Induced of plastid mutations in soybean plant (Glycine max L. Merrill) with gamma radiation and determination with RAPD

Mutation Research 556 (2004) 35–44

Induced of plastid mutations in soybean plant (Glycine max L. Merrill) with gamma radiation and determination with RAPD C ¸ imen Ataka,∗ , Sema Alikamano˘glub , Leyla Ac¸ıkc , Yasemin Canbolatb a

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Hali¸c University, Ahmet Vefik Pasa Cad: No:1, Findikzade 34280, Istanbul, Turkey b Department of Biology, Istanbul University, Faculty of Science, Istanbul, Turkey c Department of Biology, Faculty of Arts and Sciences, Gazi University, Ankara, Turkey Received 23 December 2002; received in revised form 14 June 2004; accepted 30 June 2004

Abstract The aim of our study was to induce with radiation of atrazine resistant and tolerated mutants in Coles, Amsoy-71 and 1937 soybean varieties. Atrazine that is photosynthetic inhibitor is the most important herbicide of S-triazin group, and shows toxic effect on soybean plant. For the improvement of the atrazine resistant plants with mutation breeding, the seeds belonging to the three varieties were irradiated with 200 Gy of gamma radiation dose. The irradiated seeds were sown in the field and at the end of harvesting season, every pod at node situated on the main stem was picked up separately and M2 generations were obtained. At the plants, which were obtained from M2 generation, chlorophyll mutants were determined and atrazine selection was made. The percentage of chlorophyll mutants for Amsoy-71, Coles and 1937 soybean varieties were found as 1.07, 1.48 and 1.32, respectively. At the end of atrazine selection, the percentages of atrazine resistant plants for Amsoy-71, Coles and 1937 soybean varieties were 0.80, 0.60 and 0.53, respectively. The percentages of atrazine tolerated plants were 1.07, 1.18 and 1.05, respectively as well. In our research; the differences among the mutants replying to atrazine in various concentrations were examined by using RAPD procedure as the molecular marker techniques in comparison with polymorphism. In the study done by using 14 primers; according to the amplification results, the differences between atrazine resistant plants were shown. © 2004 Elsevier B.V. All rights reserved. Keywords: Soybean; Plastid mutation; Gamma radiation; RAPD.

1. Introduction ∗

Corresponding author. Tel.: +90 212 5305024; fax: +90 212 5303535. E-mail address: [email protected] (C ¸ . Atak). 0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.06.037

Depending on the rising population, nutrition becomes a problem nowadays. For this reason, researchers do researches to obtain much more products

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from the limited agricultural fields. The damages, which were occurred by weeds and insect, cause the important crop yield losses in cultivated fields. The purpose of chemical weed control includes less crop plants injury when they are highly effective in weed control. At this position, the resistance of the culture plant for the herbicides to be used has an importance. The improvement of the varieties resisted for herbicides with the genetics studies done in culture plants has been worked. The increase of the plants variability is crosscombinations in hybridization, spontaneous mutations and induced mutations. The rate of spontaneous mutations in the nature is too low for plant breeding. Therefore, physical and chemical mutagens can be used for mutation induction in cultivated plants. It can be possible to increase the genetic variability by inducing many mutations in plants with ionized radiations at the in vivo and in vitro studies of mutation breeding. In recent years, in vitro techniques seem to offer certain advantages for plant breeding programs [1]. For the purpose of increase of the genetics variability in plants, many researchers used physical and chemical mutagens [2]. Selected mutant varieties were analyzed at the molecular level and differences in variability could be determined. At the end of the induction of ionized radiations with mutation, the numbers of released mutant varieties were derived [1]. Atrazine that is used as a weed control is the most important herbicide of S-triazine group. This herbicide, which is used for crop, plants like corn, sorghum etc. and shows toxic effect to broadleaf crops such as soybean [3]. Soybean is sensitive to triazine herbicides and acts by inhibiting photosynthesis. Atrazine is a widely used selective herbicide, which interacts with photosystem II. This herbicide blocks electron transport at PSII by binding to the QB or herbicidebinding protein encoded by the psb A gene of cp DNA [4–7]. The improvement of a soybean variety, which is resistant or tolerance to atrazine by mutation will provide the usage of soybean together with corn for intercropping and corn rotation. The determination of the plant variability at molecular level, the examination of genome structures and the establishment of plant gene maps by using molecular marker techniques were a great importance for the continuation of germplasm and plant breeding. The RAPD

method can be used for the detection of DNA damage and mutations. In this study, selection of atrazine tolerance and resistant mutants were aimed by inducing the chloroplast mutations with gamma radiation in three different varieties belonging to soybean and the differences between selected atrazine tolerance and resistant mutants and control plants were investigated comparatively with RAPD-PCR analysis.

2. Material and methods 2.1. Plant material and determination of GR50 dose In this research, Coles, Amsoy-71 and 1937 for soybean variety have been taken from Samsun Black Sea Agriculture Research Institute. The response of cell of higher plants to physical mutagens is influenced to a varying degree by numerous biological, environmental and chemical factors. Seed water content is the most important environmental factor that modifies the effectiveness (mutation per unit dose) and efficiency (ratio of mutation) of mutagens in cell of higher plants [8]. For this reason, the seeds were kept in vacuum desiccators in which contained glycerol/water mixture to adjust the seed water content. After this treatment, Coles, Amsoy-71 and 1937 soybean varieties’ seed water content were found as 12.1%, 13.6% and 11.1%, respectively. Soybean seeds were irradiated with Cs-137 source in IBL 437C irradiation facility belonging to Our Leukemia Children Foundation (dose rate/min 10 Gy) with doses of 0, 100, 200, 300,and 500 Gy of gamma rays [9–12]. On the same day, control and irradiated soybean seeds were sown into plastic boxes, which are 30 × 38 × 8 cm in dimensions. The boxes were filled with standard experimental soils. Soybean seeds were grown up in climate chamber, which has light/dark (16/8 h) day period at 26 ◦ C, under controlled conditions. In the experiments, each treatment had three parallels and 15 seeds were employed for each parallel. GR50 dose that decreases the seedling height 50%, were determined for each variety by measuring the seedling height of 14-day plants [9,13]. According to control, seedling height for Amsoy-71, Coles and 1937 soybean varieties decreased 39, 40, 30.09 and 34.82%,

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sistant and tolerated plants to atrazine and chlorophyll mutants were selected and then, they were transferred to the pots and were grown up under the controlled conditions in climate room. 2.4. Atrazine test in M2 progeny

Fig. 1. Effect of gamma radiation on seedling height of soybean varieties. GR50 doses: Coles (240 Gy), Amsoy-71 (225 Gy) and 1973 (235 Gy).

respectively at 200 Gy; 81.69, 78.48 and 79.97%, respectively, at 300 Gy. GR50 doses were calculated as 225 Gy for Amsoy-71, 240 Gy for Coles and 235 Gy for 1937 (Fig. 1). According to these results, 200 Gy dose was determined for all varieties to induce their plastid mutants. 2.2. M1 progeny In this research, 1000 seeds belonging to the each variety were irradiated with 200 Gy of gamma rays by using Cs-137 source. Control and irradiated seeds were planted into experiment fields belonging to Turkish Sugar Factories, Sarmisakli Seed Production Farm in Luleburgaz with 5 × 50 cm spacing. Every pot at node situated on the main stem of M1 plants which were grown at the harvesting time, were picked up separately [14].

Atrazine test was applied at the first leaves of 14th day seedlings. Leaf discs in 1 cm diameter were cut from the seedling leaves belonging to M2 progenies of Coles, Amsoy-71 and 1937 varieties and control groups of these varieties. The leaf discs were floated at the Petri dishes, which contained 0.8% agar and various levels of herbicide (0, 24, 36 and 48 mg/l active ingredient triazine-atrazine, Ciba-Geigy) [16,17]. Atrazine tests were replicated three times. Leaf discs were observed for 5 days in the growth chamber, which has 16 h lights and 8 h dark day period at 27 ◦ C. Atrazine reduced both chlorophyll a and chlorophyll b contents in a dose dependent way after 48 h of treatment [18]. For this reason, sensitive plant to atrazine showed chlorosis and necrosis. The leaf discs which remained green in Petri dishes containing 24 and 36 mg/l atrazine were accepted as a tolerated to atrazine and containing 48 mg/l atrazine was accepted as a resistant mutant to atrazine [16]. 2.5. RAPD analysis The differences between control and the plants showing tolerance and resistance to atrazine concentrations of soybean plants belonging to Coles, Amsoy-71 and 1973 varieties were shown with RAPD analysis.

2.3. M2 progeny 2.6. The genomic DNA isolation Mutation frequency at M2 population, which was formed by the seeds to be picked up from the lower nodes of the main stem of M1 plants concerning soybean varieties, was higher than lateral branches [14,15]. For this reason, two populations were obtained from the mixture of the seeds. Lowest node population was obtained from the mixture of the seeds harvested from first three nodes. The mixture of the seeds harvested from the upper nodes formed upper node population. In climate room, M2 plants were obtained from the seeds of low and upper node populations. On the 14th day, the number of M2 seedlings and chlorophyll mutants were determined and atrazine test was done. The re-

The DNA was extracted from leaves using the procedure described by Acık et al. [19]. 2.7. Amplification conditions Fourteen random primers were used for PCR amplifications (Table 4). Amplification for RAPD was carried out in 50 ␮l volumes containing 25 ng genomic DNA, 0.2 ␮M primer, 25 mM MgCl2 , 100 ␮M of each dATP, dGTP, dTTP, dCTP and one unit of Taq polymerase. The amplifications were performed in Techne Progene thermalcycler. It was programmed for 45

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cycles of 40 sec at 96 ◦ C (denaturation), 40 sec at 45 ◦ C (anneling) and 40 sec at 72 ◦ C (extension). After cycling completed, 15 ␮l PCR products were analyzed in 2% agorase gels. 2.8. Genetics distance determination Bands on RAPD gels were scored as either present (1) or absent (0) for all species studied. Common band analysis was conducted using the computer programme SPSS to determine the genetic distance values between them. The figures for genetic distance were then used as input data for cluster analysis to generate dendrograms.

3.2. M2 progeny 3.2.1. Selection of chlorophyll mutants The numbers and percentages of chlorophyll mutants at M2 population belonging to each three varieties were determined (Table 2). According to the obtained results; totally 14 chlorophyll mutants were determined among M2 plants belonging to three varieties. Bright yellow chlorophyll mutants died in a short time; in contradiction to this, yellowish green or dotted white mutants continued to live. Among the living chlorophyll mutants, 42.86% was fertile; 7.14% was partly fertile and 50% was sterile. The chlorophyll mutants’ percentages belonging to Amsoy-71, Coles and 1937 soybean varieties were found as 1.07, 1.48 and 1.32, respectively.

3. Results 3.1. M1 progeny The soybean seeds irradiated with 200 Gy were sown into experimental field. On the harvest day, the sterile and fertile plants in M1 progeny, which were grown up in the field were determined for each variety (Table 1). Fertile plant percentages in Amsoy-71, Coles and 1937 varieties were found as 75.98, 84.31 and 73.44, respectively. M2 population was formed with the seeds obtained from low and upper node of fertile plants.

3.2.2. Selection of atrazine mutants The number and percentages of atrazine tolerated and resistant mutants at M2 population belonging to each three varieties grow up from the seeds of the irradiated M1 plants were determined (Table 3) At the atrazine test done on leaf discs of M2 plants pertaining to the lowest nodes and upper nodes population of three soybean varieties, it was seen that damages started on the 2nd and 3rd day, on the 4th day, these damages became clearer and, yellowish, brown dots occurred on the samples. At 24 mg/l atrazine concentrations, seven plants, which remained green, were determined. Two

Table 1 Fertile plant numbers at M1 progenies of the soybean varieties irradiated with 200 Gy Varieties

Number of irradiated seeds

Number of living plants

Number of sterile plants

Fertile plants Number

%

Amsoy-71 Coles 1937

1000 1000 1000

562 612 448

135 96 119

427 516 329

75.98 84.31 73.44

Table 2 The numbers and percentages of chlorophyll mutants at M2 population belonging to each three varieties Position of nodes

Lowest nodes Upper nodes Total

Coles

Amsoy-71

Number of plants

Mutant plants Number

%

51 287 338

1 4 5

1.96 1.39 1.48

1937

Number of plants

Mutant plants Number

%

35 340 375

1 3 4

2.86 0.88 1.07

Number of plants

Mutant plants Number

%

54 326 380

2 3 5

3.70 0.92 1.32

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Table 3 The number and percentages of atrazine tolerated and resistant mutants at M2 population belonging to each three varieties Position of nodes

Selected mutants at M2 progenies

Coles Number of plants

Amsoy-71 Mutant plants Number

%

Number of plants

1937 Mutant plants Number

%

Number of plants

Mutant Number

%

Lowest nodes

Resistant to atrazine Atrazine tolerated

51

– 1

– 1.96

35

– 2

– 5.71

54

1 –

1.85 –

Upper nodes

Resistant to atrazine Atrazine tolerated

287

2 3

0.70 1.05

340

3 2

0.88 0.59

326

1 4

0.31 1.23

of these plants belong to Coles variety; two of them belong to Amsoy-71 and three of these belong to 1937 variety. At the 36 mg/l atrazine concentration, five plants remaining green were determined. Two of these belong to Coles variety; two of these belong to Amsoy71 variety and one of them belongs to 1937 variety. At 24 and 36 mg/l concentrations, the plants belonging to the leaf discs remaining green were accepted as atrazine tolerated plants. Among M2 progenies, seven plants remaining green at 48 mg/l atrazine concentration were determined. Two of these belong to Coles; three of them belong to Amsoy-71and two of them belong to 1937 variety. The plants belonging to leaf discs remaining green at 48 mg/l concentrations were accepted as atrazine resistant plants. Atrazine resistant plant percentages for Amsoy-71, Coles and 1937 soybean varieties were 0.80, 0.60 and 0.53, respectively. The plants percentages for atrazine tolerated ones were 1.07, 1.18 and 1.05, respectively. 3.3. RAPD analysis In our research, the differences among the mutants improved by applying radiation to soybean varieties were examined by using RAPD technique from the view of polymerizing as comparison. In the research, 14 primers were selected for the calculations (Table 4) and amplification results were evaluated. The control plant, which was not applied radiation at Amsoy-71 soybean variety shows amplification at five each of 14 primers according to RADP result. A1 mutant plant showing 36 mg/l atrazine tolerances at Amsoy-71 variety showed differences from each other and control groups as an amplification at 11 primers; A2 mutant plants showed 48 mg/l atrazine resistance shows differences at 13 primers; and A3 mutant plant

showing 48 mg/l atrazine resistance shows differences at 11 primers. At 1937 soybean variety, amplification was seen at nine primers, at the control groups. At 1937 variety, B1 mutant plant showing 24 mg/l atrazine tolerance and B2 mutant plant showing 48 mg/l atrazine resistance was amplification at all primers. At the same variety, B3 mutant plants resistance to 48 mg/l atrazine concentration at 12 primers, B4 and B5mutant plants at 13 primers showed differences as an amplification from control group. At Coles soybean variety which was used at control group, amplification was seen only at four each of the primers. At Coles variety, 24 mg/l atrazine tolerated C1 mutant plant and 48 mg/l atrazine resistant C4mutant plant at 11 primers; 36 mg/l atrazine tolerated C2 mutant plant at 10 primers and 48 mg/l atrazine tolerated C3 mutant plant at 12 primers showed differences from control groups as an amplification (Tables 5–7).

Table 4 Nucleotide sequences of primers that detected polymorphism Primer

Sequence

OPA-07 OPB-08 B4 B6 B7 B18 UTE OPI 18 LA 13 A4 A7 LA 11 LA 12 M13

5 -GAAACGGGTG-3 5 -GTCCACACGG-3 5 -GGACTGGAGT-3 5 -TGCTCTGCCC-3 5 -GGTGACGCAG-3 5 -CCACAGCAGT-3 5 -AGGCATCGTTGG-3 5 -TGCCCAGCCT-3 5 -CACCACGCCT-3 5 -AATCGGGCTG-3 5 -GAAACGGGTG-3 5 -TATGAAACGACGGCCAGT-3 5 -ACGACCCACG-3 5 -GAGGGTGGCGGTTCT-3

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Table 5 Amplification products and used primers at Amsoy-71 soybean variety Mutant

B18

B4

B6

B7

OPA07

OPB08

OPB118

A4

A7

LA11

LA12

LA13

UTE

M13

Control A1 A2 A3

+ + + +

− − + −

− + + +

− + + +

− − + −

− + + +

− + + +

− − + −

− + − +

+ + + +

− + + +

+ + + +

+ + + +

+ + + +

Table 6 Amplification products and used primers at 1937 soybean variety Mutant

B18

B4

B6

B7

OPA07

OPB08

OPB118

A4

A7

LA11

LA12

LA13

UTE

M13

Control B1 B2 B3 B4 B5

− + + + + +

− + + + + −

+ + + + + +

+ + + + + +

− + + + + +

+ + + + + +

− + + + + +

− + + − − +

+ + + − + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

Table 7 Amplification products and used primers at Coles soybean variety Mutant

B18

B4

B6

B7

OPA07

OPB08

OPB118

A4

A7

LA11

LA12

LA13

UTE

M13

Control C1 C2 C3 C4

− + + − +

− − − + −

− + + + +

− + + + +

− + − + −

− + + + +

− + + + +

− − − + −

− − − − +

+ + + + +

− + + + +

+ + + + +

+ + + + +

+ + + + +

In this research, RAPD marker amplified from M13 primer was compared (Fig. 2). Among the control groups and the mutants showing the atrazine tolerance and resistance, the polymorphism DNA bands were obtained. According to the RAPD assay, the genetic distance between the A1 mutant and control of Amsoy-71 soybean variety was found as 33%. The genetic distances of A2 mutant to control and A1 were found 40 and 0.77% and distance of A3 mutant to control, A1 and A2 were 25, 0.91 and 16.7%, respectively (Table 8a). A2 mutant of Amsoy-71 soybean variety was more distant to control according to the other mutants. It was seen that Amsoy-71 mutants weren’t more different from each other. According to the resulting dendrogram (Fig. 3a), A1 and A2 were almost similar to each other, A3 was less distant from these and control was rather distant. At the 1937 soybean variety, the genetic distance between the B1 mutant and control was found 0.16%.

This calculation of genetic distance for B2 mutant was same. The genetic distances of B3 mutant to control, B1and B2 were determined as 39, 43 and 43%, respectively (Table 8b). Also this calculation for B4 and B5 were same. It was seen that B1 and B2 mutants were close to control and B3, B4 and B5 mutants were more different from control (Fig. 3b). At Coles soybean variety, the genetic distance between the C1 mutant and control was found 0.16%. The genetic distances of C2 mutant to control and C1 were found as 17 and 27%, respectively. This calculation of genetic distances for C3 to control, C1 and C2 were determined as 25, 47 and 29%, respectively. Also these calculations for C4 to control, C1, C2 and C3 were found as 17, 27, 43and 29%, respectively (Table 8c). While C1 mutant was the most distant to control, it was observed that C2 and C3 mutants were different to each other. As a result, it was observed that C2 and C4 mutants were closed control and C1 and C3 mutants were most distant from control (Fig. 3c).

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Table 8 Genetic distances values based on RAPD results of atrazine tolerated and resistant mutants of soybean varieties a. Amsoy-71 Control Control A1 A2 A3

A1

A2

A3

0 0.33 0.40 0.25

0 0.077 0.091

0 0.167

0

Control

B1

B2

B3

B4

B5

0 0.16 0.16 0.39 0.39 0.39

0 0 0.43 0.43 0.43

0 0.43 0.43 0.43

0 0 0

0 0

0

Control

C1

C2

C3

C4

0 0.39 0.17 0.25 0.17

0 0.27 0.47 0.27

0 0.29 0.43

0 0.29

0

b. 1937

Control B1 B2 B3 B4 B5 c. Coles

Control C1 C2 C3 C4

Fig. 2. RAPD markers among atrazine tolerated and resistant mutants of soybean varieties revealed by the M13 primer. a: Amsoy-71, b: 1937 and c: Coles. C: Control.

4. Discussion The induction of mutations by ionizing radiation starts with the interaction of a radiation field with plants. The genetic alterations produced by ionizing radiation due to ionization end excitations of the DNA molecule. A great of different types of chemical changes are induced. There are two effects of

ionizing radiation on the heredity material: gene mutations and chromosome breaks. Induce mutations contribute by increasing genetic variability and they are used in plant breeding. New varieties were improved in many plants with X and gamma radiation, which was physical mutagen. In these varieties, some important agronomical specifics were improved such as flower and seed color, early flowering, resistant to diseases, salinity, resistant to cold and heat, resistant to lodging, yield and quality etc. [20]. In this research, the improvement of resistance to atrazine in soybean with the gamma radiation was aimed. 200 Gy gamma radiation dose which formed higher mutation rate and less physiological damage, was used for M1 plants. Due to the physiological damages caused by this radiation in M1 generation, living plants number and percentage of fertile plants are lower in each three varieties according to control. In this research, chlorophyll mutants were determined in order to show the effect of the radiation dose. It was known that many nucleus genes or a few

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Fig. 3. Dendrogram of soybean varieties derived from RAPD data.

cytoplasmic genes controlled chlorophyll mutants. Apart from this, chlorophyll mutants were caused by an interaction among cytoplasmic and nuclear genes. [21–24]. At the end of the radiation application, in M2 generation the highest chlorophyll mutant rate was found in Coles variety and the lowest chlorophyll mutant rate in Amsoy-71 variety. In M1 generation, the most physiological damage was seen in Amsoy-71 variety. Amsoy71 is the most sensitive variety to radiation among the other three varieties. When the physiologic damages increased, mutation rate has decreased. At the Coles variety the least physiologic damage has been determined in accordance with other varieties and highest chlorophyll mutant rates have been seen. In our research, atrazine resistant plants were determined, due to the mutations induced in chloroplast genes, 19 mutant plants atrazine resistant and tolerated mutant plant were selected in M2 generation. These atrazine resistance and tolerance mutants were plastidom mutations, which were induced with gamma radiation in soybean plant. Induced mutations to provide resistance to atrazine in genes located at chloroplast were obtained with ionized radiations at the crop plants [25]. The formation of mutations with X and gamma rays will be possible in DNA molecule of cytoplasmic organelles like chloroplasts and mitochondria. Cytoplasmic inher-

itance acts in a non-Mendelian manner. The cytoplasmic type of heredity is in general transferred from generation to generation, mostly via the egg cell. Therefore, the researches done with ionized radiations regarding the characters showing cytoplasmic heredity does less in comparison with the mutation research regarding the characters controlled by nucleus genes in plants. [1,26]. At the plants resisting atrazine, a change in psbA gene coded QB protein, which was involved in fotosystem II complex, is occurred [25]. The change occurred in the configuration of QB protein coded by this gene obstructs the binding of atrazine to this protein and provides the continuation of electron transfer [27]. The percentages of chlorophyll and atrazine mutants of Coles, Amsoy-71 and 1937 varieties in M2 generations were found higher in lowest node population according to mutants obtained from upper node population. These results showed that in the picking up seeds from the certain places of the plant played a great role on the increase of the frequency of mutations occurred to cpDNA in population. In our research DNA fingerprints of plants belonging to each three varieties showing resistance and tolerance to atrazine in various concentrations were taken by using 14 primer and RAPD techniques. Ferreira and Keim in their research, wrote that DNA markers assisted to prepare DNA mapsand RAPD

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system was the best analysis in forming molecular markers in soybean [28]. In RAPD system; to work with a little amount of DNA provides easiness for the analysis of the products and correct usage and low cost in techniques [29,30]. In our research, when markers obtained the end of RAPD analyses done at the plants showing resistance and different atrazine concentration and control were examined, it was seen that there was a potential polymorphism between mutants and controls. At the dendrograms done according to M13 primer, the genetically distance to control groups and mutants replying to atrazine in various concentrations in each three varieties were rather different. Randomly amplified polymorphic DNA (RAPD) markers, which can quickly detect a large number of genetic polymorphisms, have led to the creation of genetic maps in a number of woody fruit crops [31] and RAPD markers, have been used to detect mutations and DNA damage [32]. In our research, the differences among mutants replying to various doses atrazine and improved by applying radiation with soybean varieties were determined with RAPD markers. The changes occurred at chloroplast’s DNA because of the replies to atrazine. The showing tolerance and resistance to atrazine of the soybean mutant showed us that gamma radiation was effective on the plastid. In the future research, the genomic location of resistance genes will be determined at ch.DNA’s of these mutants.

Acknowledgement This work was supported by the Research Fund of The University of Istanbul. Project number: B1251/08082001.

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