eChapters

Availability of genetic variability and exploitation of this variability are two basic requirements of successful crop breeding programmes. The presence of genetic variation in a character is a must for any improvement in that character. If all the individuals in a population are identical i.e. no variation is there, selection cannot be done, so no improvement will be done. In case of crops, if variation is not present or variation is very small, variations are created through hybridization, mutation, polyploidy etc. Selection is identification and isolation of desirable plants from a variable population. A population consists of large number of heterogenous individuals with different morphological expressions. This variability is contributed by three factors: 1. Genetic constitution of individual, 2. Environmental effect on the individual and 3. Interaction effect between gene and environment. These three factors contribute towards the phenotypic variability among the individuals of the population. Assessment of magnitude of variability present in a crop species is of utmost importance as it provides the evidence for effective selection. Phenotypic variation in a population arises due to genotypic and environmental effects.

Statistics involves procedures for collection, classification, analysis, interpretation and presentation of numerical data (Gupta, 1997). Statistical investigations involves four stages ie. collection of data, compilation of data, analysis of data and drawing inferences. For any statistical analysis, first step is data collection which constitutes the foundation for statistical analysis. The required data may be obtained either through survey or an experiment. Survey is the process of collecting data from a population but have no control over the factors which may affect the population characteristics under study, whereas in experiment, data is collected while control is exercised over the factors which may affect the population characteristics under study. Experiments always and almost provide better information than the survey but surveys are necessary in natural populations. When data is not available, it is collected by any of the two methods i.e. census or survey method. In census method, data are collected for each and every unit of the population whereas in sample method a representative sample is drawn from the population on the basis of which all the conclusions are drawn about the population from that sample.

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,

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.

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.

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 10^th 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 30^th 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.

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

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.

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.

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.

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.

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.

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.

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).

Ajowan is a profusely branched winter annual herb, seed oil of which is a major source of thymol, being present to extent of 35-60%. Ajowan oil is aromatic, stimulant and carminative.  It possesses antimicrobial activity. Ajowan seeds are employed either alone or in combination with spices and condiments. It is used in pickles, confectionery and beverages.  It is a good remedy for indigestion.  A paste of crushed fruit is applied externally for relieving colic pains.  It is also used in lotions and ointments.

Camphor plant is valued for presence of camphor in its wood and leaves. Camphor is chiefly a pharmaceutical product though it is used in preparation of artificial essential oils like lavender and lavendin.  It is used as incense and in balms of various kinds.  It is employed as a masking agent in perfumery and sometimes used to achieve lift in perfume blends. Redistilled brown oil is used directly in soap perfumes for a masking effect. Oil is a source of safrole which is a starting material for production of various perfumery chemicals. In 13th century, Marco Polo noted that camphor oil was highly valued by Chinese as a medicine, scent, and embalming fluid.

“The way to health is the way to have an aromatic bath and a scented massage everyday”                        Hippocrates

Aromatherapy is the art and science of using fragrant volatile plant oils and herbs including essential oils, for psychological and physical well-being of man by promoting natural healing and health. It is an emerging art of healing through essential oil, which has got therapeutic effect on body, mind and soul.  Essential oils give calmness, emotional and hormonal balance, stress relief and rejuvenation. Medicinal and spiritual properties of scented herbs were noted by ancient Egyptians and Babylonians. Embalming of body of Egyptian Pharaohs was a practice to purify body for afterlife, whereas Egyptian priests used oils and incense as healers. Books were written in ancient Arabia indicating uses and benefits of certain aromas. Perfumed mortar was used by Babylonians to build temples and mosques, for premises cleaning and cleansing aromas. Ancient Greek believed sweet smells were of divine origin and they used to take aromatic bath to drench them in purity. Aromatic bath and scented massage were ways to health for Romans.

Essential oils are products of secondary metabolism and are secreted by specialized cells located in specified parts of most plants. By virtue of certain characteristic properties, they have wide spread use in perfumery, flavouring and medicine. Essential oils are variable mixtures of terpenoids. Since these oils are produced by plants, composition varies with species and variety of plant besides geography, climate and cultivation practices. Since, chemical components are highly reactive, physical and chemical environment to which oil is subjected to during the processes of its extraction, processing and storage have a great influence on final composition. Entire chain of activities from cultivation, extraction, processing, storage to packing has to be perfectly standardized and regulated to assure end product consistency. Being a valuable item of trade, this is important from the point of view of delivering desired activity as well as safety to consumer.

Essential, volatile or ethereal oils are mixtures composed of volatile liquid and solid compounds which vary widely in regard to their composition and boiling points. Plants owe their fragrance to presence of traces of essential oils in different parts. Numerous fragrance materials are present in roots, stems, barks, leaves, flowers, fruits and heartwoods.     
Several processes like distillation, enfleurage, maceration, expression, solvent extraction and fluid extraction are available for extraction of aroma principles. Application of these processes, either singly or in combination depends upon nature of material and essential oil or absolute intended to be recovered.

Though more than 1500 species contain volatile aromatic principles, only around 50 species serve as commercial source of essential oil and aroma-chemicals. Among these, aromatic crops under large-scale cultivation hardly exceeds two dozens. Important aromatic plants of world are classified according to economic part used, growth habit, habitat, crop duration and propagation method besides usual botanical classification.