PERFORMANCE AND MOLECULAR ANALYSIS OF POTATO LINES DEVELOPED FROM GAMMA RAYS AND EMS APPLICATIONS

An experiment was conducted in a greenhouse research station B College of Agricultural Engineering Sciences, University of Baghdad, during the fall season of 2018 with the aim of propagating and initially studying the field performance of 18 and 20 potential potato lines derived from Rivera and Arizona cv. after in vitro exposure of nodal segments to different dosages of gamma rays (0, 10, 20, and 30 Gray) and EMS (0, 10, 20, and 30 mM). Each control cultivar and their derived lines were independently cultured in plastic bags according to the RCBD, with three replications. The results showed that the highest plant height and number of leaves were obtained from Arizona derived lines which gave 60.11 cm and 25.30 leaves.plant -1 in lines 207 and 222, respectively when compared with their control that gave 38.11 cm and 13.67 leaves.plant -1 , respectively. Minitubers diameter, weight, and plant yield were in its highest values in Arizona derived lines 551, 551, and 459 which gave 35.73 mm.minituber -1 , 33.13 g. minituber -1 , and 133.8 g.plant -1 , respectively compared to their control that gave 25.35 mm.minituber, 16.8 g.minituber, and 78.57 g.plant -1 , respectively. The resulted lines were analyzed at the molecular level utilizing the inter simple sequence repeats (ISSR) markers and revealed that lines 69, 10, 68, 102, and 7 were the much distanced from its derived Rivera cultivar and gave 40.7%, 37.1%, 36.8%, 33.3%, and 30.0%, respectively while lines 551, 261, 474, and 254 were the much genetically distanced from their derived cultivar Arizona with genetic distances of 24.1%, 22.6%, 18.8%, and 17.6%, respectively.


INTRODUCTION
Potato (Solanum tuberosum L.) is one of the four major staple food including wheat, rice and corn and plays a vital role in the international food security. Therefore, improving its field performance against several biotic and abiotic stresses is crucial to elevate crop yield's potential. Mutagenesis aims at the disruption or alternation of genes and can be considered a valuable tool of genetic variations that attract plant breeders. Many phenotypic characterizations that contribute to the domestication and improvement of plant species resulted from human selection for novel traits driven from structural and regulatory genes (19). Simakove et al. (21) tested the effectiveness of multiple backcrossing scheme towards the development of commercial potato varieties. They found high efficiency of tolerant genes introgression in terms of resistance to alternaria, heat, and drought stresses. Many studies have shown the significant effect of induced mutation on plants improvement. Chepkoech et al. (8) were able to induce mutation in potato by exposing potato tubers of three commercial varieties to different dosages of gamma rays. They found that the heritability rates of four agronomical traits including plant height, shoot number, tuber number, and tuber weight were high and gave 62.51%, 52.70%, 64.30%, and 63.43%, respectively which indicates the successful induction of mutation with a high chance for the improved traits to be displayed in the future generation. Ulukapi et al. (25) noticed that high dosage of gamma rays significantly decreased some eggplant growth parameters including plant height (13.58 cm) and shoot fresh weight (1705 g.plant -1 ) compared to their control which gave 20.41 cm and 3.021 g.plant -1 , respectively. A single potato mutant with high yield potential was produced by Morshad (16) after they subjected potato callus to different types of chemical mutagens. He was able to produce 1941 variants from the applied treatments but only 331 survived under acclimatization and further 37.16% were able to survive the field condition. True potato seeds (TPS) were treated with Ethyl methanesulfonate (EMS) for different durations to induce mutation by Somalraju et al. (23) in order to develop pre-breeding germplasm. The abnormality percentage of the produced plants was 6.25% ranged from twisted stem, chlorotic and crinkle leaves, purple leaf tip, bushy growth, and slim stem. Although EMS-treated plants tend to produce more tubers per plant but no significant differences were observed between treated and untreated plants in this term. Likewise, no significant differences were noticed in tuber's weight between treated and untreated plants in spite of the higher tuber's weight exhibited in the EMS-treated plants. Khierallah and Jawad (13) Subjected 8 potato varieties to different concentrations of NaCl and found that salinity tolerance significantly differentiated among the investigated genotypes.
Taking all together, the aim of this study was to produce sufficient amount of tubers for future propagation in addition to examine some agronomical characteristics of two potato cultivars grown in the greenhouse after their exposure to different levels of gamma rays and EMS.

MATERIALS AND METHODS Lines development
Nodal segments from both cultivars were in vitro subjected within four dosages of Gamma rays at 0, 10, 20, and 30 Gray and immersed in 4 concentrations of EMS at 0, 10, 20, and 30 mM for two hours to develop potential lines. The total number of plantlets yielded from the developmental treatments were 800 came from (50 nodes * 4 dosages * 2 cultivars * 2 mutagens). The produced lines were further participated in a series of selection criteria to eliminate the ones that exhibit high mortality rates and growth abnormality.

Lines acclimatization
The selected lines were acclimatized prior to their transfer to the field. The acclimatization process was held at the Date Palm Research Unit labs / College of Agricultural Engineering Sciences / University of Baghdad for approximately one month. Plantlets were collected from the test tubes, washed thoroughly with tab water to remove the remaining growth medium, and treated with Beltanol fungicide (Fitoagricola.Co) at 1 ml.l -1 . Afterwards; plantlets were cultured in presterilized peatmoss in 100 ml pots and kept in the incubator at 25°C for two weeks. For the first week, plantlets were watered with half strength MS salts three times a day and for the second week, plantlets were watered with a solution contained balanced N, P, K, and trace elements twice a day. Subsequently, potted plantlets were transfer to the lath house for further hardening for three weeks prior to their final transfer to the greenhouse. Lines field trial Acclimatized plantlets were transferred to 15 kg plastic bags contained 1:3 (peatmoss:soil) mixture and grown in the greenhouse for 100 days to obtain sufficient line tubers. The greenhouse location was at the Research Station B -College of Agricultural Engineering Sciences -University of Baghdad. Plants were transferred to the greenhouse in the 5 th of October, 2018 where all field managements were regularly performed to ensure meeting the crop growing requirements. Potential mutagenic lines and their control cultivars were randomly distributed within each replication in which Arizona and their derived lines accounted for 21 lines while Rivera and their derived lines accounted for 19 lines with 12 plastic bags for each line that represents the experimental unit. The experiment was designed independently for each cultivar and its derived lines according to the Randomized Completely Block Design (RCBD) with three replications and means were compared according to the Least Significant Differences (LSD) test at 5% level of significance. Data collection from a pool sample of 6 randomly selected plants per experimental unit was in regard to: Plant height (cm.plant -1 ), number of stems (stems.plant -1 ), number of leaves (leaves.plant -1 ), shoot dry weight percentage (SDW%), plant yield (g.plant -1 ), minituber average weight (g,minituber -1 ), minitubers number (minitubers.plant -1 ), and minitubers diameter (mm.minituber -1 ).

DNA extraction and molecular analysis
Potato genomic DNA was extracted following the protocol established by Chaudhry et al. (6). Five inter-simple sequence repeats (ISSR) primers were obtained following the publications of Bornet et al. (5) and Mahgoub et al. (14). Genetic distances were calculated according to the following equation: Genetic distance = 1-((2nxy) / (nx+ny)) where (nxy) represents the number of bands sharing the same molecular size while nx represents the total number of band in exhibition A and ny represents the total number of band in exhibition B. The output data were analyzed using the SPSS program to obtain proximity matrix (18).

RESULTS AND DISCUSSIONS:
Three hundred explants from each cultivar (control excluded) were exposed to different dosages of gamma rays and EMS but only 18 lines from Rivera (6%) and 20 lines from Arizona (6.6%) survived the mutagenesis treatments in which their designated names are presented in Table 1. Table 1 Table 2  Nevertheless, 11 Arizona derived lines did not exhibit significant differences in this term. These results were in agreement with the findings of other researchers (1,9,17) that mutagenesis treatment of potatoes showed a significant effect in increasing plant height, number of stems, and number of shoots of produced lines. The exhibited reduction in the SDW% can be attributed to elevated water retention in the generated lines compared to their control as a result of some morphological and physiological modifications in mutants body including changes in the cuticle barrier layer (7) or controlling the stomatal conductance (15) in addition to reducing the rate of transpiration (26).

Lines Yield characteristics
The results in Table 3  The results in Table 3 (2,11) when they found that the genotypes used in their experiments had a significant effect on the yield traits which proven that the selection of suitable genotypes for mutation studies is crucial for plant improvement programs where some genotypes are more applicable for induced mutation comparing with others. Gamma rays have a biological effect on the plant cell which based on its interaction with particular molecules especially water and in result produce free radicals (12). These produced radicals can damage or modify the physical and molecular structure of the cell and therefore differentially affect plant morphology, physiology, anatomy, and biochemistry depending on the used dosages of mutagens (3). According to the morphological and yield characteristics obtained in our results, it was noticed that the two cultivars used in our study varied in their response to mutagensis applications which are in consistence with many other previous reports (2,4,10).

Molecular analysis of developed lines
The highest genetic distances between Rivera derived lines and their control were observed in lines 69, 10, 68, 102, and 7 as presented in Table 4 and gave 40.7%, 37.1%, 36.8%, 33.3%, and 30.0%, respectively while the lowest was recorded between Rivera cultivar and line 4 which gave 18.8%. Moreover, the results in Table 5 (20,24). The results were similar to the findings of Sobieh et al. (22) when they applied different gamma ray dosages on potato and found that the highest genetic distance was 39.9% while the lowest was 3.3%. We noticed that genotypes can play a vital role in responding to different types of mutagens where Rivera showed to be more genetically affected than Arizona in this manner. This was also reported by Bado et al. (4) when they irradiated 6 potato genotypes with gamma rays and found that genotypes had a significant effect on the irradiation sensitivity.