Ethyl Methane Sulphonate Induced Genetic Variability For Creating Novel Germplasm In Pepper(Capsicum Annuum CV B12)

This study was carried out to enhance genetic variability in pepper(Capsicum annuum, cv. B12) using ethyl methansulphonate(EMS). Defining the optimal mutagen dose EMS that creates the highest desired mutation frequency is considered as a valuable way in pepper breeding. For this purpose, the current study was divided into 4 parts. The first part was kill curve analysis. It was conducted by treating pepper seeds with eleven EMS concentration, including the control(0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50% v/v) and three different presoaking durations in distilled water [0(T1), 6(T2) and 12 h(T3)]. In addition, seeds were presoaked in water for 6 h then incubated for 12 h at 28°C before treating with the abovementioned EMS concentrations(T4). The effect of EMS on germination percent, cumulative germination percent, seed vigor index and seedling growth characters including height, fresh weight, growth rate and percent of injury were studied. The results showed that except for seedling injury all other tested characters decreased with increasing EMS concentration. Comparing all four presoaking treatments it was found that seed presoaking conditions influence the effectiveness of EMS concentration. There were significant differences among the tested presoaking treatments regarding germination ratio, LD50, seed vigor index, survival ratio, seedling height and weight. LD50 was detected in the cases of T1 and T3 at about 1% EMS, while it was observed at about 0.5% EMS in the cases of T2 and T4. The second part, depending on the kill curve analysis the EMS concentration 0.6% for 12 hours was chosen to mutagenize 2000 seeds for the first generation(M1). During the M1 generation, it was observed that the growth behaviors including plant height, flowering date and number of seeds per first fruit were different in the mutant population than in the wild type plants. In addition one phenotypic mutation(leaf shape and plant architecture) was observed during the M1 generation. The third part, in the M2 generation, during seedling stage, changes in the form of slow growth or chlorophyll defect(i.e., albino, pale green and yellow seedlings) were observed. At the maturity stage, there were three kinds of phenotypic mutations in three different families were characterized. The first observed change was a plant with yellow leaf color with segregation ratio(10:1), the leaves of this plant contained 62.19% less chlorophyll a and 64.06% less chlorophyll b as compared to its wild-type. The second mutation was one dwarf plant with segregation ratio(23:1), a very short stature(6 cm) with compact internodes, while leaves and stem were rough and thick. The third types of mutations occurred in four plants with different leaf shape and plant architecture having a segregation ratio of 4:1. The leaves of these plants were very thick and longer than those of wild type plants. Furthermore, the anatomical observations cleared that the leaf blade section of this mutant plant contained more xylem and collenchyma tissues in the leaf midrib of the mutant plant than wild type. In addition, its leaf blade contained thicker palisade and spongy tissues than wild type. In the fourth part, the yellow leaf color mutation was taken for molecular analysis during the M2 generation. During M2 generation sequence analysis of CaBGL11 gene indicated that there were 2 base pairs changed in the exons part and 2 base pairs in the introns part. Expression analysis indicated that CaBGL11 gene associated with yellow leaf color was mostly down-regulated in the mutant plant without any significant differences between the wild type and mutant plants in leaves and stem. In M3 generation, the yellow green mutations were grown with a segregation ratio 10:1. This yellow green plants contains lower chlorophyll a and b, carotenoids, photosynthesis rate than the wild type. Furthermore, the expression analysis of Ygl-7 gene indicated that the expression level of this gene was very high in the mutant plants leaves while it was very low in the leaves of the wild type plants. Further studies are needed for the Ca BGL11 and Ygl-7 genes function. These three types of observed mutations during M2 generation are important to use it for B12 pepper cultivar improvement.