Preface   For sustained survival, any living organism in this earth has to improve itself by evolution. One way of sustainability of an organism over centuries is by spontaneous mutation. Artificial/induced mutagenesis is effective method to enlarge genetically controlled variation, considerably within a short period. After discovery of X-rays effect on fruit fly by Muller in 1927, utilizing mutagens in crop improvement programme through induced mutation were more widely adapted by plant breeders. In 1970s and 1980s, plant breeders made substantial contribution in crop improvement through mutation breeding. In classical plant breeding programme, variation is generated by hybridization and selections are made from the resulting segregating generations. Induced mutagenesis can supplement hybridization or replace as a source of variability. Thus, mutation provides the raw material for crop evolution and it provides the fundamental variability required for crop improvement by breeding. A large number of new promising varieties in different crops have successfully been developed world wide using both physical and chemical mutagens. From 1930 to 2012, more than 3200 mutagenic plant varietals have been released as direct mutants and through crosses with mutants. A great deal of information is available in this book regarding the types of mutagens and their effects, procedures for using mutagens for crop improvement, types of mutations (micro and macro) with statistical techniques to handle the mutation population. The subject matter presented in this book will be useful for both undergraduate and post graduate students of agriculture.

Mutations are sudden heritable changes in characteristic of the organism, that is other than due to recombination (Swaminathan, 1983). Mutations occur naturally as spontaneous mutations and induced as spontaneous mutations and induced artificially by treating the biological materials with physical and chemical agents (mutagens). Experimental mutagenesis is effective method to enlarge genetically controlled variation, considerably within a short time. Darwin’s theory of evolution explains that “variation is the mainspring of evolution”. The extent of genetic variability available in the breeding population and the selection techniques determine the efficiency of any breeding programme. In classical plant breeding programme, variation is generated by hybridization and selections are made from the resulting segregating generations. Induced mutagenesis can supplement hybridization or replace as a source of variability. Thus, mutation provides the raw material for crop evolution (Mahadeva and Randerson, 1982) and it provides the fundamental variability required for crop improvement by breeding (Konzak, 1984).

A mutagen is a physical or chemical agent that changes the genetic material (DNA) of an organism and thus increases the frequency of mutations above the natural incidence. Errors during DNA replication, repair and recombination also causes spontaneous mutations naturally. Mutagens may be of chemical, physical or biological origin. The properties of each mutagen vary by causing damage to the DNA, and most often result in replication error.

Biological materials Seeds from self fertilized plants will be obtained from the concerned breeder. Hand picked, uniform sized seeds will be chosen for mutagenic treatments. Gamma irradiation Gamma irradiation will be performed in gamma chamber, exposing the seeds to gamma rays from 60Co source: well filled and hand picked uniform sized seeds with a moisture content of 8.5 per cent will be chosen for irradiation. A sample of say 120 seeds per treatment will be packed in butter paper cover and placed in 100 curie 60Co gamma cell. The treatments will be given for various duration depending on the doses required with the dose rate of say 54.05 rads/sec. Chemical mutagenesis The seeds will be soaked in water for say 6 hours before the chemical treatment. The duration of pre-treatment with water will be determined on preliminary blank experiment. In cowpea genotype Co. Vu. 623, there was about 5-10 per cent radical emergence about 12 hours after soaking in water. There was no radicle emergence before 12 hours water soaking. An initial pre-soaking period of 6 hours was therefore adopted for later treatment with mutagens. The total treatment period will be about 12 hours (6 hours with water and 6 hours with chemical mutagen). The duration of treatment with chemical mutagens will also be based on the half-life period of the mutagen solution.

Experimental mutagenesis is an important unconventional source to produce mutations in high frequencies in cultivated plants. It makes possible the achievement of desired breeding objectives or atleast decisive progress in this direction through selection of mutants. By establishing extensive collection of mutants based on productive varieties excellent sources of valuable material for theoretical studies and for breeding work can be provided. The induced mutagenesis helps in diversifying varieties and in internal rectification. The simultaneous realization of different breeding objectives may be possible through induced mutagenesis especially in leguminous crops. Stubbe (1959) stated that mutation breeding as a modern method of plant breeding has proved its justification. It mutation breeding, basic information is a prerequisite on the type of spectrum of induced mutations and relative effectiveness and efficiency of different mutagens.

Mutation in M2 The output of mutations in M2 and subsequent generations solely depends upon the complex interaction between many factors in the treated organism together with the specific modes of action of particular mutagen. These results are expressed quantitatively by the mutation rate and qualitatively by the mutation spectrum. The proportion of mutated plants to normal ones which is the mutation rate can be computed on the number of mutations per 100 M1 plants (Gustafsson, 1940), number of mutations per 100 M1 spikes (Stadler, 1928), and number of mutants per 100 M2 plants (Gaul, 1960). After comparing the three indices, Gaul (1960) has concluded that mutants per 100 M2 plants provided the best index, as it is proportional to the initial mutation rate and is independent of variations in progeny size and size of mutated sector. In Pisum, Blixt (1966) has confirmed this. Mohan Rao (1972) has reported that in barley, the M2 seedling method determined the total induced mutation frequency. This method is also superior since it accounted for the components of induced mutation frequency viz., the frequency of mutated initials in each spike primordium and the frequency of spike primordia mutated. The qualitative output of mutation in M2 and later generations is expressed as the mutation spectrum which reflects the different frequencies of different mutations. Westergard (1960) found that the alkylating chemicals induced a higher proportion of less drastic mutations such as Viridis compared to extreme mutations such as Albina than those induced by radiations because of their apparent slight effect on chromosomes. Sidorova (1966) found a positive correlation between the spectrum of chlorophyll and the spectrum of morphological mutations.

Macromutations were observed in the present set of experiments, under all the treatments (Table 12-14). As the dosage of gamma rays increased, there was a decrease in their frequency. EB showed less mutation frequency than gamma rays and EMS. But in combination with gamma rays it produced more frequency of macromutations. As the dose increased the frequency also increased. EMS as single treatment produced macromutations like that of gamma rays, but in combination with gamma rays it produced more frequency than gamma rays as individual treatment. However, as the dosage increased the mutation frequency was also reduced. Medium dose combination produced more mutations than higher and lower dose combinations of gamma rays + EMS. On M2 plant basis, gamma rays in combination with EB produced the maximum viable mutation rate (4.04), whereas gamma rays individually induced a maximum rate of 2.23 and EB as a sole mutagen generated a maximum rate of 0.79. But EMS as a single treatment showed 1.69 as viable mutation rate, in combination with gamma rays it generated as much as 2.49. Combination treatment showed a less than synergistic effect.

The induction of mutations in polygenes controlling quantitative characters can be detected by the estimation of mean and variance of progenies from normal looking mutagen treated populations. Induction of micromutations in different crop plants have been reviewed by various investigators like Gregory (1955, 1968) in peanut, Rawlings et al. (1958) in soybean, Oka et al. (1985) in rice, Brock and Latter (1961) in Trifolium, Borojevic (1966) and Scossiroli (1966) in wheat, Gaul (1961a, 1966), Astveit (1966) in barley and Brock (1967) in Arabidopsis. Though more reliable results can be obtained from the study of quantitative variation in the advanced generations than in M2 (Aastveit, 1968, and Scossiroli, 1977), an estimation of the extent of induced genetic variability in quantitative traits in M2 itself will be valuable in providing information for future selection programme. It was therefore considered worthwhile to study the shift of mean values and to gather information on induced genotypic variance heritability and genetic advance for different quantitative traits from families of M1 from different mutagenic treatments.

A detailed treatment on the concepts of mutagenic effectiveness and efficiency was given by Konzak et al. (1965). They proposed the term “effectiveness” as a measure of gene mutations in relation to the dose and “efficiency” as an estimate of the mutation rate in relation to other biological effects induced, such as lethality, injury or sterility. To obtain high efficiency, the mutagenic effect must greatly surpass other effects in the cell, such as chromosomal aberrations, and toxic effects which generally lead to damage

Germination between M1 and M2 had a non-significant relationship in germination between M1 and its corresponding M1 progenies in M2 (Table 27). Probably the reduction in the germination in M1 might be very highly and significantly influenced by the conditions prevailing in germination and physiological disturbances that would be strictly associated in M1 alone. Whereas the survival of the seedlings between M1 and M2 showed a strong positive relationship. The differential behaviour of the mutagens affecting germination and survival was also evident as discussed elsewhere. However, seedling height on 10th day sorted a differential relationship between M1 and M2. It was negative and non-significant. A non-significant relationship was exhibited for seedling height on 30th day between M1 and M2 in all the doses. It may be interpreted that extent of physiological disturbances that occurred in the early stages and the extend of recovery mechanism that operated subsequently may be the major cause for this; not withstanding the haplontic and diplontic selection that operated in the biological system. Further the type of physiological disturbances happened, whether the enzyme systems affected or a substantial amount of toxic substances produced besides the genetical causes have not been studied in the present investigation. Moreover if the reduced growth was due to mitotic arrest in the meristamatic regions of the roots of M1 plants, the disorder may not be completely inherited to its offspring’s (M2 plants), in that sense selection at the cell level damages might occur in the M1 itself and the selection for a cell with normal mitosis and extent of chromosomal damages that will be tolerated alone might be selected and hence the effect in M1 need not be always related with M2; it also depends upon the potency of the cell to recover from the mutagen induced damages. The ability of the cell in general, should be more in M1 than in M2 recover the disorder after several cell generations.

Knowledge on genetic variability of the available population is very essential for any crop improvement programme, as it will positively enhance the efficiency of selection. The variability in quantitative characters increase considerably by treating the biological materials with different mutagenic agents. It is possible to identify mutants for different characteristics which can be useful in further programme. Variability is measured by different statistics viz., phenotypic coefficient of variation (PCV) and genotypic co-efficient of variation (GCV). GCV would be more useful for the assessment of inherent variability as it exhibits the heritable portion only.

Polygenic characters exhibit a wide range of expression but the action of genes may be either additive or non-additive. According to Fisher et al. (1932), Smith (1936) Lush (1945), Panse (1946) and Mather (1949) the genetic advance can be achieved through selection of the heritable variation in only due to additive. So, selection for a particular character based on phenotypic variability without estimating the heritable part of it will not be successful. Heritability estimates along with genetic advance are normally more helpful in predicting the gain under selection than heritability estimates alone (Singh, 1997). Therefore, the heritability enters into every formula connected with breeding methods and many practical decisions are taken considering the heritability value.

The frequency distribution of the populations of M2 and M3 generation for each genotype was extracted according to Panse and Sukhatme (1961). Skewness and Kurtosis for all the characters were calculated as per the standard method. Skewness refers to the symmetry of the frequency distribution. A measure of skewness is obtained as making use of the second and their moments about the mean. It is defined as,

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