**Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains Obtained from Denizli Hospital**

Göksel Doğan, Gülümser Acar Doğanlı, Yasemin Gürsoy and Nazime Mercan Doğan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55457

### **1. Introduction**

Soon after two years of introducing methicillin, *S. aureus* strains developed resistance to methicillin by through the gain of the mecA gene (MRSA). At first *S. aureus* strains were exclusively related to hospital acquired (HA) MRSA, but as from 1990s, community acquired (CA) MRSA came into view [1].

In both HA-MRSAs and CA- MRSAs are refered to as a significant factor of serious infections in high morbidity and mortality including bacteremia, pneumonia, endocarditis, osteomyelitis and toxic shock syndrome [2,3,4,5,6]. The factors that increase the prevalence of nosocomial bacteremia are the increase in older age groups in society, life period prolongation of people with chronic diseases, widespread use of immunosuppressive drugs, increase in interventional procedures for the purposes of diagnostic and therapeutic. Generally, some of staphylococ infections are nasocomial, other infections have occured by depending on MRSAs. The colonization rate with MRSA has increased in parallel duration of hospitalization. These strains have been found resistant against penicillins, combinations of betalactam/betalactamase inhibitory, sephalosporins, combinations of monobactames and carpenemes. To identify the resistance of staphylococcus's against methycillin antibiotic, those methods as of disc diffusion, tube dilution or microdilution, agar scanning, agar dilution, automatise susceptibility tests, DNA hybridisation technics and polimeraze chain reaction have been used [7,8,9]. The aim of this study is to identify antibiotic susceptibility and specificity of MRSAs isolated from various clinic samples with various methods including disc diffusion, SDS-PAGE and DNase test.

© 2013 Doğan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Methicillin resistant** *Staphylococcus aureus*

#### **2.1. General properties**

*S. aureus* (including MRSA strains) are clusterforming, facultative aerobic, Gram-positive cocci (at 0.5-1.7 μ of diameters) with intrinsic ability to ferment carbohydrates, producing white to deep yellow pigmentation on solid culture media [2].

mase enzyme has denominated as methicillin resistant. Thus, it is clear that the resitant is chromosomal and is not with beta-lactamase enzyme inactivated the antibiotic [15,16].

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 347

PBP 2a (PBP 2'): Alhough methicillin sensitive *S. Aureus* (MSSA) has five types of penicillin binding proteins (PBP), in addition these proteins, methicillin resistant *S. Aureus* (MRSA) has also a different penicillin binding protein named as PBP 2' or PBP 2a. This protein has 78 kDa of molecular weight [17]. PBP 2a shows lower affinity against beta-lactame antibiotics then other PBPs. Hence, the enzyme is single trancepeptidase that has the ability to continue of peptidoglycan synthesis by showing high affinity in presence of beta-lactame antibiotics [13,18]. The gene encoded PBP 2a is 2.1 kb and named *mecA*. Though, all of MRSAs have this gene, but there is no this gene at MSSA strains. The emergence of methicillin resistant pheno‐ typeclly has been able to show variability among bacteria. The phenotypically expiration of methicillin resistant is possible in two ways; homogen and heterogen [17]. In homogen resistant, all of cells show high levels of resistance in presence of high methicillin consentra‐ tions by growing [19,11]. In hetrogen resistance, even though all cells have *mecA* gene had

information that needs for methicillin resitance, only some cells show resistance.

protein, while *mecI* encodes a protin suppressed the *mecA* [15,16]

**b.** Other factors affected resistant phenotype:

phenotypically [17].

cations at cell autolysis.

and ekstrakromosomal control [22,23,24,25].

**2.4. Enzymes and toxins**

**a.** *mecR1-mecI sistemi:* The *mecA* have been controled with two regulator genes. Those genes are *mecR1* and *mecI*. Also these genes are similar to *blaR1* and *blaI,* which are regulatory genes of beta-lactamase, in terms of structure, fonction and mechanism of regulation. *MecI* and *mecR1* have the same regulator role for *mecA*. *MecR1* encodes a signal stimulant

**•** Beta-lactamase plasmid: Production of beta-lactamase enzyme is encoded by *blaZ* gene and is controled by genes of *blaR1* and *blaI* which are antireceptor and receptor, recpectively. *BlaR1* which is a transmembrane protein binds to beta-lactame in presence of it and leads to start the synthesyse of beta-lactamase enzyme by providing signal transmission from out of cell to inside of cell [20]. At the same time, it has been thought that genes of *blaR1* and *blaI* have had a role for occuring of methicillin resistant

**•** Fem Factors: The obtaining sensitive strains from methicillin resistance strains by tranposones via inactivation had led to identification of genes beyond of *mec*. These genes placed out of *mec* gene region had defined as "auxiliary" or "factors essential for the expression of methicillin resistance" or shortly "fem" genes [21]. Both MRSA and MSSA strains have fem factors variously from *mec*A gene. Also, it indicated that some conditions increased the occuring of methicillin resistant were correlated with modifi‐

Staphylococci can lead to diseases by large diffusion at tissue and producing many extracel‐ lular substances. Some of these extracellular substances are enzymes, others are toxins. Most toxins are under genetic control of plasmids. Some of them can be under both kromosomal

50% of cell wall of Staphylococci has composed from peptidoglycan. Peptidoglycan chains consist of alternative polysaccharide subunits included N-acetyl glucosamines and N-acetyl muramik acid. These chains are cross linkage by pentaglycine bridges that are specific for *S.aureus* and tetra peptide chains binded with N- acetyl muramik acid. Peptidoglycan can show endotoxin properties and structural differences among strains can lead to extensive intrave‐ nous coagulation [10,11].

Most researchers demonstrated that MRSAs caused various diseases ranging from soft, superficial dermatological diseases to acute and potentially fatal systemic enervations [2,12,13]. Some MRSAs live as a normal flora member in human mucose membrans and skins, others lead to ichor, abscess formation, various piogen infections and fatal septicemia. MRSA strains have also been detected in domestic animals and birds such as horses, cattle, chickens and dogs as well as associated individuals [2].

*Staphylococcus aureus* is coagulase positive and major patogen for humans. In general patholog‐ ic Staphylocci hemolysis the blood, coagulates the plasma, and also produces various extracel‐ lular enzymes and toxins. They have developed quickly against antimicrobial drugs [14].

#### **2.2. Culture properties**

Staphylococci grows easily aerobic or microaerofilic conditions. Optimal temperature is 37°C for these bacteria. Colonies of them are orbicular, sleek, bouffant and brilliant in solid media. In general, *S.aureus* has colonie colours that change from white to golden yellow. However, in many colonies, pigment occurs by those bacteria after long incubation time. It does not occur under anaerobic conditions or in broth [14].

#### **2.3. Growing properties**

Staphylococci have produced catalase variously then Streptococci. Staphylococci fermentate carbohydrates slowly by constituting acid, but they do not produce gases. On the other hand, pathogenic Staphylococci have generated so many extracellular substances. Even though, Staphylococci are durable against conditions of dry air, temperature (50°C, 30 m) and 9% of NaCI, but they can be inactivated easly with powerful chemicals such as 3% of hexaclorofen. Other one theme, these bacteria are sensetive against many of antimicrobial drugs. Resistant separates several of categories;

Mechanism of methicillin resistant: The resistant agaist beta-lactame antibiotics (methicillin, oxacillin, nafcillin, kloxacillin and dikloxacillin) not having been hyrolysed with beta-lacta‐ mase enzyme has denominated as methicillin resistant. Thus, it is clear that the resitant is chromosomal and is not with beta-lactamase enzyme inactivated the antibiotic [15,16].

PBP 2a (PBP 2'): Alhough methicillin sensitive *S. Aureus* (MSSA) has five types of penicillin binding proteins (PBP), in addition these proteins, methicillin resistant *S. Aureus* (MRSA) has also a different penicillin binding protein named as PBP 2' or PBP 2a. This protein has 78 kDa of molecular weight [17]. PBP 2a shows lower affinity against beta-lactame antibiotics then other PBPs. Hence, the enzyme is single trancepeptidase that has the ability to continue of peptidoglycan synthesis by showing high affinity in presence of beta-lactame antibiotics [13,18]. The gene encoded PBP 2a is 2.1 kb and named *mecA*. Though, all of MRSAs have this gene, but there is no this gene at MSSA strains. The emergence of methicillin resistant pheno‐ typeclly has been able to show variability among bacteria. The phenotypically expiration of methicillin resistant is possible in two ways; homogen and heterogen [17]. In homogen resistant, all of cells show high levels of resistance in presence of high methicillin consentra‐ tions by growing [19,11]. In hetrogen resistance, even though all cells have *mecA* gene had information that needs for methicillin resitance, only some cells show resistance.

	- **•** Beta-lactamase plasmid: Production of beta-lactamase enzyme is encoded by *blaZ* gene and is controled by genes of *blaR1* and *blaI* which are antireceptor and receptor, recpectively. *BlaR1* which is a transmembrane protein binds to beta-lactame in presence of it and leads to start the synthesyse of beta-lactamase enzyme by providing signal transmission from out of cell to inside of cell [20]. At the same time, it has been thought that genes of *blaR1* and *blaI* have had a role for occuring of methicillin resistant phenotypically [17].
	- **•** Fem Factors: The obtaining sensitive strains from methicillin resistance strains by tranposones via inactivation had led to identification of genes beyond of *mec*. These genes placed out of *mec* gene region had defined as "auxiliary" or "factors essential for the expression of methicillin resistance" or shortly "fem" genes [21]. Both MRSA and MSSA strains have fem factors variously from *mec*A gene. Also, it indicated that some conditions increased the occuring of methicillin resistant were correlated with modifi‐ cations at cell autolysis.

#### **2.4. Enzymes and toxins**

**2. Methicillin resistant** *Staphylococcus aureus*

deep yellow pigmentation on solid culture media [2].

and dogs as well as associated individuals [2].

under anaerobic conditions or in broth [14].

*S. aureus* (including MRSA strains) are clusterforming, facultative aerobic, Gram-positive cocci (at 0.5-1.7 μ of diameters) with intrinsic ability to ferment carbohydrates, producing white to

50% of cell wall of Staphylococci has composed from peptidoglycan. Peptidoglycan chains consist of alternative polysaccharide subunits included N-acetyl glucosamines and N-acetyl muramik acid. These chains are cross linkage by pentaglycine bridges that are specific for *S.aureus* and tetra peptide chains binded with N- acetyl muramik acid. Peptidoglycan can show endotoxin properties and structural differences among strains can lead to extensive intrave‐

Most researchers demonstrated that MRSAs caused various diseases ranging from soft, superficial dermatological diseases to acute and potentially fatal systemic enervations [2,12,13]. Some MRSAs live as a normal flora member in human mucose membrans and skins, others lead to ichor, abscess formation, various piogen infections and fatal septicemia. MRSA strains have also been detected in domestic animals and birds such as horses, cattle, chickens

*Staphylococcus aureus* is coagulase positive and major patogen for humans. In general patholog‐ ic Staphylocci hemolysis the blood, coagulates the plasma, and also produces various extracel‐ lular enzymes and toxins. They have developed quickly against antimicrobial drugs [14].

Staphylococci grows easily aerobic or microaerofilic conditions. Optimal temperature is 37°C for these bacteria. Colonies of them are orbicular, sleek, bouffant and brilliant in solid media. In general, *S.aureus* has colonie colours that change from white to golden yellow. However, in many colonies, pigment occurs by those bacteria after long incubation time. It does not occur

Staphylococci have produced catalase variously then Streptococci. Staphylococci fermentate carbohydrates slowly by constituting acid, but they do not produce gases. On the other hand, pathogenic Staphylococci have generated so many extracellular substances. Even though, Staphylococci are durable against conditions of dry air, temperature (50°C, 30 m) and 9% of NaCI, but they can be inactivated easly with powerful chemicals such as 3% of hexaclorofen. Other one theme, these bacteria are sensetive against many of antimicrobial drugs. Resistant

Mechanism of methicillin resistant: The resistant agaist beta-lactame antibiotics (methicillin, oxacillin, nafcillin, kloxacillin and dikloxacillin) not having been hyrolysed with beta-lacta‐

**2.1. General properties**

346 Current Progress in Biological Research

nous coagulation [10,11].

**2.2. Culture properties**

**2.3. Growing properties**

separates several of categories;

Staphylococci can lead to diseases by large diffusion at tissue and producing many extracel‐ lular substances. Some of these extracellular substances are enzymes, others are toxins. Most toxins are under genetic control of plasmids. Some of them can be under both kromosomal and ekstrakromosomal control [22,23,24,25].

#### **2.5. Coagulase and clumping factor**

*S.aureus* produces coagulase being an enzyme providing coagulation of the plasma. Coagulase that can be droped out freely binds the prothrombin and initiates polymerization of fibrin. It can also accumulate the fibrin at suface of Staphylocci. It thinks that products of coagulase are similar to invasive patogenic power.

*2.10.2. Catalase test*

*2.10.3. Susceptibility tests*

oxasilin and 4% of NaCl.

MRSAs produce catalase, which converts hydrogen peroxide into water and oxygen. The

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 349

At choosing of advisable antibiotic drug associated with therapy of infection, a number of factors such as potential infectious agents, antibiotic susceptibility, host factors that may affect the activity of the drug in vivo, the location of the infection, pharmacokinetic and pharmaco‐ dynamic properties of the drug should be evaluate [26]. Generally, *in vitro* procedures applied for determination of antimicrobic activity of an antibiotic is named as susceptibility tests. Susceptibility tests apply in the cases of not foreseeable susceptibility against antibacterial agent which will be applied at treatment of aerob and facultative anaerob bacteria which are clinically important. Susceptibility against antimicrobic drugs can be detected with a lot of methods. In most, inhibitor activity of drugs (bacteriostatic) evaulates in applied method. The applied methods with this purpose can consider of; 1. dilution methods in liquid and solid media; 2. disc diffusion method; 3. gradient diffusion (E-test) method; 4. the detection of

At disc diffusion method, paper discs is absorbed a specific amount of antibiotic place onto plate inoculed with the test microorganism. Thus, antibiotic absorbed by disc diffuses into agar and inhibits the growing of bacteria at effect levels of antibiotic. At the end of this situation, a circular inhibition zone where does not grow bacteria occurs at ambient of the disc. The categories of susceptibility as of sensitive, medium and resistance identify by measuring diameter of this zone. The limit values related to these categories detect for every antimicrobic agent by regarding accescible serum levels [26,27]. For example, Staphilococci are *mec*A positive and are resistant against methicillin at Müller-Hinton agar contented 6 of μg/mL of

Molecular epedemiology of community aquired MRSA (CA-MRSA) is tolerably different from hospital acquired MRSA (HA-MRSA). CA-infections commonly cause of skin and soft tisue

Panton-Valentine leukocidin (PVL) gene encoded a toxin which is responsible from virulance of bacteria and type 4 SCC*mec* genetic component frequently are presence at CA-MRSA isolates [22,16]. HA-MRSA isolates generally have type I, II or III of staphylococcal casette chromosome (SCC*mec*) genetic component. This gene domain is responsible from showing resistant of bacteria against antibiotics made from beta-lactam and even other drugs such as clindamycin, gentamicin and florocinolon. Characterisation of the staphylococcal cassette chromosome (SCC) mec type has led to better discrimination of hospital acquired MRSA (HA-MRSA) and community acquired MRSA (CA-MRSA) [28]. SCCs are mobile elements characterized by association of a *mec complex* and *ccr genes* coding for integration into or excision from the chromosome. Three types of SCC (types I, II and III) were originally described in hospital-

catalase test differentiates the staphylococci from the streptococci [14].

enzymes which inactivate antimicrobic agents [26].

**2.11. Community and hospital acquired MRSA infections**

infections, bacteremia and endocardit [2].

Clumping Factor which is responsible for binding of organism to fibrin and fibrinogen-is a surface component of *S.aureus*. When clumbing factor comes up with plasma, *S.aureus* forms clumps. Clumping Factor is discret from coagulase [22,25].

#### **2.6. Enzymes**

Other enzymes produced by Staphylococcus's are sthapylokinase, proteinase, DNase and enzymes having different properties such as ß-lactamase.

#### **2.7. Exotoxins**

Alfa toxin is a heterogen protein acting by depending on large spectrum of eucariotic cell membrans. α toxin is a power hemolysin (a substance that causes the fragmentation of erytrosit). ß toxin have reduced the sphingomyelin, therefore it is toxic for many cells included human red blood cells. Additionally, an other toxin, δ is heterogen. It fractionates the biologic membrans and can have a role at diarrheal patients because of *S.aureus*.

#### **2.8. Leukocidin**

This toxin of *S.aureus* has two components. It affects white blood cells in humans and rabbits. These two components have moved synergistic like γ toxin at the membrane of white blood cells. The toxin is an important virulance factor in community acquired MRSA (CA-MRSA) strains.

#### **2.9. Toxic shock syndrom toxin**

Many *S.aureus* strains isolated from patients with toxic shock syndrome produce the toxic shock syndrome toxin-1 (TSST–1) also namely enterotoxin F. TSST–1 is the super antigen and binds the MHC-II molecules that leads to T cell stimulation TSST–1. This toxin is related to febrile, shock and multisystem involvement which are scope skin disease.

#### **2.10. Diagnostic laboratory tests**

#### *2.10.1. Gram staining*

*S. Aureus* bacteria appears violet colour and looks like bunch of grapes by gram staining. It is imposible to separate pathogenic organism (*S.aureus*) from saprophytic organisms (*S.epider‐ mitis*) by gram staining.

#### *2.10.2. Catalase test*

**2.5. Coagulase and clumping factor**

348 Current Progress in Biological Research

similar to invasive patogenic power.

**2.6. Enzymes**

**2.7. Exotoxins**

**2.8. Leukocidin**

**2.9. Toxic shock syndrom toxin**

**2.10. Diagnostic laboratory tests**

*2.10.1. Gram staining*

*mitis*) by gram staining.

strains.

clumps. Clumping Factor is discret from coagulase [22,25].

enzymes having different properties such as ß-lactamase.

membrans and can have a role at diarrheal patients because of *S.aureus*.

febrile, shock and multisystem involvement which are scope skin disease.

*S.aureus* produces coagulase being an enzyme providing coagulation of the plasma. Coagulase that can be droped out freely binds the prothrombin and initiates polymerization of fibrin. It can also accumulate the fibrin at suface of Staphylocci. It thinks that products of coagulase are

Clumping Factor which is responsible for binding of organism to fibrin and fibrinogen-is a surface component of *S.aureus*. When clumbing factor comes up with plasma, *S.aureus* forms

Other enzymes produced by Staphylococcus's are sthapylokinase, proteinase, DNase and

Alfa toxin is a heterogen protein acting by depending on large spectrum of eucariotic cell membrans. α toxin is a power hemolysin (a substance that causes the fragmentation of erytrosit). ß toxin have reduced the sphingomyelin, therefore it is toxic for many cells included human red blood cells. Additionally, an other toxin, δ is heterogen. It fractionates the biologic

This toxin of *S.aureus* has two components. It affects white blood cells in humans and rabbits. These two components have moved synergistic like γ toxin at the membrane of white blood cells. The toxin is an important virulance factor in community acquired MRSA (CA-MRSA)

Many *S.aureus* strains isolated from patients with toxic shock syndrome produce the toxic shock syndrome toxin-1 (TSST–1) also namely enterotoxin F. TSST–1 is the super antigen and binds the MHC-II molecules that leads to T cell stimulation TSST–1. This toxin is related to

*S. Aureus* bacteria appears violet colour and looks like bunch of grapes by gram staining. It is imposible to separate pathogenic organism (*S.aureus*) from saprophytic organisms (*S.epider‐*

MRSAs produce catalase, which converts hydrogen peroxide into water and oxygen. The catalase test differentiates the staphylococci from the streptococci [14].

#### *2.10.3. Susceptibility tests*

At choosing of advisable antibiotic drug associated with therapy of infection, a number of factors such as potential infectious agents, antibiotic susceptibility, host factors that may affect the activity of the drug in vivo, the location of the infection, pharmacokinetic and pharmaco‐ dynamic properties of the drug should be evaluate [26]. Generally, *in vitro* procedures applied for determination of antimicrobic activity of an antibiotic is named as susceptibility tests. Susceptibility tests apply in the cases of not foreseeable susceptibility against antibacterial agent which will be applied at treatment of aerob and facultative anaerob bacteria which are clinically important. Susceptibility against antimicrobic drugs can be detected with a lot of methods. In most, inhibitor activity of drugs (bacteriostatic) evaulates in applied method. The applied methods with this purpose can consider of; 1. dilution methods in liquid and solid media; 2. disc diffusion method; 3. gradient diffusion (E-test) method; 4. the detection of enzymes which inactivate antimicrobic agents [26].

At disc diffusion method, paper discs is absorbed a specific amount of antibiotic place onto plate inoculed with the test microorganism. Thus, antibiotic absorbed by disc diffuses into agar and inhibits the growing of bacteria at effect levels of antibiotic. At the end of this situation, a circular inhibition zone where does not grow bacteria occurs at ambient of the disc. The categories of susceptibility as of sensitive, medium and resistance identify by measuring diameter of this zone. The limit values related to these categories detect for every antimicrobic agent by regarding accescible serum levels [26,27]. For example, Staphilococci are *mec*A positive and are resistant against methicillin at Müller-Hinton agar contented 6 of μg/mL of oxasilin and 4% of NaCl.

#### **2.11. Community and hospital acquired MRSA infections**

Molecular epedemiology of community aquired MRSA (CA-MRSA) is tolerably different from hospital acquired MRSA (HA-MRSA). CA-infections commonly cause of skin and soft tisue infections, bacteremia and endocardit [2].

Panton-Valentine leukocidin (PVL) gene encoded a toxin which is responsible from virulance of bacteria and type 4 SCC*mec* genetic component frequently are presence at CA-MRSA isolates [22,16]. HA-MRSA isolates generally have type I, II or III of staphylococcal casette chromosome (SCC*mec*) genetic component. This gene domain is responsible from showing resistant of bacteria against antibiotics made from beta-lactam and even other drugs such as clindamycin, gentamicin and florocinolon. Characterisation of the staphylococcal cassette chromosome (SCC) mec type has led to better discrimination of hospital acquired MRSA (HA-MRSA) and community acquired MRSA (CA-MRSA) [28]. SCCs are mobile elements characterized by association of a *mec complex* and *ccr genes* coding for integration into or excision from the chromosome. Three types of SCC (types I, II and III) were originally described in hospitalacquired MRSA strains (HA-MRSA), most of them isolated before 1990. A fourth type (type IV) was recently described, first in community-acquired MRSA isolates (CA-MRSA) and then in several MRSA backgrounds, including hospital isolates [29].

To compare HA-MRSA strains with CA-MRSA, It needs to examine several aspects. Innitially, chromosomal elements for meticillin resistance in community-associated strains are chromo‐ some cassette mec (SCCmec) types IV or V, being smaller and more active than SCCmec types I–III found in hospital-acquired MRSA. In HA-MRSA, the larger gene elements are correlated with reduced bacterial ability as well as decreased toxin generation. Also, the PVL toxin is more common in CA-MRSA than in MSSA. Another thing, increased expression of certain virulence determinants which can cause more acute disease (e.g. phenol-soluble modulins-PSMs-) is available in CA MRSA. The last one,, while all S. aureus strains have an aptness to generate biofilms, it suggest that variations in biofilm matrix in CA-MRSA compared to other strains. But, there is no sufficient evidence in the literature that any of MRSA strain sample has a larger capability to cause invasive infection than MSSA strains [7].

#### **2.12. Clinic Infections**

#### *2.12.1. Skin and soft tisue infections*

CA-MRSA strains can be infected skin and soft tissue infections, which characteristically occur in healthy people without preconditions. For instance, the US had one of the biggest CA-MRSA epidemic, with one strain named USA300, being liable for most of infections. In 2005, 13.7% of all invasive MRSA infections in the US were community associated. [30]. Skin, soft tisue and bone infections seems at most. These infections can be confined from a localized infektion to more general infections such as cellulite, impedigo, folliculit, boll, carboncul and surgical wound infection. Staphylococcal bone marrow dermatitis (osteomyelitis) occurs typically at young children because of bacteremia. More severe manifestations can include necrotizing pneumonia, pyomyositis, sepsis, osteomyelitis and necrotizing fasciitis [7]. Even though, some of *S.aureus* infections corellate with development of TSS, nowadays most of patients have skin and soft tissue infections with this disease. For instance, Staphylococcal scalded skin syndrome seems at most in young children.It is less common in adults and older children. The reason is that some of toxins (exfoliative toxins; ETA and ETB) bind with GM4-like glycolipids in newborns sensitive epidermis. GM4-like glycolipids are not available in adult and children [31,32] Figure 1 shows a sample of skin and soft tisue infection of MRSA strains.

nearly every CA-MRSA strain as well as a small proportion of clinical MSSA strains. This suggests that PVL has an important role in fitness, transmissibility and virulence, but the role of PVL in the pathogenesis of CA-MRSA infections is controversial [7]. PVL is a toxin frag‐ mented speedy the white blood cells. Also, these strains can be contained other virulance

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 351

It is necessary the using of a penicillin which is resistant against β-lactamase in bacteremia, endocartidis, pneumonia and other infections which occur by *S.aureus* strains for long times. Vancomycin has been prefered at methicillin resistant strains. If the infections are because of *S.aureus* wich not produce β-lactamase, penicillin G has to choose for those infections. *S.aureus* strains izolated from clinic infections which are resistant against penicillin G always *produce* penicillinases. Those strains are generally sensitive against penicillins wich are resistant βlactamases, cephalosporins or vancomycin. Resistant of methicillin is independent from

Resistant of drugs such as penicillin, tetracyclin, aminoglycosid and, erytromycin which has detected by plasmids can be transfered to other staphylococci by transduction and conjugation. Antibiotics such as linezolid, daptomycin and dalfopristin can be used at serious staphylo‐

coccal or enterococcal infections of patients which have common drug resistance.

factors and toxins. Consequently, this kinf of infections have high rates of death.

*2.13.2. Treatment*

**Figure 1.** Skin and Soft tisue Infection of MRSA [33]

producting of β-lactamase.

#### **2.13. Systemic infections**

#### *2.13.1. Pneumonia*

At 1918 in registries, obtained from influence pandemia of young individuals, most of deaths related with bacterial super infections that *S.aureus* strains lead to those infections. Recently, *S.aureus* isolates produced PVL toxin has relevanted to infections of skin and soft tisue such as pnömonia at healthy young people. PVL is a bi-component exotoxin transmitted by bacteriophages that is encoded by two genes, *luk* FPV and *luk* S-PV. PVL genes are carried by Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 351

**Figure 1.** Skin and Soft tisue Infection of MRSA [33]

nearly every CA-MRSA strain as well as a small proportion of clinical MSSA strains. This suggests that PVL has an important role in fitness, transmissibility and virulence, but the role of PVL in the pathogenesis of CA-MRSA infections is controversial [7]. PVL is a toxin frag‐ mented speedy the white blood cells. Also, these strains can be contained other virulance factors and toxins. Consequently, this kinf of infections have high rates of death.

#### *2.13.2. Treatment*

acquired MRSA strains (HA-MRSA), most of them isolated before 1990. A fourth type (type IV) was recently described, first in community-acquired MRSA isolates (CA-MRSA) and then

To compare HA-MRSA strains with CA-MRSA, It needs to examine several aspects. Innitially, chromosomal elements for meticillin resistance in community-associated strains are chromo‐ some cassette mec (SCCmec) types IV or V, being smaller and more active than SCCmec types I–III found in hospital-acquired MRSA. In HA-MRSA, the larger gene elements are correlated with reduced bacterial ability as well as decreased toxin generation. Also, the PVL toxin is more common in CA-MRSA than in MSSA. Another thing, increased expression of certain virulence determinants which can cause more acute disease (e.g. phenol-soluble modulins-PSMs-) is available in CA MRSA. The last one,, while all S. aureus strains have an aptness to generate biofilms, it suggest that variations in biofilm matrix in CA-MRSA compared to other strains. But, there is no sufficient evidence in the literature that any of MRSA strain sample

CA-MRSA strains can be infected skin and soft tissue infections, which characteristically occur in healthy people without preconditions. For instance, the US had one of the biggest CA-MRSA epidemic, with one strain named USA300, being liable for most of infections. In 2005, 13.7% of all invasive MRSA infections in the US were community associated. [30]. Skin, soft tisue and bone infections seems at most. These infections can be confined from a localized infektion to more general infections such as cellulite, impedigo, folliculit, boll, carboncul and surgical wound infection. Staphylococcal bone marrow dermatitis (osteomyelitis) occurs typically at young children because of bacteremia. More severe manifestations can include necrotizing pneumonia, pyomyositis, sepsis, osteomyelitis and necrotizing fasciitis [7]. Even though, some of *S.aureus* infections corellate with development of TSS, nowadays most of patients have skin and soft tissue infections with this disease. For instance, Staphylococcal scalded skin syndrome seems at most in young children.It is less common in adults and older children. The reason is that some of toxins (exfoliative toxins; ETA and ETB) bind with GM4-like glycolipids in newborns sensitive epidermis. GM4-like glycolipids are not available in adult and children

[31,32] Figure 1 shows a sample of skin and soft tisue infection of MRSA strains.

At 1918 in registries, obtained from influence pandemia of young individuals, most of deaths related with bacterial super infections that *S.aureus* strains lead to those infections. Recently, *S.aureus* isolates produced PVL toxin has relevanted to infections of skin and soft tisue such as pnömonia at healthy young people. PVL is a bi-component exotoxin transmitted by bacteriophages that is encoded by two genes, *luk* FPV and *luk* S-PV. PVL genes are carried by

in several MRSA backgrounds, including hospital isolates [29].

has a larger capability to cause invasive infection than MSSA strains [7].

**2.12. Clinic Infections**

350 Current Progress in Biological Research

**2.13. Systemic infections**

*2.13.1. Pneumonia*

*2.12.1. Skin and soft tisue infections*

It is necessary the using of a penicillin which is resistant against β-lactamase in bacteremia, endocartidis, pneumonia and other infections which occur by *S.aureus* strains for long times. Vancomycin has been prefered at methicillin resistant strains. If the infections are because of *S.aureus* wich not produce β-lactamase, penicillin G has to choose for those infections. *S.aureus* strains izolated from clinic infections which are resistant against penicillin G always *produce* penicillinases. Those strains are generally sensitive against penicillins wich are resistant βlactamases, cephalosporins or vancomycin. Resistant of methicillin is independent from producting of β-lactamase.

Resistant of drugs such as penicillin, tetracyclin, aminoglycosid and, erytromycin which has detected by plasmids can be transfered to other staphylococci by transduction and conjugation. Antibiotics such as linezolid, daptomycin and dalfopristin can be used at serious staphylo‐ coccal or enterococcal infections of patients which have common drug resistance.

### **3. Material and method**

#### **3.1. MRSA isolates**

Thwenty-six isolates, which were isolated from intencive care unit (ICU) between September 2009-February 2010, were clinical isolates from ICU patients in Denizli Hospital. The isolates were identified as MRSA in hospital before obtaining. These isolates were obtained from tracheal aspirates, catheter tip, wounds, urine, blood, and effluxion, in a total of 26 strains were taken into account. Colonies were also checked by Gram-staining. Isolates were also identified by growth on Dnase test agar with methyl gren agar by DNase test. Table 1 shows the isolates and their origins.

strains at the study of antibiotic susceptibility. Tryptic soy agar (TSA) solid medium (g/l: pepton from casein 15.0; pepton from soymeal 5.0; Sodium chloride 5.0; agar-agar 15.0) was used for antibiogram tests. DNAase test agar with methyl green medium was used to

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

To determinate multiple antibiotic resistan of MRSA trains, antibiotic susceptibilities were investigated by disc diffusion method. Susceptibility to methicillin by disc diffusion had allready determined by cefoxitin (30 μg) discs before samples taking from hospital. The

and 48 h of incubation at 37 0C. In addition to cefoxitin (FOX, 30 μg), the following antibiotics were tested: vancomycin (VA, 30 μg), tetracycline (TE, 30 μg), erythromycin (E, 15 μg), clindamycin (DA, 2 μg), rifampicin (RA, 5 mcg), linezolid (LZD, 30 μg),, sulfame‐ thoxazole / trimethoprim (SXT, 25 mcg), penicillin-G (P, 10 unit), amikacin (AK, 30 mcg), seforoksimsodyum (CXM, 30 mcg), Novobiosin (NV, 5 μg), ampicillin (AM, 10 mcg) and

The method of Laemmli [34] was used by a little modification for electrophoresis. After

centrifuged for 20 min at 6000 rpm. The pellets were washed three times with sterile distilled water and stirred after adding 25 μl SDS sample buffer (0.06 M Tris, 2.5 % glycerol, 0.5 % SDS, 1.25 % β-mercaptoethanol and 0.001 % bromophenol blue). The proteins were denatured in boiling water for 10 min. Samples stayed in eppendorf tubes were upload‐

Denatured whole cell proteins were analyzed by SDS-PAGE according to Laemmli. This method used 4 % acrylamide stacking gel and 10 % acrylamide separating gel. MBI Fermentas SM0661 kit was used as molecular weight standard in SDS-PAGE. Electrophore‐ sis was performed with buffer system in a Biolab gel apparatus model V20-CDC. The gel was run at 150 V for 2 h at stacking gel, 200 V for 4 h at sperating gel until the bromophe‐ nol blue had reached the bottom. Gels were then stained with Coomassie Brilliant Blue R

were inoculated a full loop of cells to petridishes where place DNase test agar with methyl

C for 24 h. in 5 ml of TSB (Merc) media, samples were

C for 24 h. in TSB (Merc) media, 26 of MRSA strains

C'de for 15 min.

353

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457

C. Plates were read at 24

determinate the DNAase activity. All of media were autoclaved at 121 0

zone of inhibition was interpreted after 24 h of incubation at 35 0

C until using.

**3.3. Antimicrobial susceptibilities**

gentamicin (CN, 10 mcg) antibiotics.

overnight incubation at 37 0

ed to electrophoresis apparatus.

**4.1. Determination of DNase activity**

Following overnight incubation at 37 0

**4. SDS-PAGE**

250 (Sigma).

**3.4. Extraction of Whole Cell Proteins (WCPs)**

and stored at 4 0


Table shows MRSA samples obtained from 26 of patients, Denizli hospital

**Table 1.** MRSA strains and isolation resources

#### **3.2. Media**

Tryptic soy broth (TSB) medium (g/l:pepton from casein 17, pepton from soy meal 3, D(+)glucose 2.5, NACL Dipotassium hydrogen phosphate) was used for cultering MRSA strains at the study of antibiotic susceptibility. Tryptic soy agar (TSA) solid medium (g/l: pepton from casein 15.0; pepton from soymeal 5.0; Sodium chloride 5.0; agar-agar 15.0) was used for antibiogram tests. DNAase test agar with methyl green medium was used to determinate the DNAase activity. All of media were autoclaved at 121 0 C'de for 15 min. and stored at 4 0 C until using.

#### **3.3. Antimicrobial susceptibilities**

**3. Material and method**

352 Current Progress in Biological Research

Thwenty-six isolates, which were isolated from intencive care unit (ICU) between September 2009-February 2010, were clinical isolates from ICU patients in Denizli Hospital. The isolates were identified as MRSA in hospital before obtaining. These isolates were obtained from tracheal aspirates, catheter tip, wounds, urine, blood, and effluxion, in a total of 26 strains were taken into account. Colonies were also checked by Gram-staining. Isolates were also identified by growth on Dnase test agar with methyl gren agar by DNase test. Table 1 shows the isolates

**Code of Strains Source Code of Strains Source**

MRSA 1 tracheal aspirates MRSA 14 tracheal aspirates

MRSA 2 effluxion MRSA 15 tracheal aspirates

MRSA 3 tracheal aspirates MRSA 16 tracheal aspirates

MRSA 4 blood MRSA 17 tracheal aspirates

MRSA 6 catheter tip MRSA 19 tracheal aspirates

MRSA 8 blood MRSA 21 tracheal aspirates

MRSA 9 tracheal aspirates MRSA 22 catheter tip

MRSA 10 blood MRSA 23 blood

MRSA 11 urine MRSA 24 wound

MRSA 12 tracheal aspirates MRSA 25 blood

MRSA 13 tracheal aspirates MRSA 26 wound

Tryptic soy broth (TSB) medium (g/l:pepton from casein 17, pepton from soy meal 3, D(+)glucose 2.5, NACL Dipotassium hydrogen phosphate) was used for cultering MRSA

Table shows MRSA samples obtained from 26 of patients, Denizli hospital

**Table 1.** MRSA strains and isolation resources

**3.2. Media**

MRSA 5 blood MRSA 18 urine

MRSA 7 blood MRSA 20 blood

**3.1. MRSA isolates**

and their origins.

To determinate multiple antibiotic resistan of MRSA trains, antibiotic susceptibilities were investigated by disc diffusion method. Susceptibility to methicillin by disc diffusion had allready determined by cefoxitin (30 μg) discs before samples taking from hospital. The zone of inhibition was interpreted after 24 h of incubation at 35 0 C. Plates were read at 24 and 48 h of incubation at 37 0C. In addition to cefoxitin (FOX, 30 μg), the following antibiotics were tested: vancomycin (VA, 30 μg), tetracycline (TE, 30 μg), erythromycin (E, 15 μg), clindamycin (DA, 2 μg), rifampicin (RA, 5 mcg), linezolid (LZD, 30 μg),, sulfame‐ thoxazole / trimethoprim (SXT, 25 mcg), penicillin-G (P, 10 unit), amikacin (AK, 30 mcg), seforoksimsodyum (CXM, 30 mcg), Novobiosin (NV, 5 μg), ampicillin (AM, 10 mcg) and gentamicin (CN, 10 mcg) antibiotics.

#### **3.4. Extraction of Whole Cell Proteins (WCPs)**

The method of Laemmli [34] was used by a little modification for electrophoresis. After overnight incubation at 37 0 C for 24 h. in 5 ml of TSB (Merc) media, samples were centrifuged for 20 min at 6000 rpm. The pellets were washed three times with sterile distilled water and stirred after adding 25 μl SDS sample buffer (0.06 M Tris, 2.5 % glycerol, 0.5 % SDS, 1.25 % β-mercaptoethanol and 0.001 % bromophenol blue). The proteins were denatured in boiling water for 10 min. Samples stayed in eppendorf tubes were upload‐ ed to electrophoresis apparatus.

### **4. SDS-PAGE**

Denatured whole cell proteins were analyzed by SDS-PAGE according to Laemmli. This method used 4 % acrylamide stacking gel and 10 % acrylamide separating gel. MBI Fermentas SM0661 kit was used as molecular weight standard in SDS-PAGE. Electrophore‐ sis was performed with buffer system in a Biolab gel apparatus model V20-CDC. The gel was run at 150 V for 2 h at stacking gel, 200 V for 4 h at sperating gel until the bromophe‐ nol blue had reached the bottom. Gels were then stained with Coomassie Brilliant Blue R 250 (Sigma).

#### **4.1. Determination of DNase activity**

Following overnight incubation at 37 0 C for 24 h. in TSB (Merc) media, 26 of MRSA strains were inoculated a full loop of cells to petridishes where place DNase test agar with methyl green media and were applied the spot test. Inoculeted cells were incubated for overnight at 37 0 C for 24 h. After incubation, 1 N of HCl was dumped onto plates which grow MRSA colonies. It was checked whether achromatic color zone occur or not. The colonies occurring achromatic color zone were accepted as DNase positive strains, others were accepted DNase negative strains.

**Code of Strains**

**Antibiotics Dnaz**

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 355

**VA 30 TE 30 E 15 DA 2 RA 5 LZD 30 FOX 30 SXT 25 P 10 AK 30 CXM 30 NV 5 AM 10 CN 10 Activity** MRSA1 R R R R R R R R R R R R R R Positive MRSA2 R R R R R R R R R R R R R R Positive MRSA3 R R R R R R R R R R R R R R Positive MRSA4 R R R R R R R R R R R R R R Positive MRSA5 R R R R R R R R R R R R R R Positive MRSA6 R R R R R R R R R R R R R R Positive MRSA7 R R R R R R R R R R R R R R Positive MRSA8 R R R R R R R R R R R R R R Positive MRSA9 R R R R R R R R R R R R R R Positive MRSA10 R R R R R R R R R R R R R R Positive MRSA11 R R R R R R R R R R R R R R Negative MRSA12 R R R R R R R R R R R R R R Positive MRSA13 R R R R R R R R R R R R R R Positive MRSA14 R R R R R R R R R R R R R R Positive MRSA15 R R R R R R R R R R R R R R Negative MRSA16 R R R R R R R R R R R R R R Positive MRSA17 R R R R R R R R R R R R R R Positive MRSA18 R R R R R R MS R R R R R R Negative MRSA19 R R R R R R R R R R R R R R Positive MRSA20 R R R R R R R R R R R R R R Positive MRSA21 R R R R R R R R R R R R R R Positive MRSA22 R R R R R R R R R R R R R R Positive MRSA23 R R R R R R R R R R R R R R Positive MRSA24 R R R R R R R R R R R R R R Positive MRSA25 R R R R R R R R R R R R R R Positive MRSA26 R R R R R R R R R R R R R R Positive

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

R; Resistant, MS; Medium Sensetive, S; Sensetive (No sensetive strains in our study)

**Table 2.** Antibiotics susceptibility and DNase activity of MRSA strains

**Figure 2.** MRSA-24 antibiotic inhibition zones

### **5. Results**

#### **5.1. Antimicrobial susceptibilities**

Antimicrobial susceptibility testing was performed as recommended by the National Com‐ mittee on Clinical Laboratory Standards [35,36]. MRSA samples that we used in our study were activated by culturing freshly at 37 0 C for 24 h. two times. 100 μl from these activared fresh culture samples were done smear cultivation to petridishes of TSA medium. Antibiotic discs were fixed up to surface of the media by using Disc diffusion method under steril conditions. The samples were incubated at 37 0 C for 24 h. and the diameters of zone were measured.

In general, It was determinated that all of strains showed resistance against all of antibiotics. No zone of inhibition has been seen only against cefoxitin (FOX, 30 μg) antibiotic. It was observed that the zone did not occur except a few strains and strains were resistant against penicilin, amikacin (AK, 30 mcg), sulfamethoxazole / trimethoprim (SXT, 25 mcg), ampicillin (AM, 10 mcg), (CN, 10 mcg), erythromycin (E, 15 μg) and rifampicin (RA, 5 mcg) antibiotics. Beyond linezolid (LZD, 30 μg) which have started clinic using also in our country recently and used alternatively as an antimicrobial agent against glikopeptits in M-R strains, it was determinated that MRSA strains used in this study were resistant against this antibiotic. Even though it was detected that there was no resistance against vancomycin (VA, 30 μg) for MRSA infection in many studies, the sensitivity of vancomycin (VA, 30 μg) decreased significantly against MRSA strains in our study. Table 2 shows the results of antibiotic susceptibility.

#### **5.2. Determination of DNase activity**

Acording to the DNase tests, three of strains are DNase negative, the rest of 26 strains are DNase positive. Strains were 11.54% DNase negative and 88.46% DNase positive. Table 2 shows the results of DNase activity (Also, see figure 3).

#### **5.3. SDS-PAGE**

With regard to SDS PAGE analyzes whole cell proteins (WCPs) of MRSA strains showed the similar protein profile bands. Figure 4 and 5 show the band profiles obtained from WCPs (Also, see figure 4).

#### Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 355


R; Resistant, MS; Medium Sensetive, S; Sensetive (No sensetive strains in our study)

**Table 2.** Antibiotics susceptibility and DNase activity of MRSA strains

green media and were applied the spot test. Inoculeted cells were incubated for overnight at

Antimicrobial susceptibility testing was performed as recommended by the National Com‐ mittee on Clinical Laboratory Standards [35,36]. MRSA samples that we used in our study

fresh culture samples were done smear cultivation to petridishes of TSA medium. Antibiotic discs were fixed up to surface of the media by using Disc diffusion method under steril

In general, It was determinated that all of strains showed resistance against all of antibiotics. No zone of inhibition has been seen only against cefoxitin (FOX, 30 μg) antibiotic. It was observed that the zone did not occur except a few strains and strains were resistant against penicilin, amikacin (AK, 30 mcg), sulfamethoxazole / trimethoprim (SXT, 25 mcg), ampicillin (AM, 10 mcg), (CN, 10 mcg), erythromycin (E, 15 μg) and rifampicin (RA, 5 mcg) antibiotics. Beyond linezolid (LZD, 30 μg) which have started clinic using also in our country recently and used alternatively as an antimicrobial agent against glikopeptits in M-R strains, it was determinated that MRSA strains used in this study were resistant against this antibiotic. Even though it was detected that there was no resistance against vancomycin (VA, 30 μg) for MRSA infection in many studies, the sensitivity of vancomycin (VA, 30 μg) decreased significantly against MRSA strains in our study. Table 2 shows the results of antibiotic susceptibility.

Acording to the DNase tests, three of strains are DNase negative, the rest of 26 strains are DNase positive. Strains were 11.54% DNase negative and 88.46% DNase positive. Table 2

With regard to SDS PAGE analyzes whole cell proteins (WCPs) of MRSA strains showed the similar protein profile bands. Figure 4 and 5 show the band profiles obtained from WCPs (Also,

C for 24 h. two times. 100 μl from these activared

C for 24 h. and the diameters of zone were

C for 24 h. After incubation, 1 N of HCl was dumped onto plates which grow MRSA colonies. It was checked whether achromatic color zone occur or not. The colonies occurring achromatic color zone were accepted as DNase positive strains, others were accepted DNase

37 0

negative strains.

354 Current Progress in Biological Research

**5. Results**

measured.

**5.1. Antimicrobial susceptibilities**

were activated by culturing freshly at 37 0

**5.2. Determination of DNase activity**

**5.3. SDS-PAGE**

see figure 4).

shows the results of DNase activity (Also, see figure 3).

conditions. The samples were incubated at 37 0

**Figure 2.** MRSA-24 antibiotic inhibition zones

M 14 15 16 17 18 19 20 21 22 23 24 25 26

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 357

**Figure 5.** Whole cell protein profiles of MRSA strains by SDS-PAGE. Line 14-26: MRSA strains from ICU; Line M: Molec‐

Most of methods have been applied to determinate the MRSA strains. In the present study, SDS- PAGE was used for typing MRSA strains obtained from the various wards of patients in ICU. By SDS-PAGE (WCPs), all of the 26 MRSA strains, of which 11 from tracheal aspirat, 8 from blood, 2 from urine, 2 from wounds, 2 from catheter tip and the other one from effluxion were compared proteins profiles each other. Results of this study in MRSA strains by SDS-PAGE and previous studies obviusly indicate that valuable epidemiological informations can be demonstrated with electrophoretic methods. It is reported that WCPs can not be used because of the high similarities between their band patterns examined in the differentiation of MRSA strains [37]. In order to overcome this problem, it is necessary to make SDS-PAGE with the use of FPs and by N-PAGE using WCPs. Also this problem can dissolve when 5-7.5 % gel concentration uses in SDS-PAGE as opposed to 10% gel concentration [37]. İn this study we

applied only SDS-PAGE with use WCPs because of inadequate time of study term.

By using various antibiotic discs, in study of antibiotic susceptibility, generally all of the MRSA strains showed multiple drug resistance. The high level morbidity and mortality of MRSA infections has caused to trace the prevalence of multiple antibiotic resistant staphylococci mainly on the brink of MRSA. In Turkey between 1996-1999, the average of resistant against methicillin was detected 47,5 %. Vancomicin being glycopeptit antibiotic

ular weight standard in kD.

**6. Discussion**

#### **Figure 3.** DNase activity test

**Figure 4.** Whole cell protein profiles of MRSA strains by SDS-PAGE. Line 1-13: MRSA strains from ICU; Line M: Molecu‐ lar weight standard in kD.

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 357

**Figure 5.** Whole cell protein profiles of MRSA strains by SDS-PAGE. Line 14-26: MRSA strains from ICU; Line M: Molec‐ ular weight standard in kD.

### **6. Discussion**

**Figure 3.** DNase activity test

356 Current Progress in Biological Research

lar weight standard in kD.

M 1 2 3 4 5 6 7 8 9 10 11 12 13

**Figure 4.** Whole cell protein profiles of MRSA strains by SDS-PAGE. Line 1-13: MRSA strains from ICU; Line M: Molecu‐

Most of methods have been applied to determinate the MRSA strains. In the present study, SDS- PAGE was used for typing MRSA strains obtained from the various wards of patients in ICU. By SDS-PAGE (WCPs), all of the 26 MRSA strains, of which 11 from tracheal aspirat, 8 from blood, 2 from urine, 2 from wounds, 2 from catheter tip and the other one from effluxion were compared proteins profiles each other. Results of this study in MRSA strains by SDS-PAGE and previous studies obviusly indicate that valuable epidemiological informations can be demonstrated with electrophoretic methods. It is reported that WCPs can not be used because of the high similarities between their band patterns examined in the differentiation of MRSA strains [37]. In order to overcome this problem, it is necessary to make SDS-PAGE with the use of FPs and by N-PAGE using WCPs. Also this problem can dissolve when 5-7.5 % gel concentration uses in SDS-PAGE as opposed to 10% gel concentration [37]. İn this study we applied only SDS-PAGE with use WCPs because of inadequate time of study term.

By using various antibiotic discs, in study of antibiotic susceptibility, generally all of the MRSA strains showed multiple drug resistance. The high level morbidity and mortality of MRSA infections has caused to trace the prevalence of multiple antibiotic resistant staphylococci mainly on the brink of MRSA. In Turkey between 1996-1999, the average of resistant against methicillin was detected 47,5 %. Vancomicin being glycopeptit antibiotic

is very important because of methicillin resistant in staphylococcus's. Until now the resistance of glycopeptit was no informed in many studies which have done in our country [9,38]. In our study, we investigated that none of MRSA strains wree resistance against Vancomycin. Susceptibilities of antibiotic groups such as fucidic acid, sulfamethoxazole / trimethoprim, clindamycin, erithromycin, and quinolone have come into prominence because of alternativ treatment options of those antibiotics without glycopeptit antibiotics in patients having light and middle infections of MRSA and remediabled on erect pozition. SXT is an other antibiotic not to be β-lactam which uses in resistant staphilococcus's. Even though, susceptibility of SXT for MRSA strains was 81% in our study, it was 91% in study of Sengoz [19]. Also we found that rate of resistant strains for eritromycin antibiotic was 73%. In one study done in laboratory of clinic microbiology, Haydar Paşa Hospital, this rate for eritromycin was found 71%. In general it was considered that eritromycin antibiotic is not to be alternativ antibiotic option for treatment in MRSA's. Clindamycin is an alternativ antibiotic which can use in infections of staphylococcus. We found that resist‐ ant of clindamycin to MRSA strain was 23% in present study. A few studies which had been done in Turkey ; in study of Gonluugur resistant of clindamycin was 39%, in study of Dogan it was 54% [10,39].Linezolid is the first member of antibiotics which are from oksazolidinon group. Linezolids do not show the cross resistant with other antibiotics due to different effect mechanism of those antibiotics. Also, to resistant evolve is power against in vitro linezolid. Resistant evolving occurs typicaly with single nucleotid chancing in genes which encode the 23 S rRNA. In present study, susceptibility of linezolid was found 100%. It was found that linezolid antibiotic was effectiv and safe in 70% of facts with *S.aureus* infection which is not respondet or tolerant [40]. Unfortunately in our country, there is limeted number of study associated with Linezolid. This rate of resistant was 92% against gentamicin in our study.

**Author details**

\*Address all correspondence to: gksldogan@hotmail.com

Journal of Cystic Fibrosis 10; 298–306

microbials. J. Antimicrob. Chemother.; 49:999 - 1005.

mortality in a large cohort. Clin. Infect. Dis.; 31: 1170 - 1174.

, Gülümser Acar Doğanlı, Yasemin Gürsoy and Nazime Mercan Doğan

Obtained from Denizli Hospital http://dx.doi.org/10.5772/55457 359

Pamukkale University, Faculty of Arts and Sciences , Department of Biology, Denizli, Tur‐

Antibiotic Susceptibilities and SDS-PAGE Protein Profiles of Methicillin-Resistant Staphylococcus Aureus (MRSA) Strains

[1] Goss, C.H., Muhlebach, M.S., 2011. *Staphylococcus aureus* and MRSA in cystic fibrosis.

[2] Azaez Akande, O., 2010. Global trend of methicillin-resistant *Staphlococcus aureus* and emergingchallenges for control. Afr. J. Cln. Exper. Microbiol 11(3): 150-158.

[3] Rello, J., Diaz, E. 2003. Pneumonia in the intensive care unit. Crt. Care Med.; 31: 2544

[4] Graffunder. E.M., Venezia, R.A., 2002.Risk factors associated with nosocomial methi‐ cillin resistant *Staphylococcus aureus* (MRSA) infection including previous use of anti‐

[5] Gottileb, G.S., Fowler, V.G. Jr., Kong, L.K. et al. 2000. *Staphylococcus aureus* bactere‐ mia in the surgical patients: a prospective analysis of 73 postoperrative patients who developed *Staphylococcus aureus* bacteremia at a tertiary care facility. J. Am. CoIl.

[6] Mylotte, J.M., Tayara, A. 2000. *Staphylococcus aureus* bacteremia: predictors of 30 - day

[7] Watkins, R.R., David, M.Z., Salata, R.A., 2012. Current concepts on the virulence mechanisms of meticillin-resistant *Staphylococcus aureus*. Journal of Medical Microbi‐

[8] Chang, S., Sievert, D.M., Hageman, J.C., 2003. Infection with vancomycin-resistant *Staphylococcus aureus* containing the vanA resistance gene. *N Engl J Med,*; 348:1342–

[9] Chang, F.Y., MacDonald, B.B., Peacock, J.E., Jr, 2003. A prospective multicenter study of *Staphylococcus aureus* bacteremia: incidence of endocarditis, risk factors for mortali‐

ty, and clinical impact of methicillin resistance. *Medicine* ; 82:322–332.

ology Papers in Press. Published June 28, 2012 as doi:10.1099/jmm.0.043513-0.

Göksel Doğan\*

**References**


1347

Surg.; 190:50 - 57.

key

In this study, we investigated antibiotic susceptibilities, DNase activity and protein profiles by SDS-PAGE of MRSA strains. It was determinate that MRSA strains were resistant against all of antibiotics. A significant decrease in Vancomicin susceptibility is particularly notable. Moreover, the strains were 11.54% DNase negative and 88.46% DNase positive. Also strains showed similarities of band pattern for protein profiles by SDS-PAGE. Studies of Sacilik et al. (1999) and Van Belkum et al. (1997) [41] supported that dissemination of MRSA strains in Turkish hospitals probably originated from the same clone. In order to demostrate such informations and to understand clearly, it is nessesary to make more molecular genetic analyzes and studies in many zones of Turkey.

### **Acknowledgements**

We acknowledge Nilüfer AYDINLIK for her valuable assistance. We are also grateful personel of Denizli Hospital for MRSA samples.

### **Author details**

is very important because of methicillin resistant in staphylococcus's. Until now the resistance of glycopeptit was no informed in many studies which have done in our country [9,38]. In our study, we investigated that none of MRSA strains wree resistance against Vancomycin. Susceptibilities of antibiotic groups such as fucidic acid, sulfamethoxazole / trimethoprim, clindamycin, erithromycin, and quinolone have come into prominence because of alternativ treatment options of those antibiotics without glycopeptit antibiotics in patients having light and middle infections of MRSA and remediabled on erect pozition. SXT is an other antibiotic not to be β-lactam which uses in resistant staphilococcus's. Even though, susceptibility of SXT for MRSA strains was 81% in our study, it was 91% in study of Sengoz [19]. Also we found that rate of resistant strains for eritromycin antibiotic was 73%. In one study done in laboratory of clinic microbiology, Haydar Paşa Hospital, this rate for eritromycin was found 71%. In general it was considered that eritromycin antibiotic is not to be alternativ antibiotic option for treatment in MRSA's. Clindamycin is an alternativ antibiotic which can use in infections of staphylococcus. We found that resist‐ ant of clindamycin to MRSA strain was 23% in present study. A few studies which had been done in Turkey ; in study of Gonluugur resistant of clindamycin was 39%, in study of Dogan it was 54% [10,39].Linezolid is the first member of antibiotics which are from oksazolidinon group. Linezolids do not show the cross resistant with other antibiotics due to different effect mechanism of those antibiotics. Also, to resistant evolve is power against in vitro linezolid. Resistant evolving occurs typicaly with single nucleotid chancing in genes which encode the 23 S rRNA. In present study, susceptibility of linezolid was found 100%. It was found that linezolid antibiotic was effectiv and safe in 70% of facts with *S.aureus* infection which is not respondet or tolerant [40]. Unfortunately in our country, there is limeted number of study associated with Linezolid. This rate of resistant was 92% against

In this study, we investigated antibiotic susceptibilities, DNase activity and protein profiles by SDS-PAGE of MRSA strains. It was determinate that MRSA strains were resistant against all of antibiotics. A significant decrease in Vancomicin susceptibility is particularly notable. Moreover, the strains were 11.54% DNase negative and 88.46% DNase positive. Also strains showed similarities of band pattern for protein profiles by SDS-PAGE. Studies of Sacilik et al. (1999) and Van Belkum et al. (1997) [41] supported that dissemination of MRSA strains in Turkish hospitals probably originated from the same clone. In order to demostrate such informations and to understand clearly, it is nessesary to make more molecular genetic

We acknowledge Nilüfer AYDINLIK for her valuable assistance. We are also grateful personel

gentamicin in our study.

358 Current Progress in Biological Research

**Acknowledgements**

analyzes and studies in many zones of Turkey.

of Denizli Hospital for MRSA samples.

Göksel Doğan\* , Gülümser Acar Doğanlı, Yasemin Gürsoy and Nazime Mercan Doğan

\*Address all correspondence to: gksldogan@hotmail.com

Pamukkale University, Faculty of Arts and Sciences , Department of Biology, Denizli, Tur‐ key

### **References**


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[24] Mulligan, M.E., Murray-Leisure, K.A., Standiford, H.C., 1993. Methicillin- resistant *Staphylococcus aureus*: a consessus review of the microbiology, pathogenesis and epi‐ demiology with implications for prevention and management. Am J Med; 94:313-28.

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[25] Thorsherry, C., 1984. Methicillin-resistant (heteroresistant) Staphylococci Antimicro‐

[27] Novick RP, Schelievert P, Ruzin A. 2001. Pathogenicity and resistance islands of

[28] Boye, K., Bartels, M.D., Andersen, I.S., Møller, J.A., Westh, H.,2007. A new multiplex PCR for easy screening of methicillin-resistant*Staphylococcus aureus* SCC*mec* types I–

[29] Donnio, P.Y., Preney, L., Gautier-Lerestif, A.L., Avril, J.L., Lafforgue, N., 2004. Changes in staphylococcal cassette chromosome type and antibiotic resistance profile in methicillin-resistant *Staphylococcus aureus* isolates from a French hospital over an

[30] Klevens, R.M. et al., 2007. Invasive methicillin-resistant *Staphylococcus aureus* infec‐

[31] Kirca, Catar, F., 2008. *Staphylococcus aureus* suslarinda metisilin direnci tanisinda kul‐ lanilan bazi fenotipik yöntemlerin karsilastirilmasi. Uzmanlık Tezi, Gazi Universite‐

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[26] Franklin, D.L., 1998. *Staphylococcus aureus* infections. *N EnglJ Med*,; 339:520-32.

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[11] Chambers, H.F., 1988. Methicillin-resistant staphylococci. Clin Microbiol Rev;

[12] Moran, GJ., Amii, R.N., Abrahamian, F.M., Talan, D.A 2005. Methicillin resistant *Staphylococcus aureus* in community - acquired skin infections. Emerg. Infect. Dis; 11

[13] Chambers, H.F., 1997. Methicillin resistance in staphylococci: molecular and bio‐ chemical basis and clinical implications. American society for clinical microbiology.

[15] Koneman, E.W., Allen, S.D., William, M.J., Schereckenberger, P.C., Winn, W.C., 2006. Gram-positive cocci, Part I: Staphylococci and related gram-positive cocci. Winn WC Jr et al (editors). Color atlas and textbook of diagnostic microbiology, 6th ed. Lippin‐

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[23] Bronner, S., Monteil, H., Prévost. G,. 2004. Regulation of virulence determinants in *Staphylococcus aureus*: Complexity and applications. FEMS Microbiol Rev; 28:183.

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[38] Pa, W., 1997. National Committee for Clinical Laboratory Standarts. Performance standarts for antimicrobial disc susceptibility tests. Approved standart M2-A5, Na‐ tional Committee for Clinical Laboratory Standarts, 1997.

**Chapter 16**

**Plant Responses at Different Ploidy Levels**

The term "ploidy" expresses the number of sets of chromosomes in a biological cell and marked by an "X". A diploid genotype carries two paired (homologous) sets of chromosomes in the nucleus of each cell, one from each parent (Figure 1a). "Polyploidy" is the multiplication of entire sets of chromosomes. In other words, polyploid genotype has more than two homolo‐ gous sets of chromosomes in its cell. For example, tetraploid plants have four sets of chromo‐ somes in their cells (Figure 1b). Polyploidy is common among flowering plants (angiosperms) and is a major force in plant speciation [1]. Almost 47%-70% of angiosperms are polyploid [1-3].

There are differences between diploid and polyploid plants from morphological, physiologi‐ cal, cellular and biochemical aspects. Polyploid plants have bigger cells and stomatas than diploid ones that result in thicker and big leaves, larger flowers and fruits. In general, autote‐ traploids have greater vegetative volume and larger seed weight but lower reproductive fertility than diploids, and flowering and fruit formation were often later in tetraploids than in diploids as reported by Stebbins [4]. Shoots of polyploid genotypes are thicker with short internodes and wider crotch angles. As the chromosome number increased, DNA content per cell, enzyme activity per cell and cell volume all increased [5, 6]. In addition, polyploids are

Polyploid genotypes have shown resistance to biotic (pests and pathogens) and abiotic (drought and cold etc.) stress factors in some cases and this resistance enables them to have greater adaptability to wider ecological regions. This could be attributed to higher chromo‐ some number and gene expression causing to increase in the concentration of particular secondary metabolites and chemicals that are responsible for defense mechanism. This increase in the concentration of particular secondary metabolites and chemicals enable polyploid genotypes to resist against biotic and abiotic stress factors, consequently to grow in

> © 2013 Yildiz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

used as sources of variability and new genotypes for plant improvement [7, 8].

Additional information is available at the end of the chapter

Mustafa Yildiz

**1. Introduction**

http://dx.doi.org/10.5772/55785

the wide range of environments.


## **Plant Responses at Different Ploidy Levels**

### Mustafa Yildiz

[38] Pa, W., 1997. National Committee for Clinical Laboratory Standarts. Performance standarts for antimicrobial disc susceptibility tests. Approved standart M2-A5, Na‐

[39] Ridenour GA, Wong ES, Call MA, 2006. Duration of colonization with methicillin re‐ sistant*Staphylococcus aureus* among patients in the intensive care unit: implications

[40] Doganay M., 1998. Nozokomiyal sepsis: önemi ve tanımlar, Hastane İnfeksiyon Der‐

[41] Van Belkum, A., Van Leeuwen, W., Werkooyen R., Sacilik, S.C., Cokmus, C., Ver‐ brugh H., 1997. Dissemination of a single clone of methicillin-resistant *Staphylococcus*

tional Committee for Clinical Laboratory Standarts, 1997.

for intervention. *Infect Control Hosp Epidemiol,*; 27:271–278.

*aureus* among Turkish hospitals. J Clin Microbiol, 35: 978-81,

gisi; 2(4):179-81.

362 Current Progress in Biological Research

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55785

### **1. Introduction**

The term "ploidy" expresses the number of sets of chromosomes in a biological cell and marked by an "X". A diploid genotype carries two paired (homologous) sets of chromosomes in the nucleus of each cell, one from each parent (Figure 1a). "Polyploidy" is the multiplication of entire sets of chromosomes. In other words, polyploid genotype has more than two homolo‐ gous sets of chromosomes in its cell. For example, tetraploid plants have four sets of chromo‐ somes in their cells (Figure 1b). Polyploidy is common among flowering plants (angiosperms) and is a major force in plant speciation [1]. Almost 47%-70% of angiosperms are polyploid [1-3].

There are differences between diploid and polyploid plants from morphological, physiologi‐ cal, cellular and biochemical aspects. Polyploid plants have bigger cells and stomatas than diploid ones that result in thicker and big leaves, larger flowers and fruits. In general, autote‐ traploids have greater vegetative volume and larger seed weight but lower reproductive fertility than diploids, and flowering and fruit formation were often later in tetraploids than in diploids as reported by Stebbins [4]. Shoots of polyploid genotypes are thicker with short internodes and wider crotch angles. As the chromosome number increased, DNA content per cell, enzyme activity per cell and cell volume all increased [5, 6]. In addition, polyploids are used as sources of variability and new genotypes for plant improvement [7, 8].

Polyploid genotypes have shown resistance to biotic (pests and pathogens) and abiotic (drought and cold etc.) stress factors in some cases and this resistance enables them to have greater adaptability to wider ecological regions. This could be attributed to higher chromo‐ some number and gene expression causing to increase in the concentration of particular secondary metabolites and chemicals that are responsible for defense mechanism. This increase in the concentration of particular secondary metabolites and chemicals enable polyploid genotypes to resist against biotic and abiotic stress factors, consequently to grow in the wide range of environments.

© 2013 Yildiz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

However, the effects of increased ploidy level cannot be anticipated all the time. In contrast to common knowledge that polyploid individuals are superior than the diploid ones from many aspects,insomecasespolyploidplantscanhaveslowergrowthrates[9]whichcouldbeattributed todifficulties inthe cell cycleandslowcelldivision[10] causingtofewer cellnumberandsmaller organs. For example, it was reportedthatthe overall chlorophyll contentinpolyploidplants are higher than diploid ones with lower chromosome numbers [5, 6, 11], while chlorophyll a, chlorophyll b and total chlorophyll contents of tetraploid sugar beet genotypes ('AD 440' and 'CBM 315') in our study were found to be lower than diploid ones ('Agnessa' and 'Felicita') [12].

**2. Growth pattern of genotypes at different ploidy levels**

and De Veylder [17].

further developmental stages.

every time [18].

In a study conducted in sugar beet which is an important sucrose-producing crop worldwide in temperate regions and supplies about 20% of the world sugar consumption, two diploid ('Agnessa' and 'Felicita') and two tetraploid ('AD 440' and 'CBM 315') sugar beet genotypes were compared with respect to vegetative and generative characteristics such as seed germi‐ nation, seedling growth, total chlorophyll and protein contents, root and sugar yields, and sugar content [12]. The size of epidermal cells in a field of view area on upper leaf surface of sterile seedlings were counted using clear fingernail polish, clear tape, a glass slide, and a microscope at 60X magnification. From these counts, the highest results regarding cell size were recorded in tetraploid genotypes (Table 1, Figure 2). Decreased cell number in polyploid genotypes was compensated for by increased cell size as reported by Doonan [16] and, Inze

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785 365

**Genotypes Cell Number Cell Length (µm) Cell Width (µm) Approx. Cell Area (µm2)**

Diploid genotypes gave higher results than tetraploids in seed germination percentage, root length and seedling height at 4th day and root length and seedling height at 14th day (Table 2). Polyploid seeds had lower germination and emergence percentages than diploid ones due

In the first 6 weeks, diploid genotypes gave rise to the highest results with respect to plant height, root lenght, leaf length and width, approx. leaf area, plant fresh and dry weights, total chlorophyll content and protein percentage (Figure 3, Table 3). However, they were passed by tetraploid genotypes in the further stages of the development in the characters of plant height, root lenght, leaf length and width, approx. leaf area, plant fresh and dry weights (Figure 4, Table 4). These figures showed that tetraploid genotypes passed diploids vegetatively in the

Plants developed from diploid seeds were more vital and well-grown. Plant height and root length scores in diploid genotypes were good indicators for vitality and growth. Leaf area which plays an important role on the photosynthetic acticity, was found higher in diploid genotypes in the first 6 weeks. High ploidy level does not result in increased shoot growth

'Agnessa' (2X) 167.65 a 86.52 b 31.24 b 2702.88 b 'Felicita' (2X) 152.80 a 82.14 b 33.16 b 2723.76 b 'AD 440' (4X) 78.60 b 131.62 a 48.42 a 6373.04 a 'CBM 315' (4X) 72.40 b 128.24 a 52.16 a 6688.99 a

Values followed by the different letters in a column are significantly different at the 0.01 level

to their thicker seedcoat and seedling emergence strength [14, 15].

**Table 1.** Cell sizes in the upper leaf surface of 6-week-old sugar beet plants at different ploidy levels

Although there are studies reporting that seeds of tetraploid plants germinated faster with a higher percentage than those of diploids [13], in the study we conducted under greenhouse conditions, it was observed that germination and seedling growth of diploid sugar beet genotypes were much better than tetraploids. Our findings were parallel to the ones reporting that polyploid seeds might show lower germination and emergence percentage than diploids and this could be attributed to thicker seedcoat and weak seedling emergence [14] and weak embryo development [15].

**Figure 1.** Diagram for cells at different ploidy levels. (a) Diploid (indicated by 2n = 2X) cell having two homologous copies of each chromosome, (b) Tetraploid (indicated by 2n = 4X) cell having four homologous copies of each chromosome

### **2. Growth pattern of genotypes at different ploidy levels**

However, the effects of increased ploidy level cannot be anticipated all the time. In contrast to common knowledge that polyploid individuals are superior than the diploid ones from many aspects,insomecasespolyploidplantscanhaveslowergrowthrates[9]whichcouldbeattributed todifficulties inthe cell cycleandslowcelldivision[10] causingtofewer cellnumberandsmaller organs. For example, it was reportedthatthe overall chlorophyll contentinpolyploidplants are higher than diploid ones with lower chromosome numbers [5, 6, 11], while chlorophyll a, chlorophyll b and total chlorophyll contents of tetraploid sugar beet genotypes ('AD 440' and 'CBM 315') in our study were found to be lower than diploid ones ('Agnessa' and 'Felicita') [12].

Although there are studies reporting that seeds of tetraploid plants germinated faster with a higher percentage than those of diploids [13], in the study we conducted under greenhouse conditions, it was observed that germination and seedling growth of diploid sugar beet genotypes were much better than tetraploids. Our findings were parallel to the ones reporting that polyploid seeds might show lower germination and emergence percentage than diploids and this could be attributed to thicker seedcoat and weak seedling emergence [14] and weak

**Figure 1.** Diagram for cells at different ploidy levels. (a) Diploid (indicated by 2n = 2X) cell having two homologous copies of each chromosome, (b) Tetraploid (indicated by 2n = 4X) cell having four homologous copies of each chromosome

embryo development [15].

364 Current Progress in Biological Research

In a study conducted in sugar beet which is an important sucrose-producing crop worldwide in temperate regions and supplies about 20% of the world sugar consumption, two diploid ('Agnessa' and 'Felicita') and two tetraploid ('AD 440' and 'CBM 315') sugar beet genotypes were compared with respect to vegetative and generative characteristics such as seed germi‐ nation, seedling growth, total chlorophyll and protein contents, root and sugar yields, and sugar content [12]. The size of epidermal cells in a field of view area on upper leaf surface of sterile seedlings were counted using clear fingernail polish, clear tape, a glass slide, and a microscope at 60X magnification. From these counts, the highest results regarding cell size were recorded in tetraploid genotypes (Table 1, Figure 2). Decreased cell number in polyploid genotypes was compensated for by increased cell size as reported by Doonan [16] and, Inze and De Veylder [17].


Values followed by the different letters in a column are significantly different at the 0.01 level

**Table 1.** Cell sizes in the upper leaf surface of 6-week-old sugar beet plants at different ploidy levels

Diploid genotypes gave higher results than tetraploids in seed germination percentage, root length and seedling height at 4th day and root length and seedling height at 14th day (Table 2). Polyploid seeds had lower germination and emergence percentages than diploid ones due to their thicker seedcoat and seedling emergence strength [14, 15].

In the first 6 weeks, diploid genotypes gave rise to the highest results with respect to plant height, root lenght, leaf length and width, approx. leaf area, plant fresh and dry weights, total chlorophyll content and protein percentage (Figure 3, Table 3). However, they were passed by tetraploid genotypes in the further stages of the development in the characters of plant height, root lenght, leaf length and width, approx. leaf area, plant fresh and dry weights (Figure 4, Table 4). These figures showed that tetraploid genotypes passed diploids vegetatively in the further developmental stages.

Plants developed from diploid seeds were more vital and well-grown. Plant height and root length scores in diploid genotypes were good indicators for vitality and growth. Leaf area which plays an important role on the photosynthetic acticity, was found higher in diploid genotypes in the first 6 weeks. High ploidy level does not result in increased shoot growth every time [18].

It was reported that the fresh weight increase was mainly due to cell enlargement by water absorption [19] and increase in dry weight was closely related to cell division and new material synthesis [20]. Dry weight increase in diploids was due to an increase in photosynthetic activity and carbohydrate metabolism resulting from increased water uptake by longer roots. Reduced fresh weight in tetraploids could be attributed to decreased water absorption as reported by Prado et al. [21]. Sullivan and Phafter [22] reported that seedling growth was affected by genotypic differences more than ploidy in diploid and autotetraploid *Secale cereale*. Lower results in morphological characters in the first developmental stages of tetraploid genotypes could be attributed to slow cell division as reported by Comai [10].

**Genotypes**

**Germination (%) Root Length**

Values followed by the different letters in a column are significantly different at the 0.01 level

content remained constant in different ploidy levels of *Atriplex confertifolia* [26].

can show superior characteristics than tetraploid ones.

weeks after study initiation (Bar = 3 cm)

**Table 2.** Germination and seedling growth in diploid and tetraploid sugar beet genotypes

**(cm)**

'Agnessa' (2X) 75.10 a 7.40 a 6.34 a 12.67 a 9.05 a 'Felicita' (2X) 89.10 a 8.45 a 7.00 a 13.96 a 9.52 a 'AD 440' (4X) 60.10 b 5.05 b 5.74 a 7.50 b 6.21 b 'CBM 315' (4X) 60.10 b 5.86 b 5.49 a 7.52 b 7.38 b

Chlorophyll content which is accepted as an indicator of photosynthetic capacity of tissues [23-25], was again found higher in diploid plants. It was thought that this could be due to the fact that photosynthetic capacity of the tissue in diploids was higher because of higher chlorophyll content, water and nutrient uptake from the soil with their roots. Higher photo‐ synthetic capacity resulted in higher protein content in diploids. The number of phosynthetic cells per unit leaf area decreases with increasing ploidy level [26]. Although chloroplasts [27, 28] and chlorophyll content [5] are higher in polyploid genotypes, increase tendency of chlorophyll content by increasing ploidy level is not always apparent. For instance, chlorophyll

Polyploid plants may show high-ploidy syndrome that could be explained by costly cell cycle and slow cell division at higher ploidy levels. That means in some cases, diploid genotypes

**Figure 3.** Development of seedlings from seeds of (a) diploid ('Felicita') and (b) tetraploid ('AD 440') genotypes 6

**DAY 4 DAY 14**

**Height (cm) Root Length (cm) Seedling**

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785

**Height (cm)**

367

**Seedling**

**Figure 2.** Cells and stomatas from the upper leaf surface of 6-week-old sugar beet seedlings. (a) Diploid genotype 'Felicita' and (b) Tetraploid genotype 'AD 440'


Values followed by the different letters in a column are significantly different at the 0.01 level

**Table 2.** Germination and seedling growth in diploid and tetraploid sugar beet genotypes

It was reported that the fresh weight increase was mainly due to cell enlargement by water absorption [19] and increase in dry weight was closely related to cell division and new material synthesis [20]. Dry weight increase in diploids was due to an increase in photosynthetic activity and carbohydrate metabolism resulting from increased water uptake by longer roots. Reduced fresh weight in tetraploids could be attributed to decreased water absorption as reported by Prado et al. [21]. Sullivan and Phafter [22] reported that seedling growth was affected by genotypic differences more than ploidy in diploid and autotetraploid *Secale cereale*. Lower results in morphological characters in the first developmental stages of tetraploid genotypes

**Figure 2.** Cells and stomatas from the upper leaf surface of 6-week-old sugar beet seedlings. (a) Diploid genotype

'Felicita' and (b) Tetraploid genotype 'AD 440'

could be attributed to slow cell division as reported by Comai [10].

366 Current Progress in Biological Research

Chlorophyll content which is accepted as an indicator of photosynthetic capacity of tissues [23-25], was again found higher in diploid plants. It was thought that this could be due to the fact that photosynthetic capacity of the tissue in diploids was higher because of higher chlorophyll content, water and nutrient uptake from the soil with their roots. Higher photo‐ synthetic capacity resulted in higher protein content in diploids. The number of phosynthetic cells per unit leaf area decreases with increasing ploidy level [26]. Although chloroplasts [27, 28] and chlorophyll content [5] are higher in polyploid genotypes, increase tendency of chlorophyll content by increasing ploidy level is not always apparent. For instance, chlorophyll content remained constant in different ploidy levels of *Atriplex confertifolia* [26].

Polyploid plants may show high-ploidy syndrome that could be explained by costly cell cycle and slow cell division at higher ploidy levels. That means in some cases, diploid genotypes can show superior characteristics than tetraploid ones.

**Figure 3.** Development of seedlings from seeds of (a) diploid ('Felicita') and (b) tetraploid ('AD 440') genotypes 6 weeks after study initiation (Bar = 3 cm)


genotyes (Table 4). Root and sugar yields, and sugar content obtained from field trials 6 months after study initiation were again found the highest in diploids (Table 5). Polyploids flower and

> **Approx. Leaf Area (cm2)**

'Agnessa' (2X) 20.5 b 29.0 b 13.4 b 7.3 b 97.8 b 286.6 b 42.9 b 1571.5 a 20.46 a 'Felicita' (2X) 27.3 b 33.8 b 14.9 b 6.9 b 102.8 b 306.1 b 51.6 b 1533.8 a 21.77 a Mean 23.90 31.40 14.15 7.10 100.30 296.35 47.25 1552.65 21.11 'AD 440' (4X) 37.0 a 42.9 a 18.7 a 9.7 a 181.4 a 357.1 a 77.3 a 1210.6 b 16.10 b

Mean 35.00 43.85 18.40 9.25 170.35 369.10 77.45 1243.55 16.74

**Table 4.** Development of seedlings from seeds of diploid and tetraploid genotypes 10 weeks after study initiation

'Agnessa' (2X) 74.46 a 14.88 a 11.08 a 'Felicita' (2X) 68.48 a 16.21 a 11.10 a 'AD 440' (4X) 54.40 b 12.85 b 6.99 b 'CBM 315' (4X) 58.66 b 12.07 b 7.08 b

**Genotypes Root Yield (tones/ha) Sugar Content (%) Sugar Yield (tones/ha)**

**3. Regeneration capacity of genotypes at different ploidy levels under** *In*

Genetic variation is a prerequisite for successful plant breeding. *In vitro* culture techniques seem to offer certain advantages in this respect through somatic hybridization, induction of

**Vegetative Characters Generative Characters**

**Plant Dry Weight (g)**

**Total Chlorophyll Content (µg/g fresh tissue)**

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785

**Protein (%)**

369

**Plant Fresh Weight (g)**

33.0 a 44.8 a 18.1 a 8.8 a 159.3 a 381.1 a 77.6 a 1276.5 b 17.39 b

fruit later than diploids as reported by Stebbins [4].

**Root Length (cm)**

**Leaf Length (cm)**

**Leaf Width (cm)**

Values followed by the different letters in a column are significantly different at the 0.01 level

Values followed by the different letters in a column are significantly different at the 0.01 level

mutants and selection of disease free and disease resistant plants [29].

**Table 5.** Sugar content, root and sugar yields in diploid and tetraploid genotypes

**Genotypes**

'CBM 315' (4X)

*vitro* **conditions**

**Plant Height (cm)**

**Table 3.** Development of seedlings from seeds of diploid and tetraploid genotypes 6 weeks after study initiation

**Figure 4.** Development of seedlings from seeds of (a) diploid ('Felicita') and (b) tetraploid ('AD 440') genotypes 10 weeks after study initiation (Bar = 5 cm)

In general, tetraploids have higher vegetative growth but lower reproductive fertility than diploids. Thus, in our study, tetraploid genotypes passed diploid ones 10 weeks after study initiation regarding vegetative characters such as plant height, root length, leaf length and width, approximate leaf area, plant fresh and dry weights [12]. Data related to generative characters such as total chlorophyll content and protein percentage were the highest in diploid


genotyes (Table 4). Root and sugar yields, and sugar content obtained from field trials 6 months after study initiation were again found the highest in diploids (Table 5). Polyploids flower and fruit later than diploids as reported by Stebbins [4].

**Genotypes**

'CBM 315' (4X)

**Plant Height (cm)**

368 Current Progress in Biological Research

weeks after study initiation (Bar = 5 cm)

**Root Length (cm)**

**Leaf Length (cm)**

**Leaf Width (cm)**

**Approx. Leaf Area (cm2)**

'Agnessa' (2X) 18.53 a 21.40 a 5.13 a 3.04 a 16.51 a 11.81 a 2.50 a 894.07 b 18.45 a 'Felicita' (2X) 19.47 a 19.34 a 5.75 a 3.14 a 18.06 a 9.10 a 2.78 a 1035.47 a 21.07 a Mean 19.00 20.37 5.44 3.09 17.28 10.45 2.64 964.77 19.76 'AD 440' (4X) 15.01 b 13.46 b 5.00 a 2.34 b 11.70 b 6.19 b 1.18 b 815.99 b 6.33 b

Mean 15.07 14.20 4.98 2.41 12.02 5.44 1.06 747.90 4.61

**Table 3.** Development of seedlings from seeds of diploid and tetraploid genotypes 6 weeks after study initiation

**Figure 4.** Development of seedlings from seeds of (a) diploid ('Felicita') and (b) tetraploid ('AD 440') genotypes 10

In general, tetraploids have higher vegetative growth but lower reproductive fertility than diploids. Thus, in our study, tetraploid genotypes passed diploid ones 10 weeks after study initiation regarding vegetative characters such as plant height, root length, leaf length and width, approximate leaf area, plant fresh and dry weights [12]. Data related to generative characters such as total chlorophyll content and protein percentage were the highest in diploid

Values followed by the different letters in a column are significantly different at the 0.01 level

15.14 b 14.95 b 4.96 a 2.49 b 12.35 b 4.69 b 0.94 b 679.82 c 2.90 b

**Vegetative Characters Generative Characters**

**Plant Dry Weight (g)**

**Total Chlorophyll Content (µg/g fresh tissue)**

**Protein (%)**

**Plant Fresh Weight (g)**

Values followed by the different letters in a column are significantly different at the 0.01 level

**Table 4.** Development of seedlings from seeds of diploid and tetraploid genotypes 10 weeks after study initiation


Values followed by the different letters in a column are significantly different at the 0.01 level

**Table 5.** Sugar content, root and sugar yields in diploid and tetraploid genotypes

### **3. Regeneration capacity of genotypes at different ploidy levels under** *In vitro* **conditions**

Genetic variation is a prerequisite for successful plant breeding. *In vitro* culture techniques seem to offer certain advantages in this respect through somatic hybridization, induction of mutants and selection of disease free and disease resistant plants [29].

In a study conducted in sugar beet, it was aimed to examine the effect of the ploidy level on *in vitro* explant growth, adventitious shoot regeneration, rooting and plantlet establishment from petiole segments of two inbred lines ('ELK 345' - diploid and 'CBM 315' - tetraploid) [30]. Petioles were used as explant and 1 mg l-1 BAP and 0.2 mg l-1 NAA as the combination of growth regulators for shoot regeneration in accordance with studies reporting that the most responsive explant for *in vitro* culture of sugar beet was petiole [31-37] and the combination of the plant growth regulators was 1 mg l-1 BAP and 0.2 mg l-1 NAA [37]. The results clearly showed that there were sharp and statistically significant differences in all parameters examined between lines at different ploidy levels. The study was set in three parallels to confirm the accuracy of the study.

The tetraploid line 'CBM 315' had a higher fresh weight than the diploid line 'ELK 345' in all three experiments (Table 6). In all experiments, the differences in fresh weight between the diploid and the tetraploid lines were statistically significant (*p* < 0.05). Dry weight scores were again found to be higher in the tetraploid line, and the differences between these lines were statistically significant at 0.01 level in all experiments (Table 6). The highest mean of fresh and dry weights of petiole explants was recorded from the tetraploid line as 0.254 g and 0.023 g. In the diploid line, the mean fresh and dry weights of petioles were noted as 0.172 g and 0.012 g (Table 6). The difference between fresh and dry weights signifies the tissue water content. From these results, the tissue water content was calculated as 0.231 g (0.254-0.023) in the tetraploid line 'CBM 315', and 0.160 g (0.172-0.012) in the diploid line 'ELK 345'.

> cell, enzyme activity per cell, cell volume, and photosynthesis per cell all increased. It was also reported that the photosynthetic capacity of larger cells in polyploid plants are higher than

> **Table 6.** Fresh and dry weights, water and dry matter contents of petiole explants of 'ELK 345' (diploid) and 'CBM 315'

(tetraploid) lines 5 weeks after culture initiation on MS medium containing 1 mg l-1 BAP and 0.2 mg l-1 NAA

**Fresh Weight (g) Dry Weight (g) Water Content (%) Dry Matter Content**

0.168±0.009 0.226±0.011 0.012±0.001 0.023±0.002 92.83±0.410 89.76±0.410 7.17±1.147 10.24±1.147

0.161±0.091 0.251±0.028 0.012±0.001 0.022±0.002 92.34±0.361 91.34±0.275 7.66±0.361 8.66±0.275

0.187±0.017 0.284±0.019 0.013±0.0003 0.023±0.001 92.76±0.654 91.77±0.258 7.24±0.654 8.23±0.258

**'ELK 345' 2X**

**'CBM 315' 4X**

**'CBM 315' 4X**

t value 4.226\* 4.621\*\* 2.561\* 2.561\*

t value 3.120\* 4.542\*\* 2.165ns 2.165ns

t value 3.794\* 6.708\*\* 1.401ns 1.401ns

**Mean1** 0.172 0.254 0.012 0.023 92.64 90.96 7.36 9.04

**(%)**

**'CBM 315' 4X**

371

**'ELK 345' 2X**

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785

In all experiments, the highest results were obtained from petiole explants of the tetraploid line in the parameters of shoot regeneration percentage, shoot number per petiole, shoot length, total shoot number per Petri dish, number of shoots rooted, and the percentage of shoots rooted. During culture, petiole explants of the tetraploid line were observed to grow faster than the ones of the diploid line. By the end of the culture, petiole explants of the tetraploid line were bigger and well developed, and the number of shoots regenerated was also higher (Figure 5a-b) than the diploid line (Figure 5c). The differences between petiole explants of the diploid and tetraploid lines for all parameters examined were statistically significant at *p* < 0.01, with the exception of shoot regeneration percentage in all experiments

Shoot primordias on petiole explants appeared in the first week of the culture in the tetraploid line while they developed 16 days after culture initiation in the diploid line. The highest mean shoot regeneration percentage and mean shoot number per petiole was recorded as 69.99% and 20.23 in the tetraploid line while it was 45.57% and 12.61 in the diploid line (Table 7). Regenerated shoot length was again found to be higher in the tetraploid line 5 weeks after culture initiation. The mean shoot length was found as 2.8 cm in the tetraploid line, while it was 2.0 cm in the diploid line. The mean total shoot number per Petri dish, which can be determined by shoot regeneration percentage and shoot number per petiole, was recorded as

smaller cells with lower chromosome numbers [5, 6, 43].

**'ELK 345' 2X**

Significantly different from zero at \* *p* < 0.05 and \*\* *p* < 0.01

1st experiment

2nd experiment

3rd experiment

1 Mean of three experiments

**'CBM 315' 4X**

**'ELK 345' 2X**

which was different at *p <* 0.05 (Table 7, Table 8) [30].

141.77 in the tetraploid line and 57.50 in the diploid line (Table 7).

The cells with high ploidy levels have bigger vacuoles [38] that play an important role in regulating the osmotic pressure of the cell [39]. Higher osmotic pressure of the cell in polyploid plants, as reported by Tal and Gardi [40], could cause higher tissue metabolic activity by increasing water and hormone uptake from the medium. Cell enlargement by water absorp‐ tion, cell vacuolation, and turgor-deriven wall expansion is the main reason of fresh weight increase, as reported by Dale [19]. The increase in dry weight was closely related to cell division and new material synthesis [20]. Thus, increase in the fresh and dry weights of petiole explants of the tetraploid line in our study at the end of culture were chiefly due to an increase in the absorption of water and other components from the basal medium via the high cell osmotic pressure. On the contrary, lower osmotic pressure of the diploid line caused a decline in fresh and dry weights of petioles by decreasing the absorption of water and other components from the medium. Results about tissue water content clearly showed that the tetraploid line had higher osmotic pressure, which caused higher absorption of water and other components from the mediuım. Higher results of all parameters in the study could be attributed to higher cell osmotic pressure of the tetraploid line 'CBM 315'. Yildiz and Ozgen [41] have reported that increasing tissue water content, which caused higher tissue metabolic activity, resulted in higher results of all parameters examined.

The increase in ploidy level leads to a larger cell that has a higher growth rate [38]. Tetraploid genotypes had a higher water content [40] and more organic solutes than diploid genotypes [42]. Warner and Edwards [26] have reported that the chromosome number determines the size of leaves, the size of cells, the number of chloroplasts per cell, and amounts of photosyn‐ thetic enzymes and pigments in cell. As the chromosome number increased, DNA content per


1 Mean of three experiments

In a study conducted in sugar beet, it was aimed to examine the effect of the ploidy level on *in vitro* explant growth, adventitious shoot regeneration, rooting and plantlet establishment from petiole segments of two inbred lines ('ELK 345' - diploid and 'CBM 315' - tetraploid) [30]. Petioles were used as explant and 1 mg l-1 BAP and 0.2 mg l-1 NAA as the combination of growth regulators for shoot regeneration in accordance with studies reporting that the most responsive explant for *in vitro* culture of sugar beet was petiole [31-37] and the combination of the plant growth regulators was 1 mg l-1 BAP and 0.2 mg l-1 NAA [37]. The results clearly showed that there were sharp and statistically significant differences in all parameters examined between lines at different ploidy levels. The study was set in three parallels to confirm the accuracy of

The tetraploid line 'CBM 315' had a higher fresh weight than the diploid line 'ELK 345' in all three experiments (Table 6). In all experiments, the differences in fresh weight between the diploid and the tetraploid lines were statistically significant (*p* < 0.05). Dry weight scores were again found to be higher in the tetraploid line, and the differences between these lines were statistically significant at 0.01 level in all experiments (Table 6). The highest mean of fresh and dry weights of petiole explants was recorded from the tetraploid line as 0.254 g and 0.023 g. In the diploid line, the mean fresh and dry weights of petioles were noted as 0.172 g and 0.012 g (Table 6). The difference between fresh and dry weights signifies the tissue water content. From these results, the tissue water content was calculated as 0.231 g (0.254-0.023) in the

The cells with high ploidy levels have bigger vacuoles [38] that play an important role in regulating the osmotic pressure of the cell [39]. Higher osmotic pressure of the cell in polyploid plants, as reported by Tal and Gardi [40], could cause higher tissue metabolic activity by increasing water and hormone uptake from the medium. Cell enlargement by water absorp‐ tion, cell vacuolation, and turgor-deriven wall expansion is the main reason of fresh weight increase, as reported by Dale [19]. The increase in dry weight was closely related to cell division and new material synthesis [20]. Thus, increase in the fresh and dry weights of petiole explants of the tetraploid line in our study at the end of culture were chiefly due to an increase in the absorption of water and other components from the basal medium via the high cell osmotic pressure. On the contrary, lower osmotic pressure of the diploid line caused a decline in fresh and dry weights of petioles by decreasing the absorption of water and other components from the medium. Results about tissue water content clearly showed that the tetraploid line had higher osmotic pressure, which caused higher absorption of water and other components from the mediuım. Higher results of all parameters in the study could be attributed to higher cell osmotic pressure of the tetraploid line 'CBM 315'. Yildiz and Ozgen [41] have reported that increasing tissue water content, which caused higher tissue metabolic activity, resulted in

The increase in ploidy level leads to a larger cell that has a higher growth rate [38]. Tetraploid genotypes had a higher water content [40] and more organic solutes than diploid genotypes [42]. Warner and Edwards [26] have reported that the chromosome number determines the size of leaves, the size of cells, the number of chloroplasts per cell, and amounts of photosyn‐ thetic enzymes and pigments in cell. As the chromosome number increased, DNA content per

tetraploid line 'CBM 315', and 0.160 g (0.172-0.012) in the diploid line 'ELK 345'.

higher results of all parameters examined.

the study.

370 Current Progress in Biological Research

**Table 6.** Fresh and dry weights, water and dry matter contents of petiole explants of 'ELK 345' (diploid) and 'CBM 315' (tetraploid) lines 5 weeks after culture initiation on MS medium containing 1 mg l-1 BAP and 0.2 mg l-1 NAA

cell, enzyme activity per cell, cell volume, and photosynthesis per cell all increased. It was also reported that the photosynthetic capacity of larger cells in polyploid plants are higher than smaller cells with lower chromosome numbers [5, 6, 43].

In all experiments, the highest results were obtained from petiole explants of the tetraploid line in the parameters of shoot regeneration percentage, shoot number per petiole, shoot length, total shoot number per Petri dish, number of shoots rooted, and the percentage of shoots rooted. During culture, petiole explants of the tetraploid line were observed to grow faster than the ones of the diploid line. By the end of the culture, petiole explants of the tetraploid line were bigger and well developed, and the number of shoots regenerated was also higher (Figure 5a-b) than the diploid line (Figure 5c). The differences between petiole explants of the diploid and tetraploid lines for all parameters examined were statistically significant at *p* < 0.01, with the exception of shoot regeneration percentage in all experiments which was different at *p <* 0.05 (Table 7, Table 8) [30].

Shoot primordias on petiole explants appeared in the first week of the culture in the tetraploid line while they developed 16 days after culture initiation in the diploid line. The highest mean shoot regeneration percentage and mean shoot number per petiole was recorded as 69.99% and 20.23 in the tetraploid line while it was 45.57% and 12.61 in the diploid line (Table 7). Regenerated shoot length was again found to be higher in the tetraploid line 5 weeks after culture initiation. The mean shoot length was found as 2.8 cm in the tetraploid line, while it was 2.0 cm in the diploid line. The mean total shoot number per Petri dish, which can be determined by shoot regeneration percentage and shoot number per petiole, was recorded as 141.77 in the tetraploid line and 57.50 in the diploid line (Table 7).

**Figure 5.** *In vitro* shoot regeneration from petiole explants of (a-b) 'CBM 315' (4X) and (c) 'ELK 345' (2X) line 5 weeks after culture initiation (Bars = 0.5 cm in a and c, 1 cm in b).

Shoots regenerated from petiole explants of the diploid and tetraploid lines were rooted on MS medium containing 3 mg l-1 IBA for 2 weeks. The best results were observed in shoots regenerated from petiole explants of the tetraploid line in all three experiments (Table 8). From the results, it is evident that shoots regenerated from petioles of the tetraploid line were more capable of establishing new plantlets than the ones grown from petioles of the diploid line (Figure 6a). Of the 70 shoots transferred to rooting medium, 61.3 shoots (87.62%) from tetraploid line 'CBM 315' and 51.7 shoots (73.81%) from the diploid line 'ELK 345' were rooted successfully (Table 8). Transferred plants reached harvest maturity in the field and no morphological abnormalities were observed.

### **4.** *In vitro* **susceptibility of genotypes to** *Agrobacterium tumefaciens* **infection at different ploidy levels**

*Agrobacterium*-mediated transformation has been widely used for the introduction of foreign genes into plants and consequent regeneration of transgenic plants [44]. *A.tumefaciens* naturally infects the wound sites in dicotyledonous plants. Virulent strains of *A.tumefaciens*, when interacting with susceptible dicotyledonous plant cells, induce diseases known as crown gall

[45]. This strain contains a large megaplasmid (more than 200 kb), which plays a key role in tumor induction and for this reason it was named Ti (Tumor inducing) plasmid. The expression

**Table 7.** Adventitious shoot regeneration from petiole explants of 'ELK 345' (diploid) and 'CBM 315' (tetraploid) 5

weeks after culture initiation on MS medium containing 1 mg l-1 BAP and 0.2 mg l-1 NAA

**Figure 6.** *In vitro* rooting and plantlet development from petiole explants of (a) 'CBM 315' (tetraploid) and (b) 'ELK

**'CBM 315' 4X**

t value 3.957\* 8.105\*\* 6.124\*\* 5.917\*\*

t value 3.368\* 5.276\*\* 3.703\*\* 4.104\*\*

t value 3.501\* 7.889\*\* 4.583\*\* 5.847\*\* **Mean**<sup>1</sup> 45.57 69.99 12.61 20.23 2.0 2.8 57.50 141.77

**'ELK 345' 2X**

40.0±5.773 66.7±3.333 12.53±0.617 22.80±1.106 2.2±0.577 3.2±0.153 50.12±9.181 152.73±14.589

46.7±3.333 70.0±5.774 13.70±0.656 19.70±0.929 1.9±0.153 2.7±0.058 63.67±3.844 138.97±12.305

50.0±5.774 73.3±3.333 11.60±0.625 18.20±0.557 1.8±0.100 2.5±0.115 58.70±9.650 133.60±8.426

**Petiole Shoot Length (cm) Total Shoot Number per**

**'CBM 315' 4X**

**Petri Dish**

**'CBM 315' 4X**

**'ELK 345' 2X**

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785 373

**Shoot Number per**

**'ELK 345' 2X**

345' (diploid) line (Bar = 1.5 cm).

1st experiment

2nd experiment

3rd experiment

1 Mean of three experiments

**Shoot Regeneration (%)**

> **'CBM 315' 4X**

**'ELK 345' 2X**

Significantly different from zero at \* *p* < 0.05 and \*\* *p* < 0.01

**Figure 6.** *In vitro* rooting and plantlet development from petiole explants of (a) 'CBM 315' (tetraploid) and (b) 'ELK 345' (diploid) line (Bar = 1.5 cm).


Significantly different from zero at \* *p* < 0.05 and \*\* *p* < 0.01

1 Mean of three experiments

Shoots regenerated from petiole explants of the diploid and tetraploid lines were rooted on MS medium containing 3 mg l-1 IBA for 2 weeks. The best results were observed in shoots regenerated from petiole explants of the tetraploid line in all three experiments (Table 8). From the results, it is evident that shoots regenerated from petioles of the tetraploid line were more capable of establishing new plantlets than the ones grown from petioles of the diploid line (Figure 6a). Of the 70 shoots transferred to rooting medium, 61.3 shoots (87.62%) from tetraploid line 'CBM 315' and 51.7 shoots (73.81%) from the diploid line 'ELK 345' were rooted successfully (Table 8). Transferred plants reached harvest maturity in the field and no

**Figure 5.** *In vitro* shoot regeneration from petiole explants of (a-b) 'CBM 315' (4X) and (c) 'ELK 345' (2X) line 5 weeks

**4.** *In vitro* **susceptibility of genotypes to** *Agrobacterium tumefaciens*

*Agrobacterium*-mediated transformation has been widely used for the introduction of foreign genes into plants and consequent regeneration of transgenic plants [44]. *A.tumefaciens* naturally infects the wound sites in dicotyledonous plants. Virulent strains of *A.tumefaciens*, when interacting with susceptible dicotyledonous plant cells, induce diseases known as crown gall

morphological abnormalities were observed.

after culture initiation (Bars = 0.5 cm in a and c, 1 cm in b).

372 Current Progress in Biological Research

**infection at different ploidy levels**

**Table 7.** Adventitious shoot regeneration from petiole explants of 'ELK 345' (diploid) and 'CBM 315' (tetraploid) 5 weeks after culture initiation on MS medium containing 1 mg l-1 BAP and 0.2 mg l-1 NAA

[45]. This strain contains a large megaplasmid (more than 200 kb), which plays a key role in tumor induction and for this reason it was named Ti (Tumor inducing) plasmid. The expression


have been noted in rice [57], maize [58], various legumes [55], aspen [56], cucurbits [59], *Pinus* species [60],tomato [61],*Arabidopsis*[62], grape [63], andother species.Althoughsomedifferen‐ ces in transformation frequency may be attributed to environmental or physiological factors, a genetic basis for susceptibility has clearly been established in a few plant species [62, 64- 66].

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785 375

Several researchers have reported that susceptibility to *Agrobacterium* transformation of various tissues, organs and cell types within a plant may differ. De Kathen and Jacobsen [67] reported that only dedifferentiating cells near the vascular system of cotyledon and epicotyl sections of *Pisum sativum* were susceptible to *Agrobacterium* transformation. Sangwan et al. [68] showed that only dedifferentiating mesophyll cells were competent for transformation in

**Figure 7.** *In vitro* tumor formation caused by 'A281' virulent strain of *Agrobacterium tumefaciens* on leaf-disc explant

*Arabidopsis* cotyledon and leaf tissues.

of tetraploid sugar beet line 'CBM 315'

1 Mean of three experiments

**Table 8.** *In vitro* root development of shoots regenerated from petiole explants of 'ELK 345' (2X) and 'CBM 315' (4X) lines on rooting medium enriched with 3 mg l-1 IBA 2 weeks after culture initiation.

of T-DNA genes of Ti-plasmid in plant cells causes the formation of tumors at the infection site. Two genetic components of bacteria, virulence genes (*vir*) and chromosomal genes (*chv*), are directly involved in the transfer of T-DNA from *Agrobacterium* to plant cells [44]. The molecular basis of *Agrobacterium*-mediated transformation is the transfer and stable integration of a DNA sequence (T-DNA) from the *Agrobacterium tumefaciens* Ti (tumor-inducing) plasmid into the plant genome leading to plant cell transformation [46, 47].

In a study conducted by Yildiz et al. [48], it was aimed to determine the susceptibility level of two sugar beet lines to wild-type *Agrobacterium tumefaciens* infection and ploidy effect on gene transfer efficiency under *in vitro* conditions. To evaluate the susceptibility of sugar beet lines at different ploidy levels against *Agrobacterium* infection, tumor formation was scored using the virulent strains 'A281' and 'A136NC'.

Among two lines used in the study, 'CBM 315' gave the highest results in three parameters studied in 'A281' wild strain. In 'CBM 315', tumor induction percentage, tumor diameter and number of tumors per explant were scored as 94%, 3.88 mm and 7.78, respectively (Figure 7). 'CBM 315' was followed by 'ELK 345' as 62% in tumor induction percentage, 1.80 mm in tumor diameter and 4.03 in number of tumors per explant (Table 9). In 'A136NC' wild strain, the highest values in tumor induction percentage, tumor diameter and number of tumors per explant were obtained from 'CBM 315' as 96%, 4.24 mm and 8.13 whereas lowest results were recorded from line 'ELK 345' as 73%, 2.14 mm and 4.36, respectively (Table 9).

The virulence of the bacterium depends on the strain and its interaction with the host plant. Various plant species differ greatly in their susceptibility to infection by *Agrobacterium tumefaciens* or *Agrobacterium rhizogenes* [49-53].

Even within a species, different cultivars or ecotypes may show vastly different degrees of susceptibility to tumorigenesis by particular *Agrobacterium* strains [54-56]. These differences

have been noted in rice [57], maize [58], various legumes [55], aspen [56], cucurbits [59], *Pinus* species [60],tomato [61],*Arabidopsis*[62], grape [63], andother species.Althoughsomedifferen‐ ces in transformation frequency may be attributed to environmental or physiological factors, a genetic basis for susceptibility has clearly been established in a few plant species [62, 64- 66].

Several researchers have reported that susceptibility to *Agrobacterium* transformation of various tissues, organs and cell types within a plant may differ. De Kathen and Jacobsen [67] reported that only dedifferentiating cells near the vascular system of cotyledon and epicotyl sections of *Pisum sativum* were susceptible to *Agrobacterium* transformation. Sangwan et al. [68] showed that only dedifferentiating mesophyll cells were competent for transformation in *Arabidopsis* cotyledon and leaf tissues.

of T-DNA genes of Ti-plasmid in plant cells causes the formation of tumors at the infection site. Two genetic components of bacteria, virulence genes (*vir*) and chromosomal genes (*chv*), are directly involved in the transfer of T-DNA from *Agrobacterium* to plant cells [44]. The molecular basis of *Agrobacterium*-mediated transformation is the transfer and stable integration of a DNA sequence (T-DNA) from the *Agrobacterium tumefaciens* Ti (tumor-inducing) plasmid

**Table 8.** *In vitro* root development of shoots regenerated from petiole explants of 'ELK 345' (2X) and 'CBM 315' (4X)

t value 7.201\*\* 6.662\*\*

t value 3.656\* 3.709\*

t value 5.745\*\* 5.692\*\* **Mean1** 51.7 61.3 73.81 87.62

**Number of Shoots Rooted % of Shoots Rooted 'ELK 345' 2X 'CBM 315' 4X 'ELK 345' 2X 'CBM 315' 4X**

53±1.155 64±1.000 75.71±1.648 91.43±1.430

52±1.528 59±1.155 74.28±2.181 84.29±1.648

50±1.528 61±1.399 71.43±2.181 87.14±1.648

In a study conducted by Yildiz et al. [48], it was aimed to determine the susceptibility level of two sugar beet lines to wild-type *Agrobacterium tumefaciens* infection and ploidy effect on gene transfer efficiency under *in vitro* conditions. To evaluate the susceptibility of sugar beet lines at different ploidy levels against *Agrobacterium* infection, tumor formation was scored using

Among two lines used in the study, 'CBM 315' gave the highest results in three parameters studied in 'A281' wild strain. In 'CBM 315', tumor induction percentage, tumor diameter and number of tumors per explant were scored as 94%, 3.88 mm and 7.78, respectively (Figure 7). 'CBM 315' was followed by 'ELK 345' as 62% in tumor induction percentage, 1.80 mm in tumor diameter and 4.03 in number of tumors per explant (Table 9). In 'A136NC' wild strain, the highest values in tumor induction percentage, tumor diameter and number of tumors per explant were obtained from 'CBM 315' as 96%, 4.24 mm and 8.13 whereas lowest results were

The virulence of the bacterium depends on the strain and its interaction with the host plant. Various plant species differ greatly in their susceptibility to infection by *Agrobacterium*

Even within a species, different cultivars or ecotypes may show vastly different degrees of susceptibility to tumorigenesis by particular *Agrobacterium* strains [54-56]. These differences

recorded from line 'ELK 345' as 73%, 2.14 mm and 4.36, respectively (Table 9).

into the plant genome leading to plant cell transformation [46, 47].

lines on rooting medium enriched with 3 mg l-1 IBA 2 weeks after culture initiation.

the virulent strains 'A281' and 'A136NC'.

Significantly different from zero at \* *p* < 0.05 and \*\* *p* < 0.01

1st experiment

374 Current Progress in Biological Research

2nd experiment

3rd experiment

1 Mean of three experiments

*tumefaciens* or *Agrobacterium rhizogenes* [49-53].

**Figure 7.** *In vitro* tumor formation caused by 'A281' virulent strain of *Agrobacterium tumefaciens* on leaf-disc explant of tetraploid sugar beet line 'CBM 315'


genotypes [42]. Chromosome numberdetermines the size ofleaves,the size of cells,the number of chloroplasts per cell and amounts of photosynthetic enzymes and pigments in cell [26]. As chromosomenumberincreased,DNAcontentper cell, enzyme activityper cell, cellvolume and photosynthesis per cell are all increased. In general, photosynthetic capacity of larger cells in polyploid plants is higher than smaller cells with lower chromosome numbers [5, 6, 43].

In a study conducted by Yildiz et al. [73], the responses of sugar beet genotypes at different ploidy levels to salt stress were evaluated. Diploid ('Felicita') and tetraploid ('AD 440') sugar beet genotypes were grown in pots, 1-month-old seedlings were treated with NaCl at different concentrations (0, 50 and 150 mM). Four days after NaCl application, cytological observations (the number of cell and stomata in the field of view area, lengths and widths of cells and

Root lengths of both genotypes increased by increasing NaCl concentrations. Root length was recordedas7.25cmindiploidgenotype'Felicita'at150mMNaClwhileitwas7.90cmintetraploid genotype 'AD 440'. Seedling lengths also increased by increasing NaCl concentration. Seed‐ ling length was the highest in diploid genotype as 11.25 cm while it was only 7.90 in tetraploid genotype (Table 10). Damages of increasing NaCl concentration were seen clearly in the leaves

It was observed that cell number decreased by increasing NaCl concentration in both geno‐ types. However, decrease rate in cell number was higher in diploid genotype than tetraploid. This was most probably due to bigger cell size in tetraploid genotype and consequently there was few cells in the unit area. Lower cell number could be attributed to slow cell division as reported by Comai [10]. Cell length and width increased by increasing NaCl concentration. However, the highest values related to cell length and width were recorded in 150 mM NaCl concentration in diploid genotype as 40.28 μm and 29.14 μm while they were realized in 50 mM NaCl in tetraploid genotype 'AD 440' as 70.56 μm and 49.13 μm. In diploid genotype

1173.75 μm2 in 150 mM NaCl treatment. Approx. cell area was found almost two times more in diploid genotype when NaCl concentration was 150 mM. On the other hand, in tetraploid genotype 'AD 440', approx. cell area was found as 1372.14 μm2 in 0 mM NaCl (control) treatment whereas it was 3466.61 μm2 in 50 mM NaCl. The highest results in the parameters of cell length, cell width and approx. cell area were noted from 50 mM NaCl treatment in

> **Root Length (cm) Seedlings Length (cm) 0 mM NaCl 50 mM NaCl 150 mM NaCl 0 mM NaCl 50 mM NaCl 150 mM NaCl**

> > 8.75 b 5.80 b

7.25 a 7.90 a in control (0 mM NaCl) while it was

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785 377

10.75 a 7.33 ab 11.25 a 7.90 b

of seedlings. At higher NaCl concentrations, tissue necrosis was observed (Figure 8).

stomatas) and 8 days after, seedling and root lengths were recorded.

'Felicita', approx. cell area was recorded as 652.59 μm2

5.25 b 6.05 b

5.50 b 6.83 ab

Values followed by the different letters in a row are significantly different at the 0.01 level

**Table 10.** The effect of different concentrations of NaCl on sugar beet seedling development 8 days after salt

tetraploid genotype (Table 11).

**Genotype**

'Felicita' (2X) 'AD 440' (4X)

treatment

Each value is the mean of 3 replications with 12 explants

\*\* Significantly different at the 0.01 level

**Table 9.** Response of two sugar beet lines at different ploidy levels to *Agrobacterium tumefaciens* virulent strains 'A281' and 'A136NC' 4 weeks after leaf-disc inoculation

The host-range limitation is perhaps the greatest disadvantage of *Agrobacterium*-mediated transformation although it is the most common used vector for the introduction of foreign genes to many crop plants, especially to dicotyledonous. The results were in accordance with the previous studies indicating strain and genotype differences [69-71].

From the results, it could be concluded that sugar beet lines have susceptibility to *Agrobacte‐ rium* infection with different levels. Moreover, if Table 9 was examined carefully, an interesting point came to attention that the difference in tumor induction might be related to ploidy level. Actually, in both strains of *Agrobacterium*, the highest results were obtained from 'CBM 315' which was tetraploid. Analysis showed that 'CBM 315' to be more beneficial for tumor induction and more susceptible to *Agrobacterium tumefaciens*.

It was reported that increased ploidy levels resulted in bigger cell size [72]. As it is known *Agrobacterium* infects cells at wound sites and size of the cells in this sites may influence transformation efficiency. The difference between diploid and tetrapoloid sugar beet lines with respect to wild-type *Agrobacterium tumefaciens* susceptibility might be related to ploidy levels. To our knowledge, this was the first report indicating that gene transfer efficiency might be affected from cell size at wound sites. However, this finding must be verified repeatedly by detealed studies.

### **5. Plant cellular response to salt stress at different ploidy level**

The number of chlorophyll-containing chloroplasts increases from diploids to polyploids. Chlorophyll content and other proteins were shown to almost double from diploid to poly‐ ploid plants [40]. The cells with high ploidy level have bigger vacuoles and vacuole plays an important role in regulating osmotic pressure of the cell [38]. Higher cell osmotic pressure in polyploidplantscausetohightissuemetabolicactivitybyincreasingwaterandhormoneuptake from the medium. Additionally, the increase in ploidy level leads to larger cell that has high growth rate. Polyploid genotypes have a higher water content and organic solutes than diploid

genotypes [42]. Chromosome numberdetermines the size ofleaves,the size of cells,the number of chloroplasts per cell and amounts of photosynthetic enzymes and pigments in cell [26]. As chromosomenumberincreased,DNAcontentper cell, enzyme activityper cell, cellvolume and photosynthesis per cell are all increased. In general, photosynthetic capacity of larger cells in polyploid plants is higher than smaller cells with lower chromosome numbers [5, 6, 43].

**Genotype**

376 Current Progress in Biological Research

'ELK 345' (2X) 'CBM 315' (4X)

detealed studies.

**Tumor Induction (%)**

> 62 94

Each value is the mean of 3 replications with 12 explants

'A281' and 'A136NC' 4 weeks after leaf-disc inoculation

\*\* Significantly different at the 0.01 level

**Tumor Diameter (mm)**

> 1.80 3.88

the previous studies indicating strain and genotype differences [69-71].

induction and more susceptible to *Agrobacterium tumefaciens*.

**A281 A136NC**

**Tumor Induction (%)**

> 73 96

**Tumor Diameter (mm)**

> 2.14 4.24

**No of Tumors / Explant**

> 4.36 8.13

**No of Tumors / Explant**

> 4.03 7.78

t values 7.849\*\* 5.881\*\* 6.208\*\* 3.994\*\* 8.502\*\* 7.808\*\*

**Table 9.** Response of two sugar beet lines at different ploidy levels to *Agrobacterium tumefaciens* virulent strains

The host-range limitation is perhaps the greatest disadvantage of *Agrobacterium*-mediated transformation although it is the most common used vector for the introduction of foreign genes to many crop plants, especially to dicotyledonous. The results were in accordance with

From the results, it could be concluded that sugar beet lines have susceptibility to *Agrobacte‐ rium* infection with different levels. Moreover, if Table 9 was examined carefully, an interesting point came to attention that the difference in tumor induction might be related to ploidy level. Actually, in both strains of *Agrobacterium*, the highest results were obtained from 'CBM 315' which was tetraploid. Analysis showed that 'CBM 315' to be more beneficial for tumor

It was reported that increased ploidy levels resulted in bigger cell size [72]. As it is known *Agrobacterium* infects cells at wound sites and size of the cells in this sites may influence transformation efficiency. The difference between diploid and tetrapoloid sugar beet lines with respect to wild-type *Agrobacterium tumefaciens* susceptibility might be related to ploidy levels. To our knowledge, this was the first report indicating that gene transfer efficiency might be affected from cell size at wound sites. However, this finding must be verified repeatedly by

The number of chlorophyll-containing chloroplasts increases from diploids to polyploids. Chlorophyll content and other proteins were shown to almost double from diploid to poly‐ ploid plants [40]. The cells with high ploidy level have bigger vacuoles and vacuole plays an important role in regulating osmotic pressure of the cell [38]. Higher cell osmotic pressure in polyploidplantscausetohightissuemetabolicactivitybyincreasingwaterandhormoneuptake from the medium. Additionally, the increase in ploidy level leads to larger cell that has high growth rate. Polyploid genotypes have a higher water content and organic solutes than diploid

**5. Plant cellular response to salt stress at different ploidy level**

In a study conducted by Yildiz et al. [73], the responses of sugar beet genotypes at different ploidy levels to salt stress were evaluated. Diploid ('Felicita') and tetraploid ('AD 440') sugar beet genotypes were grown in pots, 1-month-old seedlings were treated with NaCl at different concentrations (0, 50 and 150 mM). Four days after NaCl application, cytological observations (the number of cell and stomata in the field of view area, lengths and widths of cells and stomatas) and 8 days after, seedling and root lengths were recorded.

Root lengths of both genotypes increased by increasing NaCl concentrations. Root length was recordedas7.25cmindiploidgenotype'Felicita'at150mMNaClwhileitwas7.90cmintetraploid genotype 'AD 440'. Seedling lengths also increased by increasing NaCl concentration. Seed‐ ling length was the highest in diploid genotype as 11.25 cm while it was only 7.90 in tetraploid genotype (Table 10). Damages of increasing NaCl concentration were seen clearly in the leaves of seedlings. At higher NaCl concentrations, tissue necrosis was observed (Figure 8).

It was observed that cell number decreased by increasing NaCl concentration in both geno‐ types. However, decrease rate in cell number was higher in diploid genotype than tetraploid. This was most probably due to bigger cell size in tetraploid genotype and consequently there was few cells in the unit area. Lower cell number could be attributed to slow cell division as reported by Comai [10]. Cell length and width increased by increasing NaCl concentration. However, the highest values related to cell length and width were recorded in 150 mM NaCl concentration in diploid genotype as 40.28 μm and 29.14 μm while they were realized in 50 mM NaCl in tetraploid genotype 'AD 440' as 70.56 μm and 49.13 μm. In diploid genotype 'Felicita', approx. cell area was recorded as 652.59 μm2 in control (0 mM NaCl) while it was 1173.75 μm2 in 150 mM NaCl treatment. Approx. cell area was found almost two times more in diploid genotype when NaCl concentration was 150 mM. On the other hand, in tetraploid genotype 'AD 440', approx. cell area was found as 1372.14 μm2 in 0 mM NaCl (control) treatment whereas it was 3466.61 μm2 in 50 mM NaCl. The highest results in the parameters of cell length, cell width and approx. cell area were noted from 50 mM NaCl treatment in tetraploid genotype (Table 11).


Values followed by the different letters in a row are significantly different at the 0.01 level

**Table 10.** The effect of different concentrations of NaCl on sugar beet seedling development 8 days after salt treatment

**Cell Number Cell Length (µm) Cell Width (µm) Approx. Cell Area (µm2)**

**0 mM NaCl**

> 20.58 b

> 25.14 b

**0 mM NaCl**

18.85 b

22.42 b

**50 mM NaCl**

> 19.28 b

> 49.13

**50 mM NaCl**

18.85 b

23.99 a

**150 mM NaCl**

29.14 a

**150 mM NaCl**

20.56 a

21.70 c

<sup>a</sup> 30.85 b

**0 mM NaCl**

Plant Responses at Different Ploidy Levels http://dx.doi.org/10.5772/55785

> 652.59 b

> 1372.1 4 b

**0 mM NaCl**

495.37 b

877.07 b

**50 mM NaCl**

> 523.25 b

> 3466.6 1 a

**Approx. Stomata Area (µm2)**

> **50 mM NaCl**

440.39 c

881.29 a

**150 mM NaCl** 379

1173.7 5 a

1383.6 2 b

> **150 mM NaCl**

538.54 a

842.82 c

**150 mM NaCl**

**150 mM NaCl**

Polyploidy is a common phenomenon in nature. There are differences between diploid and polyploid plants from morphological, physiological, cellular and biochemical aspects. Although polyploid genotypes have several advantages over diploids, the effects of increased ploidy level cannot be anticipated all the time. This was seen clearly in the studies we con‐ ducted. From one hand, diploid genotypes found superior than tetraploids in the generative characteristics such as total chlorophyll and protein contents, root and sugar yields, and sugar content under field conditions, on the other hand, regeneration capacity and susceptibility to *Agrobacterium tumefaciens* infection of polyploids were found higher under *in vtiro* conditions. Moreover, when cellular responses were examined, tetraploid genotype seemed more resistant to salt stress than diploid counterpart. Thus, it should be considered that responses of polyploid genotypes may differ from mophological, physiological, cellular and biochemical aspects. That is why, in a research study, responses of both diploid and polyploid genotypes

**Genotype**

'Felicita' (2X)

'AD 440' (4X)

**Genotype**

'Felicita' (2X)

'AD 440' (4X)

**6. Conclusion**

**0 mM NaCl**

162.70 a

84.60

**0 mM NaCl**

18.80 a

13.50 a

**50 mM NaCl**

> 124.50 b

> > **50 mM NaCl**

18.20 b

11.30 b

<sup>a</sup> 52.40 c

**150 mM NaCl**

69.30 b

**150 mM NaCl**

should be evaluated carefully for successful results.

**0 mM NaCl**

**0 mM NaCl**

**50 mM NaCl**

54.58 b 70.56 a 44.85 b

**Stomata Number Stomata Length (µm) Stomata Width (µm)**

**50 mM NaCl**

7.30 c 26.28 b 21.42 b 28.57 a

9.50 b 36.56 b 38.84 a 37.63 b

Values followed by the different letters in a row are significantly different at the 0.01 level

**Table 11.** Cellular responses to salt stress of sugar beet genotypes at different ploidy levels

70.70 c 31.71 b 27.14 b 40.28 a

**Figure 8.** Sugar beet leaf development of cv. 'Felicita' 8 days after salt treatment (a) 0 mM NaCl (control), (b) 50 mM NaCl and (c) 150 mM NaCl

Number of stomata decreased by increasing NaCl concentration and this decrease was compensated by increased stomata size as reported by Inze and De Veylder [16]. Higher NaCl concentration increased stomata length and decreased stomata width in tetraploid genotype 'AD 440'. The highest stomata area was recorded from 150 mM NaCl treatment in diploid genotype while it was noted from 50 mM in tetraploid (Table 11).

The highest cell and stomata numbers were recorded from 0 mM NaCl treatment in both genotypes. And also cell and stomata numbers decreased by increasing NaCl concentration. However, this decrease in cell and stomata numbers were observed sharper in diploid genotype than in tetraploid one. The difference in cell and stomata numbers between 0 and 150 mM NaCl treatments was higher in diploid than in tetraploid. This could be due to the fact that tetraploid genotype 'AD 440' was more resistant to salt stress than diploid genotype 'Felicita' (Table 11). In other characters (cell and stomata lengths, cell and stomata widths, approx. cell and stomata areas), the highest values were recorded from 150 mM NaCl treatment in diploid genotype while they were noted from 50 mM NaCl treatment in tetraploid genotype. Since many characters in 150 mM NaCl concentration were almost the same as in 0 mM NaCl, it could be concluded that tetraploid genotype 'AD 440' was more resistant to salt stress than diploid one.


**Table 11.** Cellular responses to salt stress of sugar beet genotypes at different ploidy levels
