Embryo rescue, Micro-propagation (somatic embryogenesis and organogenesis), Androgenic haploid plant production and

creation of and induced variations help at one or more steps involved in conventional breeding methods. These techniques

save time and increase efficiency compared with conventional methods. The benefits of these techniques are to reduce the

time needed to produce new genotypes from 6 years (in traditional methods) to two years only with more genetic

purification and lower coasts.

Biotechnological approach offers opportunities as it create variations in berseem which as such are not possible

through conventional methods. For example: somaclonal variation, somatic hybridization, and genetic transformation, etc

and Insertions of genes system may be by: chemical, electrical, and physical or micro-projectile transfer. In, quality,

improving the balance of the Egyptian clover plants used in intensive feeding production systems. Furthermore, level from

hydrocyanic and saponins which determined the level quality feed. There are several molecular techniques can be used in

berseem. The first techniques are DNA markers as Restriction fragment length polymorphism (RFLP), Amplified

fragment length polymorphism (AFLP), Random amplified polymorphic DNA (RAPD) Inter simple sequence repeat

(ISSR), simple sequence repeat (SSR), single nucleotide polymorphism(SNP) and r.RNA (gold standard) etc. The second

techniques are biochemical markers which are divided to protein and isozymes. Molecular techniques are used for

characterization of germplasm, cultivar identification, detection of hybrids and genetic mapping, quantitative trait loci

(QTL) identification and gene tagging. In addition , the biodiversity in berseem genetic resources and microorganisms need

more work. On the other hand, molecular techniques help in quality control.

However we face several challenges in berseem improvement such as most of the cultivars are synthetic

populations and variability exists within the population. However, the efforts have been made for characterizing the

varieties based on isozymes, RAPD, RFLP markers and discrimination between the varieties can be based on gene

frequencies. In addition, infrastructure: the high technology equipments and apparatuses is raising the level of capacity

building to increase more results. The sequencer, Nano-drops, Real-time-PCR and atomized PCR are helping to get

genotyping, gene identification and RNA system. Genetic mapping and gene tagging have not been attempted much.

Stress tolerance, both biotic and abiotic. Inthis paper we review some biotechnology works done on Egyptian clover in the


3/22

Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 101

following topics main tissue culture, molecular and cytological research works between have been done. Also, discuss

several paper related to origin of berseem wild types and their side presence in Egypt.

Tissue Culture

The studies on Egyptian clover based on direct and indirect somatic embryogenesis. studies on the morphogenicability of clover (Trifolium alexandrinum L.) through tissue culture (Sultan 1990). The results revealed the leaf is the best

explant to callus production as well as the low concentration from growth regulator 2,4-D. Regeneration was produced in

media have BA(benzyl adenine) for shooting and the same media have IBA(Indole butric acid) for rooting (Sultan 1990).

In the Tissue Culture Laboratory, Department of Crop Science, Faculty of Agriculture, University of Alexandria, Egypt

Barakat (1990) who successes in shoot morphogenetic development with the cultivars Sakha 4 and Giza 10 (Figure 2) on

the Murashige and Skoog (1962) medium containing 2.0 mg l -1 NAA and 0.5 mg l-1 BAP was able to induce callus from

different explants. As well as, he successes to establish cell suspension cultures from hypocotyl and cotyledons derived

callus. In addition to, Abogadallah and Quick (2010) reported new fast procedures for regeneration of Egyptian clover that

are applicable to the regeneration of various genotypes (Mescawi-ahaly, Sakha3 and Sakha4)(Figure 1). Shoots were

regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthalene acetic

acid/benzyl adenine (NAA/BA) and naphthalene acetic acid/thidiazuron (NAA/TDZ) media. They found the highest shoot

regeneration frequencies were obtained from intact cotyledons on NAA/BA (0.05 mg l-1 NAA combined with 2.0 mg l-1

BA) and NAA/TDZ (0.05 mg l-1 NAA combined with 1.0 mg l-1 TDZ) media (66.2 and 43.1% respectively) compared to

18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively. 21.0% shoot regeneration frequency

was observed for hypocotyls on NAA/BA (2.0 mg l-1 NAA combined with 0.5 mg l-1 BA) medium but no regeneration was

obtained on NAA/TDZ medium ( Figure 1). Rooting of the regenerated shoots was induced on indole butyric acid (IBA:

0.24 mg l-1) or NAA (2.0 mg l-1) media where IBA medium supported significantly higher frequencies of rooting as well as

survival of the whole plantlets after transfer to soil, the time scale of whole plantlet regeneration ranged from 7.5 weeks for

intact and wounded cotyledons to 10 weeks for hypocotyl explants (Figure 1)

Fig. 1: Steps in the regeneration of Mescawi-ahaly clover from intact cotyledons (ae), wounded cotyledons (fi)

and hypocotyls (jn).(a) 3 days old seedling from which intact cotyledons were dissected by cutting at the arrow. (b)

Dissected cotyledons immediately beforeculturing. (c) Small shoots regenerated on the cotyledon stalks after 2 weeks from

culture initiation. (d) Same as c but at higher magnification.e Fully developed shoots after 4.5 weeks from culture

initiation.(f) Cotyledons were obtained by cutting at the arrow. (g)Wounded cotyledons after 3 days of culturing. (h) Shoot

regeneration on wounded cotyledons after 3 weeks of culturing. (i) Fully developed shoots after 4.5 weeks and


4/22

102 Ehab Mohamed Zayedimmediately before sub culturing onto rooting medium. (j) The 5 days old seedling used as source of hypocotyl explants,

arrows delimit the part of seedling used as explant. (k) Isolated hypocotyl explant.(l) Small shoots developing on

hypocotyls explant after 4 weeksof culturing. (m) Fully developed shoots after 6 weeks from culture initiation. (n) As in m

but shoots separated .Source :Abogadallah and Quick (2010).

The tissue culture system is divided two main methods direct and indirect somatic embryogenesis,and direct and

indirect organogenesis (Figure 1). The most publication talk about somatic embryogenesis. Aly et al.,(1994) established

somatic embryogenesis tissue culture system and showed the different steps to produce plantlets. On the other hand, the

organogenesis was studied in Bhowal et al.,(2011). They established a protocol for direct organogenesis was developed

for this particular plant of berseem by inoculating the explants (shoot tip, cotyledonary node without cotyledons, cotyledon

and hypo-cotyledon) on different media like MS and L2. Shoot tip was found to be the best responsive explants for direct

organogenesis. Subsequently, it was found that 75% of explants produced shoots on L2 media supplemented with BAP (1

mg l-1

) and IAA (0.1 mg l-1

). The shoots were produced after 5 days of inoculation. Direct organogenesis from the shoot

tip gives a pure line which can be utilized in genetic improvement of this crop plant. In beside that, Masoud and

Hamta(2008) studied the potential of somaclonal variation in berseem clover.

Figure 2: First Division of Berseem Cotyledon Protoplasts (Barakat 1990)

The protocol for in vitro screening for salt tolerance in Trifolium alexandrinum using direct germination of mature

embryos excised from the flower buds of three ecotypes of berseem (Saidi, Fahl and Miskawi) was conducted by Kuldip

Dwivedi et al.,(2007). The calluses were sub-cultured on 6 differentiation media for induction of somatic embryogenesis;

green somatic structures developed after 6-8 weeks of sub culturing, cotyledon-derived calluses differentiated better than

hypocotyl-derived calluses in the work of Kaur et al.,(2005).

The first report on the development of interspecific hybrids between T. alexandrinum and T. resupinatum, using

embryo rescue; T. resupinatum is an important donor species for traits such as resistance to root and stem rot diseases, and

tolerance to alkalinity for the potential improvement ofT. alexandrinum; T. alexandrinum, as female parent, was crossed

with T. resupinatum and embryos were excised 10-12 days after pollination and cultured in vitro on EC3 medium theseresults obtained by Kaushal et al.,(2005). They found the plants were late flowering with pollen fertility more than 90%.

Three out of five hybrid plants set seed following selfing.

Also, the embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T.

constantinopolitanum Roy et al., (2004). They showed the hybrids intermediate morphological features with reduced

pollen fertility (55-65%) . On the other hand, Roy et al.,(2005) studied the differences in callus induction frequency were

found to be statistically significant for both explant and media as well as their interaction; the calluses from petiole

explant-'D' medium combination showed very good organogenetic and embryogenetic potential on sub-culture. Also, they

found 50% of sub-cultures showed shoot induction and a large number of them showed root induction in rooting media, a


5/22


suitable protocol for regeneration and transfer of plantlets in vitro in this genotype of T. apertum has been developed for

first time. Moreover, Kaushal et al., (2004). Studied EC3 medium with MS basal salts containing only kinetin (0.5 mgl-1)

and moderate sucrose (3%) was the best for embryo germination. EC4 media with high adenine and high sucrose supported

high callusing and reduced germination.

Transformation

Most transformation systems in berseem were used Agrobacterium .Transformation was established system in

2001 according to Tanaka et al., (2001). Agrobacterium rhizogenes, which used as carrier of vectors to transfer genes in

berseem. They used more advanced techniques to ensure transformed plant. They found also, in hairy roots of berseem

(Figure 3), a rolB expression seemed to influence the intrinsic growth capacity and to call for the proper level in order that

the hairy roots grew vigorously, while the endogenous IAA seemed to directly affect the growth capacity on medium

without exogenous auxin. In addition to, whole plant was obtained from the shoot when they were cultured in Florilate

two months (Figure 3); the generated plants displayed the hairy root syndrome such as dwarfing and alternation of leaf

shape according to Hiroshi et al,.(2004).

Figure 3: Transformed Berseem According to Tanaka et al., (2001)

Berseem was transformed with the Arabidopsis HARDY gene that belongs to the stress-related AP2/ERF

(APETALA2/ethylene responsive element binding factors) superfamily of transcription factors by Abogadallah et al.,

(2011). They found the fresh weights of the transgenic lines L2 and L3 were improved by 42 and 55% under drought

stress and by 38 and 95% under salt stress compared to the wild type, respectively, the dry weights were similarly

improved; L2 and L3 had thicker stems and in case of L3, more xylem rows per vascular bundle, which may have made L3

more resistant to lodging in the field. Field performance of L2 and L3 under combined drought and salt stress was

significantly better than that of the wild type in terms of fresh and dry weights (40 %, 46 % and 31%, 40 %, respectively).

The results of them provide further evidence for the efficiency of overexpression of a single gene in improving tolerance to

abiotic stress under field conditions.

Biochemical Genetics (Isozymes and Protein)

Field evaluation of agronomic character of berseem clover (Trifolium alexandrinum L.) seeds of five Egyptian

clover varieties, Helaly, Serw 1, Gemmiza 1, Giza 6 and Sakha 4 were planted during 2007 at Giza Agricultural Research

Farm, Egypt (Tarrad and Zayed 2009). The highest total dry matter yield of 15.86 t ha -1 was recorded for Gemmiza 1

variety. In conclusion, the investigation has shown variation between five varieties in morphological characters in plant

height, leaf stem ratio, number of leaves and main stem diameter. The highest total dry matter yield of 15.86 t ha-1was

recorded for Gmmiza1 variety as compared to the other varieties which could be a result of the maximum plant height


6/22

104 Ehab Mohamed Zayed(79.8 cm) and the more number of leaves(10.73). To confirm the obtained results in the field for mentioned varieties,

polyacrylamide gel electrophoresis (PAGE) was performed for native and SDS protein and isozyme variations (Figure 4).

Figure 4: Isozymes Seedling Polymorphism Based on PAGE Electrophoreses and SDS-PAGE Protein Banding

Patterns Analysis of the Five Egyptian Clover (Tarrad And Zayed 2009)

The protein and isozymes were used to compare among four Egyptian clover cultivars on, isozymes and seed

protein electrophoresis analysis. of four Egyptian clover varieties, i.e. Serw 1, Gemmiza 1, Giza 6 and Fahl were planted

in Petri dishes in six replicates per cultivar, having taken the following attributes on the seedling after 10,15 and 20 days in

the study of Zayed et al.,(2012). They indicated that the isozyme and protein analysis using PAGE are suitable for

maintaining and determining the genetic relationship in berseem cultivars.

Furthermore, the differences on the molecular level (proteins and isozyme bands) reflect the genetic change

(Figure 5) caused by plasma radiation treatments (Tarrad et al., 2011).

Field experiments were conducted at Atomic Energy Authority (AEA) Farm at Anshas, Egypt during 2009-2010

on multicut Egyptian clover cv. Gemmiza 1 (Gm) and single cut Fahl. Seeds were subjected to six treatments of non-

thermal plasma pulse (pulsed atmosphericpressure plasma jet [APPJ] apparatus) (Tarrad et al., 2011).

The plasma radiation (ozone, UV, visible light) was injected into the seed samples at different varying durations

(number of pulses). Treatments were 1, 3, 5, 7, 9, and 11 pulses and non-treated control. They revealed prominent effect of

application of non-thermal plasma technique on morphological characters as well as fresh and dry yield on both cultivars.

The highest values of dry and fresh yields were 10.0 and 63.0 t fed -1 (fed; Feddan= 4200 m2) under 9 pulse

(Tarrad et al., 2011). The 7 pulse treatment gave the highest values for Fahl dry and fresh matter 21.3 and 104.6 gram/10plants respectively. Thus, non-thermal plasma radiation of clover seeds led to important results(Tarrad et al., 2011). It

increase total DY for Gm clover cultivar to 10.0 t fed-1 in response to 9 pulse PR treatment.

It represented about 14.9 % significant (p>0.49) increase over the total DMY of non-treated control (Tarrad et al.,

2011). Also, Fahl clover cultivar had remarkable fresh weight (FW) of 104.6 g as well as dry weight (DW) of 21.3 g in

response to 7 pulse PR treatment compared to other treatments under study(Tarrad etal., 2011).

So, plasma radiation (PR) treatments could be used as a novel tool to affect yield and quality of both clover

cultivars (Tarrad et al., 2011); berseem ecotypes response to the non-thermal plasma radiation (Figure 5).


7/22


Figure 5: Isozymes and Protein Banding in Two Ecotypes of Egyptian Clover and their Mutants of Plasma

Radiation Treatments Labels of 1 to 7 for Gm and 8 to 14 for Fahl; Samples from Left to Right Control, 1, 3, 5, 7, 9

and 11 Pulses of PR Treatments in Ach Cultivar (Tarrad et al., 2011)

In the other investigation, comparison biochemical studies(Figure 6) among four berseem (Trifolium

alexandrinum L.) cultivars refereeing to cutting type (Zayed etal., 2012). They indicated that the isozyme and protein

analysis using PAGE are suitable for maintaining and determining the genetic relationship in Egyptian clover cultivars

(Figure 6). They obtained results, the five isozymes and protein were produced different total bands 28 (42.9%

polymorphism; pol.) in peroxidase 7 bands (28.6% pol.). in esterase 2 bands (0 % pol.), in acid phosphatase 4 bands (25%

pol.), in alkaline phosphatase 3 bands(66.7 % pol.), in Superoxide dismutase (SOD) 1 band (0% pol.) and in protein 11

bands (53.2% pol.). It was found that 11 bands were produced from four Egyptian clover, its ranged from 120 to 14.4

KDa.

Figure 6: Marker Polymorphism of the Four Varieties Egyptian Clover (1= Fahl, 2 = Gemmiza-1, 3 = Giza-6 and 4

= Serw-1) Based on (A) Seed Protein SDS-PAGE, (B) Isozymes Peroxidase, (C) Isozymes Esterase,(D) Isozymes

Acid Phosphatase, (E) Isozymes Alkaline Phosphatase and (F) Isozymes Superoxide Dismutase (SOD). M = Marker

Protein (Zayed et al., 2012)

Furthermore, enzymes analysis was performed on 10% native-polyacrylamide gel electrophoresis (Native-PAGE)

(Figure 7), to detect variation in Peroxidase, - and - Esterase Abd-Elnaby et al., (2012).They found the differences

between the Egyptian clover hybrids at the molecular level. Two Egyptian clover genotypes, multi-cut variety Sakha-4

belonging to type Miskawi and single cut berseem variety Fahl were reciprocally crossed. F1, F2, BC and reciprocals along

with the two parents were studied and analyzed.

Enzymes analysis was performed on 10% native-polyacrylamide gel electrophoresis (Native-PAGE), to detect

variation in Peroxidase, - and - Esterase. Electrophoresis profile of peroxidase, -Esterase and - Esterase revealed 2, 9


8/22

106 Ehab Mohamed Zayedand 9 bands, respectively (Figure 7). The polymorphism percentages were 50, 33.3 and 33.3% in the same order. Total

polymorphism percentage was 27.8 for the all three enzymes. Superior enzyme was demonstrating the extent of the

difference between genotypes (Abd-Elnaby et al., 2012).

Figure 7: Electrophoretic Enzyme Banding Pattern of-Esterase, - Esterase and Peroxidase for Egyptian Clover

hybrids :1=PM , 2= BC1MF, 3= BC2MM, 4 F2MF, 5=F1MF, 6= F1MM, 7= F2 MM, 8= PF(Abd-Elnaby et al., 2012)

Figure 8: Protein Analysis of Six Egyptian Clover Varieties, Lane M: Protein Marker; 1-6: 1=Fahl, 2=Sakha,

3=Gemmiza1, 4=Serw, 5=Giza 6 and 6=Helaly, the Arrows Indicated to the Unique Bands; the Arrows were

Indicated to Variation and Unique Bands, Azab et al.,(2011)

Moreover, Azab et al.,(2011) found SDS-PAGE analysis for the water soluble proteins in the six Egyptian clover

revealed a total number of 19 bands with molecular weights (MW) ranging from about 12.24 to 121.2 KDa and this six

Egyptian clover cultivars cannot be uniquely identified (fingerprinted) with cultivar protein markers(Figure 8). The use of

protein data for Trifolium cultivar identification and differentiation is suggested by Sheidai et al.,(1999). They revealed

differences among species/populations ofTrifolium repens, T. pratense, T. alexandrinum, T. fragiferum, T. subterraneum,

T. resupinatum and T. hybridum using analysis morphometric and SDS-PAGE seed-protein. Cluster analysis and

ordination of the species/accessions based on principal component analysis of morphological and protein data provided

evidence for the species inter-relationship and inter-population differences. Important work established by Sammour

(1999). He analysed by SDS-PAGE seed extracts/homogenates ofT. alexandrinum cv. Miskawi, T. arvense var. arvense, T.

pratense var. maritimum, T. rubens, T. medium subsp. medium, T. pignantii and T. pannonicum subsp. elongatum (sect.

Trifolium), T. glomeratum, T. pallescens, T. repens var. repens, and T. alpinum (sect. Lotoidea), T. spumosum (sect.

Mistyllus), T. fragiferum var. modestum and T. tomentosum var. tomentosum (sect. Vesicaria).


9/22


He showed that the taxa of section Vesicaria are highly homogeneous, and the delimitation of the sections

Vesicaria and Mistyllus agreed with a previous one based on morphological and chromosomal characters. They also

showed that the taxa of section Lotoidea are highly heterogeneous. The heterogeneity encountered in this section was

attributed to the primitive nature of its members.

Also, he found that the data showed that the taxa of section Trifolium were divided into two groups; the first

included T. rubens, T. arvense var. arvense, T. pratense var. maritimum, T. alexandrinum cv. Miskawi, and the second

contained T. medium subsp. medium, T. pignantii and T. pannonicum subsp. elongatum. The failure of the hybridization

between T. pratense var. maritimum from the first group and T. medium subsp. medium from the second suggests the

separation of the studied taxa into two subsections. The successful hybridization between T. repens var. repens and T.

pallescens in combination with the cluster analysis data suggest the possible hybridization between taxa of sections

Trifolium andLotoidea. As well as, the application of protein SDS-PAGE was used by Zahran (1992). He studied thirty

isolates of root-nodule bacteria, indigenous in the salt-affected soil of Egypt in berseem and other crops; the result

indicates that root-nodule bacteria of broad bean may have greater survival ability in saline soil. Most isolates synthesized

specific LPS (smooth, ladder-like type) and protein, which were different from LPS and protein of other Rhizobiumspecies. These specific forms of LPS and protein could be a prerequisite for the bacterial isolates to tolerate salt stress

conditions. Kaushal et al.,(2005) found the hybridity of the plants was also confirmed through Est, PRX, SOD, GOT

isozymes and protein banding patterns. Moreover, Roy et al., (2004). showed the hybrids between Trifolium

alexandrinum and T. constantinopolitanum using a biochemical characterization by isozymes to confirm hybridity.

On the other work of protein SDS-PAGE, Seedher and Narula (1993) extracted protein from crushed and de-

oiled Trifolium alexandrinum seeds using different extractants (water, 5% NaCl or NH3/NH4Cl buffer of pH 9.2), followed

by (NH4)2SO4, and the protein extracts and fractions were studied using the following methods; solubility, SDS-PAGE,

electrometric titrations or molecular exclusion chromatography (MEC). The total seed protein content was 10.71%. The

solubility data showed that precipitation at the pH of minimum solubility of 5.8 left 43% of the total proteins unrecovered.The number of charged amino acid residues in the extracts and protein fractions were calculated. All protein fractions

contained adequate amounts of the essential amino acids, histidine and lysine, but were deficient in arginine. Moreover,

Seedher and Narula(1991) showed protein SDS-PAGE patterns which contained single bands for the globulin, albumin

and prolamin fractions of T. alexandrinum seed protein and for the globulin fraction of M. sativa protein. All proteins

except M. sativa pseudoglobulin and prolamin contained adequate amounts of essential amino acids such as histidine,

lysine and arginine. In the work on berseem and goats, Kholif et al.,(2006). added Lysine and methionine (2.5 g/head/day)

in 2 forms were to the control diet consisting of berseem clover and concentrates (30:70, DM basis). Supplementation of

lysine and methionine in protected form increased the milk protein content. Moreover, milk protein fractions (true protein

nitrogen, non-casein nitrogen and casein nitrogen) increased with experimental rations compared to the control ration.

SDS-PAGE (silver staining) showed patterns of proteins with a total of 9-10 proteins.

Molecular Genetics (RAPD, ISSR, SSR and AFLP)

Molecular characterization of berseem was carried out on two berseem cultivars namely Fahl and Helaly were

analyzed based on seed soluble protein pattern as well as RAPD, ISSR and AFLP generated DNA profiles. For AFLP

analysis, DNA of Egyptian clover cultivars were digested with EcoRI + MseI restriction enzymes. Among the molecular

markers used ISSR showed highest level of molecular variance (24.5%). In terms of allele frequency (p), level of

difference between two cultivars was variable Zayed et al., (2010). They indicated that relationship study between two

cultivars can be better performed using Cubic, Quadratic model.


10/22

108 Ehab Mohamed ZayedRegression Relationship between Fahl and Helaly

Based on SPSS analysis, a high correlation coefficient (0.9) value was obtained indicating a strong correlation and

the direct relationship between the Fahl and Helaly.Coefficient of determination (R2) shows how different Helaly is from

Fahl, and significant relationship betweencultivars (Fahl and Helaly) as depicted by non-moral value F-large of the value

of F-indexed. This indicated thatassumption (H0) and nihilistic regression coefficient equalto zero, i.e., the regression

model is non-significant. Rhodes (1991) and Nolan et al. (2001) studied regression analysis of clover content in each

grazing period showedstrong effects of grazing regime which was generallypositive relationships. Curve fitting We could

not determine the relationship between cultivarstherefore by presuming the number of forms as therelationship and by

taking closest of the relationshipbetween them; we could draw a linear relationshipbetween variables as inferred. This

shows that the relationship between Fahl and Helaly take cubic or quadratic relationship, since it hasthe largest value of R2

=.920. The best model to represent the relationship between two cultivars at molecular basis of the relationship is Cubic,

Quadratic(Figure 9) . This illustratedthe form of relationships which shows the coefficient of determination is equal in

relationships needed.

Figure 9: Model Description Showing Dependent Variable (Fahl) and Independent Variable

(Helaly) Zayed et al., (2010)

Figure 10: Dendrogram Based on Cluster Analysis of Isozymes, Protein, and RAPD Data Showing Relatedness

among Egyptian Clover Cultivars; Tarrad and Zayed (2009)

RAPD was conducted using 8 arbitrary 10-mer primers. Combined analysis based on four isozymes, PAGE

protein electrophoresis and RAPD analyses revealed highest similarity of 0.85 between the two varieties Sakha 4 and


11/22


Gemmiza 1, while the lowest similarity (0.53)(Figure 10) was observed between Giza 6 and Helaly. Combined analysis

based on four isozymes PAGE, protein electrophoresis, and RAPD-PCR analyses revealed the highest similarity (0.85)

between the two varieties Sakha 4 and Gemmiza 1, while the lowest similarity index (0.532) (Figure 10) was observed

between the two genotypes Giza 6 and Helaly (Tarrad and Zayed 2009).

Table 1: Total Number of RAPD-PCR Band Products by OP-Primers in five Egyptian Clover Cultivars. Tarrad

and Zayed (2009)

Primers

Total

no ofband

Helaly Serw 1 Gemmiza 1 Giza 6 Sakha 4

Total

no ofband

Total

Positive

Polymorphic

%

Polymorphic

OP-A19 13 1,2,3,4,5,

6,7,8,13

1,3,4,5,6,7,

8,9,10,11,12

1,3,4,5,8,

9,10,11,12

1, 3,5,8,9,

10,11,12,13

1,3,4,5,

8,9,11,12

47 7 14.9

OP-BO7 10 4,5 2,4,5,6,7,9 2,4,6,7,8,9 2,4,6,7,

8,9,10

2,4,6,7,

8,9,10

28 4 14.3

OP-B05 12 8,9,10,11,12

1,2,3,4,5,6,7,8,9,10,11, 12

1,2,3,4,5,6,7,8,10,11,12

4,5,6,7,8,9,10,11,12

1,2,3,4,5,6,7,8,10

46 0 0

OPD01 15 1,4,6,

8,9,10

1,2,3,4,5,6,7,

8,9,11,

12,14,15

1,2,3,4,5,6,

7,8,9,10,

12,13,15

1,2,3,4,5,6,7,

8,9,10,11,12,

13,15

1,2,3,4,5,6,8,

11,12,14,15

56 4 7.1

OP-F06 13 0 1,4,10 1,2,3,4,5,6,7,

8,9,10,11,12

1,3,4,5,6,7,8,

9,10,11,12,13

1,3,4,5,6,7,

8,9,10,11

37 4 10.8

OP-C15 12 2,5,9,10 2,4,5,6,7,

8,9,10,12

2,5,8,9,

10,11,12

2,3,4,5,6,7,8,

9,10,11,12

1,2,3,4,5,

6,8,12

38 7 18.4

OP-AO 5 11 4,5,6,

7,8,10

4,5,6,7,

8,9,10

1,3,4,5,6,7,

8,9,10,11

2,4,5,6,7,

8,9,10

3,5,6,7,

8,9,10

38 5 13.2

OP-DO7 14 3,8,13 1,2,3,4,5,6,8,

9,10,11,

12,13,14

1,2,3,4,5,8,

9,10,11,13

1,2,3,5,7,8,

9,10,11,13

1,2,4,

6,7,8,9

43 7 16.3

Total no

of band100

Internal Simple Sequence Repeat (ISSR) technique in two ecotypes of Egyptian clover ( Zayed et al data under

publisher ). Two ecotypes of Egyptian clover (Trifolium alexandrinum L) were used in the present study variety Fahl is

prevalent in whole of Egypt and is good for single cut as it has poorregeneration ability, whereas Serw1, Giza 6 and

Gemmiza 1 are distributed in Egypt and can give 5-6 cuts of good fodder. The results revealed the polymorphism level

revealed by ISSR primers. HB-10 ISSR-primer was higher than that of the rest ISSR primers. The Fahl mono-cut ecotype

had 29 present bands, 2 absent bands and two unique bands.

The multicut ecotype Gemmiza 1, Giza 6 and Serw1 were given 20 present, 11 absent; 21 present and 10 absent

and 23 present and 11 absent respectively. The three unique bands were appeared in two ecotypes. Fahl was given two

with HB-11 and HB-13; Serw-1 multi-cut cultivar had one unique bands with HB-08. Similarity indices among the Four

Egyptian clover cultivars based on ISSR analysis was estimated the highest value was appeared between Fahl and

Gemmiza1 as well as Giza 6 and Serw 1 followed by Fahl and Serw 1. The lowest similarity value was appeared between

Gemmiza 1 and Serw 1 followed by Gemmiza 1 and Giza 6 (Figure 11).


12/22

110 Ehab Mohamed Zayed

Figure 11: Illustrated ISSRPCR Reaction 6 HB-Primers, HB-08, 09,10,11,12 and HB-13 with Two Ecotypes (Mono

and Multicut), 1= Fahl(Monocut),2=Gemmiza 1(Multicut),3=Giza 6 (Multicut) and 4 = Serw 1(Multicut) (Zayed et

al., Unpublished Data)

In addition to, Identification features molecular differences between Egyptian clover hybrids. ISSR-PCR

reactions were conducted using five primers. Amplification was performed in 25l reaction volume.

The goal of this study was to examine the differences and identification between the Egyptian clover hybrids at

the molecular level to help breeders of Egyptian clover (Figure 12) (Abd El-Naby et al.,2010).

Figure 12: ISSR Profile of Egyptian Clover Hybrids 1= PM, 2= BC1MF, 3= BC2MM, 4 F2MF, 5=F1MF, 6= F1MM,

7= F2 MM, 8= PF by Primers HB10, HB11, HB12, HB13 and HB15 and M=marker. Abdel Enaby et al.2012

Heat and forest tolerance:In the study field performance of commercial berseem clover (Trifolium alexandrinum

L.) cultivars under high temperature conditions.

The RAPD-PCR help in the compare variations among five berseem cultivars in heat temperature conditions

(Zayed etal., 2011 and Azab etal., 2011).


13/22


Average soil temperatures every 15 days before, during and after the cultivation of

Egyptian clover

0.0

10.0

20.0

30.0

40.0

50.0

60.0

16/5/2007

16/6/2007

16/7/2007

16/8/2007

16/9/2007

16/10/2007

16/11/2007

16/12/2007

16/1/2008

16/2/2008

16/3/2008

16/4/2008

16/5/2008

Months before, during and after the cultivation of Egyptian clover

Averagetemperaturesevery15days

Deap.5cm

Deap.10cm

Deap.20cm

Cultivated date

Cut 1st date

Cut3 rd date

33.3

17.4

20.6

Cut 2nd

25.9

16.0

18.8

18.0

23.5

20.1

26.9

32.6

34.0

Figure 13: Average Soil Temperatures at Experimental Site during Berseem Growth Season

(5 Nov 2007 17 Apr 2008)

Heat stress due to high temperature is a serious threat to crop production worldwide. This investigation

determined field performance of six cultivars from berseem, i.e., Helaly, Serw 1, Gemmiza 1, Giza 6, Sakha 4 and Fahl

under Shandwel Agricultural Research Station Farm, Egypt at high temperature. The results showed that Giza 6 performed

the best with total fresh yield (78.35 t ha -1) followed by Sakha 4 (74.7 t ha-1). In dry matter yield the cultivars Giza 6 (multi

cut) and Helaly (multi cut) had highest values 11.28 and 11.57 t ha-1, respectively and significant differences among

cultivars were recorded. The values of crude protein and crude fiber and ash content per cent differed in different cuts.Fahl cultivar showed no major effect of air and soil temperature and humidity (Figure 13).

Egyptian clover as well as all clover, ecology and geography play important roles. We can look in detail at DNA

sequence variation and at the same time have a thorough understanding of the plant's ecology. The frosts could cause cell

rupture and the release of cyanide leading to auto toxicity. The genetic basis behind cyanide production in clover plants

boils down to just two genes according to Olsen etal.,(2007 and 2008) (Figure 14). They reported two genes that were

responsible forest tolerance and cyanide production.

Figure 14: Showed Cyanide Pomp Vacuoles

Improvement the adverse effect of salt stress in Egyptian clover (Trifolium alexandrinum L.) by ASA application

through some biochemical and RT-PCR marker was carried out by Ammonia et al., (2012). Additionally, semi-quantitative

reverse transcription-PCR (RT-PCR) enabled to determine transcript levels of each of the pyrroline-5-carboxylate

synthetase (P5CS), delayed-response (RD29) and alcohol dehydrogenase (ADH) genes. The results showed that, there

were changes in the expressions level of RD29, ADH and P5CS genes under salinity stress compared with control.

The results revealed that salt induced deleterious effect in Egyptian clover seedlings and could bealleviated by

AsA treatment. Moreover, AsA can be used as a signal molecule to investigate plant defense to salt stress. Application of

Terror Cells Which being Tolerant Forest by HCNCyanide "bomb" in Cell


14/22

112 Ehab Mohamed ZayedDNA (RAPD) and ultrastructure to detect the effect of cadmium stress in Egyptian clover in the study Ali (2012). She

found in conclusion, biochemical, molecular and ultrastructure changes in Egyptian clover could be used as a useful

biomarker assay for the detection of genotoxic effects of Cd stress on plants. However, it is necessary to be further

confirmed and optimized in the future research.

Figure 15: RAPD Analysis of Six Egyptian Clover Varieties with Six Primers OP-a12, OP-L12, OP-C05, OP-C12,

OP-Z01 and OPL and M: DNA Marker; 1-6: 1=Fahl, 2=Sakha, 3=Gemmiza1, 4=Serw, 5=Giza 6 and 6=Helaly, the

Arrows Indicated to the Unique Bands; the Arrows were Indicated to Variation and Unique Bands Azab et al.,

(2011)

The highest number of unique markers at 794, 614, 543, 489 and 367 bp of primers OP-L12. As well as, 545 bp

and 243 bp of primer OP-A12. The cultivar Serw 1 scored four unique markers, bands no. 10 of primer OP-C13 at 200 bp,

band no.5 of primer OP-F09 at 337 bp and bands no 1, 4 of primer OP-L12 at 794 bp and 543 bp. Also, cultivar Gemmiza-

1 showed three unique markers at bands no. 8 (243 bp) of primer OP-A12, and band no. 3, 5 had size respectively 614 and

489 bp of primer OP-L12. Whereas, the cultivars Sakha- 4, Helaly and Fahl give one unique marker at band no. 3 had size

545 bp of primer OP-A12, no.8 had size 281 bp of primer OP-Z01 and no.7 had size 367 bp of primer OP-L12,

respectively(figure 15) (Azab et al.,2011).

Deferent work by molecular markers and its applications were used in Zeze et al., (2007) and Zeze et al., (2008)

isolated A mycorrhizal fungal inoculum "Aoufous Complex" in Morocco was shown to enhance Trifolium alexandrinum

resistance in water deficit situation. They confirmed efficiency of this inoculums in this study. In order to identify this

inoculum, specific primers were used to amplify the 18S subunit. The AluI RFLP typing of the PCR products revealed a

single pattern showing no diversity.


15/22


Phylogenetic analyses of seven sequences including other glomeromycetes allowed an unambiguous identification

of the "Aoufous Complex" as Glomus mosseae strains. Furthermore, new information will be useful in identifying and

producing improved recombinant strn by plasmid transfer according to Kuykendall et al., (1994). They determined the

symbiotic competence ofRhizobium strains with lucerne (Medicago sativa) of berseem. Symbiotic effectiveness varied

significantly among the R. meliloti strs but not among the berseem symbionts. Only one str., R. meliloti ARC 104, wassymbiotically ineffective. RFLP analysis using a ribosomal RNA operon probe, with three different enzyme digests,

produced four related groups which correlated with plasmid profiles. Tester and Romola (2003) explored the mechanism

of sodium tolerance. The wild type used in a few studies on Rhizobium for example Yadav et al.,(1998 and 1999), and

Perrine-Walker et al.,(2007).

Figure 16: Comparison between Fahl and Helaly-Miskawi in Molecular Level Zayed et al., (2010)

AFLP analyses in two combinations were produced 204 bands by ratio of polymorphism 16.7(Figure 16)(Zayed et

al., 2010). Estimates of genetic similarity using genetic fingerprinting data are a useful tool in plant breeding, breeders to

make informed decisions regarding the selection of germplasm to be used in crossing schemes. Fingerprints themselves are

also useful to breeders per se for protection of their own varieties and for seed producers (Dan -Milbourne, et al., 1997 and

Vos, et al., 1995).

Fernandez, et al., 2002; Greene, et al., 2004 and Bussell, et al., 2005, reported that the appropriate level of the

application of arbitrarily amplified dominant markers, which include RAPD, ISSR, AFLP are useful below taxonomic

levels at which the variable sequences can provide sufficient in formation and might be useful for phylogenetic analysis.Cytogenetic

Cytological comparison of Egyptian clover (Trifolium alexandrinum L) Soliman, et al., (2010). Cytological

studies including chromosome number and karyotype analysis have been carried out on two cultivars Helaly and Fahl in

berseem. The somatic chromosome counts for two cultivars were 2n = 16. Karyotype analysis showed differences in

chromosome morphology. They observed Chromosomes nsm (+) in cultivar Helaly. The karyotype formula for Helaly

was 2 nsm (+) + 2 nsm (-) + 12 nm. For Fahl, 6 nsm (-) + 10 nm were recorded. Helaly had highest A1 and A2 where Fahl

had highest TF %, S%, Syi index and Rec index (Figure 17 and 18).


16/22

114 Ehab Mohamed ZayedKaryotype analysis revealed that cultivar Helaly is advanced one whereas cultivar Fahl is primitive. Chromosomal

abnormalities were observed at mitotic division, which was higher in cultivar Fahl. In conclusion of them, the Egyptian

clover cultivar Fahl (monocut) differed from cultivar Helaly (multicut) with respect to chromosome morphology and

karyotype.

These differences might be due to chromosome deletions or due to different levels of condensation anddifferential contraction of chromosomes as suggested by El-Nahas(2000). Most species ofTrifolium are diploid (2n=16)

and only 16% of the 248 species are polyploid (Majumdar et al., 2004). 70% of the known polyploids occur in the

subgenusAmoria, which is considered to be most primitive and unspecialized subgenera. Polyploid cells were recorded in

cultivar Fahl cultivar only(Figure 17 and 18). Gupta and Gupta (1978) pointed out that the reduction in the chromosome

length is often associated with increasing evolution and that the occurrence of polyploidy is an evidence for its advanced

state. Moscone (1992) also pointed out that polyploidy is an important event during the evolution.

Figure 17: Karyotyping and Idiogrammatic Helay and Fahl; Soliman, et al., (2010)

Figure 18: Chromosomal Abnormalities of the two Varieties of T. alexandrinum L. (A): Non Congression at

Metaphase, (B): Polyploidy and(C): Late Separation at Anaphase (X=1000) Soliman, et al., (2010)

Cytological and biochemical comparisons among four berseem cultivars concerning cutting type (Zayed et al.,

2012). This investigation was comparing four Egyptian clover varieties (two ecotypes i.e. mono- and multi cut) on

cytology studies (karyotype). Seeds of four Egyptian clover varieties, i.e. Serw 1, Gemmiza 1, Giza 6 and Fahl were

planted on Petri dishes in six replicates per variety, having taken the following attributes on the seedling every few days

Idiogrammatic Representation

of the karyotype of Trifolium

alexandrinum cv Helaly

Idiogrammatic Representation

of the karyotype of Trifolium

alexandrinum cv Fahl

The karyotype ofTrifolium

alexandrinum cv Fahl and

Helaly


17/22


(10, 15 and 20 days) as well as, the mean measurements of three cells for each seed were used to construct the karyotype.

The results revealed the cultivars of berseem were diploid. It showed a chromosome number (2n=16). Single cutting Fahl

was significant highest value (4.65) while, Gemmiza -1 cultivar was significant lowest value (2.45) in shoot length. The

cultivar Serw-1 was given significant highest value (5.20 cm) while, Gemmiza -1 cultivar was significant lowest value

(3.30 cm) in radial length. Conclusion of this investigation: In comparison of 4 berseem cultivars regarding cut-type(mono-cut & multi-cut), cultivar Fahl (mono-cut) is distinguished by significant highest value of shoot length after 15 & 20

days from germination date and a unique protein band of molecular weight 120 KDa as well as presence of nearly sub

telocentric (-) in karyotype formula. Chromosome numbers were compared in a total of 91 plants ofT. alexandrinum cv.

Sacromontes, of which 21 had been cloned in vitro (C), 64 regenerated via somatic embryogenesis (R-1) and 6 regenerated

using callus from flowers of regenerated plants (R-2) according to Curn (1993).

He studied cloned plants were genetically stable and had the basic chromosome number 2n = 16. Regenerated

plants exhibited instances of abnormal mitosis involving the occurrence of lagging chromosomes (3.1% of mitoses) and

aneuploid chromosome numbers arose in 14 plants (1.1% of mitoses). In R-2 plants, chromosomal irregularities were

relatively frequent and included the occurrence of 2 sterile tetraploid plants showing a prostrate and compact habit andslow growth.

Furthermore, Masoud and Hamta (2008) investigated cytogenetical analysis of the tissue culture regenerated

plants showed the presence of different somatic chromosome numbers ranging from 2n = 16 to 2n = 48, the latter

chromosome number only occurred in 12 weeks culture time-period.

Roy et al., (2004). showed the hybrids between Trifolium alexandrinum and T. constantinopolitanum had a

chromosomal complement of 2n=16,biochemical characterization using isozymes confirmed hybridity. Roy et al.,(2005)

studied the cytology of the tissue culture plants that revealed normal meiotic pairing with 8 bivalents. The cytological

karyotyping was used to compare among four berseem cultivars on i.e. Serw 1, Gemmiza 1, Giza 6 and Fahl Zayed et

al.,(2012).

Wild Types

Morphological and description of berseem tall erect herb with narrow elliptical leaflet. Heads dense, not umbel-

like, white flowers.Place of locations area: (1) The Nile region, including the delta, the valley and the Faiyum,(2)The oases

of the libyan desert(west nile),(3) The Mediterranean coastal strip from El-Sollum to Rafah, and(4) The Isthmic desert,

i.e. el-Tih and the region north of WadiTumilat (sues cannel). In my opinion, old local varieties grown in farmer fields

might include some of berseem wild types (Figure 19).

Despite Badr et al., (2008) studied the origin and ancestry for Egyptian clover, Trifolium alexandrinum, was

examined using AFLP data but Viva (1974) collected the wild type and she tokens about the places and locations of wild

types and wild relatives. So, the origin of Egyptian clover needs to many of the assertions and more inventory of berseem

family to emphasize the fact the difference.

Badr et al., (2008) supported a close relationship of berseem accessions from Syria and Egypt to T. apertum, T.

berytheum, and T. salmoneum. However, cross ability and geographic distributions suggest that T. apertum is an unlikely

progenitor. They, appeared T. salmoneum to be the most probable progenitor for Syrian material of Egyptian clover,

although a close relationship to T. berytheum was also revealed. They found ability of these species to cross freely

indicates that T. salmoneum and T. berytheum may be regarded as the primary ancestors from, which man domesticated

Egyptian clover through artificial selection in Syria. Also, they followed domestication; the earlier forms of the crop

species could have been taken into rain-fed cultivation in Palestine and irrigated cultivation in Egypt.


18/22

116 Ehab Mohamed ZayedFurthermore, they found the domestication of berseem may be analogous to other crops, such as barley and wheat,

which were also domesticated in the Fertile Crescent and taken into cultivation in the Nile Valley. It appears that genetic

improvement of the crop occurred in Egypt after cultivation, and that the varieties that were developed in Egypt were later

distributed worldwide.

Table 2: Wild Type of the Trifolium in Egypt and Wild Relative of Berseem

Scientific NameNo.Scientific NameNo.

T. alexandrinum L.12T. fragiferum L.1

T. medium L.13T. resupinatum L.2

T. lappaceum L.14T. tomentosum L.3

T. scabrum L.15T. patens4

T. incarnatum L.16T. campestre5

T. stellatum L.17T. procumbens L.6

T. formosum18T. philistaem7

T. angustifolium L.19T. xerocephalum8

T. dichroanthum20T. nervulosum9

T. purpureum21T. nigrescens10

T. desvauxii22T. repens L.11

ViviTackholm (1974). Flora of Egypt

Figure 19: Showed Map Location of Berseem Wild Type in Egypt

Our long term goals: we aim to resolve many of the problems: Fertility and Sterility genes and thus find a solution

to the isolation distances. Understand the relationship between dodder and Berseem of molecular signals, genes responsible

for resistance to varying the stress, Genetic map of Egyptian Clover Genome. The application of molecular breeding

concept to berseem clover: Marker adds selection, Genes of wild type, Industry Application of Egyptian clover and

bioinformatics. Furthermore, suggest


19/22


1. International network of Egyptian clover2. International Center for training and exchange expertise3. International journal of Egyptian clover4. Protection of our cultivars and landraces

REFERENCES

1. Abdel Elnaby, Zienab M.;E.M. Zayed and S. S. Abo Fteih (2012). Biochemical and molecular differencesbetween Egyptian clover hybrids. Egyptian Journal of Biotechnology(41) June 104-118.

2. Abogadallah, G. M.; Nada, R. M.; Malinowski, R.; Quick,(2011). Overexpression of HARDY, an AP2/ERF genefrom Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic

Trifolium alexandrinum L.Planta; 233: 6, 1265-1276.

3. Abogadallah, G. M.; Quick, W. P.(2010). Fast Versatile Re-generation ofTrifolium alexandrinum L., Plant Cell,Tissue and Organ Culture; 100: 1, 39-48.

4. Aly, M. A.M., Khalil M.F.M.,Moghieb,R.E.A.,Yousif,A.S, El-Sharkawy, A.M.A.(1994).Somatic embryogenesisin berseem (Trifolium alexandrinum L.).The influence genetic background. Egypt J. Genetic and cytology 23,151-

161.

5. Aly, Amina A., Ahmed F. Khafaga And Gadalla N. Omar (2012). Improvement the Adverse Effect of Salt Stressin Egyptian clover (Trifolium alexandrinum L.) By ASA Application through Some Biochemical and RT-PCR

Markers. Journal of Applied Phytotechnology in Environmental Sanitation, 1 (2): 91-102.

6. Aly, Amina A.(2012). Application of DNA (RAPD) and ultrastructure to detect the effect of cadmium stress inEgyptian clover and Sudan grass plantlets.J. of Stress Physiology & Biochemistry, 8 : (1), pp. 241-257

7. Azab M.M., E.M.R. Metwali, A.F. Khafaga and E.M. Zayed(2011). Field performance and molecular profile ofcommercial Egyptian clover (Trifolium alexandrinum L.) varieties under high temperature conditions .Middle-

East Journal of Scientific Research 7 (5): 652-662.

8. Badr Abdelfattah, Hanaa H. El-Shazly, Linda E. Watson (2008). Origin and ancestry of Egyptian clover(Trifolium alexandrinum L.) as revealed by AFLP markers. Genetic Resources and Crop Evolution 55, 1, 21-31.

9. Barakat, M. N.(1990). In vitro cultures of tissues, cells and protoplasts Trifolium alexandrinium L. (Egyptianclover). Euphytica; 48: 2, 103-110.

10. Bhowal M., Cherian K.J. and Das L. (2011). Direct organogenesis in fodder crop Trifoliumalexandruim L., J.Environ. Res. Develop., 5(4), 892-897.

11. Bussell, J.D.,M. Waycott and J.A. Chappill(2005) Arbitrarily amplified DNA as characters for phylogeneticinference. Prospective in Plant Ecology, Evolution and Systematic,7:3-26

12. Curn, V.(1993).Sbornik - Jihoceska Univerzita Zemedelska Fakulta, Ceske Budejovice. Fytotechnicka Rada; 10:1, 137-145.

13. Dan Milbourne, Rhonda Meyer, John E. Bradshaw, Eileen Baird, Nicky Bonar, Jim Provan, Wayne Powell andRobbie Waugh(1997). Comparison of PCR-based marker systems for the analysis of genetic relationships in

cultivated potato. Molecular Breeding 3: 127136.


20/22

118 Ehab Mohamed Zayed14. El-Nahas, A. I. (2000). Chromosomal criteria in relation to seed protein of some Egyptian cultivars ofVicia faba.

Egypt. J.Biotechnol 7: 276-292.

15. ENAL [Egyptian National Agriculture Library](2012). Journals and books @OVID and CAB data base.16. Fernandez ME, Figueiras AM and Benito C(2002). The use of. ISSR and RAPD markers for detecting DNA17. polymorphism. Theor. Appl. Genet.; 104: 84551.18. Greene SL, Gritsenko M and Vandemark G(2004). Relating morphologic and RAPD marker variation to

collection siteenvironment in wild population of red clover (Trifoliumpratense)Genet Res. Crop Evol; 51: 643

53.

19. Gupta, R. and P. K. Gupta. 1978. Karyotypic studies in the genusCrotalaria linn. Cytologia 43:357-369.20. Indra K. Vasil(2008). A history of biotechnology: from the cell theory of Schledin and Schwann to biotech

crop.(2008). Plant cell report, 27:1423-1440.

21. James, C (2007). Global statues of commercialized biotech/GM Crops 2007. ISAAA Brief 37.22. Kaur, G. P.; Bhardwaj, B. L.; Gosal, S. S. (2005). Induction of somatic embryogenesis and plant regeneration in

Trifolium alexandrinum L. Crop Improvement; 32: 1, 1-6.

23. Kaushal, P.; Malaviya, D. R.; Roy, A. K.; Kumar, B (2004).Plant Cell Biotechnology and Molecular Biology; 5:3/4, 109-114. 13.

24. Kaushal, P.; Malaviya, D. R.; Roy, A. K.; Kumar, B.; Tiwari, A(2005). Trifolium alexandrinum T.resupinatum interspecific hybrids developed throughembryo rescue. Plant Cell, Tissue and Organ Culture; 83:

2, 137-144.

25. Kholif, S. M.; Foda, M. I.; Kholif, A. M.(2006). Indian Journal of Dairy Science; 59: 5, 281-286. 25.26. KuldipDwivedi; Malaviya, D. R.; Roy, A. K.; Kaushal, P.(2007).Range Management and Agroforestry; 28: 2B,

280-282.

27. Kuykendall, L. D.; Abdel-Wahab, S. M.; Hashem, F. M.; Berkum, P. van(1994). Letters in AppliedMicrobiology; 19: 6, 477-482.

28. Majumdar, S., S. Banerjee and K. Kumar (2004). Meiotic behavior of chromosomes in PMCs and karyotype ofTrifoliumrepens L. from Darjeeling Himalaya.Acta BiologicaCracoviensia Series Botanica 46: 217-220.

29. Masoud, S.; Hamta, A.(2008).Caryologia; 61: 4, 392-396.30. Moscone, E. A. 1992. Meiotic chromosome studies in Solanaceae from Argentina.Darwiniana 31:261-297.31. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures.

Physiology Plant 15:473497.

32. Nolan, T., J. Connolly, and M. Wachendorf (2001). Mixed Grazing and Climatic Determinants of White Clover(Trifolium repens L.) Content in a Permanent Pasture.Annals of Botany 88 (Special Issue): 713-724.

33. Olsen, K. M., B.L. Sutherland and Linda L. Small(2007). Molecular evaluation of the Li/li chemical defensepolymorphism in white clover (T. repens L.) Mol. Ecol. 16:4180-4193.


21/22


34. Olsen, K. M., Shin Chung Hsu, Linda L. Small(2008). Evidence in molecular basis of the Ac/ac Adaptivecynogenesis polymorphism in white clover (T. repens L.).Genetics 179:57-526.

35. Perrine-Walker, Francine M.; Prayitno, Joko; Rolfe, Barry G. ; Weinman, Jeremy J. and Hocart, CharlesH.(2007). Journal of Experimental Botany. 58(12):3343-3350.

3366.. Rhodes I. (1991) Progress in white clover breeding. In: White clover Development in Europe, Reur TechnicalSeries, no. 19, pp. 1-9. FAO, Rome.

3377.. Roy, A. K.; AparnaTiwari; Malaviya, D. R.; PankajKaushal; Bijendra Kumar(2005).Cytologia; 70: 2, 153-159.38. Roy, A. K.; Malaviya, D. R.; Kaushal, P.; Kumar, B.; Tiwari, A.(2004). Interspecific hybridization ofTrifolium

alexandrinum T. constantinopolitanum usingembryo rescue. Plant Cell Reports; 22: 9, 705-710. 28.

39. Sammour, R. H.(1999). SDS-PAGE analysis of the seed protein of some Trifolium taxa. Plant Varieties & Seeds;12: 1, 11-21.

40. Seedher, N.; Narula, V.(1993).Plant Physiology & Biochemistry (New Delhi); 19: 2, 93-98.41. Seedher, N.; Sharma, A.(1991).Indian Journal of Plant Physiology; 34: 4, 291-296.42. Sheidai, M.; Hamta, A.; Jaffari, A.; Noori-Daloii, M. R.(1999).Plant Genetic Resources Newsletter; 1999. 120,

52-54.

43. Soliman, Magda I., E. M. Zayed and G. A. Ramadan(2010).Cytological comparison of two cultivars of Egyptianclover (Trifolium alexandrinum L) Range Mgmt. & Agroforestry 31 (1) : 7-10.

44. Sultan, Fadia Mohamed Mohamed(1990). MSc. Thesis, Ain Shams University. Faculty of Agriculture.Department of Agronomy.

45. Tanaka N., Y. Fujikawa, M.A.M. Aly, H. Saneoka, K. Fujita and I. Yamashita(2001). Proliferation and rol geneexpression in hairy root lines of Egyptian clover. Plant Cell, Tissue and Organ Culture 66: 175182.

46. Tarrad M. M. , G. Abd El-Nasser Ahmed and E. M. Zayed(2011). Response of Egyptian clover ecotypes to thenon-thermal plasma radiation. Range Mgmt. & Agroforestry 32 (1) :9-14.

47. Tarrad M. M. and E. M. Zayed(2009). Morphological, biochemical and molecular characterization of Egyptianclover (Trifolium alexandrinum L.) varieties. Range Mgmt. & Agroforestry 30 (2) : 115-121.

48. Tester, Mark and Davenport, Romola (2003). Annals of Botany. 91(5):503-527.49. Vivi Tackholm (1974). Flora of Egypt.50. Vos P, Hogers R, Bleeker M, Rerjans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau

M: AFLP(1995). a new technique for DNA fingerprinting. Nucl Acids Res 23: 44074414.

51. Yadav, A S ; Vasudeva, M ; Upadhyay, K K ; Sawhney, S K and Vashishat, R K (1999). Letters in AppliedMicrobiology. 28(6):466-470.

52. Yadav, A. S. ; Vashishat, R. K. ; Kuykendall, L. D. and Hashem, F. M.(1998). Letters in AppliedMicrobiology. 26(1):22-26.

53. Zahran, H. H.(1992).Journal of Basic Microbiology; 32: 4, 279-287.


22/22

120 Ehab Mohamed Zayed54. Zayed E.M., Magda I. Soliman, G. A. Ramadan and M. M. Tarrad(2010). Molecular characterization of two

cultivars of Egyptian clover (Trifolium alexandrinum L.). Range Mgmt. & Agroforestry 31 (2) : 140-143.

55. Zayed E. M., E. M. R. Metwali, A. F. Khafaga and M. M. Azab(2011). Field performance of commercialEgyptian clover (Trifolium alexandrinum L.) cultivars under high temperature condition. Range Mgmt. &

Agroforestry 32 (2) : 87-91.

56. Zayed, M.E., Metwali, E.M.R., N.O. Gad-Allaha and R.M. Shoaib(2012). Comparison Cytological AndBiochemical Studies Among Four Egyptian Clover (Trifoliumalexandrinum L.) Cultivars Refereeing To Cutting

Type. Australian Journal of Basic and Applied Sciences, 6(3): 622-629.

57. Zeze, A.; Brou, Y. C.; Meddich, A.; Marty, F.(2008). African Journal of Agricultural Research; 3: 1, 078-083.58. Zeze, A.; Brou, Y. C.; Medich, A.; Marty, F.(2007). African Journal of Biotechnology; 6: 13, 1524-1528.

9.agri sci - ijasr - applications full

Documents