## 3 Discussion  
Presently, improvement in the yield and quality of medicinal plants is a more important feature than the yield of the crop because of the pressing demand for medicinal plants in various industrial sectors. Since the genetic improvement program of any organism requires wide genetic variability in the population, the approach of induced mutagenesis is used to widen the genetic variation and diversity of medicinal plants so that medicinal plant with an improved yield and active principle can be developed. Improvement of medicinal plants to give them higher yields and more active ingredients through induced mutagenesis has been reported over the last several years, though these represent only 0.2% of the total crop varieties developed through mutation breeding [3]. In our earlier study, _Nigella sativa_ L. was treated with ethyl methane sulfonate, \(\gamma\)-rays, and a combination of treatments to explore mutagen-induced genetic variability using chemical and physical mutagens; this study reported nine lines of mutants [15; 16]. These mutant plants showed significant variability in their growth habits, shapes, and the colors of their leaves; the characteristics of their flowers and seeds; their seed thymoquinone content; and their seed yield [15]. Induced mutagenesis has many advantages because of its easy processing and requirement of little genetic information from the plant. Induced mutagenesis by EMS and \(\gamma\)-rays is currently the most suitable and prevalent method to induce point mutations. This approach has successfully generated mutants of several medicinal plants. Narcotic 'opium poppy' has been genetically converted into non-narcotic'seed poppy' using EMS and \(\gamma\)-rays [17]. A distinct _Catharanthus roseus_ mutant with unique inflorescence (monogenic recessive with leafless inflorescence architecture and increased flower frequency) and salt tolerance features has been developed through chemical mutagenesis [18]. EMS-induced macro-mutants of C. _roseus_ were developed with high root and leaf alkaloids [19]. Mutagenesis has played a crucial role in improving some medicinal plants, such as _Atropa_, _Crocus_, _Papaver_, and _Psoralea_[20]. Eleven mutant lines of _Trigorella foenum-gracum_ L. were identified to have better yield potential and higher diosogenin content by [PERSON] and [PERSON] [21] using EMS and gamma rays. These reports confirm that induced mutations could be used for the genetic improvement of _Nigella sativa_. However, the use of random chemical mutagenesis as a genetic approach has some limitations because of difficulty in identifying the genes involved in the expression of the phenotypic traits of mutants [22]. To understand the phenotypic changes in the morphological and yield mutant lines of _N. sativa_ at the molecular level, a proteomics approach was applied using 2-DE to determine the proteins involved in the expressing phenotypic traits. _Nigella sativa_ contains a large number of interfering metabolites. These metabolites cause streaking of the gel and change the plant's heterogeneity. Analysis of differentially expressed proteins through 2D gels requires that proteins be well separated with no streaking, background staining, or smearing. The phenol-based method was found to be better than other methods in the present study in terms of the quantity of the isolated protein. The quality of the isolated protein from the three methods of protein isolation was measured by separating the protein in a 2D gel system. The greatest numbers of protein spots have been observed in proteins isolated using the phenol-based method. Therefore, the phenol-based method of protein isolation was also used in this study. This method of protein isolation is preferred also over other protein isolation methods because of its high clean-up capacity. This method also hinders the molecular interaction between proteins and other interfering compounds [23]. The IPG strips with pH ranges of 3-6, 3-10, 4-7, and 7-10 were chosen to optimize the maximum resolution of the protein spots.