Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 39-56, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2338 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sobieh, S.S., & Eid Eliwa, N. (2024). Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba reveal the alle- viation effect of gamma radiation and putrescine. Caryologia 77(1): 39-56. doi: 10.36253/caryologia-2338 Received: October 19, 2023 Accepted: May 18, 2024 Published: July 8, 2024 Copyright: © 2024 Sobieh, S.S., & Eid Eliwa, N. This is an open access, peer-reviewed article published by Firenze University Press (https://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID SSS: 0000-0002-1286-7352 Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba reveal the alleviation effect of gamma radiation and putrescine Shaimaa S. Sobieh1,*, Noha Eid Eliwa2 1 Botany Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt 2 Natural Products Research Dept., National Center for Radiation Research and Technol- ogy, Egyptian Atomic Energy Authority, Egypt *Corresponding author. Email: shimaa.sobieh@women.asu.edu.eg Abstract. Climate change caused increasing in soil salinity worldwide. Therefore, it is critical to enhance the capability of plant to tolerate salinity stress. For this goal, putrescine and irradiation by gamma radiation were used to improve the salt toler- ance of Vicia faba (the most important human crop). The results indicated depres- sion in mitotic division and all growth parameters associated with the induction of micronucleus (MN) when salinity increased to 100mM, while there was increase in mitotic aberrations. NaCl decreased total soluble sugar, while total N%, total free amino acid, proline and protein contents showed slight increase with increasing salin- ity stress. Putrescine and gamma radiation mitigated the effect of salinity on cell divi- sion and growth parameters. Salt stress decreased the expression of gene encoding cyclin-dependent kinase 2 (cdc2). Both putrescine and gamma radiation increased cdc2 expression. The genetic diversity has been detected among control and treated V. faba using ISSR and SCoT markers. Ten primers had successfully generated 129 reproducible polymorphic amplicons that were suitable for studying the genetic diver- sity between studied genotypes. ISSR markers provided more discriminating data and were more informative than SCoT markers. Besides, cluster analysis using UPGMA and PAC successfully explained the genetic diversity within studied genotypes. These findings emphasize the efficiency of putrescine and gamma radiation for alleviating the negative impact of salt stress. Moreover, prove the importance of assessing mitotic activity, chromosomes behavior, physiological parameters, the expression level of cdc2 and molecular diversity in V. faba under stress to improve the salt tolerance of it. Keywords: Vicia faba, salt stress, putrescine, gamma radiation, growth parameters, cell division, mitotic aberrations, micronucleus, cdc2 expression, ISSR& SCoT. INTRODUCTION The significant threats in the world today are caused by climate change. In the twenty-first century, salinity is one of the key issues brought on by cli- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2338 https://doi.org/10.36253/caryologia-2338 https://www.fupress.com/caryologia https://www.fupress.com/caryologia https://orcid.org/0000-0002-1286-7352 mailto:shimaa.sobieh@women.asu.edu.eg 40 Shaimaa S. Sobieh, Noha Eid Eliwa mate change and one of the greatest risks to plants and crop yields globally, its negative impacts are increased under the severe changes in climate, particularly in arid and semiarid regions. According to recent statistics, salt stress has abused more than 45 million hectares of irri- gated land worldwide, and this number is always rising (Isayenkov and Maathuis 2019). Additionally, salt stress generates a significant amount of reactive oxygen spe- cies, which damage some molecules such as lipids, pro- teins, and DNA, cause breakdown of cell membrane sys- tems and terminate some enzymatic processes (Demi- ral and Türkan 2005). Plants use several mechanisms to manage salt damage and regulate cellular homeosta- sis and growth to overcome this problem. According to Isayenkov and Maathuis (2019), salt tolerance frequently involves the activation of cell signaling pathways that result in the production of antioxidant enzymes, and osmo-protective metabolites such amino acids and car- bohydrates. Vicia faba L. (2n = 12) or faba bean is one of the most important leguminous crops produced worldwide. Faba bean is the fourth most significant leguminous plant in the world (FAOSTAT 2018). Approximately 2.56 million hectares of land are harvested each year to yield 4.56 million tons of dry V. faba grains (FAOSTAT 2022). Eighty percent of the dried faba bean grains are pro- duced in Asia and Africa (FAOSTAT 2022). In the Mid- dle East, North Africa, the Mediterranean region, the Nile Valley, and Ethiopia, V. faba is regarded as a crucial food crop for human nutrition and cattle feed because mature Vicia faba seeds are a excellent source of pro- tein, carbohydrates, cellulose, vitamin C, and minerals (Qahtan et al. 2021). Polyamines (PAs) play a crucial role during envi- ronmental stress. They are low molecular weight growth regulators and present as aliphatic amines. According to FAO Statistics (2021) polyamines are multifunctional polycationic plant growth regulators that have an impact on several physiological, metabolic, and developmen- tal processes. Wisniewski et al. (2014) reported that PAs could control DNA replication, cell division, seed germi- nation, and development. Putrescine, spermidine, and spermine are typical polyamines found in plants (Gupta et al. 2013). One of the main polyamines, putrescine, is crucial for plant growth and differentiation as well as stress responses (Sequera-Mutiozabal et al. 2017). The positive charges of PAs can help stabilize cell mem- branes under environmental stress by attaching to the negatively charged phospholipids and proteins that make up the membranes (Kuznetsov and Shevyakova 2007), Additionally, PAs enhance antioxidant systems, regulate some gene expression (Matkovics et al. 1993) and cause scavenging of free radicals (Velikova et al. 2000). Useful mutations are the modifications of the geno- typic structure to improve the species’ variability and help them to respond better to different range of stresses (Spencer et al. 2018). Physical mutagenic agents includ- ing ultraviolet light, protons, neutrons, alpha and beta particles, and ionizing radiation (X-rays and gamma rays) can cause useful mutations. Gamma radiation can directly cause physical, biological, and chemical changes in cells (Ludovici et al. 2020). It can indirectly affect free radical production and directly trigger specific altera- tions in the genome (Caplin and Willey 2018). Molecular markers are considered an effective method for analyzing and identifying genetic variability within and/or between genotypes. The degree of poly- morphism affects how discriminatory power, which in turn determine how markers are categorized. To ascer- tain the genetic diversity among species, cultivars, and treatments, polymorphism is used. In recent decades, research on the genetic diversity of genotypes has been used in numbers of disciplines, including genetics, ecol- ogy, botany, biology, and others (Chesnokov and Arte- myeva 2015). Inter-Simple Sequence Repeat (ISSR) markers are Polymerase Chain Reaction PCR (PCR) approaches for measuring the genetic diversity in plants (Ziêtkiewicz et al. 1994). These markers amplify the inter-SSR sequences of varied sizes. Start codon targeted (SCoT) was generated to start a trend away from ran- dom DNA markers and toward gene-targeted markers based on the short on served region flanking the ATG of plant genes. Since the SCoT marker is frequently reliable, it is understood that factors other than annealing tem- perature and primer length can affect repeatability (Col- lard and Mackill 2009). Since no specific knowledge of the genome sequence was required for the SCoT mark- ers design, it was possible to apply it to plants without genome references (Xiong et al. 2011). In V. faba and many other plant species, both markers are frequently used to assess genetic diversity (Albrifcany et al. 2022). The most vulnerable stages of growth, germination, and seedling development are partially affected by cell cycle suppression. Salinity reduces the cell division fre- quency. It also causes defects in the chromosomes struc- ture and caused induction of micronuclei (Souguir et al. 2018). Micronucleus Test (MN Test) is used to evaluate the genotoxic potential of substances. Biotic and abiotic stress cause cell cycle inhibition which in turn causes harmful effect on plant growth. Although the molecular interactions that link the cell cycle machinery to perception of stress are not fully understood, recent studies indicated the involvement of cyclin dependent kinases (cdcs) in the plant response 41Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba machinery (Kitsios and Doonan 2011). Cell divisions in eukaryotic cells are controlled by a family of protein kinases, the cyclin-dependent kinases (cdcs). The activity of cdcs is regulated by cyclins, or to the cdc inhibitors. The activity and localization of different cdc complexes regulate many of the actions during the cell cycle (Nigg 1995). In plants and animals many cdcs are present and regulate the G1/S and the G2/M transitions (Magyar et al. 1997). Different plant species contain one or two cdc gene, which contains a fully conserved PSTAIRE sequence motif (Hirt and Heberle-Bors 1994). Mutant cdc gene in Arabidopsis (cdc2a-At) arrest cell cycle at G1/S and/or G2/M points, demonstrating its ability to control both checkpoints (Hemerly et al. 1995). From the previous, plants must develop mecha- nisms to adapt to the variable environment conditions. Therefore, this study was conducted to declare the role of gamma radiation and putrescine in the alleviation of the harmful effect of salinity on V. faba plants by track- ing the physiological, genetical, and molecular changes and affirm the involvement of cdc2 in the signaling con- trol of stress tolerance. MATERIALS AND METHODS Seeds of bean (Vicia faba) (cv. Giza 2) were obtained from the Agriculture Research Center, Ministry of Agri- culture, Giza, Egypt. Radiation treatments Dry seeds irradiation was performed at National Center for Radiation Research and Technology (NCR- RT). Cairo, Egypt. By using (Co60) as source of γ-rays at dose levels 10 Gy with dose rate of 0.623 rad/sec. Putrescine treatments Putrescine solutions were prepared at levels of the solutions (5 mMol) were sprayed on leaves of the seed- lings after the appearance of first leave and repeated eve- ry seven days until harvest after 50 days. Experimental design A pot experiment was conducted under field condi- tions at a wire house at NCRRT, Cairo, Egypt. The seeds were irradiated with 10 Gy radiation dose and then three seeds were grown in each plastic pot, (40 × 35 cm, height × diameter), containing equal quantities of sandy loam soil, commercial peat and clay. The seeds were left to grow inside the greenhouse under natural lighting. A full concentration of Hoagland’s nutrient solution was used to irrigate pots (Hoagland and Arnon, 1950). Randomized complete block design with three repli- cations was used for the experimental design. Salt solu- tion (250 ml) was applied at 15 days after seedling emer- gence twice per weak. Foliar spray of putrescine was applied after 2day from salinity treatments. It repeated twice a week. Either: 1) Full strength Hoagland’s solution (control). 2) Full strength Hoagland’s solution +50 mMNaCl. 3) Full strength Hoagland’s solution + 100 mM NaCl. 4) Full strength Hoagland’s solution + 5 mM putres- cine (foliar spray). 5) Full strength Hoagland’s solution + 5 mM putres- cine (foliar spray) + 50 mM NaCl. 6) Full strength Hoagland’s solution + 5 m M putres- cine (foliar spray) + 100 mM NaCl. 7) Full strength Hoagland’s solution + 10 Gy. 8) Full strength Hoagland’s solution + 10 Gy + 50 mM NaCl. 9) Full strength Hoagland’s solution + 10 Gy +100 mM NaCl. After 50 days seven growth parameters were meas- ured. Physiological analysis Extraction of plant extract. Fresh grinded leaves (0.1g) were accurately weighted then extracted by in 80 % aqueous ethanol for 6 h (AOAC 1984). The extract was filtered and completed to 50 ml in a measuring flask with ethanol 80% for further experiments. Determination of total soluble sugar. Total soluble sugar of the fresh leaves was quantified using a modified phenol-sulfuric acid assay (Zhang 1993). Determination of total free amino acid. Total free amino acid of fresh leaves was measured according to Rosen (1957). Determination of proline content. Proline content was evaluated using the method of Bates et al. (1973). Determination of Nitrogen (N) %. The leaf N con- centration (LNC) was assessed according to the method Wild et al. (1985). Determination of total soluble protein content. Total soluble proteins were estimated according to Lowery et al. (1951). 42 Shaimaa S. Sobieh, Noha Eid Eliwa Genetical analysis Quantitative analysis of mitotic division and chro- mosomal aberrations. To get rid of the soil remnants, the roots of each sample group were taken and rinsed under running water. Root tips (3 to 4 cm in length) were stored in 70% ethyl alcohol at 4°C until usage after being treated in Carnoy’s solution for 24 hours. After being hydrolyzed in 1N HCl, the roots were stained for one hour with basic Fuchsin stain. The root tips that were darkly discolored were crushed in a drop of 45% acetic acid. For each treatment group and control group, 1000 cells on 10 slides were used to score and compute the percentage of mitotic division (MI) and frequency of mitotic abnormalities (Xavier et al., 2023). Quantitative analysis of micronucleus. Cell with micronuclei (MN) reveals the genotoxicity effect of treat- ment. Cells with intact cell wall that contains MN along- side the nucleus were scored. The frequency of MN was scored in 5000 cells and expressed as the percentage of the number of cells with micronuclei per 1000 cells per slide (Xavier et al. 2023). MN was scored to prove geno- toxic effect of salinity stress and the mitigating effect of both putrescine and gamma radiation. Cyclin dependent kinase 2 (cdc2) gene expression quantification using qReal-Time PCR. Pure RNA was extracted from V. faba root for all experiment groups using total RNA Purification Kit (Qiagen, Rneasy Kit) following the manufacturer protocol. RNA input for all samples was adjusted to 1000 µg and cDNA was synthesized using Reverse Transcription kit (Thermo Fisher Scientific, RevertAid RT Reverse Transcription Kit). Specific primers for cdc2 gene F 5’ACTCTCAT- AGGGTTCTCC3’ (Tm 54°C); R 5’ CTCGGTACCA- GAGAGTAA3’ (Tm 54°C) were used. The amplifica- tion protocol was as follow, 1 cycle at 95ºC for 10 min followed by 40 twostep cycles 95 ºC for 15 sec, and 60 ºC for 60 sec. Results were normalized by housekeeping actin gene using the following primer F 5’ CTTCCCAA- GATAGTAGGAG3’ (Tm 55°C) and R 5’ CTTAGACT- GTGCCTCATC3’ (Tm 54°C). The expression level of genes was calculated in relation to 2- ΔΔ ct according to Livak and Schmittgen (2001). Molecular fingerprint using ISSR-PCR and SCoT- PCR. DNA was isolated from V. faba fresh leaves accord- ing to cetyltrimethylammonium bromide (CTAB) method. The quality of DNA was checked at 260/280 nm and on 1% agarose gel. DNA was used for ISSR and SCoT techniques according to Ziêtkiewicz et al. (1994) and Collard and Mackill (2009) respectively. Ten prim- ers (Table1) five for each technique were screened against each DNA sample to analyze molecular diversity among all treated and control groups. Polymerase chain reac- tion (PCR) was accomplished in an automated thermal cycle (model Techno 512, Stafford, UK system) using Dream Taq green master Mix (Thermo-Scientific). The PCR reaction volume was 25 μl including 2.5 μl of dNTPs (2.5 mM), 1.5 μl of MgCl2 (25 mM), 2.5 μl of 10× buffer, 2.0 μl of primer (2.5 μM), 2.0 μl of template DNA (50 ng μl/1), 0.3 μl of Taq polymerase (5 U μl/μ1) and 14.7 μl of sterile ddH2O. PCR reaction for both fin- gerprints was 1 cycle at 95°C for 5 min followed by 40 cycles of 1 min at 95°C, 1 min at annealing temperature 56°C and 2 min at 72°C, followed by 1 cycle at 72°C for 10 min. PCR products were resolved on a 1.5% agarose gel with 100 – 3000bp DNA Ladder, GeneDirex, Inc (100bp DNA Ladder H3 RTU Ready-to-Use). Data and cluster analyses Binary data matrix for the bands (0 for absent 1 for present bands) were analyzed. Polymorphism percent- age, total number of amplicons (TNA), total polymor- phic amplicons (TPA), monomorphic amplicons (MA) and unique amplicon (UA) were scored. Four param- eters, polymorphic information content (PIC), effec- tive multiplex ratio (EMR), marker index, and resolving power (RP) were examined the efficiency of ISSR and SCoT markers as well as the genetic difference among genotypes under study. PIC, Rp, EMR and MI were cal- culated according Venkatesan et al (2021). The genetic similarity coefficient was used to con- struct the phylogenetic tree. Genetic similarity coeffi- cient between two genotypes and principal component analysis (PCA) was achevied using the Paleontologi- cal Statistics Software Package for Education and Data Analysis (PAST) version 4.03 (Hammer et al. 2001). Table 1. ISSR and SCoT primers nucleotide sequences. Analysis Primer Nucleotide sequence of the used primer 5’ to 3’ ISSR UBC810 GTGTGTGTGTGTGTGTCA UBC818 CAC ACA CAC ACA CAC AG UBC849 GAGAGAGAGAGAGAGAT UBC-823 TCTCTCTCTCTCTCTCC UBC-817 CACACACACACACACAA SCoT SCoT -31 CCATGGCTACCACCGCCT SCoT -34 ACCATGGCTACCACCGCA SCoT -13 ACGACATGGCGACCATCG SCoT -14 ACGACATGGCGACCACGC SCoT -52 ACAATGGCTACCACTGCA 43Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba Statistical analysis of data Statistical analysis was carried out according to Sne- decor and Cochran (1980). The individual comparisons between the obtained data were carried using M-STAT computer software program at p≤ 0.05. RESULTS Physiological analysis Figure 1A declared the effect of salinity, putres- cine, and gamma radiation on the stem length of V. faba plants. Increasing NaCl concentration from 50 mM to 100 mM decreased stem length from 33.71 to 31.11 cm compared to the control (36.50 cm). Foliar spraying with putrescine increased stem length to (37.72 cm) compared to the control plants. It is obvious that gamma radiation gave the longest stem length (48.68 cm) above all treat- ments used especially with 10Gy + 50 mM NaCl treat- ed plants. The same trend was observed with the root length i.e. salinity reduced root length to 2.4 cm with 100m M NaCl treated plants. Putrescine and radiation alleviated the harmful effect of salinity as they gave 4 cm with 5m M treated plants and 6.5 cm with 10 Gy + 50 m M NaCl treated plants. In Fig. 1C number of leaves decreased to 12 leaves with increasing salinity concen- tration to 100 mM. While putrescine and radiation had a non-significant effect on the number of leaves. On the other hand, radiation increased shoot fresh and dry weight above the control or putrescine treatments (Fig. 1 D, E). The maximum shoot fresh and dry weight was 36.8 g and 7.1 g in 10 Gy + 50 Mm NaCl treated plants respectively. The same effect of radiation appeared in root fresh weight and dry weight (Fig. 1 F, G). Figure 2A showed the effect of salinity, putrescine, and radia- tion on total soluble sugars (TSS) of V. faba plants. It is cleared that salinity reduced TSS% from 1.89% to 1.52% as salinity increased from zero to 100 Mm NaCl. Foliar spraying of putrescine has a positive effect in TSS % i.e increased to 2.64%with 5 mM put + 50 mM NaCl treat- ed plants. The highest TSS% was observed with radia- tion in all treatments used especially with 10 Gy+50 mM NaCl treated plants (2.508%). Fig. 2B cleared the influ- ence of salinity on total free amino acid. The maximum increment (0.1221g/ 100 g) reached by 100 mM NaCl treated plants. Radiation also increased the total amino acid with 10 Gy + 100 mM NaCl treated plants (0.2089 g/ 100 g). Salinity showed positive effect on proline contents of leaves, the content of proline increased as salin- ity increased, it reached 7.004 mg/100g in plants treat- ed with 100 mM NaCL compared to zero NaCl 2.8600 mg/100g. This increment is still observed with putres- cine treatments. There was a decrease in proline content (7.5840) by 10Gy +100mM NaCl than with 10 Gy treated plants (9.278). Concerning N% contents in leaves of V. faba plant, N% increased in 50 mM NaCl treated plants and 10 Gy + 50 m M NaCl treated plants to 0.407% and 0.438% respectively. Fig. 2E illustrates that protein con- tent increased with increasing salinity concentration, the maximum increase observed with 50 mM NaCl treated plants. Putrescine and radiation increased protein con- tents of V. faba leaves. Treatment with both 50 mM NaCl+ 5mM putrescine and 50 mM + 10 Gy showed the maximum value of protein contents (1.56 and 2.74 respectively). Genetical analysis Quantitative analysis of mitotic division and chromosom- al aberrations The mitotic index (MI) is a very important indicator for the rate of mitotic division. Salinity stress by 50mM and 100mM NaCl caused significant decrease in MI of V. faba meristematic root cells reached 5.58±0.14 and 4.98±0.20 respectively as compared with control (Fig. 3A). Alleviation effect of both putrescine and gamma radiation (at low dose 10 Gy) was obvious specifically with concentration of 50mM NaCl. Foliar application of salt stressed V. faba with 5mM putrescine caused high- ly significant increase in MI as compared with salin- ity stressed V. faba, but MI was still less than control. While gamma radiation mitigated the harmful effect of NaCl and enhanced the mitotic division and caused highly significant increase in MI as compared with control and salt stressed plants. The maximum MI was 7.84±0.21, which has attained after treatment with 10 Gy + 50mMNaCl. The changes in mitotic index frequencies were asso- ciated with significantly increment in mitotic aberrations percentage under salinity stress. The frequency of mitot- ic abnormality after treatment with 50mM NaCl was 20.64±0.75, while the ratio of mitotic abnormality after treatment with 100mM NaCl was 31.21±0.26. As well as all treatments with gamma radiation and putrescine sig- nificantly increased the ratio of mitotic abnormality but less than those of salt stress treatment (Fig. 3B). Only one exception, treatment with 10 Gy with 100mM NaCl caused higher frequency of mitotic aberration reached 32.44±0.39 (the maximum ratio), while the minimum ratio was produced after the foliar application by putres- cine only. 44 Shaimaa S. Sobieh, Noha Eid Eliwa Additionally, the reduction of MI after salinity stress was accompanied with significant increase in prophase index (Fig. 3C). The maximum prophase accumulation was 48.16±0.14 achieved by treatment with 100mMNaCl. Concerning treatment with putrescine, phase indices showed non-significant accumulation of prophase and displayed normal contribution of phases as compared with control. While treatment with gamma radiation reflected general accumulation in metaphase. The maxi- mum metaphase accumulation was 42.97±0.23 achieved by treatment with 10 Gy. Several types of chromosomal aberrations were observed after all treatments as compared with control. There were three main classes of chromosomal abnor- malities (Fig. 4i-xii). The first class was chromosome stickiness, the second class was clastogenic aberrations including breakage, bridge, and ring chromosome, and finally chromosome disturbance including disturbance, A B C D E F G CD DEF F C CD EF B A DE 0 20 40 60 Control 50 mM NaCl 100 mM NaCL St em le ng th (c m ) Stem length LSD=2.802 control Putrescine Radiation D D G C D E A B F 0 5 10 Control 50 mM NaCl 100 mM NaCL Ro ot l en gt h (c m ) Root length LSD=0.2706 control Putrescine Radiation AB C C AB BC BC A BC C 0 5 10 15 20 Control 50 mM NaCl 100 mM NaCL N o. o f l ea ve s No. of leaves LSD=2.295 control Putrescine Radiation F G H E B D B A C 0 10 20 30 40 Control 50 mM NaCl 100 mM NaCLSh oo t f re sh w t. g ) Shoot fresh wt. LSD=1.025 control Putrescine Radiation G H I E B F C A D 0 2 4 6 8 Control 50 mM NaCl 100 mM NaCL Sh oo t d ry w t. g ) Shoot dry wt. LSD=0.2188 control Putrescin e Radiation E F G D B CD B A C 0 10 20 30 40 Control 50 mM NaCl 100 mM NaCL Ro ot fr es h w t. g ) Root fresh wt. LSD=1.596 control Putrescine Radiation F G H E D E B A C 0 2 4 6 8 Control 50 mM NaCl 100 mM NaCL Ro ot d ry w t. g ) Root dry wt. LSD=0.2808 control Putrescine Radiation Figure 1. Effect of salinity, putrecine and gamma radiation on growth of bean plant (Non identical letters indicate significant difference). 45Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba multipolarity, diagonal. Chromosome stickiness was more pronounced by salt stress. Although the alleviation effect of gamma radiation on MI, clastogenic aberration was more obvious after gamma radiation treatment. Quantitative analysis of micronucleus Meristematic cells of V. faba under salt stress and gamma radiation showed significant production of MN. The formation of MN was significant at level p≤0.001. Micronucleus was observed in interphase cells and in several phases (Fig. 4 xiii-xvi). The percentage of MN for- mation was concentration dependent. Treatment with 10 Gy+100mM NaCl exhibited the highest frequency of MN (18.46±0.11). Foliar application of putrescine alleviated the toxic effect of NaCl and produced the least MN percentage 3.47±0.26 after treatment with 5mM putrescine (Fig. 5). Cyclin dependent kinase 2 (cdc2) gene expression quanti- fication using qReal-Time PCR Significant changes were recognized in the expres- sion level of cdc2 gene in V. faba root. Marked sig- A B C D E CD D F E A EF B A BC 0 0,5 1 1,5 2 2,5 3 Control 50 mM NaCl 100 mM NaCL To ta l s ug ar % Total sugar % LSD=0.218 control Putrescine Radiation F CD C B DE D CD CD A 0 0,05 0,1 0,15 0,2 0,25 Control 50 mM NaCl 100 mM NaCLTo ta l f re e am in o ac id g /1 00 g Total free amino acid LSD=0.01678 control Putrescine Radiation F E C F F D A A B 0 2 4 6 8 10 Control 50 mM NaCl 100 mM NaCL Pr ol in e c on te nt m g/ 10 0g Proline content LSD=0.4344 control Putrescine Radiation B E F I H G C A D 0 0,1 0,2 0,3 0,4 0,5 Control 50 mM NaCl 100 mM NaCL N % N% LSD=0.01678 control Putrescine Radiation E B C H G F D A C 0 0,5 1 1,5 2 2,5 3 Control 50 mM NaCl 100 mM NaCL Pr ot in c on te nt Protein content LSD=0.05307 control Putrescine Radiation Figure 2. Effect of salinity, putrescine and gamma radiation on physiological contents of bean plants (Non identical letters indicate signifi- cant difference). 46 Shaimaa S. Sobieh, Noha Eid Eliwa nificant down regulation was recorded after treatment with 50 and100mM NaCl (Fig. 6). Conversely, marked upregulation in cdc2 expression level was noticed after the combination between NaCl and putrescine or with10Gy radiation. Exposure of V. faba to 5mM putrescine+50mM NaCl and 10Gy+ 50mM NaCl increased the cdc2 expression level 2-folds and 4-fold respectively. In contrast, both 5mMputrescine and 10Gy with 100mM NaCl caused downregulation of cdc2 gene expression level (0.965 and 1.04 respectively), which were more than those of salt stressed only. Molecular fingerprint using ISSR-PCR and SCoT-PCR) ISSR and SCoT markers were used to investigate the molecular variations in osmotic stressed V. faba treated with putrescine as well as gamma radiation. Ten ISSR and SCoT-primers (5 for each analysis) succeeded in amplifying 126 amplicons with a range between 2840 to 135 bp (Fig.7). Eighty amplicons of 126 were reproduc- ible polymorphic amplicons, 25 amplicons of 80 were unique amplicons, while 46 were reproducible mono- morphic amplicons. The maximum total number of 0 1 2 3 4 5 6 7 8 Con tro l 50 mM N aC l 10 0m M N aC l 5m M Put. 5m M Put.+ 50m M NaC l 5m M Put.+ 100 mM NaC l 10 G ra y 10 G ra y+ 50 mM N aC l 10 G ra y+ 10 0m M N aC l B CD D CD BC CD BC A B Fr eq ue nc y of M I Treatment Figure 3A. Change in mitotic index of osmotic stressed V. faba treated by gamma radiation and putrescine. Non identical letters indicate significant difference at p≤0.001 LSD= 0.6671. G C B F F D E D A 0 10 20 30 40 Control 50mM NaCl 100mM NaCl 6mM Put. 6mM Put.+50mMNaCl 6mM Put.+100mMNaCl 10 Gray 10 Gray+50mM NaCl 10 Gray+100mM NaCl Percentage of mitotic aberrations T re at m en t Figure 3B. Frequency of mitotic abnormalities of osmotic stressed V. faba treated by gamma radiation and putrescine. Non identical letters indicate significant difference at p≤0.001 LSD=0.7180. 0 5 10 15 20 25 30 35 40 45 50 Control 50mM NaCl 100mM NaCl 5mM Put. 5mM Put.+50m MNaCl 5mM Put.+100m MNaCl 10 Gy 10 Gy+50mM NaCl 10 Gy+100m M NaCl Prophase 25,5 36 48,16 20,5 25,57 28,26 32,82 38,92 42,47 Metaphase 24,39 29,66 22,44 34,88 31,48 27,42 42,79 37,78 32,43 Anaphase 20,43 15,97 19,36 29,27 22,36 20,89 14,22 10,98 14,38 Telophase 29,68 18,37 10,04 15,35 20,59 23,43 10,17 12,19 10,2 g d a h g f e c b h f i c e g a b d c e d a b c f g f a d h e c b g f g fr eq ue nc y of p ha se in di ce s Prophase Metaphase Anaphase Telophase Figure 3C. Change in mitotic phases of osmotic stressed V. faba treated by putrescine and gamma radiation. Non identical letters for each phase indicate significant difference at p≤0.001. 47Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba i ii iii iv v vi vii viii ix x xi xii xiii xiv xv xvi Figure 4. Types of chromosomal aberrations formed in V. faba by different treatments. i: Sticky metaphase and anaphase, ii: Sticky meta- phase, iii: Sticky anaphase, iv: Sticky anaphase with bridge, v: Disturbance anaphase with multibridge, vi: Disturbance anaphase with lag- gard, vii: Disturbance metaphase, viii: C-anaphase, ix: Multipolarity, x: Metaphase with laggard, xi: Anaphase with laggard, xii: Metaphase with ring chromosome, xiii-xiv: Interphase with micronucleus, xv: Metaphase with micronucleus, xvi: Anaphase with micronucleus. H E D G F E C B A 0 2 4 6 8 10 12 14 16 18 20 Control 50mM NaCl 100mM NaCl 5mM Put. 5mM Put.+50mMNaCl 5mM Put.+100mMNaCl 10 Gy 10 Gy+50mM NaCl 10 Gy+100mM NaCl Percentage of micronucleus T re at m en t Figure 5. Frequency of micronucleus in osmotic stressed V. faba treated by putrescine and gamma radiation. Non identical letters indicate significant difference at p≤0.001 LSD=0.1914. 1B D F C B B,C E A B 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 Con tro l 50 mM N aC l 10 0m M N aC l 5m M Put. 5m M Put.+ 50m M NaC l 5m M Put.+ 100 mM NaC l 10 G y 10 G y+ 50 mM N aC l 10 G y+ 10 0mM N aC l Cd c2 e xg re ss io n fo ld le ve l Treatment Figure 6. Change in cdc2 gene expression level of osmotic stressed V. faba treated by putrescine and gamma radiation. Non identical letters indicate significant difference at p≤0.001 LSD=0.9360. 48 Shaimaa S. Sobieh, Noha Eid Eliwa amplicons was 18 amplicons for primer SCoT-34 with 83.33 % percentage of polymorphism, while the mini- mum number of amplicons was 9 produced by both primers UBC810 and SCoT-52 with 66.6% polymor- phism for the two primers. The minimum percentage of polymorphism was 50% achieved by UBC849 primer (Table 2). ISSR and SCoT markers performance The polymorphism information contents (PIC), or heterozygosity index displays the capability of each marker for revealing the frequency of polymorphism or genetic diversity between the different genotypes. It was calculated for each locus depending on the number of alleles and the allele frequency. Table 2 revealed that PIC values of ISSR markers was ranged from 0.10 to 0.80 with average of 0.45, which reflected intermediate level of polymorphisms for the used ISSR sites in the different treated genotypes of V. faba. Three primers (UBC810, UBC849 and UBC-817) were highly informative, PIC value > 0.5 and two primers (UBC818 and UBC-817) were low informative, PIC value < 0.25. While The PIC value of SCoT markers ranged from 0.09 to 0.65 with average of 0.25, which reflected low level of polymor- phisms. Four primers were low informative, PIC value < 0.25 and only one primer SCoT -13 was highly informa- tive, PIC value > 0.5 reached 0.65. The resolving power (Rp) is the most effective parameter used to discriminate effectiveness of the primer to reveal genetic diversity level among individu- als. Rp value of ISSR was ranged from 1.82 (UBC849) to 4.70 (UBC-823), Rp value of SCoT was ranged from 2.75(SCoT-13) to 10.55 (SCoT-34). Table 2 shows that UBC-823 generated the high- est number of amplicon (14) and highest RP value (4.70) with 57.14% polymorphism frequency, as well primers SCoT-34 targeted the highest number of amplicons (18) with a polymorphism percent of 83.33% and highest RP value (10.55). This demonstrates that these two primers (UBC-823 and SCoT-34) had high-edifying and discrim- inative abilities in determining genetic diversity. EMR was calculated as the total number of poly- morphic loci for each primer. Marker is more efficient when the EMR value was higher. SCoT-34 gave the high- est EMR value (12.5) among all used ISSR and SCoT primers (Table 2). Marker index is a statistical tool to describe the capability of each primer to discriminate polymorphic loci among the used genotypes. The maximum marker index value was 3.15 achieved by ISSR marker UBC-817 (Table 2). Treatment specific ISSR and SCoT primer Twenty positive marker amplicons were produced by the 10 loci of ISSR and SCoT. All ISSR primers were successful in generating 13 amplicons extending from 260 to 1600 pb, while 3 SCoT primers (SCoT-13, SCoT- 14, and SCoT-52) were able to target 7 amplicons ranging from 515 to 2090 (Table 3). These primers could be con- sidered as marker related specific treatments. Table 2. ISSR-PCR and SCoT-PCR amplicons from osmotic stressed V. faba treated by putrescine and gamma radiation. Analysis Primer Range of amplicon molecular size (bp) TNA MN TPA UA Polymorphism % PIC RP EMR Marker index ISSR UBC810 1480-170 9 3 6 2 66.66% 0.61 2.64 4 2.5 UBC818 2050-188 10 4 6 2 60.00% 0.10 4.22 3.6 0.36 UBC849 1600-155 10 5 5 3 50.00% 0.80 1.82 2.5 2 UBC-823 1960-160 14 6 8 4 57.14% 0.10 4.70 4.9 0.45 UBC-817 2250-275 13 5 8 4 61.53% 0.64 2.96 4.9 3.15 Total 56 23 33 15 2.25 16.34 19.9 8.46 Mean 11.2 4.6 6.6 3 59.07% 0.45 2.27 3.98 1.69 SCoT SCoT -31 2840-135 17 8 9 0 52.94 0.16 6.16 4.76 0.76 SCoT -34 2050-233 18 3 15 0 83.33 0.20 10.55 12.5 2.5 SCoT -13 2090-320 13 6 7 4 53.84 0.65 2.72 3.77 2.45 SCoT -14 1935-310 13 3 10 3 76.92 0.15 4.68 8.10 1.22 SCoT -52 2100-240 9 3 6 3 66.66 0.09 5.14 7.69 0.69 Total 70 23 47 10 1.25 29.25 36.82 7.62 Mean 14 4.6 9.4 2 66.74 0.25 5.85 7.36 1.52 49Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba Cluster analysis The dendrogram generated using UPGMA depends on ISSR and SCoT data revealed that all treatments and control form one cluster (cluster I), only radiated plants with 100NaCl formed a distinct cluster (cluster II) (Fig 8A). The first cluster separated into 2 subclusters. One of them included all other radiated treatments and 100 mM NaCl treatment. On the other subcluster control present in sub-subcluster alone and 50mM NaCl and all putres- cine treatments are presents in other sub-subcluster. This pattern of clustering proved the genetic variability of 10Gy+100mMNaCl genotype from control and other treatments. Principle component analysis (PCA) follows the same pattern of UPGMA dendrogram and demonstrated the genetic diversity of 10Gy+100mMNaCl genotype (Fig 8B). DISCUSSION Fabaceous plants are a good answer for expanding populations, improving human and animal food, and enhancing soil fertility (Castro-Guerrero et al. 2016). Present results show that growth parameters such as stem length, root length, number of leaves, shoot fresh UBC810 UBC818 UBC849 UBC-823 UBC-817 SCoT -31 SCoT -34 SCoT -13 SCoT-14 SCoT -52 Figure 7. ISSR-PCR and SCoT-PCR profiles from osmotic stressed V. faba treated by putrescine and gamma radiation. M: Marker, 1:Con- trol, 2:50mmNaCl, 3:100mM NaCl, 4:5mM Put., 5:5mM Put.+ 50mMNaCl, 6:5mM Put.+ 100mMNaCl, 7:10Gy, 8:10Gy+ 50mMNaCl, 9: 10Gy+ 100mMNaCl. 50 Shaimaa S. Sobieh, Noha Eid Eliwa weight, shoot dry weight, root fresh weight and root dry weight were decreased by salinity stress. These findings concur with those made on several crops by Sadak and Abd Elhamid (2013). Putrescine application or gamma radiation treat- ment of plants were successful in accelerating develop- ment in saline environments. This is in line with the findings of Zhao and Qin (2004), who discovered that exogenous application of putrescine improved root development in barley seedlings under salt stress. Fur- thermore, Khosroshahi and Ashari (2008) demonstrated that strawberry, apricot, peach, and sweet cherry fruits’ soluble solids content, weight loss, and titratable acidity were all improved by the foliar application of polyam- ines, such as putrescine. Many physiological processes and plant response to biotic and abiotic stresses are reg- ulated by substances like putrescine in plants (Alcázar et al. 2010). As they activate or inhibit vegetative growth, blooming, fruiting (Harez and Abbas 2015). The efficiency of metabolic processes in plants can be increased by polyamine compounds, including putrescine. Additionally, the physiological functions of plants are improved because of the roots’ increased ability to absorb nutrients from the soil. These present favorable results of putrescine can be explained by these two factors (Youssef et al. 2007). Moreover, putrescine can accelerate growth, cell division and elongation (El- Bassiouny and Mostafa 2008) by boosting the levels of endogenous growth regulators including auxins, cyto- kinin, and gibberellins while lowering the efficiency and quantity of growth inhibitors like abscisic acid (ABA). A form of electromagnetic wave called gamma irra- diation has a good effect on molecular penetration and can ionize materials by igniting their electrons (UNSC 2000). Ionized cells might be distinguished by the dis- ruption of host DNA that resulted in noticeable chang- es in hereditary features. Because DNA has the capac- ity to repair itself after damage due to the mechanism of proofreading, the perturbation of DNA might be transitory rather than permanent, which would have less severe effects (Ali et al. 2016). Low levels of gam- ma radiation may have physiological effects on plant growth due to an interaction between gamma rays and cell-based compounds that results in the production of free radicals. According to, the effect of low doses can be summed up by the acceleration of cell division, cell expansion, enzyme activity, tolerance against biotic or abiotic stress, and the increase in plant yield. The Table 3. Specific positive amplicons of ISSR and SCoT loci for osmotic stressed V. faba treated by gamma radiation and putrescine. Analysis Treatment Primer Molecular size of positive marker amplicon (bp) ISSR 50mM NaCl UBC-823 1600 100mM NaCl UBC810 950 UBC818 925 UBC849 470 5mMPut+100mMNaCl UBC810 170 UBC-817 1270,1055 10Gy+50mM NaCl UBC849 390,260 10Gy+100mM NaCl UBC810 641 UBC818 188 UBC-823 920 UBC-817 1455 SCoT 5mMPutrescine SCoT -52 1600 10Gy SCoT -52 1430 10Gy+50mM NaCl SCoT -13 2090,885,515 SCoT -52 1294 10Gy+100mM NaCl SCoT -14 712 A B Cl us te r I Cl us te r I I Figure 8. A) UPGMA dendrogram based on ISSR and SCoT mark- ers data. B) Scatter plot of PCA using ISSR and SCoT markers. 51Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba resulted free radicals can alter the primary components of cells (El-Beltagi et al. 2011). According to Aly et al. (2019), utilizing gamma rays increased all growth met- rics in eggplant. Low doses of gamma radiation increase plant height which may be related to the ability of radia- tion to stimulate cell division and other crucial process- es that improve nucleic acid synthesis and activation of RNA or protein synthesis (Asare et al. 2017). Regarding the impact of salinity on the chemi- cal components of the V. faba plant, rising salinity lev- el resulted in a decrease in total soluble sugar. Salinity may inhibit photosynthesis activity and/or increase con- sumption of carbohydrates, according to the reduction in leaf photosynthetic pigments (Hassanein et al., 2009). Additionally, one of the alterations brought by salt and drought stress in plants is proline buildup, which is fre- quently thought to be implicated in stress resistance processes. The stabilization of proteins and membranes against the denaturation impact of excessive concen- trations of salts and other damaging solutes may play a role in maintaining the structure of macromolecules and other organelles (Munns, 2002). With rising salin- ity levels, salt stress caused declines in total nitrogen and increment in proline and free amino acids (Abdelhamid et al. 2013). These results are very similar to those found by Taie et al. (2013). The decrease in protein synthesis and/or the rise in its breakdown can be linked to the reduction in total nitrogen. Strong osmo-protectants known as compatible osmolytes (a group of tiny molecules that includes pol- yamines, glycinebetaine and TSS) help to mitigate the negative consequences of osmotic stress. Putrescine foli- ar spraying treatment resulted in a significant increase in the percentage of total carbohydrates and nitrogen. This increase in growth may be attributable to this sub- stance’s ability to stimulate physiological processes that were improved vegetative growth (El-Bassiouny and Mostafa, 2008). Additionally, putrescine increased the levels of proline, soluble sugars, and amino acids in wheat plants (Hussein et al. 2023). Moreover, putres- cine’s protective action on wheat’s Rubisco protein may be the cause of variance in protein expression in plants developing under water deficiency stress (Hassan et al., 2020). Also, radiation offers protection from the effects of salt stress. One of the key defensive responses of plant cells to gamma irradiation stress is the development of defense systems (Jan et al., 2012). The increase in the content of soluble protein is one of the plant’s defenses against gamma irradiation damages, Hanafy and Ageeb (2018) discovered that leaves produced from irradiated seed- lings with lower doses of gamma rays have higher total protein and proline levels. Additionally, Afrin et al. (2019) discovered that onion bulbs with low gamma ray doses had the highest nitrogen content. According to Jităreanu et al. (2013), the mitot- ic index is the significant biological parameter that indicates the frequency of cell division and meristem growth. The current findings showed that the negative influence of NaCl on mitotic division of tested V. faba meristematic, which was directly related to the salt con- centration. This mitodepressive effect on MI was accom- panied by an increase in chromosomal abnormalities and the induction of micronucleus rate (Souguir et al. 2022). Mitotic depression may be due to decrease in cyc- lin dependent kinases (cdcs) activity (Zhao et al. 2014), accumulation of cells at G1 phase inhibiting DNA syn- thesis or arresting the cell in G2, hindering the cell to enter M phase (Mahfouz and Rayan 2017). Foliar appli- cation of putrescine and irradiation with gamma radia- tion mitigated the harmful effect of NaCl causing signifi- cant increase in MI frequencies with low mitotic abnor- malities percentage as demonstrated before. Previously treatment with putrescine showed enhancement in MI after 6h that supports that polyamine is essential constit- uents of the cell and implicates in cell growth and pro- liferation (Gömürgen et al. 2005). El-Azab et al. (2018) proved that exposure to low levels of gamma radiation may stimulate ROS at a very low rate, which can speed up the passage of the cell cycle from G0 to G1, activating plant cell cycle machinery. The frequencies of the various division stages changed because of treatment with NaCl. Following salt stress, prophase frequency increased consider- ably, whereas gamma radiation exposure significantly increased cell accumulation during metaphase. Putres- cine caused non-significant changes in frequencies of the different phases. These variations in the frequencies of mitosis phases show that NaCl and gamma radia- tion affects the relative length of each phase of division as compared with the control. The prophase accumula- tion proves the toxic strength of a treatment caused by delaying in breakdown of nuclear membrane or delay- ing in chromosome compression because of block- ing dividing cells at Chfr point that inhibits prophase/ metaphase transition (Sobieh et al. 2014). While the accumulation of dividing cells in metaphase may be due to spindle apparatus disruption leading to prevention of metaphase/anaphase transition (Sobieh and Fahmy 2021). Chromosome stickiness was the most frequent form of abnormality seen with NaCl treatments. Chro- mosome stickiness indicates that NaCl has harmful effects that are permanent and cause cell death (Gömür- gen et al. 2005). 52 Shaimaa S. Sobieh, Noha Eid Eliwa Stickiness may be due to changes in nucleosomes formation and/or absent of specific non-histone proteins implied in chromosome organization essential for chro- mosome segregation and chromatid separation (Potapova and Gorbsky 2017). Chromosome bridge/or multibridge are developed due to strong adhesion between chroma- tin fibers which stick sister chromatids at metaphase and hold them together, this strong connection may prevent the correct separation of joined chromatids during mito- sis (Bordin et al. 2023). The formation of spindle distur- bance and multipolarity by gamma radiation may be resulted from inhibition of spindle fiber formation fol- lowed by the random distribution of the chromosomes in the cytoplasm (Singh and Roy, 2017). Moreover, the presence of ring chromosome could be result from loss of telomeric part of chromosome (Khanna and Sharma 2013). The induction of laggards indicates the clastogenic effect of any treatment causing loss of some genetic mate- rial (Sobieh et al. 2016). Laggards usually form micro- nuclei (El-Azab et al. 2018). As well, micronuclei can be formed when lagging free chromosomes cannot reach to the cell poles in the correct time to be included in the major nucleus (Utsunomiya et al., 2002). Micronu- clei often serve as a marker of chromosomal instability or reflect sensitivity due to single gene polymorphisms as suggested by Luzhna et al. (2013). Direct relationship for MN induction and salt ions shows the mitodepressive effects of higher salt concentrations. It is also an indica- tor of cell division sensitivity to ion level, which may interfere with the cell cycle regulatory machines. Cl− ions can induce variation of genome arrangements and muta- tions (Boyko et al. 2010) The highly positive relationship between gamma radiation and induction of MN could be due to that gamma radiation can produce chromosomal breaks in two chromosomes that tend to reunite forming a MN (Pampalona et al. 2016). Many checkpoints control the cell cycle of eukary- otic cells. Before entering mitosis, cell cycle checkpoints measure the size of the cell, ensure accurate chromosome replication, and ensure chromosome integrity. The meta- phase checkpoint then starts the correct segregation dur- ing mitosis through the mitotic spindle. The reversible phosphorylation of the regulatory protein cyclins is neces- sary for the cell cycle checkpoint. the activity Cdcs fam- ily mediates this phosphorylation (Fouad and Hafez 2018). Therefore, Cdcs are regulatory proteins that regulate tran- scription and restrict cell division in response to undesir- able conditions (Ding et al. 2020). Cdcs are divided into eight types based on the putative cyclin-binding domains. One of them, CDCA, is encoded in V. Faba by the cdc2 gene (Binarova et al. 1998). It has previously been demon- strated that CDCAs are involved in both G1/S and G2/M transitions (Hemerly et al. 1995). To promote MI in Arabi- dopsis, cdc2 expression is upregulated prior to cell division (Hemerly et al. 1993). The reduction of salt’s influence on the cdcs activities and consequently mitotic division was verified by the current data, where there was a consider- able downregulation in cdc2 expression level after salt stress. Reduction in Cdcs and cyclin activity under salt stress was previously reported by Qi and Zhang (2020). In general, the downregulation of cdc genes expression is a stress response (Kitsios and Doonan 2011) leading to cell cycle arrest, prolonged S-phase progression, or delayed entry into mitosis (De Veylder et al. 2007). Previously, treatment of V. faba root tips with bohemine or roscovi- tine (inhibitors of cdc2-k gene) led to characteristic abnor- malities in mitosis leading to prolonged prophase with intact nuclear envelope (Binarova et al. 1998). On the contrary, the alleviation effect of both putrescine and gamma radiation caused highly sig- nificant upregulation in mitotic division rate alongside with cdc2 expression level. The low concentration of NaCl with both putrescine and gamma radiation caused upregulation of the expression level for 2 and 4-folds respectively. This upregulation of cdc2 expression level accompanied with increase in mitotic division is like the results of Fouad and Hafez (2018), who found increase in mitotic division associated with increase in cdc2 expres- sion level in Allim cepa. Two fingerprint markers ISSR and SCoT were used to evaluate the genetic diversity among the eight treatment groups of V. faba and control. Present data show that ISSR and SCoT molecular markers have produced differ- ent patterns of DNA polymorphism and discriminated molecular variations among treated and control V. faba. This reveals the incidence of modifications at the molec- ular level among V. faba plants by different treatment used. Afiah et al. (2016) proved that ISSR was beneficial in realizing the genetic variation among V. faba genotypes and V. faba genotypes under salt stress respectively. The SCoT molecular marker technique is effective in assessing genetic variations between control and stress treated V. faba (Essa et al. 2023) and between control and mutants V. faba caused by gamma irradiation. These variations caused changes at the molecular and phenotypic levels. In Vigna unguiculata the appearance and the disappear- ance of amplicons in the mutant genotypes compared to control proved the impact of gamma irradiation on phe- notypic and genotypic traits (Vanmathi et al. 2021). More- over, gamma irradiation had the capabilities of induction genetic variation in the genotypes of Cowpea varieties, as assessed by RAPD and ISSR markers (Badr et al. 2014). In the same concern ISSR fingerprints differentiate the ama- ranth mutant from Ficha cultivar and K-433 hybrid and 53Physiological, genetical changes and cdc2 gene expression for osmotic stressed Vicia faba showed that the genetic diversity produced may be part of the complex response to the gamma-radiance (Žiarovská et al 2013). Besides, modifications in control and gamma radiated treatments might be produced by inter microsat- ellite length polymorphism (Aly et al. 2019). A comparison of the level of polymorphism and dis- criminating efficacy of ISSR and SCoT showed that each of the two techniques can detect genetic variation among control and treated V. faba. But the results revealed that the ISSR markers were more efficient than SCoT for dif- ferentiating the genetic variation. ISSR markers effec- tively assessed the genetic diversity among the V. faba and produced a wide range of PIC and medium RP value, which indicates the presence of specific alleles in some genotypes, which can assist the differentiation of these genotypes from the others (Serry et al. 2019). PIC and Rp are reported to be better informative factors than MI to describe the discriminative power of a primer to distin- guish various genotypes (Shingote et al. 2019(. Although ISSR was more efficient, both two markers produced high level of polymorphism. Therefore, a combination of ISSR (spanning selected repetitive sequences) and SCoT (targeting the start codon sequences of the DNA) can be considered as best markers for more expressive and dependable investigation of genetic variability as clari- fied by cluster analysis using UPGMA dendrogram and PCA scatter plot. They revealed high genetic variability of 10Gy+100mM NaCl. So, it can be concluded that gamma radiation was an effective means for initiation mutation in V. faba. These changes could be successfully identified by ISSR and SCoT analysis. Therefore, the recent progress in mutation breeding studies in relation with new tech- nologies is quite critical to influence and improve plant breeding programs to overcome the climate changes. CONCLUSION Salinity is a severe problem, which affect plant growth and productivity. Plants have developed highly advanced stress tolerance mechanisms to adapt stresses. The application of putrescine or irradiated plants with gamma radiation were effective in enhancing growth, cell division under saline conditions and alleviating the harmful effect of salinity. ACKNOWLEDGEMENTS The authors are grateful to express their gratitude to the Egyptian Atomic Energy Authority (EAEA) Cairo- Egypt for supporting this research. REFERENCES Abdelhamid MT, Sadak MSH, Schmidhalter URS, EL- Saady M. 2013. 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