Using of Chemical Mutagenesis in Genetic Variation Induction to Drought Tolerance and It’s Effect on DNA Methylation and Gene Expression in Tissue Culture and Field of Maize (Zea mays L.)

Abstract

The study aimed to investigate the chemical mutagen efficiency EMS in inducing genetic variation for drought tolerance and evaluating this at the tissue, cellular, molecular and morphological levels in two maize inbred lines and their single hybrid. Tissue culture study was applied according to the completely randomized design (CRD) with factorial arrangement in ten replications which included the sterilization experiment using 0, 2, 4 and 6% of sodium hypochlorite NaOCl; callus induction experiment by adopting 0, 0.5, 1, 1.5, 2, and 2.5 mg l-1 of 2,4-D; drought tolerance and selection experiment treated with 0, -0.14, -0.36, -0.65, -1.03 and -1.48bar of drought stress; chemical mutagenesis experiment using the concentrations 0, 0.5, 1, 1.5, 2, and 2.5% of EMS and immersion durations 30, 60, 90 and 120 min; EMS effect on drought tolerance using 1.5% of EMS and 0, -0.14, -0.36, -0.65, -1.03 and -1.48bar of drought, in addition to the regeneration experiment which included 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mg l-1 of BAP. Cellular tests were included the examination of chromosomal variations induced by 0, 50, 60 and 70 mM EMS concentrations in the soaked seeds and the next generation seeds (F1) of the investigated genotypes. The seeds germination experiment was conducted according to the completely randomized design (CRD) with factorial arrangement in three replications where the effect of 0, 50, 60 and 70 mM of EMS mutagen was tested. The field study was applied according to RCBD design with split-split plot arrangement in three replications, included three irrigation intervals 7, 9 and 11 days occupied the main plot, while the sub plots contained the three maize genotypes, and the four EMS concentrations (0, 50, 60 and 70 mM) were located in sub-sub plots, and the results are summarized as follows: 1. Tissue culture: Results of the statistical analysis showed highly significant differences between most of the studied factors, as the concentration of 6% NaOCl for duration 35 min. showed the lowest contamination percent (0%). The highest fresh weight of callus was achieved in addition 1.5 mg l-1 of 2,4-D to Murashige and Skoog medium (MS), 0bar PEG, control treatment using the selection method, inbred 18, 1% EMS concentration, 90 min. immersion duration, as well as the interactions of (inbred 18 x 1% EMS), (inbred 18 and of 90 min. duration), (1% EMS and 90 min. duration), (inbred 18 x 1% EMS x 90 min. duration), (1.5% EMS x 90 min. duration x -0.14bar PEG) and (1.5% EMS x -1.03bar PEG) which reached 145.5, 221.57, 1462.1, 222.4, 167.47, 181.21, 160.46, 198.05, 174.02, 192. 39, 211.07, 209.1 and 325.96 mg, respectively. The best regeneration of plantlets resulted in 2 mg l-1 a concentration of BAP. 2. Cytogenetic: Inbred 18 showed the highest mean of cell division in both, soaked and next generation seeds (F1) (168 and 179 cells, respectively). The highest number of abnormal cells exhibited by inbred 5 in the soaked seeds and the hybrid in the next generation seeds (26 cells and 16 cells, respectively). The concentration of 60 mM EMS produced the highest number of divided cells and abnormal cells in the soaked seeds (145.6 and 23.3 cells, respectively). Furthermore, the same concentration gave the highest total mean of divided cells in the next generation seeds (148.3 cells), meanwhile, the 70 mM concentration of EMS showed the highest number of abnormal cells (14.6 cells). The highest chromosomal aberration was observed in the soaked seeds, with a general mean of 0.92, and its highest value (1.09 deviations) exhibited by inbred 18 at 60 mM of EMS. As for the next generation seeds, it reached a lower mean (0.89 deviations), and its highest value (1.6 deviations) was revealed by inbred 18 at 50 mM of EMS. Most of the deviations were in the form of chromosomal conjugations, chromosomal bridges, and lingering chromosomes. 3. Molecular genetic/DNA methylation: The single hybrid achieved the highest number of methylated alleles at drought stress -1.03bar, and inbred 5 was distinguished by showing no methylated alleles under 0, -0.65 and -1.03bar of drought stress, whereas inbred 18 and single hybrid were the least methylated treatments in the tissue samples. In field, inbred 5 has the highest number of methylated alleles at 70 mM EMS and the shorter irrigation interval,. However, no either methylated or unmethylated alleles were detected in inbred 18 that subjected to 70 mM EMS and 7 day irrigation interval, and the single hybrid treated with 60 mM EMS and 7 and 11 days intervals, as well as the b hybrid subjected to 70 mM EMS and 7 days interval. The number of diagnosed unmethylated alleles within the investigated three genomes ranged between 0 and 2 alleles. 4. Molecular genetic/Gene expression: Inbred 5 and the interaction treatments of (inbred 5 x 1.5% EMS), (hybrid x drought stress -1.03bar) and (inbred 18 x 1.5% EMS x drought stress -1.03bar) exhibited outperformed level of Cu/ZnSOD gene expression in the tissue samples by exhibiting 9.83 58.39, 5.85, and 68.52 copies, respectively. The MnSOD gene expression was in its highest levels in the single hybrid and the interaction treatments of (hybrid x 1.5% EMS), (hybrid x drought stress - 1.03bar) and (inbred 5 x 1.5% EMS x drought stress -1.03bar) producing 0.22, 38.35, 18.92 and 171.21 copies, respectively. In field, inbred 18 and the interactions of (inbred 18 x 70 mM), (inbred 18 x 11 days) and (hybrid x 70 mM) achieved the highest level of Cu/ZnSOD expression (39.54, 29.33, 14.34 and 18.88 copies, respectively). As for MnSOD gene expression, inbred 18 and interactions of (inbred 5 x 60 mM), (inbred 18 x interval 11 days) and (hybrid x 70 mM x interval 11 days) achieved the highest level of gene expression reached 98.33, 61.92, 71.84 and 120 .59 copies, respectively. 5. Germination percentage: Results of the statistical analysis showed significant differences within genotype and EMS concentrations. Inbred line 18 and 0 mM were outperformed in germination percentage (86.75 and 100%, respectively). 6. Morphological characterization: Results of the statistical analysis showed significant differences in most of the studied morphological traits. Inbred 5, 60 mM EMS concentration, 9 days irrigation interval, and interactions of (inbred 5 x 60 mM), (inbred 5 x 9 days), (60 mM x 9 days) and (inbred 18 x 60 mM x 9 days) were outperformed in 50% silking days (71.94, 70.48, 72.83, 69.55, 70.91, 68.44 and 67.33 days, respectively). The single hybrid, 0 mM EMS concentration, shorter irrigation interval and the interactions of (hybrid x 0 mM), (hybrid x 7 days interval), (0 mM x 7 days interval) and (hybrid x 0 mM x 7 days interval) were outperformed in most other traits, most notably was the plant height (154.47 153.41, 145.69, 169.56, 163.50, and 161 cm, respectively), number of grains per ear-1 (770.30, 707.20, 720.10, 930.90, 968, 878.20 and 1113.30 grain ear-1, respectively) and plant yield (162.08, 159.56, 164.50, 180.78, 184.58, 176, and 199.67 g plant-1, respectively). Generally, tissue culture of mature embryos could be an effective tool in developing new maize genotypes, furthermore, EMS found to be valuable mutagen in inducing the desired genetic variability via tissue culture and/or seeds soaking.