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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.
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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
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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
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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
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 103
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
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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).
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 105
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
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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).
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 107
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.
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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
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 109
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).
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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).
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 111
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
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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.
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 113
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).
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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
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 115
(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.
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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
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Applications of Biotechnology on Egyptian Clover [(Berseem)( Trifolium Alexandrinum L.)] 117
1. International network of Egyptian clover2. International Center for training and exchange expertise3. International journal of Egyptian clover4. Protection of our cultivars and landraces
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