Comparative Studies on Mutagenic Effectiveness and Efficiency in Horsegram [Macrotyloma uniflorum (Lam) Verdc.]

Two popular horsegram cultivars of Southern India viz., PAIYUR 2 and CRIDA1-18R were utilized for mutagenic studies. The seeds of PAIYUR 2 were procured from Tamil Nadu Agricultural University (TNAU), India and CRIDA1-18R from Central Research Institute for Dryland Agriculture, India. The well filled seeds were uniformly dried to a moisture content of 10-12 per cent. The seeds were irradiated with 100 Gy, 200 Gy, 300 Gy and 400 Gy doses of gamma rays (G), electron beam (EB) and its combinations. The irradiation treatments were imposed at Bhabha Atomic Research Center (BARC), Trombay, Mumbai, India. High frequency of mutations was reported at lower doses of EMS (0.2% and 0.3%) in horsegram (Bolbhat et al., 2012). Hence, combination treatments involving four doses of G with 0.3 per cent EMS were chosen to explore the combined effect of physical and chemical mutagens in horsegram. A part of gamma irradiated seeds were soaked in distilled water for 10 hours and the pre-soaked seeds were treated with 0.3 per cent of EMS prepared in sodium phosphate buffer maintaining pH of 7.0 for 4 hours with intermittent shaking.

A total of 34 treatment combinations including control (untreated seeds) constituted M₁ generation. Four replications with 25 seeds each were sown in roll towel method to study the effect of mutagenesis on seedling traits viz., seed germination (%), root length (cm), shoot length (cm) and seedling height (cm). A total of 500 seeds from each treatment were sown in a randomized block design with two replications at Department of Pulses, TNAU during rabi 2018. Observations on survival (%), plant height (cm) on 30^th day and maturity, pollen fertility (%) and seed fertility (%) were recorded. Seeds of individual M₁ plants were harvested separately and forwarded to M₂ generation following plant to progeny row method. Chlorophyll mutants were observed in M₂ population periodically from seedling stage to maturity as suggested by Bolbhat et al., (2012). The chlorophyll mutants were classified according to the pattern developed by Gustaffson (1940) and Blixt (1972). Parameters recorded in M₁ and M₂ generation were utilized to determine the effectiveness and efficiency of mutagenic doses (Konzak et al., 1965).
 
Mutagenic effectiveness
 
Physical mutagen
Chemical mutagen
Combinations
Mutagenic efficiency
Mutation frequency was estimated as percentage of M₁ plant progenies segregating for chlorophyll mutants (Gaul, 1964). The per cent injury, lethality and sterility represent the per cent reduction of plant height at 30^th day, plant survival and seed fertility respectively in M₁ generation.

Several classes of physical and chemical mutagens differ in their efficiency in inducing mutations (Wani 2009) either directly or indirectly affecting cellular components. The mutagenic effect of any ionizing radiation depends upon the amount of energy lost to biological material of its path which is defined as linear energy transfer (LET). Among ionizing radiations, G and EB possess low LET value of around 0.2 KeV µm^-1. (Magori et al.,  2010). Earlier attempts on G led to release of superior mutants but the mutagenic effect of EB remains unexplored in horsegram. Studies on EB would also produce desirable mutations as it exhibit comparatively high relative biological effectiveness (RBE) due to increased absorption dose rate than G and other radiation methods (Zhu et al., 2008). Chemical mutagens have also contributed equally in development of mutants as that of ionizing radiations (Ganapathy et al., 2008). Point mutations i.e., base pair changes are often induced by chemical mutagens which results in altered gene product due to change in amino acid composition (Shu et al., 2012).

The degree of any mutagenic effect can be quantitatively determined by plant injuries in M₁ generation (Table 1). Drastic reduction in seed germination was observed in both varieties with increased dosage rate. The decline may be the result of altered metabolic activities due to enzymatic disturbances involved in seed germination (Kulkarni, 2011). A similar decline in survival percent was recorded at higher mutagenic doses (Kulkarni and Mogle 2013). Seedlings are highly sensitive to mutagenic effect and provide an easy means to measure injury. Traits viz., shoot and root length gradually declined with increased mutagenic dose in all treatments. The maximum reduction was recorded in combination treatment of G+EMS (400 Gy+0.3%). The inhibitory effect had occurred due to interrupted mitotic division leading to reduced formation of cells contributing seedling growth. Of the two seedling traits studied, the mutagenic treatments exerted greater effect on root length rather than shoot length. Girija et al., (2013) reported high percent reduction of root length in cowpea due to cytological changes (anaphasic bridges, laggards, stickiness) occurring in root cell on treatment of seeds with higher mutagenic doses. Vegetative traits viz., plant height at 30^th day and maturity was reduced with increased mutagenic dose in all treatments and the highest reduction was observed at 400 Gy of G+EB combination in both the cultivars. Reduction in plant height can be attributed to slow rate of cell division, altered amylase and peroxidase activity (Cherry and Lessman, 1967). An inverse relationship existed between pollen fertility level and dosage rate. Pollen sterility results due to aberrant genetic and physiological damages which are induced by chromosomal breaks and point mutations (Rana and Swaminathan, 1964). Similar decline in trend was registered for seed fertility with increased mutagenic dosage (Ramya et al., 2014).



Gaul (1964) insisted the use of chlorophyll mutation as markers for exploring the gene action of mutagenic factors. Wide spectrum of chlorophyll deficient mutants was observed in M₂ population which includes: albina, albina-green, striata, chlorina, xantha, viridis, xanthaviridis, maculata and tigrina (Fig 1). Average frequency of total chlorophyll mutants was varying in magnitude from 0.73% to 1.31% in PAIYUR 2 and 0.94% to 1.18% in CRIDA1-18R (Table 2). Among the chlorophyll mutants, chlorina recorded the highest frequency followed by striata, viridis, xantha and albina in both varieties (Fig 2a). The maximum occurrence of chlorina was noticed in combination of G+EMS treatment (44.91% - PAIYUR 2 and 37.55% - CRIDA1-18R). A steady decline in chlorophyll mutants was observed at higher doses in all mutagenic treatments except G in this study. The decrease in mutation frequency at higher doses can be an event of chromosomal aberrations or saturation (Mehraj et al., 1999). EB and its combination with G induced rare mutant types viz., albina-green and maculata in PAIYUR 2. A chlorophyll deficient mutant xanthaviridis was observed at all mutagenic treatments except G+EB combination whereas tigrina (rare type) was observed only at one combination dose (G+EMS:300 Gy+0.3%) in CRIDA1-18R cultivar. Among the four mutagenic treatments, EB induced maximum frequency of chlorophyll mutants followed by combinations in both varieties (Fig 2b). The improved efficiency of EB may be attributed to high absorbed dose rate which can deliver high density free radicals in a shorter time period leading to increased double stranded DNA breaks resulting in high mutagenic effects (Zhu et al., 2008).







 

Knowledge on relative biological effectiveness and efficiency of a mutagen is an essential pre-requisite to determine the recovery of high frequency of desirable mutants (Smith 1972). Mutagenic effectiveness refers to the rate of mutations induced per unit dose of a mutagen (dose or time × concentration), whereas mutagenic efficiency depicts the proportion of mutations as against undesirable biological damages viz., lethality, seedling injury and sterility. The effectiveness ranged from 0.14% (G+EB: 400 Gy) to 1.45% (G+EB:100 Gy) in PAIYUR 2 and 0.15% (G+EB:400 Gy) to 1.74% (G+EB:100 Gy) in CRIDA1-18R (Table 3). Maximum effectiveness was noticed at the lowest concentration of mutagenic dose in single and combination treatments (Shirsat et al., 2010). The lowest dose viz., 100 Gy of G and G+EB can be employed in future mutation breeding programme for obtaining high frequency of desirable mutants. On an average, EB and its combination were found to be the most effective mutagen in both varieties (Fig 3a). Similar results were reported by Joshi-Saha et al.  (2015) in chickpea. A sharp decline in effectiveness was observed at higher doses in all mutagenic treatments studied. The order of mutation rate for effectiveness was: EB (0.63%) and G+EB (0.63%) > G+EMS (0.45%) > G (0.33%) in PAIYUR 2 and G+EB (0.71%) > EB (0.57%) > G+EMS (0.47%) > G (0.42%) in CRIDA1-18R.



The percentage of lethality, injury and sterility increased steadily with high doses of mutagen while the efficiency was found to be declining with higher doses. Similar results were observed by Kavithamni et al., (2008) in soybean. The efficiency with relation to lethality decreased at higher mutagenic doses in all treatments except G in both varieties. Mutagenic doses viz., EB:200 Gy (3.30%) in PAIYUR 2 and G+EB:100 Gy (3.63%) in CRIDA1-18R scored maximum value for efficiency in terms of lethality. The highest efficiency was noticed in G:100 Gy (16.72%) in PAIYUR 2 and G+EMS:100 Gy+0.3% (3.53%) in CRIDA1-18R with reference to injury whereas, EB:200 Gy (13.17%) and G+EB:100 Gy (14.43%) showed improved efficiency for sterility in PAIYUR 2 and CRIDA1-18R respectively. Higher efficiency at lower and moderate doses of mutagenic agent is due to relatively low biological damage, which increases at faster rate on higher doses than the mutation induced (Konzak et al., 1965). EB recorded high mutation rate for sterility in both varieties. With respect to lethality and injury, varied mutagenic efficiency was registered between varieties. EB and G exhibited high mutation rate for lethality and sterility in PAIYUR 2 whereas G+EMS was found to efficient in CRIDA1-18 R (Fig 3b). The order of mutation rate for efficiency is as follows: