Abstract: A comparative study of the frequency and spectrum of chlorophyll mutations induced by MMS (0.01, 0.02, 0.03, 0.04 and 0.05%), gamma rays (5, 10, 20, 30 and 40 kR) and their combinations (10 kR+0.01%, 10k R+ 0.02%, 10 kR+0.03%, 10 kR+0.04% and 10 kR+0.05%) in M2 generation was made in 2 varieties of Vicia faba L. viz., minor and major. Six different types of chlorophyll mutants viz., albina, chlorina, maculata, tigrina, virescence and xantha were identified in the treated populations. Chlorophyll mutation frequency was calculated on plant population basis. Frequency of chlorina mutants was highest followed by xantha and other types in both the varieties. Combination treatments in general proved to be most effective followed by MMS and gamma rays in inducing maximum frequency of chlorophyll mutations (Gamma rays + MMS> MMS> Gamma rays). A significant variation in varietal response was observed. The co-efficient of interaction was less than additive, but synergistic effect was also observed.
INTRODUCTION
Chlorophyll mutations are considered as the most dependable indices for evaluating the efficiency of different mutagens in inducing the genetic variability for crop improvement and are also used as genetic markers in basic and applied research. The occurrence of chlorophyll mutations after treatments with physical and chemical mutagens have been reported in several crops (Swaminathan et al., 1962; Prasad and Das, 1980; Sharma and Sharma, 1981; Reddy and Gupta, 1989; Kharkwal, 1998a; Mitra and Bhowmik, 1999; Solanki, 2005). Combined treatments of physical and chemical mutagens have been reported to alter the mutation frequency and spectrum and also result in additive and synergistic effect (Sharma, 1970; Khalatkar and Bhatia, 1975; Gautam et al., 1992; Singh et al., 1999; Wani and Anis, 2004; Bhat et al., 2005). The chlorophyll mutation frequency in M2 generation is the most dependable index for evaluating the genetic effects of mutagenic treatments (Kharkwal, 1998a; Waghmare and Mehra, 2001). Improvement in the frequency and spectrum of mutations in a predictable manner and thereby achieving desired plant characteristics for their direct or indirect exploitation in the breeding programme is an important goal of mutation research. The selection of effective and efficient mutagen(s) is very essential to recover a high frequency and spectrum of desirable mutations (Solanki and Sharma, 1994; Kharkwal, 1998b; Mahapatra, 1983). In the present investigation, the effect of gamma rays and Methylmethane Sulphonate (MMS) applied singly as well as in combination on the frequency and spectrum of chlorophyll mutations was studied in 2 varieties of broad bean, one of the most dominating pulse crop of this sub continent.
MATERIALS AND METHODS
Breeder seeds of 2 broad bean varieties viz., minor and major were obtained from the Genetics Division, IARI, New Delhi. Dry (10-12% moisture content) and healthy seeds of both the varieties were irradiated from a 60Co source at Nuclear Research Division at IARI, New Delhi with a dose of 5, 10, 20, 30 and 40 kR. Another set of presoaked seeds in distilled water for 12 h was treated with MMS at different concentrations (0.01, 0.02, 0.03, 0.04 and 0.05%) prepared in sodium phosphate buffer with 7.0 pH for 6 h with constant intermittent shaking. A portion of seeds irradiated at 10 kR gamma ray doses were also treated with MMS independently at 0.01, 0.02, 0.03, 0.04 and 0.05% for 6 h. A total of 16 treatment combinations (including control) were evaluated separately for each variety in CRBD with 3 replications during the rabi season of 2003-2004 and 2004-2005 at the Agriculture Farm, Aligarh Muslim University, Aligarh. Each treatment consisted of 300 seeds with 100 seeds in each plot of 3x6 m size. The seed-to-seed and row-to-row distance was maintained at 15 and 40 cm, respectively. Each M1 plant was harvested separately and M2 generation was raised from a composite sample of 30 seeds obtained from each M1 harvested plant of a treatment. M2 population was also evaluated in CRBD with 3 replications, each replication plot consisting of 500 seeds with seed-to-seed and row-to-row distance being same as in M1 in both the varieties. The treated as well as control populations were carefully screened for various chlorophyll mutations (Gustaffson, 1940).
The interaction coefficient (K) of each combined treatment was worked out as under (Sharma and Swaminathan, 1969):
Where a and b stand for 2 mutagens and K is a hypothetical interaction coefficient. Any deviation from the unit value of K would reveal either a synergistic effect (K>1) or antagonistic (K<1).
RESULTS AND DISCUSSION
Based on the intensity of pigmentation at the seedling stage, 6 types of chlorophyll mutants viz., albina, chlorina, maculata, tigrina, virescence and xantha were recorded in the segregating M2 plants in both the varieties of broad bean. Most of the chlorophyll mutant types (albina, chlorina, maculata and xantha) were lethal and survived for 25-35 days only. Maculata and tigrina types survived till the maturity, but produced very few seeds only possibly due to very high pollen sterility (75-85%). Significant variation in the spectrum and frequency of different chlorophyll mutations was recorded among the different treatments and also between the 2 varieties ( Table 1). Chlorophyll development seems to be controlled by many genes located on several chromosomes which could be adjacent to centromere and proximal segment of chromosome (Swaminathan, 1964; Goud, 1967). Mutations in these chlorophyll genes are reflected in the M2 and subsequent generations in the form of different types of mutants. In the present study, chlorophyll mutation frequency of all the types has increased linearly with the increase in the dose of the mutagen, with the exception in combination doses where 10 kR+0.04% of MMS showed comparatively more frequency than 10 kR+0.05% of MMS in both the varieties. In case of MMS a slight reduction in chlorophyll mutation frequency was also recorded at 0.03% of MMS. Among the different mutagens used, the maximum frequency was obtained at 0.05% MMS, 40 kR gamma rays and 10 kR+ 0.04% of MMS in both the varieties. However, in case of different types of mutations, the frequency of chlorina, xantha and tigrina was comparatively high than other types in almost all the concentrations of the mutagens. Combination treatment of 10 kR with 0.01, 0.02, 0.03, 0.04 and 0.05% of MMS increased total chlorophyll mutation frequency as well as frequency of different types of chlorophyll mutants as compared to separate treatments of MMS and gamma rays. Among different combination treatments, 10 kR+0.04% of MMS induced maximum frequency of chlorophyll mutations as compared to other combination treatments in both the varieties. It seems that strong mutagens reach their saturation point even at a lower dose in the genotypes having mutable allelic sites and any further increase in the dose does not add to their mutation frequency. It has also been suggested that with the increase in the mutagen dose beyond a certain point, the strong mutagens become more toxic than higher doses of relatively weaker mutagens. Similar observations were made by Kharkwal (1998b) in chickpea and Mitra and Bhowmik (1999) in Nigella sativa.
The occurrence of chlorophyll mutations in the present study showed the predominance of chlorina followed by xantha and other types in both the varieties. The frequency of albina and other types was comparatively more in MMS than in gamma rays, although their highest frequency was obtained in the combination treatments. It is generally believed that ionizing radiations produce a high frequency of albina type, whereas, chemical mutagens produce higher frequency of chlorina and xantha types (Gupta and Yashvir, 1975). Kharkwal (1998a) obtained higher frequency of albina followed by chlorina and xantha among EMS treatments in chickpea. Contrary to this, in the present investigation higher frequency of chlorina followed by xantha and other types were obtained in both the varieties.
Among the mutagens, combination treatments in general induced maximum frequency
of chlorophyll mutations indicating their greater effectiveness, whereas MMS
proved to be more effective than gamma rays. The order of effectiveness was,
therefore, gamma rays + MMS > MMS > Gamma rays in both the varieties of
Vicia faba L. The greater effectiveness of combined treatments in inducing
chlorophyll mutation has been reported by many workers (Sharma, 1970; Gautam
et al., 1992; Singh et al., 1999). Most of the combination treatments
showed less than additive effects.
| Table 1: | Spectrum and frequency of chlorophyll mutations in M2 generation in Vicia faba L. |
| - = Not found | |
However, additive and synergistic effects were also obtained at 10 kR+0.04% of MMS in both the varieties. Similar findings have been reported by several workers (Khalatkar and Bhatia, 1975; Gautam et al., 1992). Prasad and Das (1980) reported that if 2 different mutagens compete for the same site, the effects are completely independent of each other, then the result could be either additive or less than additive. The synergistic effect of combined treatments has been attributed by Sharma (1970) to either (i) the first mutagen treatment makes accessible otherwise non-available sites for reaction to the second mutagen or (ii) premutational lesions induced by the first mutagen became fixed on repair enzymes. Both these pathways should yield a frequency of mutations higher than the total of 2 mutagens. In the present investigation, the induction of high frequency of chlorophyll mutations may be attributed to the method of treatment and efficient scoring and handling of the mutaginized populations. High frequency of different chlorophyll mutations in var. minor as compared to var. major indicated influence of genetic background and response to the different mutagens a finding observed by most of the research workers in several crops. It is therefore, concluded that although the chlorophyll mutations do not have any economic value due to their lethal nature, such a study could be useful in identifying the threshold dose of a mutagen that would increase the genetic variability and number of economically useful mutants in the segregating generations.
ACKNOWLEDGMENT
The authors thank Director, Nuclear Research Division, IARI, New Delhi for irradiation of seeds and Chairman Department of Botany, Aligarh Muslim University, Aligarh, for providing necessary facilities for completion of this research.