The combination of mutation breeding and “in vitro mutagenesis” is called mutagenesis. The source for most breeding material begins with mutations, whether the mutation occurs in modern cultivars, a landrace, a plant accession, a wild related species, or in an unrelated organism. It has been found to make induction and the selection of induced somatic mutations more effectively and regeneration of mutant plants. Mutations in a broad sense include all those heritable changes, which alter the phenotype of an individual. Genetic variation is the starting point of any breeding programme. Genetic variation may already be present in nature, may be obtained after several years of selection. Spontaneous somatic mutations have played an essential role in the speciation and domestication of plants. Unfortunately, the rate of occurrence of spontaneous mutation is too low to satisfy practical breeding needs. Plant breeding using the conventional procedure is time consuming and sometime for a number of plant species (Ehsanpour and Jones 2001). New technology such as tissue culture, gene transformation and mutation breeding technology are possible solutions to improve the productivity of modern agro ecosystem (D. Ramakrishna and T Shasthree 2016; Rudulier et al. 1984). Hence the combined use of mutation induction and in vitro technology is more efficient, because it speeds up the production of mutants as a result of an increased propagation rate and a greater number of generations per unit time and space (Espino 1986; Murpurgo 1997). The principle of in vitro mutagenesis is to device a scheme by which we can induce DNA lesions in a certain population of cells maintained in vitro and allow these cells to divide rapidly so that the repair mechanism introduces minor errors in the nucleotide sequence of the DNA. As a result, such cells have mutations in specific genes and if whole plants were regenerated from such cells, one would obtain mutant plant lines. The mutagens cause various kinds of DNA damage, such as deletion or duplication of nucleotide segments of DNA in the chromosomes. Plant growth regulators and in particular a cytokinin-enriched medium can increase the recovery rate of mutated cells (T. Shasthree et al. 2012). Although the callus maintains its regeneration capacity for a longer period, prolonged subculturing may lead to higher frequency of mutants, especially in higher concentration of 2,4-D (Jureti and Jelaska 1991). Mutagenic agents such as radiations and certain chemicals can be used to induce mutations at a higher frequency and generate genetic variation from which desired mutants may be selected (Stover and Buddenhagen 1986; Van Harten 1998; De Langhe 1987). Proposed mutation techniques as tool for crop improvement. Several researchers suggested the use of mutagenic agent for induction of resistance to several diseases (Panton and Menendez 1972). Gamma irradiation is the main physical mutagen used to induce genetic variation. Novak (1986), Shasthree (2009) described the dose response of tissue-cultured shoot tips to gamma irradiation. Stotzky (1964) reported on the effect of gamma rays on seed germination and seedling survival in the diploid Musa balbisiana. However, a longer incubation period coupled with low concentration is preferred because it decreases damage causing hydrolytic by-products and thus improves the mutagenic efficiency (Walther 1969 and Savin 1968). Among physical mutagens frequently used on plant cell cultures are X-rays, UV- irradiation and γ-rays (CO⁶⁰) after determining the most suitable dosage and period of exposure. Ultraviolet light (UV) is convenient for mutagenesis. UV mutagenesis is important to incubate cells in dark so as to reduce photo- inducible DNA repair. Subjecting the minimum unit of explants to mutagen can be achieved through induction of mutation under in vitro condition of tissue culture and the opportunity for the mutated cells to survive get increased (Broerties et al., 1976). When callus exposed to UV-C the effect of drought stress on growth was lower than unexposed calluses. This could be due to genetic changes or epigenetic changes. The possibility of UV-C response in Medicago calluses for selection to osmatic stress using Poly Ethylene Glycol (PEG) added to MS medium according to diffusion based metho (Girma and Kreig 1992; Dami and Hughes 1997). The effect of UV-C doses on plant cells (e.g. protoplast and cell suspension) has already been tested in content of DNA mutagenesis in Arabidopsis thaliana with increasing UV-C doses, (Danon and Gallois, 1998). UV radiation can affect many aspects of plant process at the physiological and DNA level (Kolb et al. 2001; Woodall and Stewart 1998). Chemical mutagenesis is one of the major approaches for induction of mutation (Kharkwal et al. 2004). Chemical mutagen represents a powerful tool to enhance variability in plants for selection of new cultivars. Ethyl methane sulphonate (EMS) 50 mg/l most commonly used chemical. N-methyl-N-nitro-N-nitrosoguanine (NG), another mutagen, has successfully been used in deriving cell lines resistant to amino acid analogues. Heinz (1973) used methyl methane sulphonate (50 mg/l) and EMS to bring about mutations in sugarcane. EMS and 5-bromo-2- deoxyuridine have been used for broadening the range of alkaloid content of Nicotiana sylvestris. When plants are treated with EMS or a mutating chemical, lots of mutations arise in the DNA, but even these are difficult to detect with molecular marker methods (Bouman and De Klerk 2001).

There are very few reports on in vitro chemical mutagenesis. George and Rao (1980) studied the effect of EMS on cotyledon cultures of mustard plants. Gosh et al. (1984) reported the effect of EMS of legume tissue culture. Mustafa et al., (1991) reported induction of multiple shoots from cotyledon culture of cucumber (Cucumis sativus) by MMS. Most of the mutations recovered in tomato were identical with the already known spontaneous or induced mutations, but some mutations were entirely new e.g., jointless pedicel mutant in tomato which is highly valued for mechanical picking. Murugan and Subramanian (1993), observed variability in progenies in Cowpea varieties upon radiation with gamma rays and the analysis of variance between progeny and within progeny were highly significant (Lawhale 1982; Kharkwal 2001). In order to obtain tetraploid plants from diploid day-lily callus, colchicines has been used (Chen and Geoden-Kallemeyn 1979). For an efficient mutation breeding programme, the knowledge on efficiency of mutagens being used is of basic importance (Singh et al. 1997, Kumar and Narayan 2005). Mutation frequency, mutagenic effectiveness and efficiency were calculated on M₂ generation as per (Konzak et al., 1965). Mahana et al., (1990) observed several chlorophyll and morphological mutants in Cowpea (Cicer arientium) upon treatment with EMS. Relatively higher mutation frequency both for chlorophyll and morphological mutations were observed for EMS and MMS.

There were no reports of in vitro mutagenic studies in Citrullus colocynthis L. In the present investigation the effect of gamma rays on morphogenesis rooting efficiency, caulogenesis and number of shoot production were studied. Several crop plants were subjected to mutation in vitro (both physical and chemical) for desirable characters such as yield, quality, resistance, tolerance (Evolva et al., 1983). These were considerable work on in vitro mutagenesis especially followed gamma-irradiation on seeds, explants callus culture and seedling for their morphological and physiological variations.

Citrullus colocynthis plants were used in the present investigation and seeds were collected from river valleys of Koonoor Warangal, Andhra Pradesh, Basara Nizamabad Telangana State and Bharathidasan University, Thiruchunapally, Tamilnadu, India. The collected plants were maintained in the department green house at Kakatiya University. Leaf, cotyledon, stem and node explants were collected from the green house grown plants and washed thoroughly in running tap water for 5 to 10 min under aseptic conditions. Explants were surface-sterilized by dipping in 0.1% fresh aqueous mercuric chloride (HgCl2) solution for 1 to 2 min and subsequently washed thoroughly with double-distilled water to remove traces of HgCl2. The pH of the medium was adjusted to 5.6 to 5.8 by using either 0.1N NaoH or 0.1N HCl before autoclaving. About 10 ml of the medium was dispensed in each culture tube and sealed with nonabsorbent cotton plugs prior to autoclaving at 121°C for 15 min under 15 psi. Sterilized cotyledon and leaf explants were cultured on MS sterilized medium supplemented with various concentrations of auxins and cytokinine and incubated at 25 ± 1°C under a 16/8-h (light/ dark) photoperiod provided by cool white fluorescent tubes (Crompton India Ltd.) with light intensity of 2,000 lux. After 5 weeks on induction media, explants and callus (EC) were exposed to 5 kR, 10 kR, 15 kR and 20 kR, of gamma radiation (GR) in a gamma cell (60Co source) installed at the instrumentation department, Kakatiya University. After irradiation, EC was transferred to fresh medium. For ethyl methane sulphonate (EMS) treatments, the appropriate amounts of EMS was mixed separately in MS basal medium and dissolved thoroughly for each treatment. The pH of the medium was adjusted to 6.0 EMS, respectively, prior to adding the mutagens. The solutions were filter- sterilized with sterile Millipore 0.45 µm membrane filter in the laminar air flow chamber. The EC was treated with 1, 2, 3, 4 and 5 mM EMS for 30 min. The doses/ concentrations of both physical and chemical mutagens were selected. The treated EC were thoroughly rinsed with sterile MS liquid medium (basal) to rinse out any excess mutagens and blotted dry on sterile tissue paper. Then untreated EC (controls) and treated EC were cultured on above mentioned induction medium for 2 ~ 3 weeks to assess the survival rate of ECs and then plated on maturation medium containing MS salts, 3% (m/v) sucrose and different levels of plant growth regulators (PGRs) such as 0.5 to 2.5 mg/l BAP, 0.5 to 2.0 mg/l 2,4-D, 1.0 to 1.5 mg/l IAA, 1.0 to 2.0 mg/l NAA, 0.5 to 2.0 mg/l TDZ, 1.0 to 2.0 L-glutamic acid, 1.0 to 1.5 kn, 10 to 20% Coconut milk (CM) and 0.7% agar. The observations were made the callus texture, color, shoots and root ratio from treated explants and callus of C. colocynthis. Morphological variations of the regenerated plants during the developmental stages were noted with reference to the control. Cultures were incubated for 10 to 15 d and plantlets with well developed roots were transferred to plastic cups containing autoclaved mixture of sand, red soil, and vermicompost (1:1:1). Each plantlet was covered with a polythene bag and the cups were maintained in controlled temperature and 60% relative humidity. After hardening, well established plants (R₀ plants) were transferred to the pots (40 × 45 cm), and grown to maturity under recommended agricultural practices. R₀ plants produced normal flowers and viable seeds in pods. Each treatment consisted of at least 20 explants and the experiment was repeated thrice. The data was analysised using ANOVA.

Effect of Gamma rays

Optimal level mutagenic agents such as gamma radiation and certain chemicals, like ethyl methane sulphonate (EMS) have been playing an important role in the crop growth, development and enhancement of secondary metabolites in plants. Moreover, lower levels of gamma irradiation and ethyl methane sulphonate (EMS) are reduced regeneration capacity of callus and the higher dose of gamma radiation and EMS was lethal to micropropagated plants of Citurullus colocynthis. So this paper could be helpful for understanding the effect of gamma irradiation and ethyl methane sulphonate in plant system

The Principal Investigator Dr. T. Shasthree is thankful to UGC New Delhi for financial assistance in the form of Major Research Project Vide F. No.: 41-530/2012 (SR) during July 2012- July 2015 for this work.